中华眼镜蛇毒组分联合活化免疫细胞对hu-Balb/c-nu-KG1a白血病的治疗作用
|
摘要
研究背景与目的
急性髓系白血病(acute myeloblastic leukemia, AML)是一种以成髓细胞失控性增殖为特征的危及生命的恶性疾病,AML的临床进展和预后与侵袭细胞类型、基因遗传学改变及克隆的生物学特性紧密相关。临床上针对AML治疗的化疗药物或靶向药物虽然能够提高完全缓解率(CR),但基于AML中存在具有自我更新和增殖的干细胞群,故患者体内有可能存在无法清除的白血病微小克隆而导致缓解后复发。因此,寻找有效治疗AML的天然或合成药物已经成为肿瘤研究者研究的目标之一。
AML的发生历经了免疫监视、相互对抗和免疫逃逸的免疫选择和编辑过程,机体的免疫细胞无法对其进行杀伤。NKG2D是免疫细胞表面的激活性受体,在NK细胞、NK／T细胞、Tγδ细胞、DC细胞、IDC细胞等多种细胞表面表达。这些免疫细胞可以通过NKG2D分子识别肿瘤细胞表面的相应NKG2D配体而被激活,表达TRAIL、Fas L、颗粒酶、穿孔素,从而诱导靶细胞凋亡或直接杀伤,产生有效的抗肿瘤免疫应答。研究证实,多种药物具有上调肿瘤细胞表面的NKG2D配体表达的作用,因而提高肿瘤细胞被NK细胞杀伤的敏感性,对肿瘤细胞进行有效清除。
蛇毒毒素在肿瘤防治方面具有良好的效果。研究发现,蛇毒毒素对人卵巢癌、膀胱癌、侵袭性前列腺癌、急性早幼粒白血病等均具有抑制作用,作用机制与调节肿瘤细胞凋亡相关基因表达有关。本实验所用中华眼镜蛇毒组分(Naja Naja Actra Venom Component, NNAVC)即是从中华眼镜蛇毒液中提取的具有抗肿瘤作用的成分。
本研究以人急性髓系白血病细胞株KG1a细胞为研究对象,以NNAVC和活化免疫细胞为干预因素,探讨：①NNAVC与活化免疫细胞联合对白血病模型鼠体内实验研究；②NNAVC对KG1a细胞增殖抑制及凋亡诱导作用；③NNAVC诱导KG1a细胞的相关分子机制；④NNAVC与活化免疫细胞联合对KGla细胞的杀伤作用及二者共同作用的关联性。
本研究通过体内外实验对NNAVC在AML治疗方面的作用进行初步探讨,为寻求难治性复发性白血病的治疗策略提供理论和实验依据。
方法
第一章NNAVC单独及与活化免疫细胞联合对白血病模型鼠体内实验研究
选用Balb/c-nu小鼠为实验动物,尾静脉注射KG1a细胞建立Balb/c-nu小鼠白血病模型,以外周血及骨髓细胞形态学、荷白血病细胞小鼠骨髓原代细胞培养、FISH检测Y染色体、膜抗原检测、免疫组化检测人特异性抗原等证明白血病模型的成功建立；选用正常Balb/c-nu小鼠,腹腔注射不同剂量的NNAVC,以小鼠体重、行为变化及组织病理切片评价NNAVC的毒副作用；制备KG1a白血病模型,观察NNAVC单独及与活化免疫细胞联合对白血病模型生存时间的影响。
第二章NNAVC对KG1a细胞增殖抑制及凋亡诱导作用
用台盼蓝拒染法测定NNAVC对KG1a细胞生长曲线的影响；倒置显微镜下观察NNAVC作用前后KG1a细胞形态变化；以MTT法测定NNAVC处理后KG1a细胞的增殖活性,并计算半数抑制浓度(IC50)；甲基纤维素半固体培养法检测NNAVC对KG1a细胞体外克隆形成能力的影响；PI染色流式细胞仪检测细胞周期分布；采用Annexin V/PI双标记,荧光显微镜观察细胞凋亡形态,流式检测仪检测NNAVC诱导KG1a细胞的凋亡率；DNA Ladder实验对NNAVC能够诱导KG1a细胞凋亡进行验证。
第三章NNAVC诱导KGla细胞的相关分子机制
以RT-PCR方法检测凋亡通路调节分子Bcl-2、Bcl-xl、Bax、Bak、Bad、Bid、PUMA、NOXA、p53、NF-kB的mRNA表达水平；用人细胞色素C ELISA检测试剂盒检测NNAVC处理后KG1a细胞内细胞色素C的蛋白表达水平；以分光光度法检测Caspase-8、Caspase-9、Caspase-3蛋白表达；以流式细胞术检测TRAIL死亡受体和诱骗受体蛋白表达。
第四章NNAVC与活化免疫细胞联合对KGla细胞的杀伤作用
用淋巴细胞分离液分离健康供者外周血单个核细胞(PBMC),体外用细胞因子刺激培养活化免疫细胞；CCK-8法检测不同浓度NNAVC对’PBMC和活化免疫细胞的细胞毒作用；采用不同健康供者来源的活化免疫细胞序贯杀伤KG1a细胞,LDH杀伤活性检测试剂盒测定KG1a细胞经不同次数活化免疫细胞诱导杀伤作用后,对不同效靶比时活化免疫细胞杀伤敏感性的变化；LDH释放法检测NNAVC联合活化免疫细胞对KG1A细胞的细胞毒性作用；甲基纤维素半固体培养法检测NNAVC单独及与活化免疫细胞联合对KG1A细胞体外克隆形成能力的影响；以流式细胞术检测NNAVC作用后KG1a细胞表面NKG2D配体表达的变化。
结果
第一章NNAVC单独及与活化免疫细胞联合对白血病模型鼠体内实验研究
1.1 KGla细胞白血病模型的鉴定
1.1.1白血病模型的细胞形态：
根据每周一次外周血涂片分析,小鼠经尾静脉注射KG1a细胞后4-5周开始在外周血发现白血病细胞,形态与KG1a细胞具有高度一致性。随着时间的延长,白血病细胞数量逐渐增多。
1.1.2来源于白血病模型小鼠骨髓细胞的原代培养
来源于白血病模型小鼠骨髓细胞培养3-4周后开始进入对数生长期,细胞形态与KG1a细胞相似,可进行稳定传代。
1.1.3 KG1a细胞白血病模型小鼠骨髓培养细胞表面标志
流式细胞仪检测正常小鼠骨髓细胞、KG1a细胞和白血病模型小鼠骨髓培养细胞的CD34和CD38抗原表达情况。正常小鼠骨髓细胞群中几乎无法检测到此两种抗原(CD34-CD38-为99.76%)；KG1a细胞CD34+CD38-的表达率为32.74%,CD34+CD38+的表达率为66.87%；白血病模型小鼠骨髓培养细胞CD34+CD38-的表达率为83.06%,CD34+CD38+的表达率为16.72%。以上结果提示模型组小鼠骨髓内有人白血病细胞存在,且浸润的KG1a细胞大部分处于干细胞水平,应具有较强致病力,证明KGla细胞白血病模型建立成功。
1.1.4白血病模型小鼠骨髓培养细胞染色体检测
荧光原位杂交(FISH)实验结果显示,正常小鼠的骨髓细胞内无法检测到Y染色体,KG1a细胞内性染色体为XY,来源于白血病模型小鼠骨髓的培养细胞内性染色体也为XY。本实验所用小鼠均为雌性,而KG1a细胞是从一位男性AML患者体内获得,因此,以上结果提示模型小鼠体内已经存在人KG1a细胞。
1.1.5白血病模型小鼠多器官浸润检测
根据病理切片HE染色和免疫组化检测,观察白血病模型小鼠体内多脏器的白血病细胞浸润情况。结果显示,模型小鼠肝脏、脾脏、肺脏、肾脏等器官均有白血病细胞存在,其中肝脏的浸润情况最为严重；免疫组化检测发现,浸润的白血病细胞表面表达人特异性CD34和CD45抗原,说明模型小鼠体内的白血病细胞是实验操作获得的人源性细胞,而非小鼠自发产生的。
1.2 NNAVC对Balb/c-nu小鼠的毒性作用
1.2.1小鼠生存质量
在注射NNAVC期间,各组小鼠精神状态良好、饮食活动正常,无明显躁动、惊恐不安、萎靡不振、拒食等现象,注射部位皮肤无红肿破溃或感染化脓表现,实验时间内各组小鼠无死亡。
1.2.2 NNAVC对小鼠体重及脏器质量的影响
观察结果显示,各组小鼠在实验前后体重变化差异有显著性意义(F=15.624,P=0.002)；不同处理组间小鼠体重的差异无显著性意义(F=0.361,P=0.704)：NNAVC作用10天后,各药物组小鼠体重均较给药前有所增加,但与阴性对照组相比,体重变化无显著性差异(P=0.425,P=0.654)。
根据公式计算各组小鼠心脏、肝脏、肾脏、肺脏、脾脏的脏／体系数,结果表明,NNAVC高、低剂量组的各组脏／体系数与阴性对照组相比差异无统计学意义(P=0.124,P=0.435,P=0.352,P=0.403,P=0.663)。脏／体系数是反映药物对动物脏器毒性作用的指标之一,因此,本实验结果提示此两种剂量的NNAVC对正常Balb/c-nu小鼠的体重和脏器质量无明显毒性作用。
1.2.3组织病理学变化
光镜下观察,药物组与对照组相比,各组织器官无明显病理学改变。
1.3. NNAVC和活化免疫细胞对白血病模型生存率及生存时间的影响
1.3.1小鼠生存质量
制备白血病模型期间,各组小鼠精神状态良好、饮食活动正常,注射部位皮肤及周围组织无炎症感染表现。观察小鼠生存期期间发现,死亡前一周左右小鼠出现饮食饮水量下降、活动减少、萎靡不振等现象,部分小鼠体表有红色斑点出现(见于面部、背部、后肢或臀部,数量一般为1-2个,小鼠体温正常,无创伤感染表现),体重持续下降,一周内死亡
1.3.2各组KG1a细胞白血病模型小鼠生存时间观察
观察接种KG1a细胞后各组小鼠生存时间：阴性对照组小鼠在接种细胞后52天内全部死亡,其中位生存时间是50(34,66)天；NNAVC低剂量(0.04mg/kg)组和高剂量(0.08mg/kg)组的中位生存时间分别是43(41,45)天和61(35,87)天；活化免疫细胞组小鼠中位生存时间为52(38,66)天；联合组(NNAVC高剂量与活化免疫细胞联合)小鼠中位生存时间为71(58,84)天；阳性对照组(柔红霉素2.5mg/kg)小鼠中位生存时间为53(39,67)天。除联合组小鼠生存期显著高于阴性对照组(P=0.025)外,其余各处理组生存期与阴性对照组相比均无显著性差异(P=0.277,P=0.197,P=0.341,P=0.197)；NNAVC低剂量(0.04mg/kg)组小鼠生存时间低于高剂量组,二者比较差异有显著性意义(P=0.025)；联合组与NNAVC高剂量(0.08mg/kg)组小鼠的存活时间比较差异无统计学意义(P=0.110),但与活化免疫细胞组比较差异有显著性(P=0.025)。以上结果表明,白血病小鼠生存期随着NNAVC药物剂量增加而延长,而活化免疫细胞与高剂量NNAVC联合应用的疗效优于二者各自单独作用的效果。
1.3.3 NNAVC和活化免疫细胞对白血病模型小鼠脏器质量的影响
各组白血病小鼠经处理后,至生存期结束时各器官脏／体系数变化数据显不,NNAVC低剂量组心脏系数与正常对照组相比较差异有显著性意义(P=0.001)；各组肝脏系数无明显变化(F=2.176,P=0.108)；阴性对照组、NNAVC低剂量组及高剂量组小鼠脾脏系数与正常对照组比较差异有显著性意义(P=0.000,P=0.000,P=0.013)；阳性对照组的肺脏系数和肾脏系数均有增高趋势,与正常对照组相比差异有统计学意义(P=0.000,P=0.011),活化免疫细胞组肾脏系数与正常对照组相比差异也有统计学意义(P=0.009),其余各组肺脏系数和肾脏系数差异无显著性(P>0.05)。而与阴性对照组(即白血病模型组)比较,活化免疫细胞组、联合组及阳性对照组的脾脏系数均升高,差异有统计学意义(P=0.000,P=0.000,P=0.000)；活化免疫细胞组和阳性对照组的肾脏系数与阴性对照组相比差异有显著性意义(P=0.008,P=0.009)。
第二章NNAVC对KG1a细胞增殖抑制及凋亡诱导作用
2.1 NNAVC作用后KG1a细胞生长曲线的变化
台盼蓝拒染法结果显示,低浓度NNAVC (0.625μg/ml)组KG1a细胞的生长曲线斜率明显低于对照组,表现出有效的增殖抑制作用；药物终浓度达1.25μg/ml时,细胞增殖相当缓慢,生长曲线趋于平整；当NNAVC浓度≥2.5 u g/ml时,KG1a细胞在24h内几乎全部死亡。提示NNAVC可以有效抑制KG1a细胞的生长增殖,并且随着其浓度的增加、作用时间的延长,增殖抑制作用逐渐增强,抑制效应具时间和剂量依赖性。
2.2 Zeiss倒置显微镜进行细胞形态学观察结果
显微镜镜下观察,KG1a细胞加药前状态良好,细胞壁完整、光滑透亮、胞浆均匀、细胞形态规则。加入NNAVC作用后连续动态观察24h,结果显示KG1a细胞作用6h后开始出现细胞壁不完整、细胞形状不规则、胞浆颗粒增多等改变,随着NNAVC作用时间的延长,发生上述形态改变的细胞数量逐渐增多。至NNAVC作用24h后,可以看到明显的细胞死亡,细胞质空泡、固缩、细胞破碎等形态学变化。
2.3 NNAVC对KG1a细胞的细胞毒作用
细胞毒性实验结果显示,NNAVC不同作用浓度与作用时间之间有交互效应(F=11.868,P=0.000)；不同NNAVC浓度对KG1a细胞的抑制率之间比较差异有显著性意义(F=934.427,P=0.000)；两个作用时间NNAVC刘‘KG1a细胞的抑制率比较有显著性差异(F=1074.672,P=0.000)。
NNAVC在0.6μg/ml至1.2μg/ml的浓度范围内,在相同作用时间时,对KG1a细胞的生长抑制作用具有递增趋势,差异比较有统计学意义(F=428.725,P=0.000；F=550.172,P=0.000)；在相同浓度条件下,NNAVC处理KG1a细胞在24h与48h时间点的抑制率具有显著性差异(t=-11.549,P=0.000；t=-14.190,P=0.000；t=-13.219,P=0.000；t=-13.194,P=0.000；t=-10.218,P=0.001；t=-15.272,P=0.000；t=-10.436,P=0.000；),抑制率随着作用时间的延长而增加。说明NNAVC对KG1a细胞的增殖抑制作用具有剂量依赖性和时间依赖性。
NNAVC对KG1a细胞IC50浓度在24h为0.8806(0.8654-0.8959)μg/ml,48h为0.7037(0.6875-0.7187)μg/ml.
2.4 NNAVC对KG1a细胞集落形成能力的影响
用甲基纤维素半固体培养体系进行培养,KG1a细胞接种后第1-2天为分散的单个细胞,第7天即可见克隆形成,7-14天之间集落数量不断增多。各组细胞的集落形态：正常KG1a细胞形成的集落数量多,呈密集型集落；NNAVC作用后KG1a细胞形成的集落数量明显减少,细胞团内有死细胞出现,高剂量组(NNAVC 0.8μg/ml)集落较为松散,细胞碎片较多。
分别对各组细胞在培养7天及14天后克隆形成数量进行统计,结果：NNAVC作用浓度与克隆培养时间之间有交互效应(F=70.623,P=0.000)；不同处理组之间克隆形成数量差别有统计学意义(F=204.351,P=0.000)；各组细胞培养不同时间所形成的克隆数量差异有显著性意义(F=305.877,P=0.000)。
各组细胞在培养14天后的克隆数均显著高于培养7天时的数量,差异比较有统计学意义(t=-13.784,P=0.000；t=-8.615,P=0.000；t=-6.728,P=0.003)；在各观察时间点,不同浓度NNAVC对KG1a细胞集落形成能力均有影响,药物作用后克隆数明显降低,组内比较差异有显著性意义(F=34.357,P=0.001；F=172.368,P=0.000)；NNAVC0.8μg/ml组在各观察时间的克隆形成数量与0.7μg/m1组相比无统计学意义(P=0.411,P=0.185),但前者克隆数低于后者。根据结果分析提示,用药后各药物组细胞的增殖性下降,说明NNAVC可以在一定程度上抑制KG1a细胞的集落形成能力,从而抑制其增殖。
2.5 NNAVC对KG1a细胞生长周期的影响
根据流式检测结果,加入NNAVC作用24 h后,细胞周期变化如下：在细胞周期的不同时段,各组细胞的比例具有显著性差异(F=339.698,P=0.000；F=6.439,P=0.032；F=127.007,P=0.000)；NNAVC 0.7μg/ml组和0.8μg/ml组中处于G0／G1期细胞比例分别上升为58.67%和62.20%,与对照组52.93%相比差异有显著性意义(P=0.032,P=0.002),而处于G2/M期的细胞比例分别下降为5.33%和3.13%,与对照组相比有显著性差异(P=0.000,P=0.000)；0.8μg/ml组S期细胞比例为34.70%,与对照组36.90%相比差异有统计学意义(P=0.012)。NNAVC 0.7μg/ml组和0.8μg/ml组之间处于G0／G1期和S期的细胞比例比较差异无显著性(P=0.048,P=0.079),处于G2／M期细胞比例比较差异有统计学意义(P=0.003)。加药组细胞G1期前均有亚二倍体峰出现。以上结果提示,NNAVC可能通过诱导KG1a细胞生长周期阻滞于G0／G1期,减少G2／M期细胞百分含量而抑制其生长增殖,并且NNAVC还具有促使细胞凋亡的作用。
2.6荧光显微镜观察细胞凋亡
荧光显微镜下观察,正常细胞形态饱满、细胞着色均匀,无亮色小体,说明细胞的胞膜完整,没有凋亡小体出现。NNAVC处理组细胞发生核固缩、破裂,细胞膜出泡,有凋亡小体出现。其细胞凋亡数随药物浓度增高及作用时间的延长而逐渐增多。
2.7流式检测仪检测细胞凋亡率
检测结果发现,KG1a细胞经NNAVC处理后,早期凋亡率和晚期凋亡率均有所增加,其中0.7μg/ml浓度组早期凋亡率和晚期凋亡率分别增加到39.63%和22.23%,与对照组相比差异有统计学意义(P= 0.049, P= 0.034); 0.8μg/ml浓度组早期凋亡率和晚期凋亡率分别增加到40.73%和41.50%,与对照组相比有显著性差异(P=0.040,P=0.016)；而0.6μg/ml浓度组凋亡率均无明显变化；两两比较显示,0.7μg/ml和0.8μg/ml浓度组间早期凋亡率的差异无统计学意义(P=1.000),晚期凋亡率相比差异有显著性意义(P=0.021)。以上结果证实NNAVC具有诱导KG1a细胞凋亡的作用。
2.8 DNA Ladder检测
根据琼脂糖凝胶电泳结果可见,正常对照组没有梯状条带(DNA Ladder)出现,DNA无明显弥散,为大片段的基因组DNA；经NNAVC处理后的KG1a细胞有典型的180-200 bp或其整倍数的梯状条带形成。提示NNAVC可以引起KG1a细胞发生DNA片段化,证实其具有诱导凋亡作用。
第三章NNAVC对KG1a细胞凋亡相关基因和蛋白表达的影响
3.1 NNAVC对KG1a细胞凋亡相关基因mRNA水平表达的影响
RT-PCR法检测各组KG1a细胞内凋亡相关基因表达的变化：NNAVC不同浓度组细胞内Bcl-2、Bcl-xL、Bax、Bid、Bad、p53、PUMA、NOXA和NFκB等基因的表达差异有统计学意义(F=5.774,P=0.021；F=20.652,P=0.000；F=9.780,P=0.005；F=6.795,P=0.014；F=18.389,P=0.001；F=246.243,P=0.000；F=10.467,P=0.004；F=7.153,P=0.012；F=47.767,P=0.000)；三种不同浓度NNAVC作用后,Bcl-2基因的表达均有下降,与对照组相比有显著性差异(P=0.035,P=0.004, P=0.028),各药物组之间两两比较差异无显著性(P=0.162,P=0.884,P=0.202)；抑凋亡基因的Bcl-xL在药物作用后表达有所上升,并表现出一定的剂量依赖性,其中0.7μg/ml组和0.8μg/ml组的变化幅度与对照组相比有统计学意义(P=0.001,P=0.000),但两浓度组之间差异无显著性(P=0.202)；在0.7μg/ml组和0.8μg/ml组,促凋亡基因Bax表达水平与对照组相比差异有统计学意义(P=0.003,P=0.018),而各组细胞Bak基因表达差异无统计学意义(F=0.406,P=0.753)；正常KG1a细胞几乎不表达抑癌基因p53,在不同浓度NNAVC作用后,细胞内p53的表达水平均得到显著升高(P=0.000,P=0.000,P=0.000),p53基因相关的PUMA和NOXA两种基因的表达变化无明显趋势；在本实验中检测到,NNAVC 0.6μg/ml组和0.7μg/ml组的NFκB表达水平与对照组相比有所下降,统计有显著性差异(P=0.000,P=0.000)。综上所述,NNAVC对KG1a细胞内各促／抑凋亡基因的表达具有不同的调节作用,提示其发挥作用的机制与凋亡密切相关。
3.2 NNAVC对KG1a细胞胞浆内细胞色素C表达影响
结果显示,0.7μg/ml组细胞在NNAVC作用0h-12h间,细胞色素C的表达水平呈急速上升状态,之后表达量明显下调；0.8μg/ml组细胞在药物作用0h-6h间,胞浆内细胞色素C的含量呈上升趋势,在药物作用6h-12h间细胞色素C的含量无明显变化,之后表达逐渐下降,至24h时间点,两药物组细胞色素C的表达水平相近,但仍略高于0h时间点。数据分析：NNAVC不同浓度处理与作用时间两因素存在交互效应(F=51.868,P=0.000)；NNAVC不同浓度及作用时间对各组KGla细胞内细胞色素C蛋白含量的影响均有显著性差异(F=82.331,P=0.001；F=552.286,P=0.000)。在两种NNAVC浓度处理时,在不同作用时间点,细胞色素C含量变化组内比较差异有显著性意义(F=316.021,P=0.001；F=278.700,P=0.000)；NNAVC作用6h时间点,0.7μg/ml组细胞色素C含量低于0.8μg/ml组(P=0.018)；在1 2h和18h时间点,0.7μg/m1组细胞色素C含量均明显高于0.8μg/ml组,差异比较有显著性意义(P=0.001；P=0.026)。
3.3分光光度法检测Caspase-9蛋白表达
分光光度法检测细胞内caspase-9蛋白表达水平的变化：NNAVC不同浓度处理与作用时间两因素存在交互效应(F=23.751,P=0.000)；NNAVC不同浓度处理后KG1a细胞内caspase-9蛋白含量有显著性差异(F=23.982,P=0.001)；作用时间对各组KGla细胞内caspase-9蛋白含量的影响有显著性差异(F=180.774,P=0.000)。在NNAVC作用0h-12h之间,caspase-9蛋白表达水平随作用时间的延长而升高,12h后缓慢下降；NNAVC对KG1a细胞内caspase-9蛋白表达水平的影响同样具有浓度依赖性。结果提示在NNAVC通过激活caspase-9诱导KG1a细胞凋亡。
3.4 NNAVC对KG1a细胞表面TRAIL死亡受体和诱骗受体表达的影响
死亡受体DR4在不同浓度NNAVC作用后,表达率随着药物浓度的增加而减少,组间比较差异有显著性意义(F=170.107,P=0.000),各浓度组组间两两比较差异有统计学意义(P≤0.005)；死亡受体DR5在0.6μg/ml和0.7μg/ml浓度时表达有所升高,差异与对照组相比有统计学意义(P=0.000；P=0.000)；诱骗受体DcR1在各组细胞的表达率比较差异有显著性意义(F=37.002,P=0.000),0.8μg/ml组的表达率下降与其他两浓度组比较差异有显著性(P=0.000,P=0.000)；诱骗受体DcR2在三种NNAVC浓度作用下,表达率随药物浓度的增加而降低,但与对照组相比,0.6μg/ml组和0.7μg/ml组细胞DcR2的表达率是升高的,差异有统计学意义(P=0.000；P=0.000)。
3.5分光光度法检测Caspase-8蛋白表达
不同处理后KGla细胞内caspase-8蛋白表达水平的变化显示：NNAVC不同浓度处理与作用时间两因素存在交互效应(F= 51.653, P=0.000); NNAVC不同浓度处理后KG1a细胞内caspase-8蛋白含量有显著性差异(F=47.400,P=0.000)；作用时间对各组KG1a细胞内caspase-8蛋白含量的影响有显著性差异(F=189.867,P=0.000)。
各浓度组caspase-8蛋白表达均具有由低到高、再逐渐下降的过程,提示NNAVC可以在短时间内使caspase-8蛋白活化,进而发挥诱导凋亡的效应。
3.6 NNAVC对KG1a细胞Caspase-3蛋白表达的影响
NNAVC不同浓度处理与作用时间两因素存在交互效应(F=29.694,P=0.000); NNAVC不同浓度处理后KG1a细胞内caspase-3蛋白含量有显著性差异(F=202.507,P=0.000)；作用时间对各组KGla细胞内caspase-3蛋白含量的影响有显著性差异(F=205.361,P=0.000)。
在同一NNAVC浓度作用下,细胞内caspase-3蛋白表达呈现低—高—低的趋势,不同时间点差异有显著性意义(F=36.236,P=0.020；F=28.489,P=0.025;F= 175.320,P=0.003); NNAVC分别作用12h和24h时间点时,caspase-3蛋白表达水平随药物浓度的增加而上升,差异比较有统计学意义(F=177.938,P=0.000；F=42.728,P=0.000)。结果提示NNAVC通过激活caspase-3诱导KG1a细胞凋亡,caspase-3蛋白在NNAVC作用6h后就被激活,12h表达量最高,活性升高具有剂量依赖性。
第四章NNAVC与活化免疫细胞联合对KG1a细胞的杀伤作用
4.1 NNAVC对PBMC和活化免疫细胞的细胞毒作用
CCK-8检测结果显示,NNAVC不同作用浓度时,PBMC和活化免疫细胞活性的差异无显著性意义(F=4.025,P=0.062)；不同NNAVC浓度对细胞毒性作用的差异有显著性意义(F=4.629,P=0.016)。NNAVC在(0.5-2.0)μg/ml的浓度范围内对PBMC无明显的毒性作用,差异与对照组相比无统计学意义(F=0.626,P=0.618); NNAVC≤1.0μg/ml时对活化免疫细胞无毒性作用,2.0μg/ml NNAVC作用后活化免疫细胞活性的差异有显著性意义(P=0.003)。提示在1.0μg/ml浓度以下,NNAVC对PBMC和活化免疫细胞均无明显的毒性作用。
4.2活化免疫细胞对KG1a细胞的杀伤作用
活化免疫细胞在不同效靶比时对诱导杀伤后的KG1A细胞再次杀伤作用比较LDH杀伤检测试剂盒检测结果：活化免疫细胞对各组靶细胞的杀伤活性结果显示,在不同效靶比时,各组靶细胞之间的差异有显著性意义(F=199.354,P=0.000)；细胞杀伤活性均数分别为50.04%、3.95%、36.59%、23.63%、33.27%和8.35%。不同效靶比之间细胞杀伤率比较差异有统计学意义(F=345.299,P=0.000)；除E组外,活化免疫细胞对其余各组靶细胞的杀伤率随着效靶比的升高而增加,在各效靶比作用下,活化免疫细胞对各靶细胞的杀伤活性组间比较差异有显著性意义(F=25.711,P=0.000；F=53.466,P=0.000；F=43.224,P=0.000；F=135.884,P=0.000)。与正常对照组相比,活化免疫细胞对A-E组细胞的杀伤率总体呈下降趋势,提示经过不同来源活化免疫细胞的杀伤作用后,KG1a细胞对活化免疫细胞的敏感性下降。
4.3 NNAVC联合活化免疫细胞对KG1a细胞的作用
单独应用NNAVC对KG1a细胞的杀伤率为40.61%；单独加入活化免疫细胞,在效靶比为10：1和20：1的情况下,对KG1a细胞的杀伤率分别为34.55%和41.35%,二者比较差异无统计学意义(P=0.414)；NNAVC分别与两种比例的活化免疫细胞联合应用时,对KG1a细胞的杀伤率为56.21%和85.59%,高于这两种干预因素各自单独作用的杀伤率,差异有显著性意义(P=0.018,P=0.000); NNAVC在体系中的作用浓度对两种比例的活化免疫细胞均无明显的细胞毒作用,杀伤率分别为4.35%和1.33%。以上结果提示,NNAVC在对活化免疫细胞无明显毒性作用的浓度时,可以与活化免疫细胞联合杀伤KG1a细胞,作用效果优于二者单独作用,但并未显示二者有协同作用。
4.4 NNAVC作用后的KG1a细胞对活化免疫细胞的敏感性
LDH释放测定法显示结果显示：活化免疫细胞在不同效靶比间对各组靶细胞杀伤率的差异有显著性意义(F=148.593,P=0.000)；各组靶细胞在两种不同的效靶比的活化免疫细胞作用下,细胞活性间的差异有显著性意义(F=8.721,P=0.001)。在效靶比为10：1的情况下,活化免疫细胞对各组靶细胞的杀伤率组间比较差异有统计学意义(F=12.015,P=0.002)；活化免疫细胞在效靶比为20：1时对各组靶细胞杀伤率组间比较无显著性差异(F=1.177,P=0.337)；各组靶细胞在两种效靶比作用下,组内比较有显著性差异(P=0.003,P=0.010,P=0.010,P=0.001)。以上结果说明,活化免疫细胞对各组靶细胞的杀伤活性随着效靶比的升高而增强,NNAVC作用后的KG1a细胞对活化免疫细胞仍有较强的的杀伤敏感性。
4.5 NNAVC联合活化免疫细胞对KG1A细胞体外克隆抑制实验
经过不同处理作用后,KG1a细胞接种后7天所形成的克隆情况：两个处理组克隆形成数量的差异组间比较有显著性意义(F=108.614,P=0.000)；不同NNAVC作用浓度之间克隆形成数量的差异有统计学意义(F=270.741,P=0.000); NNAVC浓度与不同处理方法之间有交互效应(F=9.956,P=0.003)。在相同NNAVC浓度作用后,单用NNAVC对KG1a细胞克隆形成能力的抑制作用明显弱于NNAVC与活化免疫细胞联合组,两处理组克隆形成数量比较差异有统计学意义(t=9.141,P=0.001；t=8.273,P=0.001)；经同样处理因素作用后,细胞克隆形成数量随NNAVC浓度的增加而减少,比较差异有统计学意义(F=58.886,P=0.000：F=426.629,P=0.000)。
经过不同处理后的KG1a细胞接种后14天形成的克隆数量结果显示：在各药物浓度作用后,两个处理组克隆形成数量的差异有显著性意义(F=26.059,P=0.000)；不同NNAVC浓度间克隆形成数量的差异有统计学意义(F=174.366,P=0.000); NNAVC浓度与不同处理方法之间交互效应无显著性意义(F=2.623,P=0.114)。在相同NNAVC浓度作用后,NNAVC和活化免疫细胞联合作用后细胞形成的克隆数量与NNAVC单独作用后细胞形成的克隆数量比较,差异有显著性意义(t=5.822,P=0.004；t=6.554,P=0.003)；在相同处理条件下,各组细胞克隆形成数量随NNAVC浓度的增加而减少,与对照组相比差异有统计学意义(F=49.972,P=0.000；F=167.300,P=0.000)。
NNAVC与活化免疫细胞联合应用后,KG1a细胞形成的克隆数量明显低于NNAVC单独作用组,提示活化免疫细胞可以与NNAVC共同作用杀伤KG1a细胞的克隆形成细胞,使得其克隆数量显著降低。
4.6 NNAVC对KG1a细胞表面NKG2D配体表达的影响
流式细胞仪检测结果显示：不同处理组细胞表面MICA、MICB、ULBP1、ULBP2和ULBP3的表达率组间比较差异有显著性意义(F=3654.504,P=0.000；F=978.748,P=0.000；F=1328.553,P=0.000；F=455.271,P=0.000；F=223.263,P=0.000); 0.6μg/ml的NNAVC作用于KG1a细胞后,ULBP3表达升高,与对照组相比差异有显著性(P=0.003),其余NKG2D配体表达均低于对照组,差异有显著性意义(P=0.000,P=0.000,P=0.000,P=0.000)；0.7μg／ml的NNAVC作用后,KG1a细胞的ULBP1、ULBP2、ULBP3表达增加,差异与对照组相比有统计学意义(P=0.000,P=0.000,P=0.000),MICB表达无明显差异(P=1.000)；0.8μg/ml的NNAVC也可以诱导细胞表面的ULBP1、ULBP2、ULBP3表达增加,与对照组相比差异有统计学意义(P=0.000,P=0.001,P=0.000)；ULBP1-3在三种药物浓度组细胞的表达水平两两比较差异均有显著性(P=0.000)。提示ULBP1-3有可能是NNAVC的调节靶点。
结论
1. NNAVC能够抑制KG1a细胞增殖自我更新能力,使KG1a细胞的生长周期阻滞于G0/G1期,并诱导细胞凋亡。
2. NNAVC能够上调Bax、Bad、p53、NOXA mRNA表达,下调Bcl-2、NF-KBmRNA表达。
3. NNAVC促使KG1a细胞线粒体内细胞色素C释放到胞浆中,激活caspase-9,从而激活caspase-3而引起凋亡效应。
4.在对KG1a细胞有效浓度范围内,对活化免疫细胞无明显毒性作用。
5. NNAVC无法清除的KG1a细胞可以采用活化免疫细胞进行杀伤。
6. NNAVC与活化免疫细胞能够联合应用,同时给予或序贯给予均可有效提高对KG1a细胞的杀伤率,有望最大限度地清除AML细胞。
7. NNAVC (0.08mg/kg)与活化免疫细胞联合作用能够有效延长hu-Balb/c-nu-KG1a白血病(Balb/c裸鼠荷人KG1a细胞白血病模型)动物的生存期。
本研究的创新点
首次发现中华眼镜蛇毒组分与活化免疫细胞可以联合应用于急性髓系白血病的治疗研究,并且中华眼镜蛇毒组分在有效杀伤白血病细胞的同时,对活化的免疫细胞无明显毒副作用。
研究价值
中华眼镜蛇毒组分与活化免疫细胞联合能够更有效地杀伤KG1a细胞,对白血病模型动物具有治疗作用,本研究为临床白血病的治疗、延长患者生存时间提供了新的研究方向。
Background and Objective
Acute myeloblastic leukemia (AML) is a malignant disease that can threaten one's life, characterized by the rapid growth of lymphomyelocyte, and its clinic progression and prognosis have a close connection with invasive cell types, the change of gene genetics and clonal biology. Chemotherapeutics or targeted drugs can improve the complete remission (CR) of AML patients in clinical medicine, but because AML have self-renewal and proliferative stem cell groups,leukemic microclone cannot be removed in patients which possibly leads to the relapse of leukemia. Therefore, one of the goals of cancer research has been, and continues to be, the discovery of natural and synthetic products for AML prevention or treatment.
AML has experienced the immunoselection and editing process of immunosurveillance, confrontation and immune escape, which makes AML cells not being killed by immune cells.NKG2D is the activated receptor in the surface of immunoctye, which can be expressed by many kinds of cells, just like NK cells, NK/T cells, Tγδcells, DC cells, IDC cells. These immunocytes can be activated through the corresponding ligand in the surface of tumor cells recognized by NKG2D molecules, express the molecules,such as TRAIL、FasL, granzyme, porforin, then induce the targeted cell apoptosis or kill them. Many researches have suggested that some drugs can up-regulated the expression of NKG2D ligand in the surface of tumor cells, and then improve the sensitivity of tumor cells killed by NK cells, which leads to effectively remove tumor cells.
Snake venom has the excellent effect on the prevention and therapy of tumor. The research has shown that snake venom has the inhibitory action on human ovary cancer, carcinoma of bladder, invasive prostatic carcinoma, acute promyelocytic leukemia, etc., the mechanisms of which are related to modulate the apoptosis gene expression. Naja Naja Actra Venom Component (NNAVC), isolated from the Naja Naja Actra Venom, was chosen in the experiment because of the effect of its anti-tumor.
Human acute myeloid leukemia(AML) cell line, KG1a cell was chosen as experimental subject, NNAVC and activated immune cells were used, the following would be discussed in the research:①The experimental research of leukemic model treated with NNAVC or NNAVC combined with activated immune cells in mice;②the action of proliferative inhibition and apoptosis induced by NNAVC on KG1a cell;③the molecular mechanisms related to KG1a cell apoptosis induced by NNAVC;④The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells and their synergic mechanism.
The research aimed to discuss the therapeutic action of NNAVC on AML in vivo and in vitro, which will provide the theory and experimental foundation of therapy strategy for refractory and relapsed leukemia.
Methods
Chapter 1 The experimental research of leukemia model treated with NNAVC alone or NNAVE combined with activated immune cells in mice.
The experiments were performed on Balb/c-nu mice weighing 18-22g,and Balb/c-nu mice were injected into KGla cells through vena caudalis to establish Balb/c-nu mice leukemic model, which was assessed by the morphology of peripheral blood and marrow cell, marrow primary cell culture in mice with leukemic cells, Y chromosome by FISH, membrane antigen detection, human specific antigen by immunohistochemisty, and etc. The normal Balb/c-nu mice were injected into different doses of NNAVC through intraperitoneal injection, and then the weight of mice,the change of behavior and pathological section were assessed for the side-effect of NNAVC; the survival times of leukemic model treated with NNAVC or NNAVE combined with activated immune cells were observed after KG1a leukemic model was established. Statistical analysis
The calculation was performed using SPSS 13.0 software package. The data presented as the mean±standard deviation. Comparison in experiments was performed using one-way analysis of variance (ANOVA), independent-samples t-test and repeated measurement of analysis of variance to assess the statistical significance of differences between groups. Differences with P<0.05 were considered to be significant.
Chapter 2 The action of proliferative inhibition and apoptosis induced by NNAVC on KGla cell
Trypan blue exclusion was used to measure the growth curve; of NNAVC on KG1a cells; KG1a cell morphological change could be seen by inverted microscope; MTT assay was used to detect the proliferative activity of KG1a cell after NNAVC treatment, and half inhibitory concentration(IC50) was calculated; the colony forming ability of KGla in vitro was measured by methylcellulose semisolid method; flow cytometry was used to measure the influence of NNAVC on KGla cell cycle; the morphology of apoptotic cells was observed by fluorescence microscope and KGla apoptotic rate was measured by flow cytometry after KGla cell was stained with Annexin V/PI; the apoptosis of KG1a cell was further proved by DNA Ladder.
Chapter 3 The effects of apoptosis-related genes and protein expression of NNAVC against KGla cells
The mRNA expression levels of molecules related to apoptosis, such as Bcl-2,Bcl-xl,Bax,Bak,Bad,Bid,PUMA,NOXA,p53,NF-kB,etc.,were measured by RT-PCR; cytochrome C level was measured according to human cytochrome C ELISA detecting kit; expressions of Caspase-8,Caspase-9 and Caspase-3 were detected by spectrophotometric method; expressions of death receptors and decoy receptors of TRAIL were analyzed by flow cytometry.
Chapter 4 The cytotoxic effect of NNAVC combined with activated immune cells on KGla cells
Peripheral blood mononuclear cells(PBMC) were separated from the healthy donors using lymphocyte separating medium, which were stimulated by cytokine to culture activated immune cells in vitro. The cytotoxic effects of NNAVC with different concentrations on PBMC and activated immune cells were measured by CCK-8 assay; activated immune cells from different healthy donors killed KGla cells by turns, the sensitivity of activated immune cells to kill KGla at different effector-to-target(E:T) cell ratios was detected by LDH cytotoxicity assay after KG1a cells were killed for different times by activated immune cells; The cytotoxic effects of NNAVC combined with activated immune cells on KG1a cells were measured by LDH releasing assay; the colony forming abilities of KGla treated with NNAVC alone or NNAVC combined with activated immune cells in vitro were measured by methylcellulose semisolid method; the expressions of NKG2D ligand in the surface of KG1a were measured by flow cytometry.
Results
Chapter 1 The experimental research of leukemic model treated with NNAVC alone or NNAVE combined with activated immune cells in mice.
1.1 Identification of leukemic model
1.1.1 The cell morphology of leukemic model
Leukemic cells, which were similar to KG1a cells, were found in the peripheral blood after mice were injected into KG1a cells four weeks later according to the peripheral blood film once every week. The amounts of leukemic cells increased gradually with the time going.
1.1.2 The primary culture originated from marrow cells in leukemic model
The primary culture originated from marrow cells in leukemic model developed to exponential phase of growth about three to four weeks later, the cells were similar to KGla cells, could go down to the future generation steadily.
1.1.3 The markers on the surface of culture cell in leukemic cells
The CD34 and CD39 antigen expressions of normal marrow cell, KGla cell and cells in the leukemic model were measured by flow cytometry. The CD34 and CD39 antigen expressions of normal marrow cell could not be measured(CD34-CD38-,99.76%);Those of KGla cell were 32.74% (CD34+CD38-); Those of cells in the leukemic model were 83.06% (CD34+CD38-),16.72% (CD34+CD38+)。The results indicated that the model was successful because human leukemic cells were found in the marrow of leukemic model, and which were in the stem cell stage.
1.1.4 The measurement of marrow culture cell chromosome in model
Y chromosome could not be measured in the normal mice, sex chromosome in KG1a cells was XY, that of cells in the leukemic model was XY as well.The female mice were chosen in the experiment and KG1a cell came from a man with AML, therefore, those findings confirmed human KG1a cell existed in the model.
1.1.5 The measurement of organs affected in the model
The organs affected in the model were measured according to HE staining and immunohistochemistry assay. The research showed that leukemic cells existed in the liver, spleen, lung, kidney and other organs of model mice ,especially liver most affected; The expressions of human specific CD34 and CD45 antigens on the surface of leukemic cells were found, which suggested that the leukemic cells in the model came from human leukemic cell, not from the body of mice spontaneously.
1.2 The toxic effects of NNAVC on Balb/c-nu mice
1.2.1 The survival quality in mice
The mice had a good condition without restlessness, scare, apositia and no death during the experiment.
1.2.2 The changes of body and organ weight in mice
The change of body weight in the experiment had significant deviation (F= 15.624, P=0.002);However, there were no significance between NNAVC-treated groups (F= 0.361, P= 0.704);The body weight in NNAVC-treated groups increased after 10 days, but comparing with negative control,that was no significant(P=0.425, P= 0.654).
There were no significant in organ weight to body weight between high-dose group and low-dose group (P= 0.124, P= 0.435, P= 0.352, P= 0.403, P= 0.663).Organ weight to body weight coefficient was considered to be one of the indexes of toxic effect, so the results indicated that body and organ weight in Balb/c-nu mice treated with NNAVC were not significant.
1.2.3 The change of tissue pathology
Pathological changes of organs were not significant comparing with control in light microscope.
1.3 The effects of survival rate and time of NNAVC and activated immune cells on model mice.
1.3.1 The survival quality of mice
The mice had a good condition with normal diet during the experiment. The mice had abnormal behaviors with descending water-drinking, decreasing activity and etc. One week before death and red speckle appeared on the surface of body partially in the model, their body weight began to reduce till to death in a week.
1.3.2 The survival time of mice in the model
The medium survival time of mice in the negative control was 50 (34,66) days and died after mice were inoculated KGla cells for 52 days; That in NNAVC 0.04mg/kg and 0.08mg/kg were 43(41,45)days和61 (35,87)days; That in activated immune cells alone or combined with NNAVC 0.08mg/kg were 52(38,66) days, 71 (58,84) days, respectively. That in positive control(daunorubicin,2.5mg/kg) was 53 (39,67) days. That in activated immune cells combined with NNAVC 0.08mg/kg was longer than negative control(P=0.025), that in other groups was not significant comparing with negative control (P= 0.277, P=0.197, P= 0.341, P = 0.197); That in NNAVC 0.04mg/kg was significantly less than NNAVC 0.08mg/kg (P=0.025);That was not significant between NNAVC 0.08mg/kg and NNAVC combined with activated immune cells (P= 0.110).The results suggested that the survival time in leukemic mice was longer with the concentration of NNAVC increasing, and NNAVC combined with activated immune cells had a better effect than NNAVC alone or NNAVC combined with activated immune cells alone.
1.3.3 The organs weight of NNAVC and activated immune cells on leukemic model mice
The heart weight to body weight coefficient was significant comparing with control (P=0.001),but the coefficient of liver was not significant (F=2.176, P =0.108);That of spleen in every group(negative, NNAVC low dose and high dose) was significant comparing with control (P=0.000, P=0.000, P= 0.013);That of lung and kidney in the psositive group had a increasing tendencycomparing with control (P= 0.000, P=0.011);That of kidney in activated immune cells-treated group was significant as well (P= 0.009),while other groups (P>0.05).The coefficient of spleen in activated immune cells-treated group, NNAVC combined with activated immune cells, positive control were higher comparing with negative control (P=0.000, P=0.000, P=0.000); The coefficient of kidney in activated immune cells-treated group, positive control were significant comparing with negative control (P=0.008, P=0.009)
Chapter 2 The action of proliferative inhibition and apoptosis induced by NNAVC on KGla cell
2.1 The influence of NNAVC on the growth curve of KGla
Trypan blue exclusion assay showed that the slope of growth curve in the low concentration group(0.625μg/ml)of NNAVC was lower than control group, that suggested NNAVC had obvious inhibition on KG1a cells; the growth of KG1a cells was so slow that the curve was almost flat when the concentration was up to 1.25μg/ml;The KGla almost died out when more than 2.5μg/ml.These results showed that NNAVC could obviously inhibit the proliferation of KGla cells in a time- and dose-dependent manner.
2.2 Morphological view of KG1a cells could be seen by Zeiss inverted microscope
KG1a cells were in good condition with the integrity,smooth and lucency of cellwall; even cytoplasm; regular shape before NNAVC treatment.KGla cells began to emerge bad integrity of cellwall, irregular shape, more particles after the 6th hour of NNAVC, more cells changed with longer action time, when the action time was the 24th hour or longer, the smaller volume ,deformation,vacuole within the cell, cell pyknosis and other morphological changes were seen by inverted microscope.
2.3 The cytotoxic effects of NNAVC on KGla cells
The cytotoxic results showed that there were reciprocal effects existing between the different concentration and time of NNAVC (F= 11.868, P= 0.000); The inhibitory rates of different NNAVC concentrations against KGla cells were statistically significant (F= 934.427, P=0.000); The inhibitory rates of two time groups of NNAVC against KGla cells were also statistically significant (F= 1074.672, P= 0.000)
The growth inhibition of NNAVC against KGla cells in the same action time significantly increased between the concentration of 0.6μg/ml to 1.2μg/ml (F= 428.725, P=0.000; F= 550.172, P=0.000);The inhibitory rates of NNAVC on KG1a cells at the same concentration were significant (t=-11.549, P=0.000; t =-14.190, P= 0.000; t=-13.219, P= 0.000; t=-13.194, P= 0.000; t=-10.218, P=0.001; t=-15.272, P=0.000; t=-10.436, P= 0.000;) at the 24th and 48th hour, and higher inhibitory rate with longer action time. These findings suggested that NNAVC could obviously inhibit the proliferation of KGla cells in a time- and dose-dependent manner.
The half inhibitory concentration(IC5o) of NNAVC against KG1a cells was 0.8806 (0.8654-0.8959)μg/ml at the 24th hour,0.7037 (0.6875-0.7187)μg/ml at the 48th hour。
2.4 The influence of colony-forming ability of NNAVC against KGla cells
Methylcellulose semisolid method was used in the experiment, the KGla cells were dispersed on the 1st day to 2nd day, the colony was began to form on the 7th day and gradually increased on the 7th to 14th day. The morphology of colony in every group :the amount of colony forming was more in the control group, which was intensive colony; the amount of colony forming significantly decreased in the NNAVC-treated group and dead cells existing in the cells, while the colony was less and more cell debris in the high concentration group(0.8μg/ml).
The amounts of colony forming in every group on the 7th and 14th day were statistically performed, and the results showed that the reciprocal effect existed between the NNAVC concentration and culture time (F=70.623, P= 0.000);The amounts of colony forming among different groups were significant(F=204.351, P = 0.000) and the culture time were also significant (F=305.877, P= 0.000)
The amount of colony after the 14th day of culture was more than that on the 7th day (t=-13.784, P=0.000; t=-8.615, P=0.000; t=-6.728, P= 0.003). Different concentrations of NNAVC had an effect on colony-forming ability of KG1a cells in the designated time, and the amount of colony was significantly less inside the group (F= 34.357, P= 0.001; F=172.368, P= 0.000); The amount of colony forming in the group of NNAVC 0.8μg/ml had no significant difference comparing with in the group of NNAVC 0.7μg/ml (P=0.411, P=0.185),but the former was less than latter. These findings indicated that the proliferative ability of KG1a cells was lower in the NNAVC groups, NNAVC could inhibit the colony forming ability of KGla cells in some extent,which caused proliferative inhibition.
2.5 Cell cycle effects of NNAVC on KGla
The proportions of KGla cells at the different stage of cell cycle in every group were significant (F= 339.698, P= 0.000; F=6.439, P= 0.032; F= 127.007, P=0.000);The proportions of cells at the G0/G1 stage at the concentration of 0.7μg/ml and 0.8μg/ml were up to 58.67% and 62.20%,respectively,which was significant comparing with the control group(52.93%,P=0.032, P= 0.002),while the proportion at the G2/M stage decreased to 5.33% and 3.13%,respectively (P= 0.000, P= 0.000).The proportion at the S stage at the concentration of 0.8μg/ml was 34.70% comparing with control group(36.90%,P= 0.012).There were no significant difference at the G0/G1 and S stage between the concentration of 0.7μg/ml group and 0.8μg/ml group (P=0.048, P= 0.079),while the proportion at the G2/M stage was significant (P=0.003),and subdiploid peak ahead of G1 stage appeared in the treated group. The results suggested that NNAVC could induce cell cycle arrest of KGla cells at the G0/G1 stage and reduce the contents at the G2/M stage to inhibit the proliferation.
2.6 The observation of cell apoptosis by fluorescent microscope
KGla cells were in good condition with the integrity of cellwall and no apoptotic body emerged in the control group. The vacuole within the cell, cell pyknosis and rupture, apoptotic body appeared in the NNAVC-treated group, and more apoptotic cells emerged with higher concentrations and longer action time.
2.7The apoptotic rates measurement of KGla cells treated with NNAVC
The early and late apoptotic rates increased after NNAVC treatment, and the early and late apoptotic rates in the 0.7μg/ml concentration group increased to 39.63 % and 22.23%,respectively(P= 0.049, P= 0.034);those in the 0.8μg/ml concentration group were 40.73% and 41.50%(P= 0.040, P= 0.016);while no significance in the 0.6μg/ml concentration group. The early apoptotic rates between the 0.7¨g/ml and 0.8μg/ml concentration groups were not significant(P=1.000),but the late apoptotic rates were significant(P=0.021).The results confirmed that NNAVC Could induce the apoptosis of KG1a cells.
2.8 DNA Ladder measurement
There was no DNA Ladder in the control group,which showed the genomic DNA；DNA Ladder was seen in the NNAVC-treated group,which consisted of 180-200bp or more.These results indicated that NNAVC could lead to the DNA fragment of KG1a cells,that further confirmed the apoptotic induction of NNAVC.
Chapter 3 The effects of apoptosis-related genes and protein expression of NNAVC against KG1a cells
3.1 The effects of apoptosis-related mRNA expression levels of NNAVC against KGla cells
The results showed that the apoptosis-related genes in the different concentration of NNAVC groups,such as Bcl-2,Bcl-xL,Bax,Bid,Bad, p53,PUMA,NOXA and NF-KB,etc.,were significant,respectively(F=5.774,P= 0.021；F=20.652,P=0.000；F=9.780,P=0.005；F=6.795,P=0.014；F= 18.389,P=0.001; F=14.816,P=0.001；F=246.243,P=0.000；F=10.467, P=0.004；F=7.153,P=0.012；F=47.767,P=0.000)；The expression level of Bcl-2 gene descended comparing with control group(P=0.035,P=0.004, P= 0.028),while there were no significance among the groups(P=0.162,P=0.884, P=0.202)；The expression level of Bcl-xL gene rose up in a dose-dependent manner, especially 0.7μg/ml and 0.8μg/ml(P=0.003,P=0.018)；The expression level of Bak gene had no significant deviation in all groups(F=0.406,P=0.753)；The expression level of P53 gene,which was not expressed in normal KG 1 a cell,had obviously risen up (P=0.000,P=0.000,P=0.000),while PUMA and NOXA genes related to P53 had no change; The expression level of NF-κB gene in the concentration of 0.6μg/mland 0.7μg/ml descended (P= 0.000, P= 0.000).Taken together, NNAVC could induce the KGla cells apoptosis by modulating the balance between pro-and anti-apoptosis genes.
3.2 The effects of cytochrome C of NNAVC on KGla cells
The results showed that the expression level of cytochrome C had an up-and-down trend in the concentration of 0.7μg/ml within 12h;while that in the concentration of 0.8μg/ml rose up within 6h,and unchanged within 6h to 12h, then descended within 12h to 24h, at last that was very close between two groups. there were reciprocal effects existing between the different concentration and time of NNAVC (F= 51.868, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of cytochrome C (F= 82.331, P= 0.001;F=552.286, P=0.000); The content of cytochrome C in 0.7μg/ml group less than that in 0.8μg/ml group (P=0.018) at the 6th hour; However, the content of cytochrome C in 0.7μg/ml group more than that in 0.8μg/ml group (P=0.001; P = 0.026) at the 12th and 18th hour.
3.3 The measurement of caspase-9 protein by spectrophotometric method
There were reciprocal effects existing between the different concentration and time of NNAVC (F=23.751, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-9 protein (F= 23.982, P=0.001;F=180.774, P=0.000); The expression level of caspase-9 rose up within 12h and gradually descended after 12h; NNAVC had an effect on expression level of caspase-9 of KGla cells in a concentration-dependent manner. The results suggested that NNAVC could induce the apoptosis of KGla cells by activating caspase-9.
3.4 The effects of death receptors and decoy receptors expression levels of NNAVC on KGla cells
The expression level of death receptor 4(DR4) decreased with the NNAVC concentration increasing,and there were significant among the groups (F=170.107, P=0.000) or between groups (P≤0.005); The expression level of DR5 rose up in the concentration of 0.6μg/ml and 0.7μg/ml (P= 0.000; P=0.000); The expression level of decoy receptor 1(DcRl) had obvious significance among the groups (F=37.002, P=0.000),that in the concentration of 0.8μg/ml descended (comparing with other groups, P=0.000, P=0.000); The expression level of DcR2 decreased with the NNAVC concentration increasing, however, that in the concentration of 0.6μg/ml and 0.7μg/ml rose up (P=0.000; P=0.000)
3.5 The measurement of caspase-8 protein by spectrophotometric method
There were reciprocal effects existing between the different concentration and time of NNAVC (F=51.653, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-8 (F=47.400, P = 0.000; F= 189.867, P= 0.000). The expression level of caspase-8 had a rise-and-down process in groups. These showed that NNAVC could activate the caspase-8 in a short time to initiate the apoptotic process.
3.6 The effects of caspase-3 expression levelof NNAVC on KGla cells
There were reciprocal effects existing between the different concentration and time of NNAVC (F=29.694, P= 0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-3 (F=202.507, P = 0.000; F=205.361, P= 0.000).
The expression level of caspase-3 had a rise-and-down tendency in groups, and there were significant deviance in times (F= 36.236, P= 0.020; F=28.489, P= 0.025; F= 175.320, P=0.003). The expression level of caspase-3 rose up with the NNAVC concentration increasing at the 12th and 24th hour (F= 177.938, P= 0.000; F= 42.728, P= 0.000).The results indicated that NNAVC could activate the caspase-3 to induce the apoptosis of KGla cells, and caspase-3 was activated at the 6th hour,most at the 12th hour,in a dose-dependent manner.
Chapter 4 The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells
4.1 The cytotoxic effects of NNAVC against PBMC and activated immune cells
The cell viabilities of PBMC and activated immune cells were not significant difference at various concentrations of NNAVC treatment (F=4.025, P=0.062); The cytotoxic effects of cells treated with different concentration of NNAVC were significant (F=4.629, P=0.016).The toxic effect of NNAVC (0.5-2.0μg/ml) on PBMC was almost rare comparing with the control group (F= 0.626, P=0.618);There was no toxic action of NNAVC on activated immune cells when NNAVC was less than 1.0μg/ml, but toxic effect at the concentration of 2.0μg/ml was significant (P=0.003).That indicated that there were no toxic effects of NNAVC against PBMC and activated immune cells when the concentration was less than 1.0μg/ml.
4.2 The cytotoxic effects of activated immune cells on KGla cells
The results showed that the cytotoxic effects of activated immune cells on KG1a cells at the different effector-to-target(E:T) cell ratios were significant deviation (F=199.354, P=0.000),and the mean of cell activities were 50.04%,3.95 %,36.59%,23.63%,33.27% and 8.35% ,respectively. The cytotoxicity had significant deviation at the different E:T cell ratios (F=345.299, P=0.000); The cytotoxic effects of activated immune cells on targeted cells increased with the E:T cell ratios rising except for E group, there were significant deviation among the groups (F=25.711, P=0.000; F=53.466, P=0.000; F=43.224, P=0.000; F=135.884, P=0.000). The cytotoxic effects of activated immune cells on targeted cells in A to E groups had a descending tendency, which suggested that the sensitivity of KG1a cells to activated immune cells decreased after those were killed by different activated immune cells.
4.3 The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells
The cytotoxicity of NNAVC alone on KGla cells was 40.61%;while the cytotoxicity of activated immune cells alone at the E:T cell ratios of 10:1 and 20:1 on KG1a cells were 34.55% and 41.35%,respectively(P=0.414). The cytotoxicity of NNAVC combined with activated immune cells at the E:T cell ratios of 10:1 and 20:1 on KGla cells were 56.21% and 85.59%,which was significantly higher than NNAVC alone or activated immune cells alone (P= 0.018, P= 0.000). The cytotoxicity of NNAVC on activated immune cells at the E:T cell ratios of 10:1 and 20:1 was very low (4.35% and 1.33%, respectively).The results indicated that the cytotoxicity of NNAVC combined with activated immune cells on KGla cells was better than NNAVC or activated immune cells alone, but it didn't mean the synergism of NNAVC and activated immune cells.
4.4 The sensitivity of KGla treated with NNAVC to activated immune cells
The cytotoxicity of activated immune cells at the different E:T cell ratios on KG1a cells treated with NNAVC was significant (F=148.593, P=0.000); The cell activities of activated immune cells at the E:T cell ratios of 10:1 and 20:1 on targeted cells treated with NNAVC was significant (F=8.721, P=0.001). The cytotoxicity of activated immune cells at the E:T cell ratio of 10:1 on targeted cells treated with NNAVC was significant among groups (F=12.015, P=0.002); However, that at the E:T cell ratios of 20:1 was not significant among groups (F=1.177, P=0.337);The targeted cells within the group were significant (P=0.003, P= 0.010, P= 0.010, P= 0.001) at the E:T cell ratios of 10:1 and 20:1.The results showed that the cytotoxicity of activated immune cells on targeted cells treated with NNAVC was stronger with higher E:T cell ratio, KG1a treated with NNAVC to activated immune cells was still very sensitive.
4.5 The colony-forming abilities of NNAVC alone or combined with activated immune cells on KGla cells
The colony-forming amounts of NNAVC alone or combined with activated immune cells on KG1a cells were significant (F= 108.614, P=0.000) after KG1a was inoculated for 7 days; The different concentrations of NNAVC were significant as well (F=270.741, P=0.000); There were reciprocal effects existing between the different concentrations and treatment strategy of NNAVC (F=9.956, P= 0.003) The inhibitory effect of colony-forming abilities of NNAVC alone on KGla cells was significantly worse than the NNAVC combined with activated immune cells group (t=9.141, P=0.001; t=8.273, P=0.001); The colony-forming amounts significantly decreased with the concentration of NNAVC increasing (F=58.886, P = 0.000; F=426.629, P= 0.000)
The colony-forming amounts of NNAVC alone or combined with activated immune cells on KG1a cells were significant (F= 26.059, P=0.000) after KG1a was inoculated for 14 days; The different concentrations of NNAVC were significant as well (F= 174.366, P=0.000); There were not reciprocal effects existing between the different concentrations and treatment stategy of NNAVC (F=2.623, P=0.114). The inhibitory effect of colony-forming abilities of NNAVC alone on KGla cells was significantly worse than the NNAVC combined with activated immune cells group (t=5.822, P=0.004; t=6.554, P=0.003); The colony-forming amounts significantly decreased with the concentration of NNAVC increasing (F=49.972, P = 0.000; F=167.300, P= 0.000).
The colony-forming amounts of NNAVC combined with activated immune cells on KGla cells less than NNAVC alone, which suggested that NNAVC combined with activated immune cells could decrease the amount of colony by killing the colony-forming cells of KG1a.
4.6 The effects of NNAVC on expressions of NKG2D ligand on the surface of KGla cells
The expressions of MICA,MICB,ULBP1,ULBP2 and ULBP3 in KGla cells treated with NNAVC were significant between groups (F=3654.504, P=0.000; F = 978.748, P= 0.000;F= 1328.553, P=0.000;F=455.271,P= 0.000;F=223.263, P= 0.000); The expressions of ULBP3 in KGla cells treated with 0.6μg/ml NNAVC significantly rose up (P=0.003),however, those of other ligands were significantly less than control group (P=0.000, P=0.000, P=0.000, P=0.000); The expressions of ULBP1,ULBP2 and ULBP3 except for MICB in KGla cells treated with 0.7μg/ml NNAVC significantly rose up comparing with control (P= 0.000, P=0.000, P=0.000).Those treated with 0.8μg/ml NNAVC were as well (P= 0.000, P=0.001, P=0.000); The expressions of ULBP1,ULBP2 and ULBP3 were significant between groups (P= 0.000).The results indicated that ULBP1,ULBP2 and ULBP3 could be the target of NNAVC in KG1a cells.
Conclusion
1. NNAVC can obviously inhibit the proliferation of KGla cells by inducing apoptosis, cell cycle arrest of KG1a cells at the G0/G1 stage and the inhibition of colony-forming cells.
2. NNAVC could upregulated the expression of Bax, Bad, p53 and NOXA at mRNA level, and downregulated the expression of Bcl-2 and NF-KB.
3. NNAVC could induce cytochrome-c released into the endochylema, activate the caspase-9 and caspase-3, the intrinsic apoptosis pathway was involved in the action of NNAVC to KG1a.
4. At the range of effective concentration of NNAVC to KG1a cells,the cytotoxicity of NNAVC on activated immune cells was less than on KG1a cells.
5. The KG1a cells that are survived after NNAVC's effection could be killed by activated immune cells.
6. NNAVC can combine with activated immune cells to kill KGla cells whether co-incubated or in one-after-another way.
7. NNAVC(0.08mg/kg) combined with activated immune cells can increase the survival time of hu-Balb/c-nu-KG1a leukemic mice.
Innovation of our research
Our research firstly discovered the fact that NNAVC combined with activated immune cells could be used to treat acute myeloid leukemia, and NNAVC had no side effect on activated immune cells when they killed leukemic cells.
Values of our research
Our research demonstrated NNAVC combined with activated immune cells could kill the KGla cells more effectively and treat hu-Balb/c-nu-KGla leukemic mice,whose survival times were increased.The research provided a new insight for the the therapy of AML and long
急性髓系白血病(acute myeloblastic leukemia, AML)是一种以成髓细胞失控性增殖为特征的危及生命的恶性疾病,AML的临床进展和预后与侵袭细胞类型、基因遗传学改变及克隆的生物学特性紧密相关。临床上针对AML治疗的化疗药物或靶向药物虽然能够提高完全缓解率(CR),但基于AML中存在具有自我更新和增殖的干细胞群,故患者体内有可能存在无法清除的白血病微小克隆而导致缓解后复发。因此,寻找有效治疗AML的天然或合成药物已经成为肿瘤研究者研究的目标之一。
AML的发生历经了免疫监视、相互对抗和免疫逃逸的免疫选择和编辑过程,机体的免疫细胞无法对其进行杀伤。NKG2D是免疫细胞表面的激活性受体,在NK细胞、NK／T细胞、Tγδ细胞、DC细胞、IDC细胞等多种细胞表面表达。这些免疫细胞可以通过NKG2D分子识别肿瘤细胞表面的相应NKG2D配体而被激活,表达TRAIL、Fas L、颗粒酶、穿孔素,从而诱导靶细胞凋亡或直接杀伤,产生有效的抗肿瘤免疫应答。研究证实,多种药物具有上调肿瘤细胞表面的NKG2D配体表达的作用,因而提高肿瘤细胞被NK细胞杀伤的敏感性,对肿瘤细胞进行有效清除。
蛇毒毒素在肿瘤防治方面具有良好的效果。研究发现,蛇毒毒素对人卵巢癌、膀胱癌、侵袭性前列腺癌、急性早幼粒白血病等均具有抑制作用,作用机制与调节肿瘤细胞凋亡相关基因表达有关。本实验所用中华眼镜蛇毒组分(Naja Naja Actra Venom Component, NNAVC)即是从中华眼镜蛇毒液中提取的具有抗肿瘤作用的成分。
本研究以人急性髓系白血病细胞株KG1a细胞为研究对象,以NNAVC和活化免疫细胞为干预因素,探讨：①NNAVC与活化免疫细胞联合对白血病模型鼠体内实验研究；②NNAVC对KG1a细胞增殖抑制及凋亡诱导作用；③NNAVC诱导KG1a细胞的相关分子机制；④NNAVC与活化免疫细胞联合对KGla细胞的杀伤作用及二者共同作用的关联性。
本研究通过体内外实验对NNAVC在AML治疗方面的作用进行初步探讨,为寻求难治性复发性白血病的治疗策略提供理论和实验依据。
方法
第一章NNAVC单独及与活化免疫细胞联合对白血病模型鼠体内实验研究
选用Balb/c-nu小鼠为实验动物,尾静脉注射KG1a细胞建立Balb/c-nu小鼠白血病模型,以外周血及骨髓细胞形态学、荷白血病细胞小鼠骨髓原代细胞培养、FISH检测Y染色体、膜抗原检测、免疫组化检测人特异性抗原等证明白血病模型的成功建立；选用正常Balb/c-nu小鼠,腹腔注射不同剂量的NNAVC,以小鼠体重、行为变化及组织病理切片评价NNAVC的毒副作用；制备KG1a白血病模型,观察NNAVC单独及与活化免疫细胞联合对白血病模型生存时间的影响。
第二章NNAVC对KG1a细胞增殖抑制及凋亡诱导作用
用台盼蓝拒染法测定NNAVC对KG1a细胞生长曲线的影响；倒置显微镜下观察NNAVC作用前后KG1a细胞形态变化；以MTT法测定NNAVC处理后KG1a细胞的增殖活性,并计算半数抑制浓度(IC50)；甲基纤维素半固体培养法检测NNAVC对KG1a细胞体外克隆形成能力的影响；PI染色流式细胞仪检测细胞周期分布；采用Annexin V/PI双标记,荧光显微镜观察细胞凋亡形态,流式检测仪检测NNAVC诱导KG1a细胞的凋亡率；DNA Ladder实验对NNAVC能够诱导KG1a细胞凋亡进行验证。
第三章NNAVC诱导KGla细胞的相关分子机制
以RT-PCR方法检测凋亡通路调节分子Bcl-2、Bcl-xl、Bax、Bak、Bad、Bid、PUMA、NOXA、p53、NF-kB的mRNA表达水平；用人细胞色素C ELISA检测试剂盒检测NNAVC处理后KG1a细胞内细胞色素C的蛋白表达水平；以分光光度法检测Caspase-8、Caspase-9、Caspase-3蛋白表达；以流式细胞术检测TRAIL死亡受体和诱骗受体蛋白表达。
第四章NNAVC与活化免疫细胞联合对KGla细胞的杀伤作用
用淋巴细胞分离液分离健康供者外周血单个核细胞(PBMC),体外用细胞因子刺激培养活化免疫细胞；CCK-8法检测不同浓度NNAVC对’PBMC和活化免疫细胞的细胞毒作用；采用不同健康供者来源的活化免疫细胞序贯杀伤KG1a细胞,LDH杀伤活性检测试剂盒测定KG1a细胞经不同次数活化免疫细胞诱导杀伤作用后,对不同效靶比时活化免疫细胞杀伤敏感性的变化；LDH释放法检测NNAVC联合活化免疫细胞对KG1A细胞的细胞毒性作用；甲基纤维素半固体培养法检测NNAVC单独及与活化免疫细胞联合对KG1A细胞体外克隆形成能力的影响；以流式细胞术检测NNAVC作用后KG1a细胞表面NKG2D配体表达的变化。
结果
第一章NNAVC单独及与活化免疫细胞联合对白血病模型鼠体内实验研究
1.1 KGla细胞白血病模型的鉴定
1.1.1白血病模型的细胞形态：
根据每周一次外周血涂片分析,小鼠经尾静脉注射KG1a细胞后4-5周开始在外周血发现白血病细胞,形态与KG1a细胞具有高度一致性。随着时间的延长,白血病细胞数量逐渐增多。
1.1.2来源于白血病模型小鼠骨髓细胞的原代培养
来源于白血病模型小鼠骨髓细胞培养3-4周后开始进入对数生长期,细胞形态与KG1a细胞相似,可进行稳定传代。
1.1.3 KG1a细胞白血病模型小鼠骨髓培养细胞表面标志
流式细胞仪检测正常小鼠骨髓细胞、KG1a细胞和白血病模型小鼠骨髓培养细胞的CD34和CD38抗原表达情况。正常小鼠骨髓细胞群中几乎无法检测到此两种抗原(CD34-CD38-为99.76%)；KG1a细胞CD34+CD38-的表达率为32.74%,CD34+CD38+的表达率为66.87%；白血病模型小鼠骨髓培养细胞CD34+CD38-的表达率为83.06%,CD34+CD38+的表达率为16.72%。以上结果提示模型组小鼠骨髓内有人白血病细胞存在,且浸润的KG1a细胞大部分处于干细胞水平,应具有较强致病力,证明KGla细胞白血病模型建立成功。
1.1.4白血病模型小鼠骨髓培养细胞染色体检测
荧光原位杂交(FISH)实验结果显示,正常小鼠的骨髓细胞内无法检测到Y染色体,KG1a细胞内性染色体为XY,来源于白血病模型小鼠骨髓的培养细胞内性染色体也为XY。本实验所用小鼠均为雌性,而KG1a细胞是从一位男性AML患者体内获得,因此,以上结果提示模型小鼠体内已经存在人KG1a细胞。
1.1.5白血病模型小鼠多器官浸润检测
根据病理切片HE染色和免疫组化检测,观察白血病模型小鼠体内多脏器的白血病细胞浸润情况。结果显示,模型小鼠肝脏、脾脏、肺脏、肾脏等器官均有白血病细胞存在,其中肝脏的浸润情况最为严重；免疫组化检测发现,浸润的白血病细胞表面表达人特异性CD34和CD45抗原,说明模型小鼠体内的白血病细胞是实验操作获得的人源性细胞,而非小鼠自发产生的。
1.2 NNAVC对Balb/c-nu小鼠的毒性作用
1.2.1小鼠生存质量
在注射NNAVC期间,各组小鼠精神状态良好、饮食活动正常,无明显躁动、惊恐不安、萎靡不振、拒食等现象,注射部位皮肤无红肿破溃或感染化脓表现,实验时间内各组小鼠无死亡。
1.2.2 NNAVC对小鼠体重及脏器质量的影响
观察结果显示,各组小鼠在实验前后体重变化差异有显著性意义(F=15.624,P=0.002)；不同处理组间小鼠体重的差异无显著性意义(F=0.361,P=0.704)：NNAVC作用10天后,各药物组小鼠体重均较给药前有所增加,但与阴性对照组相比,体重变化无显著性差异(P=0.425,P=0.654)。
根据公式计算各组小鼠心脏、肝脏、肾脏、肺脏、脾脏的脏／体系数,结果表明,NNAVC高、低剂量组的各组脏／体系数与阴性对照组相比差异无统计学意义(P=0.124,P=0.435,P=0.352,P=0.403,P=0.663)。脏／体系数是反映药物对动物脏器毒性作用的指标之一,因此,本实验结果提示此两种剂量的NNAVC对正常Balb/c-nu小鼠的体重和脏器质量无明显毒性作用。
1.2.3组织病理学变化
光镜下观察,药物组与对照组相比,各组织器官无明显病理学改变。
1.3. NNAVC和活化免疫细胞对白血病模型生存率及生存时间的影响
1.3.1小鼠生存质量
制备白血病模型期间,各组小鼠精神状态良好、饮食活动正常,注射部位皮肤及周围组织无炎症感染表现。观察小鼠生存期期间发现,死亡前一周左右小鼠出现饮食饮水量下降、活动减少、萎靡不振等现象,部分小鼠体表有红色斑点出现(见于面部、背部、后肢或臀部,数量一般为1-2个,小鼠体温正常,无创伤感染表现),体重持续下降,一周内死亡
1.3.2各组KG1a细胞白血病模型小鼠生存时间观察
观察接种KG1a细胞后各组小鼠生存时间：阴性对照组小鼠在接种细胞后52天内全部死亡,其中位生存时间是50(34,66)天；NNAVC低剂量(0.04mg/kg)组和高剂量(0.08mg/kg)组的中位生存时间分别是43(41,45)天和61(35,87)天；活化免疫细胞组小鼠中位生存时间为52(38,66)天；联合组(NNAVC高剂量与活化免疫细胞联合)小鼠中位生存时间为71(58,84)天；阳性对照组(柔红霉素2.5mg/kg)小鼠中位生存时间为53(39,67)天。除联合组小鼠生存期显著高于阴性对照组(P=0.025)外,其余各处理组生存期与阴性对照组相比均无显著性差异(P=0.277,P=0.197,P=0.341,P=0.197)；NNAVC低剂量(0.04mg/kg)组小鼠生存时间低于高剂量组,二者比较差异有显著性意义(P=0.025)；联合组与NNAVC高剂量(0.08mg/kg)组小鼠的存活时间比较差异无统计学意义(P=0.110),但与活化免疫细胞组比较差异有显著性(P=0.025)。以上结果表明,白血病小鼠生存期随着NNAVC药物剂量增加而延长,而活化免疫细胞与高剂量NNAVC联合应用的疗效优于二者各自单独作用的效果。
1.3.3 NNAVC和活化免疫细胞对白血病模型小鼠脏器质量的影响
各组白血病小鼠经处理后,至生存期结束时各器官脏／体系数变化数据显不,NNAVC低剂量组心脏系数与正常对照组相比较差异有显著性意义(P=0.001)；各组肝脏系数无明显变化(F=2.176,P=0.108)；阴性对照组、NNAVC低剂量组及高剂量组小鼠脾脏系数与正常对照组比较差异有显著性意义(P=0.000,P=0.000,P=0.013)；阳性对照组的肺脏系数和肾脏系数均有增高趋势,与正常对照组相比差异有统计学意义(P=0.000,P=0.011),活化免疫细胞组肾脏系数与正常对照组相比差异也有统计学意义(P=0.009),其余各组肺脏系数和肾脏系数差异无显著性(P>0.05)。而与阴性对照组(即白血病模型组)比较,活化免疫细胞组、联合组及阳性对照组的脾脏系数均升高,差异有统计学意义(P=0.000,P=0.000,P=0.000)；活化免疫细胞组和阳性对照组的肾脏系数与阴性对照组相比差异有显著性意义(P=0.008,P=0.009)。
第二章NNAVC对KG1a细胞增殖抑制及凋亡诱导作用
2.1 NNAVC作用后KG1a细胞生长曲线的变化
台盼蓝拒染法结果显示,低浓度NNAVC (0.625μg/ml)组KG1a细胞的生长曲线斜率明显低于对照组,表现出有效的增殖抑制作用；药物终浓度达1.25μg/ml时,细胞增殖相当缓慢,生长曲线趋于平整；当NNAVC浓度≥2.5 u g/ml时,KG1a细胞在24h内几乎全部死亡。提示NNAVC可以有效抑制KG1a细胞的生长增殖,并且随着其浓度的增加、作用时间的延长,增殖抑制作用逐渐增强,抑制效应具时间和剂量依赖性。
2.2 Zeiss倒置显微镜进行细胞形态学观察结果
显微镜镜下观察,KG1a细胞加药前状态良好,细胞壁完整、光滑透亮、胞浆均匀、细胞形态规则。加入NNAVC作用后连续动态观察24h,结果显示KG1a细胞作用6h后开始出现细胞壁不完整、细胞形状不规则、胞浆颗粒增多等改变,随着NNAVC作用时间的延长,发生上述形态改变的细胞数量逐渐增多。至NNAVC作用24h后,可以看到明显的细胞死亡,细胞质空泡、固缩、细胞破碎等形态学变化。
2.3 NNAVC对KG1a细胞的细胞毒作用
细胞毒性实验结果显示,NNAVC不同作用浓度与作用时间之间有交互效应(F=11.868,P=0.000)；不同NNAVC浓度对KG1a细胞的抑制率之间比较差异有显著性意义(F=934.427,P=0.000)；两个作用时间NNAVC刘‘KG1a细胞的抑制率比较有显著性差异(F=1074.672,P=0.000)。
NNAVC在0.6μg/ml至1.2μg/ml的浓度范围内,在相同作用时间时,对KG1a细胞的生长抑制作用具有递增趋势,差异比较有统计学意义(F=428.725,P=0.000；F=550.172,P=0.000)；在相同浓度条件下,NNAVC处理KG1a细胞在24h与48h时间点的抑制率具有显著性差异(t=-11.549,P=0.000；t=-14.190,P=0.000；t=-13.219,P=0.000；t=-13.194,P=0.000；t=-10.218,P=0.001；t=-15.272,P=0.000；t=-10.436,P=0.000；),抑制率随着作用时间的延长而增加。说明NNAVC对KG1a细胞的增殖抑制作用具有剂量依赖性和时间依赖性。
NNAVC对KG1a细胞IC50浓度在24h为0.8806(0.8654-0.8959)μg/ml,48h为0.7037(0.6875-0.7187)μg/ml.
2.4 NNAVC对KG1a细胞集落形成能力的影响
用甲基纤维素半固体培养体系进行培养,KG1a细胞接种后第1-2天为分散的单个细胞,第7天即可见克隆形成,7-14天之间集落数量不断增多。各组细胞的集落形态：正常KG1a细胞形成的集落数量多,呈密集型集落；NNAVC作用后KG1a细胞形成的集落数量明显减少,细胞团内有死细胞出现,高剂量组(NNAVC 0.8μg/ml)集落较为松散,细胞碎片较多。
分别对各组细胞在培养7天及14天后克隆形成数量进行统计,结果：NNAVC作用浓度与克隆培养时间之间有交互效应(F=70.623,P=0.000)；不同处理组之间克隆形成数量差别有统计学意义(F=204.351,P=0.000)；各组细胞培养不同时间所形成的克隆数量差异有显著性意义(F=305.877,P=0.000)。
各组细胞在培养14天后的克隆数均显著高于培养7天时的数量,差异比较有统计学意义(t=-13.784,P=0.000；t=-8.615,P=0.000；t=-6.728,P=0.003)；在各观察时间点,不同浓度NNAVC对KG1a细胞集落形成能力均有影响,药物作用后克隆数明显降低,组内比较差异有显著性意义(F=34.357,P=0.001；F=172.368,P=0.000)；NNAVC0.8μg/ml组在各观察时间的克隆形成数量与0.7μg/m1组相比无统计学意义(P=0.411,P=0.185),但前者克隆数低于后者。根据结果分析提示,用药后各药物组细胞的增殖性下降,说明NNAVC可以在一定程度上抑制KG1a细胞的集落形成能力,从而抑制其增殖。
2.5 NNAVC对KG1a细胞生长周期的影响
根据流式检测结果,加入NNAVC作用24 h后,细胞周期变化如下：在细胞周期的不同时段,各组细胞的比例具有显著性差异(F=339.698,P=0.000；F=6.439,P=0.032；F=127.007,P=0.000)；NNAVC 0.7μg/ml组和0.8μg/ml组中处于G0／G1期细胞比例分别上升为58.67%和62.20%,与对照组52.93%相比差异有显著性意义(P=0.032,P=0.002),而处于G2/M期的细胞比例分别下降为5.33%和3.13%,与对照组相比有显著性差异(P=0.000,P=0.000)；0.8μg/ml组S期细胞比例为34.70%,与对照组36.90%相比差异有统计学意义(P=0.012)。NNAVC 0.7μg/ml组和0.8μg/ml组之间处于G0／G1期和S期的细胞比例比较差异无显著性(P=0.048,P=0.079),处于G2／M期细胞比例比较差异有统计学意义(P=0.003)。加药组细胞G1期前均有亚二倍体峰出现。以上结果提示,NNAVC可能通过诱导KG1a细胞生长周期阻滞于G0／G1期,减少G2／M期细胞百分含量而抑制其生长增殖,并且NNAVC还具有促使细胞凋亡的作用。
2.6荧光显微镜观察细胞凋亡
荧光显微镜下观察,正常细胞形态饱满、细胞着色均匀,无亮色小体,说明细胞的胞膜完整,没有凋亡小体出现。NNAVC处理组细胞发生核固缩、破裂,细胞膜出泡,有凋亡小体出现。其细胞凋亡数随药物浓度增高及作用时间的延长而逐渐增多。
2.7流式检测仪检测细胞凋亡率
检测结果发现,KG1a细胞经NNAVC处理后,早期凋亡率和晚期凋亡率均有所增加,其中0.7μg/ml浓度组早期凋亡率和晚期凋亡率分别增加到39.63%和22.23%,与对照组相比差异有统计学意义(P= 0.049, P= 0.034); 0.8μg/ml浓度组早期凋亡率和晚期凋亡率分别增加到40.73%和41.50%,与对照组相比有显著性差异(P=0.040,P=0.016)；而0.6μg/ml浓度组凋亡率均无明显变化；两两比较显示,0.7μg/ml和0.8μg/ml浓度组间早期凋亡率的差异无统计学意义(P=1.000),晚期凋亡率相比差异有显著性意义(P=0.021)。以上结果证实NNAVC具有诱导KG1a细胞凋亡的作用。
2.8 DNA Ladder检测
根据琼脂糖凝胶电泳结果可见,正常对照组没有梯状条带(DNA Ladder)出现,DNA无明显弥散,为大片段的基因组DNA；经NNAVC处理后的KG1a细胞有典型的180-200 bp或其整倍数的梯状条带形成。提示NNAVC可以引起KG1a细胞发生DNA片段化,证实其具有诱导凋亡作用。
第三章NNAVC对KG1a细胞凋亡相关基因和蛋白表达的影响
3.1 NNAVC对KG1a细胞凋亡相关基因mRNA水平表达的影响
RT-PCR法检测各组KG1a细胞内凋亡相关基因表达的变化：NNAVC不同浓度组细胞内Bcl-2、Bcl-xL、Bax、Bid、Bad、p53、PUMA、NOXA和NFκB等基因的表达差异有统计学意义(F=5.774,P=0.021；F=20.652,P=0.000；F=9.780,P=0.005；F=6.795,P=0.014；F=18.389,P=0.001；F=246.243,P=0.000；F=10.467,P=0.004；F=7.153,P=0.012；F=47.767,P=0.000)；三种不同浓度NNAVC作用后,Bcl-2基因的表达均有下降,与对照组相比有显著性差异(P=0.035,P=0.004, P=0.028),各药物组之间两两比较差异无显著性(P=0.162,P=0.884,P=0.202)；抑凋亡基因的Bcl-xL在药物作用后表达有所上升,并表现出一定的剂量依赖性,其中0.7μg/ml组和0.8μg/ml组的变化幅度与对照组相比有统计学意义(P=0.001,P=0.000),但两浓度组之间差异无显著性(P=0.202)；在0.7μg/ml组和0.8μg/ml组,促凋亡基因Bax表达水平与对照组相比差异有统计学意义(P=0.003,P=0.018),而各组细胞Bak基因表达差异无统计学意义(F=0.406,P=0.753)；正常KG1a细胞几乎不表达抑癌基因p53,在不同浓度NNAVC作用后,细胞内p53的表达水平均得到显著升高(P=0.000,P=0.000,P=0.000),p53基因相关的PUMA和NOXA两种基因的表达变化无明显趋势；在本实验中检测到,NNAVC 0.6μg/ml组和0.7μg/ml组的NFκB表达水平与对照组相比有所下降,统计有显著性差异(P=0.000,P=0.000)。综上所述,NNAVC对KG1a细胞内各促／抑凋亡基因的表达具有不同的调节作用,提示其发挥作用的机制与凋亡密切相关。
3.2 NNAVC对KG1a细胞胞浆内细胞色素C表达影响
结果显示,0.7μg/ml组细胞在NNAVC作用0h-12h间,细胞色素C的表达水平呈急速上升状态,之后表达量明显下调；0.8μg/ml组细胞在药物作用0h-6h间,胞浆内细胞色素C的含量呈上升趋势,在药物作用6h-12h间细胞色素C的含量无明显变化,之后表达逐渐下降,至24h时间点,两药物组细胞色素C的表达水平相近,但仍略高于0h时间点。数据分析：NNAVC不同浓度处理与作用时间两因素存在交互效应(F=51.868,P=0.000)；NNAVC不同浓度及作用时间对各组KGla细胞内细胞色素C蛋白含量的影响均有显著性差异(F=82.331,P=0.001；F=552.286,P=0.000)。在两种NNAVC浓度处理时,在不同作用时间点,细胞色素C含量变化组内比较差异有显著性意义(F=316.021,P=0.001；F=278.700,P=0.000)；NNAVC作用6h时间点,0.7μg/ml组细胞色素C含量低于0.8μg/ml组(P=0.018)；在1 2h和18h时间点,0.7μg/m1组细胞色素C含量均明显高于0.8μg/ml组,差异比较有显著性意义(P=0.001；P=0.026)。
3.3分光光度法检测Caspase-9蛋白表达
分光光度法检测细胞内caspase-9蛋白表达水平的变化：NNAVC不同浓度处理与作用时间两因素存在交互效应(F=23.751,P=0.000)；NNAVC不同浓度处理后KG1a细胞内caspase-9蛋白含量有显著性差异(F=23.982,P=0.001)；作用时间对各组KGla细胞内caspase-9蛋白含量的影响有显著性差异(F=180.774,P=0.000)。在NNAVC作用0h-12h之间,caspase-9蛋白表达水平随作用时间的延长而升高,12h后缓慢下降；NNAVC对KG1a细胞内caspase-9蛋白表达水平的影响同样具有浓度依赖性。结果提示在NNAVC通过激活caspase-9诱导KG1a细胞凋亡。
3.4 NNAVC对KG1a细胞表面TRAIL死亡受体和诱骗受体表达的影响
死亡受体DR4在不同浓度NNAVC作用后,表达率随着药物浓度的增加而减少,组间比较差异有显著性意义(F=170.107,P=0.000),各浓度组组间两两比较差异有统计学意义(P≤0.005)；死亡受体DR5在0.6μg/ml和0.7μg/ml浓度时表达有所升高,差异与对照组相比有统计学意义(P=0.000；P=0.000)；诱骗受体DcR1在各组细胞的表达率比较差异有显著性意义(F=37.002,P=0.000),0.8μg/ml组的表达率下降与其他两浓度组比较差异有显著性(P=0.000,P=0.000)；诱骗受体DcR2在三种NNAVC浓度作用下,表达率随药物浓度的增加而降低,但与对照组相比,0.6μg/ml组和0.7μg/ml组细胞DcR2的表达率是升高的,差异有统计学意义(P=0.000；P=0.000)。
3.5分光光度法检测Caspase-8蛋白表达
不同处理后KGla细胞内caspase-8蛋白表达水平的变化显示：NNAVC不同浓度处理与作用时间两因素存在交互效应(F= 51.653, P=0.000); NNAVC不同浓度处理后KG1a细胞内caspase-8蛋白含量有显著性差异(F=47.400,P=0.000)；作用时间对各组KG1a细胞内caspase-8蛋白含量的影响有显著性差异(F=189.867,P=0.000)。
各浓度组caspase-8蛋白表达均具有由低到高、再逐渐下降的过程,提示NNAVC可以在短时间内使caspase-8蛋白活化,进而发挥诱导凋亡的效应。
3.6 NNAVC对KG1a细胞Caspase-3蛋白表达的影响
NNAVC不同浓度处理与作用时间两因素存在交互效应(F=29.694,P=0.000); NNAVC不同浓度处理后KG1a细胞内caspase-3蛋白含量有显著性差异(F=202.507,P=0.000)；作用时间对各组KGla细胞内caspase-3蛋白含量的影响有显著性差异(F=205.361,P=0.000)。
在同一NNAVC浓度作用下,细胞内caspase-3蛋白表达呈现低—高—低的趋势,不同时间点差异有显著性意义(F=36.236,P=0.020；F=28.489,P=0.025;F= 175.320,P=0.003); NNAVC分别作用12h和24h时间点时,caspase-3蛋白表达水平随药物浓度的增加而上升,差异比较有统计学意义(F=177.938,P=0.000；F=42.728,P=0.000)。结果提示NNAVC通过激活caspase-3诱导KG1a细胞凋亡,caspase-3蛋白在NNAVC作用6h后就被激活,12h表达量最高,活性升高具有剂量依赖性。
第四章NNAVC与活化免疫细胞联合对KG1a细胞的杀伤作用
4.1 NNAVC对PBMC和活化免疫细胞的细胞毒作用
CCK-8检测结果显示,NNAVC不同作用浓度时,PBMC和活化免疫细胞活性的差异无显著性意义(F=4.025,P=0.062)；不同NNAVC浓度对细胞毒性作用的差异有显著性意义(F=4.629,P=0.016)。NNAVC在(0.5-2.0)μg/ml的浓度范围内对PBMC无明显的毒性作用,差异与对照组相比无统计学意义(F=0.626,P=0.618); NNAVC≤1.0μg/ml时对活化免疫细胞无毒性作用,2.0μg/ml NNAVC作用后活化免疫细胞活性的差异有显著性意义(P=0.003)。提示在1.0μg/ml浓度以下,NNAVC对PBMC和活化免疫细胞均无明显的毒性作用。
4.2活化免疫细胞对KG1a细胞的杀伤作用
活化免疫细胞在不同效靶比时对诱导杀伤后的KG1A细胞再次杀伤作用比较LDH杀伤检测试剂盒检测结果：活化免疫细胞对各组靶细胞的杀伤活性结果显示,在不同效靶比时,各组靶细胞之间的差异有显著性意义(F=199.354,P=0.000)；细胞杀伤活性均数分别为50.04%、3.95%、36.59%、23.63%、33.27%和8.35%。不同效靶比之间细胞杀伤率比较差异有统计学意义(F=345.299,P=0.000)；除E组外,活化免疫细胞对其余各组靶细胞的杀伤率随着效靶比的升高而增加,在各效靶比作用下,活化免疫细胞对各靶细胞的杀伤活性组间比较差异有显著性意义(F=25.711,P=0.000；F=53.466,P=0.000；F=43.224,P=0.000；F=135.884,P=0.000)。与正常对照组相比,活化免疫细胞对A-E组细胞的杀伤率总体呈下降趋势,提示经过不同来源活化免疫细胞的杀伤作用后,KG1a细胞对活化免疫细胞的敏感性下降。
4.3 NNAVC联合活化免疫细胞对KG1a细胞的作用
单独应用NNAVC对KG1a细胞的杀伤率为40.61%；单独加入活化免疫细胞,在效靶比为10：1和20：1的情况下,对KG1a细胞的杀伤率分别为34.55%和41.35%,二者比较差异无统计学意义(P=0.414)；NNAVC分别与两种比例的活化免疫细胞联合应用时,对KG1a细胞的杀伤率为56.21%和85.59%,高于这两种干预因素各自单独作用的杀伤率,差异有显著性意义(P=0.018,P=0.000); NNAVC在体系中的作用浓度对两种比例的活化免疫细胞均无明显的细胞毒作用,杀伤率分别为4.35%和1.33%。以上结果提示,NNAVC在对活化免疫细胞无明显毒性作用的浓度时,可以与活化免疫细胞联合杀伤KG1a细胞,作用效果优于二者单独作用,但并未显示二者有协同作用。
4.4 NNAVC作用后的KG1a细胞对活化免疫细胞的敏感性
LDH释放测定法显示结果显示：活化免疫细胞在不同效靶比间对各组靶细胞杀伤率的差异有显著性意义(F=148.593,P=0.000)；各组靶细胞在两种不同的效靶比的活化免疫细胞作用下,细胞活性间的差异有显著性意义(F=8.721,P=0.001)。在效靶比为10：1的情况下,活化免疫细胞对各组靶细胞的杀伤率组间比较差异有统计学意义(F=12.015,P=0.002)；活化免疫细胞在效靶比为20：1时对各组靶细胞杀伤率组间比较无显著性差异(F=1.177,P=0.337)；各组靶细胞在两种效靶比作用下,组内比较有显著性差异(P=0.003,P=0.010,P=0.010,P=0.001)。以上结果说明,活化免疫细胞对各组靶细胞的杀伤活性随着效靶比的升高而增强,NNAVC作用后的KG1a细胞对活化免疫细胞仍有较强的的杀伤敏感性。
4.5 NNAVC联合活化免疫细胞对KG1A细胞体外克隆抑制实验
经过不同处理作用后,KG1a细胞接种后7天所形成的克隆情况：两个处理组克隆形成数量的差异组间比较有显著性意义(F=108.614,P=0.000)；不同NNAVC作用浓度之间克隆形成数量的差异有统计学意义(F=270.741,P=0.000); NNAVC浓度与不同处理方法之间有交互效应(F=9.956,P=0.003)。在相同NNAVC浓度作用后,单用NNAVC对KG1a细胞克隆形成能力的抑制作用明显弱于NNAVC与活化免疫细胞联合组,两处理组克隆形成数量比较差异有统计学意义(t=9.141,P=0.001；t=8.273,P=0.001)；经同样处理因素作用后,细胞克隆形成数量随NNAVC浓度的增加而减少,比较差异有统计学意义(F=58.886,P=0.000：F=426.629,P=0.000)。
经过不同处理后的KG1a细胞接种后14天形成的克隆数量结果显示：在各药物浓度作用后,两个处理组克隆形成数量的差异有显著性意义(F=26.059,P=0.000)；不同NNAVC浓度间克隆形成数量的差异有统计学意义(F=174.366,P=0.000); NNAVC浓度与不同处理方法之间交互效应无显著性意义(F=2.623,P=0.114)。在相同NNAVC浓度作用后,NNAVC和活化免疫细胞联合作用后细胞形成的克隆数量与NNAVC单独作用后细胞形成的克隆数量比较,差异有显著性意义(t=5.822,P=0.004；t=6.554,P=0.003)；在相同处理条件下,各组细胞克隆形成数量随NNAVC浓度的增加而减少,与对照组相比差异有统计学意义(F=49.972,P=0.000；F=167.300,P=0.000)。
NNAVC与活化免疫细胞联合应用后,KG1a细胞形成的克隆数量明显低于NNAVC单独作用组,提示活化免疫细胞可以与NNAVC共同作用杀伤KG1a细胞的克隆形成细胞,使得其克隆数量显著降低。
4.6 NNAVC对KG1a细胞表面NKG2D配体表达的影响
流式细胞仪检测结果显示：不同处理组细胞表面MICA、MICB、ULBP1、ULBP2和ULBP3的表达率组间比较差异有显著性意义(F=3654.504,P=0.000；F=978.748,P=0.000；F=1328.553,P=0.000；F=455.271,P=0.000；F=223.263,P=0.000); 0.6μg/ml的NNAVC作用于KG1a细胞后,ULBP3表达升高,与对照组相比差异有显著性(P=0.003),其余NKG2D配体表达均低于对照组,差异有显著性意义(P=0.000,P=0.000,P=0.000,P=0.000)；0.7μg／ml的NNAVC作用后,KG1a细胞的ULBP1、ULBP2、ULBP3表达增加,差异与对照组相比有统计学意义(P=0.000,P=0.000,P=0.000),MICB表达无明显差异(P=1.000)；0.8μg/ml的NNAVC也可以诱导细胞表面的ULBP1、ULBP2、ULBP3表达增加,与对照组相比差异有统计学意义(P=0.000,P=0.001,P=0.000)；ULBP1-3在三种药物浓度组细胞的表达水平两两比较差异均有显著性(P=0.000)。提示ULBP1-3有可能是NNAVC的调节靶点。
结论
1. NNAVC能够抑制KG1a细胞增殖自我更新能力,使KG1a细胞的生长周期阻滞于G0/G1期,并诱导细胞凋亡。
2. NNAVC能够上调Bax、Bad、p53、NOXA mRNA表达,下调Bcl-2、NF-KBmRNA表达。
3. NNAVC促使KG1a细胞线粒体内细胞色素C释放到胞浆中,激活caspase-9,从而激活caspase-3而引起凋亡效应。
4.在对KG1a细胞有效浓度范围内,对活化免疫细胞无明显毒性作用。
5. NNAVC无法清除的KG1a细胞可以采用活化免疫细胞进行杀伤。
6. NNAVC与活化免疫细胞能够联合应用,同时给予或序贯给予均可有效提高对KG1a细胞的杀伤率,有望最大限度地清除AML细胞。
7. NNAVC (0.08mg/kg)与活化免疫细胞联合作用能够有效延长hu-Balb/c-nu-KG1a白血病(Balb/c裸鼠荷人KG1a细胞白血病模型)动物的生存期。
本研究的创新点
首次发现中华眼镜蛇毒组分与活化免疫细胞可以联合应用于急性髓系白血病的治疗研究,并且中华眼镜蛇毒组分在有效杀伤白血病细胞的同时,对活化的免疫细胞无明显毒副作用。
研究价值
中华眼镜蛇毒组分与活化免疫细胞联合能够更有效地杀伤KG1a细胞,对白血病模型动物具有治疗作用,本研究为临床白血病的治疗、延长患者生存时间提供了新的研究方向。
Background and Objective
Acute myeloblastic leukemia (AML) is a malignant disease that can threaten one's life, characterized by the rapid growth of lymphomyelocyte, and its clinic progression and prognosis have a close connection with invasive cell types, the change of gene genetics and clonal biology. Chemotherapeutics or targeted drugs can improve the complete remission (CR) of AML patients in clinical medicine, but because AML have self-renewal and proliferative stem cell groups,leukemic microclone cannot be removed in patients which possibly leads to the relapse of leukemia. Therefore, one of the goals of cancer research has been, and continues to be, the discovery of natural and synthetic products for AML prevention or treatment.
AML has experienced the immunoselection and editing process of immunosurveillance, confrontation and immune escape, which makes AML cells not being killed by immune cells.NKG2D is the activated receptor in the surface of immunoctye, which can be expressed by many kinds of cells, just like NK cells, NK/T cells, Tγδcells, DC cells, IDC cells. These immunocytes can be activated through the corresponding ligand in the surface of tumor cells recognized by NKG2D molecules, express the molecules,such as TRAIL、FasL, granzyme, porforin, then induce the targeted cell apoptosis or kill them. Many researches have suggested that some drugs can up-regulated the expression of NKG2D ligand in the surface of tumor cells, and then improve the sensitivity of tumor cells killed by NK cells, which leads to effectively remove tumor cells.
Snake venom has the excellent effect on the prevention and therapy of tumor. The research has shown that snake venom has the inhibitory action on human ovary cancer, carcinoma of bladder, invasive prostatic carcinoma, acute promyelocytic leukemia, etc., the mechanisms of which are related to modulate the apoptosis gene expression. Naja Naja Actra Venom Component (NNAVC), isolated from the Naja Naja Actra Venom, was chosen in the experiment because of the effect of its anti-tumor.
Human acute myeloid leukemia(AML) cell line, KG1a cell was chosen as experimental subject, NNAVC and activated immune cells were used, the following would be discussed in the research:①The experimental research of leukemic model treated with NNAVC or NNAVC combined with activated immune cells in mice;②the action of proliferative inhibition and apoptosis induced by NNAVC on KG1a cell;③the molecular mechanisms related to KG1a cell apoptosis induced by NNAVC;④The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells and their synergic mechanism.
The research aimed to discuss the therapeutic action of NNAVC on AML in vivo and in vitro, which will provide the theory and experimental foundation of therapy strategy for refractory and relapsed leukemia.
Methods
Chapter 1 The experimental research of leukemia model treated with NNAVC alone or NNAVE combined with activated immune cells in mice.
The experiments were performed on Balb/c-nu mice weighing 18-22g,and Balb/c-nu mice were injected into KGla cells through vena caudalis to establish Balb/c-nu mice leukemic model, which was assessed by the morphology of peripheral blood and marrow cell, marrow primary cell culture in mice with leukemic cells, Y chromosome by FISH, membrane antigen detection, human specific antigen by immunohistochemisty, and etc. The normal Balb/c-nu mice were injected into different doses of NNAVC through intraperitoneal injection, and then the weight of mice,the change of behavior and pathological section were assessed for the side-effect of NNAVC; the survival times of leukemic model treated with NNAVC or NNAVE combined with activated immune cells were observed after KG1a leukemic model was established. Statistical analysis
The calculation was performed using SPSS 13.0 software package. The data presented as the mean±standard deviation. Comparison in experiments was performed using one-way analysis of variance (ANOVA), independent-samples t-test and repeated measurement of analysis of variance to assess the statistical significance of differences between groups. Differences with P<0.05 were considered to be significant.
Chapter 2 The action of proliferative inhibition and apoptosis induced by NNAVC on KGla cell
Trypan blue exclusion was used to measure the growth curve; of NNAVC on KG1a cells; KG1a cell morphological change could be seen by inverted microscope; MTT assay was used to detect the proliferative activity of KG1a cell after NNAVC treatment, and half inhibitory concentration(IC50) was calculated; the colony forming ability of KGla in vitro was measured by methylcellulose semisolid method; flow cytometry was used to measure the influence of NNAVC on KGla cell cycle; the morphology of apoptotic cells was observed by fluorescence microscope and KGla apoptotic rate was measured by flow cytometry after KGla cell was stained with Annexin V/PI; the apoptosis of KG1a cell was further proved by DNA Ladder.
Chapter 3 The effects of apoptosis-related genes and protein expression of NNAVC against KGla cells
The mRNA expression levels of molecules related to apoptosis, such as Bcl-2,Bcl-xl,Bax,Bak,Bad,Bid,PUMA,NOXA,p53,NF-kB,etc.,were measured by RT-PCR; cytochrome C level was measured according to human cytochrome C ELISA detecting kit; expressions of Caspase-8,Caspase-9 and Caspase-3 were detected by spectrophotometric method; expressions of death receptors and decoy receptors of TRAIL were analyzed by flow cytometry.
Chapter 4 The cytotoxic effect of NNAVC combined with activated immune cells on KGla cells
Peripheral blood mononuclear cells(PBMC) were separated from the healthy donors using lymphocyte separating medium, which were stimulated by cytokine to culture activated immune cells in vitro. The cytotoxic effects of NNAVC with different concentrations on PBMC and activated immune cells were measured by CCK-8 assay; activated immune cells from different healthy donors killed KGla cells by turns, the sensitivity of activated immune cells to kill KGla at different effector-to-target(E:T) cell ratios was detected by LDH cytotoxicity assay after KG1a cells were killed for different times by activated immune cells; The cytotoxic effects of NNAVC combined with activated immune cells on KG1a cells were measured by LDH releasing assay; the colony forming abilities of KGla treated with NNAVC alone or NNAVC combined with activated immune cells in vitro were measured by methylcellulose semisolid method; the expressions of NKG2D ligand in the surface of KG1a were measured by flow cytometry.
Results
Chapter 1 The experimental research of leukemic model treated with NNAVC alone or NNAVE combined with activated immune cells in mice.
1.1 Identification of leukemic model
1.1.1 The cell morphology of leukemic model
Leukemic cells, which were similar to KG1a cells, were found in the peripheral blood after mice were injected into KG1a cells four weeks later according to the peripheral blood film once every week. The amounts of leukemic cells increased gradually with the time going.
1.1.2 The primary culture originated from marrow cells in leukemic model
The primary culture originated from marrow cells in leukemic model developed to exponential phase of growth about three to four weeks later, the cells were similar to KGla cells, could go down to the future generation steadily.
1.1.3 The markers on the surface of culture cell in leukemic cells
The CD34 and CD39 antigen expressions of normal marrow cell, KGla cell and cells in the leukemic model were measured by flow cytometry. The CD34 and CD39 antigen expressions of normal marrow cell could not be measured(CD34-CD38-,99.76%);Those of KGla cell were 32.74% (CD34+CD38-); Those of cells in the leukemic model were 83.06% (CD34+CD38-),16.72% (CD34+CD38+)。The results indicated that the model was successful because human leukemic cells were found in the marrow of leukemic model, and which were in the stem cell stage.
1.1.4 The measurement of marrow culture cell chromosome in model
Y chromosome could not be measured in the normal mice, sex chromosome in KG1a cells was XY, that of cells in the leukemic model was XY as well.The female mice were chosen in the experiment and KG1a cell came from a man with AML, therefore, those findings confirmed human KG1a cell existed in the model.
1.1.5 The measurement of organs affected in the model
The organs affected in the model were measured according to HE staining and immunohistochemistry assay. The research showed that leukemic cells existed in the liver, spleen, lung, kidney and other organs of model mice ,especially liver most affected; The expressions of human specific CD34 and CD45 antigens on the surface of leukemic cells were found, which suggested that the leukemic cells in the model came from human leukemic cell, not from the body of mice spontaneously.
1.2 The toxic effects of NNAVC on Balb/c-nu mice
1.2.1 The survival quality in mice
The mice had a good condition without restlessness, scare, apositia and no death during the experiment.
1.2.2 The changes of body and organ weight in mice
The change of body weight in the experiment had significant deviation (F= 15.624, P=0.002);However, there were no significance between NNAVC-treated groups (F= 0.361, P= 0.704);The body weight in NNAVC-treated groups increased after 10 days, but comparing with negative control,that was no significant(P=0.425, P= 0.654).
There were no significant in organ weight to body weight between high-dose group and low-dose group (P= 0.124, P= 0.435, P= 0.352, P= 0.403, P= 0.663).Organ weight to body weight coefficient was considered to be one of the indexes of toxic effect, so the results indicated that body and organ weight in Balb/c-nu mice treated with NNAVC were not significant.
1.2.3 The change of tissue pathology
Pathological changes of organs were not significant comparing with control in light microscope.
1.3 The effects of survival rate and time of NNAVC and activated immune cells on model mice.
1.3.1 The survival quality of mice
The mice had a good condition with normal diet during the experiment. The mice had abnormal behaviors with descending water-drinking, decreasing activity and etc. One week before death and red speckle appeared on the surface of body partially in the model, their body weight began to reduce till to death in a week.
1.3.2 The survival time of mice in the model
The medium survival time of mice in the negative control was 50 (34,66) days and died after mice were inoculated KGla cells for 52 days; That in NNAVC 0.04mg/kg and 0.08mg/kg were 43(41,45)days和61 (35,87)days; That in activated immune cells alone or combined with NNAVC 0.08mg/kg were 52(38,66) days, 71 (58,84) days, respectively. That in positive control(daunorubicin,2.5mg/kg) was 53 (39,67) days. That in activated immune cells combined with NNAVC 0.08mg/kg was longer than negative control(P=0.025), that in other groups was not significant comparing with negative control (P= 0.277, P=0.197, P= 0.341, P = 0.197); That in NNAVC 0.04mg/kg was significantly less than NNAVC 0.08mg/kg (P=0.025);That was not significant between NNAVC 0.08mg/kg and NNAVC combined with activated immune cells (P= 0.110).The results suggested that the survival time in leukemic mice was longer with the concentration of NNAVC increasing, and NNAVC combined with activated immune cells had a better effect than NNAVC alone or NNAVC combined with activated immune cells alone.
1.3.3 The organs weight of NNAVC and activated immune cells on leukemic model mice
The heart weight to body weight coefficient was significant comparing with control (P=0.001),but the coefficient of liver was not significant (F=2.176, P =0.108);That of spleen in every group(negative, NNAVC low dose and high dose) was significant comparing with control (P=0.000, P=0.000, P= 0.013);That of lung and kidney in the psositive group had a increasing tendencycomparing with control (P= 0.000, P=0.011);That of kidney in activated immune cells-treated group was significant as well (P= 0.009),while other groups (P>0.05).The coefficient of spleen in activated immune cells-treated group, NNAVC combined with activated immune cells, positive control were higher comparing with negative control (P=0.000, P=0.000, P=0.000); The coefficient of kidney in activated immune cells-treated group, positive control were significant comparing with negative control (P=0.008, P=0.009)
Chapter 2 The action of proliferative inhibition and apoptosis induced by NNAVC on KGla cell
2.1 The influence of NNAVC on the growth curve of KGla
Trypan blue exclusion assay showed that the slope of growth curve in the low concentration group(0.625μg/ml)of NNAVC was lower than control group, that suggested NNAVC had obvious inhibition on KG1a cells; the growth of KG1a cells was so slow that the curve was almost flat when the concentration was up to 1.25μg/ml;The KGla almost died out when more than 2.5μg/ml.These results showed that NNAVC could obviously inhibit the proliferation of KGla cells in a time- and dose-dependent manner.
2.2 Morphological view of KG1a cells could be seen by Zeiss inverted microscope
KG1a cells were in good condition with the integrity,smooth and lucency of cellwall; even cytoplasm; regular shape before NNAVC treatment.KGla cells began to emerge bad integrity of cellwall, irregular shape, more particles after the 6th hour of NNAVC, more cells changed with longer action time, when the action time was the 24th hour or longer, the smaller volume ,deformation,vacuole within the cell, cell pyknosis and other morphological changes were seen by inverted microscope.
2.3 The cytotoxic effects of NNAVC on KGla cells
The cytotoxic results showed that there were reciprocal effects existing between the different concentration and time of NNAVC (F= 11.868, P= 0.000); The inhibitory rates of different NNAVC concentrations against KGla cells were statistically significant (F= 934.427, P=0.000); The inhibitory rates of two time groups of NNAVC against KGla cells were also statistically significant (F= 1074.672, P= 0.000)
The growth inhibition of NNAVC against KGla cells in the same action time significantly increased between the concentration of 0.6μg/ml to 1.2μg/ml (F= 428.725, P=0.000; F= 550.172, P=0.000);The inhibitory rates of NNAVC on KG1a cells at the same concentration were significant (t=-11.549, P=0.000; t =-14.190, P= 0.000; t=-13.219, P= 0.000; t=-13.194, P= 0.000; t=-10.218, P=0.001; t=-15.272, P=0.000; t=-10.436, P= 0.000;) at the 24th and 48th hour, and higher inhibitory rate with longer action time. These findings suggested that NNAVC could obviously inhibit the proliferation of KGla cells in a time- and dose-dependent manner.
The half inhibitory concentration(IC5o) of NNAVC against KG1a cells was 0.8806 (0.8654-0.8959)μg/ml at the 24th hour,0.7037 (0.6875-0.7187)μg/ml at the 48th hour。
2.4 The influence of colony-forming ability of NNAVC against KGla cells
Methylcellulose semisolid method was used in the experiment, the KGla cells were dispersed on the 1st day to 2nd day, the colony was began to form on the 7th day and gradually increased on the 7th to 14th day. The morphology of colony in every group :the amount of colony forming was more in the control group, which was intensive colony; the amount of colony forming significantly decreased in the NNAVC-treated group and dead cells existing in the cells, while the colony was less and more cell debris in the high concentration group(0.8μg/ml).
The amounts of colony forming in every group on the 7th and 14th day were statistically performed, and the results showed that the reciprocal effect existed between the NNAVC concentration and culture time (F=70.623, P= 0.000);The amounts of colony forming among different groups were significant(F=204.351, P = 0.000) and the culture time were also significant (F=305.877, P= 0.000)
The amount of colony after the 14th day of culture was more than that on the 7th day (t=-13.784, P=0.000; t=-8.615, P=0.000; t=-6.728, P= 0.003). Different concentrations of NNAVC had an effect on colony-forming ability of KG1a cells in the designated time, and the amount of colony was significantly less inside the group (F= 34.357, P= 0.001; F=172.368, P= 0.000); The amount of colony forming in the group of NNAVC 0.8μg/ml had no significant difference comparing with in the group of NNAVC 0.7μg/ml (P=0.411, P=0.185),but the former was less than latter. These findings indicated that the proliferative ability of KG1a cells was lower in the NNAVC groups, NNAVC could inhibit the colony forming ability of KGla cells in some extent,which caused proliferative inhibition.
2.5 Cell cycle effects of NNAVC on KGla
The proportions of KGla cells at the different stage of cell cycle in every group were significant (F= 339.698, P= 0.000; F=6.439, P= 0.032; F= 127.007, P=0.000);The proportions of cells at the G0/G1 stage at the concentration of 0.7μg/ml and 0.8μg/ml were up to 58.67% and 62.20%,respectively,which was significant comparing with the control group(52.93%,P=0.032, P= 0.002),while the proportion at the G2/M stage decreased to 5.33% and 3.13%,respectively (P= 0.000, P= 0.000).The proportion at the S stage at the concentration of 0.8μg/ml was 34.70% comparing with control group(36.90%,P= 0.012).There were no significant difference at the G0/G1 and S stage between the concentration of 0.7μg/ml group and 0.8μg/ml group (P=0.048, P= 0.079),while the proportion at the G2/M stage was significant (P=0.003),and subdiploid peak ahead of G1 stage appeared in the treated group. The results suggested that NNAVC could induce cell cycle arrest of KGla cells at the G0/G1 stage and reduce the contents at the G2/M stage to inhibit the proliferation.
2.6 The observation of cell apoptosis by fluorescent microscope
KGla cells were in good condition with the integrity of cellwall and no apoptotic body emerged in the control group. The vacuole within the cell, cell pyknosis and rupture, apoptotic body appeared in the NNAVC-treated group, and more apoptotic cells emerged with higher concentrations and longer action time.
2.7The apoptotic rates measurement of KGla cells treated with NNAVC
The early and late apoptotic rates increased after NNAVC treatment, and the early and late apoptotic rates in the 0.7μg/ml concentration group increased to 39.63 % and 22.23%,respectively(P= 0.049, P= 0.034);those in the 0.8μg/ml concentration group were 40.73% and 41.50%(P= 0.040, P= 0.016);while no significance in the 0.6μg/ml concentration group. The early apoptotic rates between the 0.7¨g/ml and 0.8μg/ml concentration groups were not significant(P=1.000),but the late apoptotic rates were significant(P=0.021).The results confirmed that NNAVC Could induce the apoptosis of KG1a cells.
2.8 DNA Ladder measurement
There was no DNA Ladder in the control group,which showed the genomic DNA；DNA Ladder was seen in the NNAVC-treated group,which consisted of 180-200bp or more.These results indicated that NNAVC could lead to the DNA fragment of KG1a cells,that further confirmed the apoptotic induction of NNAVC.
Chapter 3 The effects of apoptosis-related genes and protein expression of NNAVC against KG1a cells
3.1 The effects of apoptosis-related mRNA expression levels of NNAVC against KGla cells
The results showed that the apoptosis-related genes in the different concentration of NNAVC groups,such as Bcl-2,Bcl-xL,Bax,Bid,Bad, p53,PUMA,NOXA and NF-KB,etc.,were significant,respectively(F=5.774,P= 0.021；F=20.652,P=0.000；F=9.780,P=0.005；F=6.795,P=0.014；F= 18.389,P=0.001; F=14.816,P=0.001；F=246.243,P=0.000；F=10.467, P=0.004；F=7.153,P=0.012；F=47.767,P=0.000)；The expression level of Bcl-2 gene descended comparing with control group(P=0.035,P=0.004, P= 0.028),while there were no significance among the groups(P=0.162,P=0.884, P=0.202)；The expression level of Bcl-xL gene rose up in a dose-dependent manner, especially 0.7μg/ml and 0.8μg/ml(P=0.003,P=0.018)；The expression level of Bak gene had no significant deviation in all groups(F=0.406,P=0.753)；The expression level of P53 gene,which was not expressed in normal KG 1 a cell,had obviously risen up (P=0.000,P=0.000,P=0.000),while PUMA and NOXA genes related to P53 had no change; The expression level of NF-κB gene in the concentration of 0.6μg/mland 0.7μg/ml descended (P= 0.000, P= 0.000).Taken together, NNAVC could induce the KGla cells apoptosis by modulating the balance between pro-and anti-apoptosis genes.
3.2 The effects of cytochrome C of NNAVC on KGla cells
The results showed that the expression level of cytochrome C had an up-and-down trend in the concentration of 0.7μg/ml within 12h;while that in the concentration of 0.8μg/ml rose up within 6h,and unchanged within 6h to 12h, then descended within 12h to 24h, at last that was very close between two groups. there were reciprocal effects existing between the different concentration and time of NNAVC (F= 51.868, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of cytochrome C (F= 82.331, P= 0.001;F=552.286, P=0.000); The content of cytochrome C in 0.7μg/ml group less than that in 0.8μg/ml group (P=0.018) at the 6th hour; However, the content of cytochrome C in 0.7μg/ml group more than that in 0.8μg/ml group (P=0.001; P = 0.026) at the 12th and 18th hour.
3.3 The measurement of caspase-9 protein by spectrophotometric method
There were reciprocal effects existing between the different concentration and time of NNAVC (F=23.751, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-9 protein (F= 23.982, P=0.001;F=180.774, P=0.000); The expression level of caspase-9 rose up within 12h and gradually descended after 12h; NNAVC had an effect on expression level of caspase-9 of KGla cells in a concentration-dependent manner. The results suggested that NNAVC could induce the apoptosis of KGla cells by activating caspase-9.
3.4 The effects of death receptors and decoy receptors expression levels of NNAVC on KGla cells
The expression level of death receptor 4(DR4) decreased with the NNAVC concentration increasing,and there were significant among the groups (F=170.107, P=0.000) or between groups (P≤0.005); The expression level of DR5 rose up in the concentration of 0.6μg/ml and 0.7μg/ml (P= 0.000; P=0.000); The expression level of decoy receptor 1(DcRl) had obvious significance among the groups (F=37.002, P=0.000),that in the concentration of 0.8μg/ml descended (comparing with other groups, P=0.000, P=0.000); The expression level of DcR2 decreased with the NNAVC concentration increasing, however, that in the concentration of 0.6μg/ml and 0.7μg/ml rose up (P=0.000; P=0.000)
3.5 The measurement of caspase-8 protein by spectrophotometric method
There were reciprocal effects existing between the different concentration and time of NNAVC (F=51.653, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-8 (F=47.400, P = 0.000; F= 189.867, P= 0.000). The expression level of caspase-8 had a rise-and-down process in groups. These showed that NNAVC could activate the caspase-8 in a short time to initiate the apoptotic process.
3.6 The effects of caspase-3 expression levelof NNAVC on KGla cells
There were reciprocal effects existing between the different concentration and time of NNAVC (F=29.694, P= 0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-3 (F=202.507, P = 0.000; F=205.361, P= 0.000).
The expression level of caspase-3 had a rise-and-down tendency in groups, and there were significant deviance in times (F= 36.236, P= 0.020; F=28.489, P= 0.025; F= 175.320, P=0.003). The expression level of caspase-3 rose up with the NNAVC concentration increasing at the 12th and 24th hour (F= 177.938, P= 0.000; F= 42.728, P= 0.000).The results indicated that NNAVC could activate the caspase-3 to induce the apoptosis of KGla cells, and caspase-3 was activated at the 6th hour,most at the 12th hour,in a dose-dependent manner.
Chapter 4 The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells
4.1 The cytotoxic effects of NNAVC against PBMC and activated immune cells
The cell viabilities of PBMC and activated immune cells were not significant difference at various concentrations of NNAVC treatment (F=4.025, P=0.062); The cytotoxic effects of cells treated with different concentration of NNAVC were significant (F=4.629, P=0.016).The toxic effect of NNAVC (0.5-2.0μg/ml) on PBMC was almost rare comparing with the control group (F= 0.626, P=0.618);There was no toxic action of NNAVC on activated immune cells when NNAVC was less than 1.0μg/ml, but toxic effect at the concentration of 2.0μg/ml was significant (P=0.003).That indicated that there were no toxic effects of NNAVC against PBMC and activated immune cells when the concentration was less than 1.0μg/ml.
4.2 The cytotoxic effects of activated immune cells on KGla cells
The results showed that the cytotoxic effects of activated immune cells on KG1a cells at the different effector-to-target(E:T) cell ratios were significant deviation (F=199.354, P=0.000),and the mean of cell activities were 50.04%,3.95 %,36.59%,23.63%,33.27% and 8.35% ,respectively. The cytotoxicity had significant deviation at the different E:T cell ratios (F=345.299, P=0.000); The cytotoxic effects of activated immune cells on targeted cells increased with the E:T cell ratios rising except for E group, there were significant deviation among the groups (F=25.711, P=0.000; F=53.466, P=0.000; F=43.224, P=0.000; F=135.884, P=0.000). The cytotoxic effects of activated immune cells on targeted cells in A to E groups had a descending tendency, which suggested that the sensitivity of KG1a cells to activated immune cells decreased after those were killed by different activated immune cells.
4.3 The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells
The cytotoxicity of NNAVC alone on KGla cells was 40.61%;while the cytotoxicity of activated immune cells alone at the E:T cell ratios of 10:1 and 20:1 on KG1a cells were 34.55% and 41.35%,respectively(P=0.414). The cytotoxicity of NNAVC combined with activated immune cells at the E:T cell ratios of 10:1 and 20:1 on KGla cells were 56.21% and 85.59%,which was significantly higher than NNAVC alone or activated immune cells alone (P= 0.018, P= 0.000). The cytotoxicity of NNAVC on activated immune cells at the E:T cell ratios of 10:1 and 20:1 was very low (4.35% and 1.33%, respectively).The results indicated that the cytotoxicity of NNAVC combined with activated immune cells on KGla cells was better than NNAVC or activated immune cells alone, but it didn't mean the synergism of NNAVC and activated immune cells.
4.4 The sensitivity of KGla treated with NNAVC to activated immune cells
The cytotoxicity of activated immune cells at the different E:T cell ratios on KG1a cells treated with NNAVC was significant (F=148.593, P=0.000); The cell activities of activated immune cells at the E:T cell ratios of 10:1 and 20:1 on targeted cells treated with NNAVC was significant (F=8.721, P=0.001). The cytotoxicity of activated immune cells at the E:T cell ratio of 10:1 on targeted cells treated with NNAVC was significant among groups (F=12.015, P=0.002); However, that at the E:T cell ratios of 20:1 was not significant among groups (F=1.177, P=0.337);The targeted cells within the group were significant (P=0.003, P= 0.010, P= 0.010, P= 0.001) at the E:T cell ratios of 10:1 and 20:1.The results showed that the cytotoxicity of activated immune cells on targeted cells treated with NNAVC was stronger with higher E:T cell ratio, KG1a treated with NNAVC to activated immune cells was still very sensitive.
4.5 The colony-forming abilities of NNAVC alone or combined with activated immune cells on KGla cells
The colony-forming amounts of NNAVC alone or combined with activated immune cells on KG1a cells were significant (F= 108.614, P=0.000) after KG1a was inoculated for 7 days; The different concentrations of NNAVC were significant as well (F=270.741, P=0.000); There were reciprocal effects existing between the different concentrations and treatment strategy of NNAVC (F=9.956, P= 0.003) The inhibitory effect of colony-forming abilities of NNAVC alone on KGla cells was significantly worse than the NNAVC combined with activated immune cells group (t=9.141, P=0.001; t=8.273, P=0.001); The colony-forming amounts significantly decreased with the concentration of NNAVC increasing (F=58.886, P = 0.000; F=426.629, P= 0.000)
The colony-forming amounts of NNAVC alone or combined with activated immune cells on KG1a cells were significant (F= 26.059, P=0.000) after KG1a was inoculated for 14 days; The different concentrations of NNAVC were significant as well (F= 174.366, P=0.000); There were not reciprocal effects existing between the different concentrations and treatment stategy of NNAVC (F=2.623, P=0.114). The inhibitory effect of colony-forming abilities of NNAVC alone on KGla cells was significantly worse than the NNAVC combined with activated immune cells group (t=5.822, P=0.004; t=6.554, P=0.003); The colony-forming amounts significantly decreased with the concentration of NNAVC increasing (F=49.972, P = 0.000; F=167.300, P= 0.000).
The colony-forming amounts of NNAVC combined with activated immune cells on KGla cells less than NNAVC alone, which suggested that NNAVC combined with activated immune cells could decrease the amount of colony by killing the colony-forming cells of KG1a.
4.6 The effects of NNAVC on expressions of NKG2D ligand on the surface of KGla cells
The expressions of MICA,MICB,ULBP1,ULBP2 and ULBP3 in KGla cells treated with NNAVC were significant between groups (F=3654.504, P=0.000; F = 978.748, P= 0.000;F= 1328.553, P=0.000;F=455.271,P= 0.000;F=223.263, P= 0.000); The expressions of ULBP3 in KGla cells treated with 0.6μg/ml NNAVC significantly rose up (P=0.003),however, those of other ligands were significantly less than control group (P=0.000, P=0.000, P=0.000, P=0.000); The expressions of ULBP1,ULBP2 and ULBP3 except for MICB in KGla cells treated with 0.7μg/ml NNAVC significantly rose up comparing with control (P= 0.000, P=0.000, P=0.000).Those treated with 0.8μg/ml NNAVC were as well (P= 0.000, P=0.001, P=0.000); The expressions of ULBP1,ULBP2 and ULBP3 were significant between groups (P= 0.000).The results indicated that ULBP1,ULBP2 and ULBP3 could be the target of NNAVC in KG1a cells.
Conclusion
1. NNAVC can obviously inhibit the proliferation of KGla cells by inducing apoptosis, cell cycle arrest of KG1a cells at the G0/G1 stage and the inhibition of colony-forming cells.
2. NNAVC could upregulated the expression of Bax, Bad, p53 and NOXA at mRNA level, and downregulated the expression of Bcl-2 and NF-KB.
3. NNAVC could induce cytochrome-c released into the endochylema, activate the caspase-9 and caspase-3, the intrinsic apoptosis pathway was involved in the action of NNAVC to KG1a.
4. At the range of effective concentration of NNAVC to KG1a cells,the cytotoxicity of NNAVC on activated immune cells was less than on KG1a cells.
5. The KG1a cells that are survived after NNAVC's effection could be killed by activated immune cells.
6. NNAVC can combine with activated immune cells to kill KGla cells whether co-incubated or in one-after-another way.
7. NNAVC(0.08mg/kg) combined with activated immune cells can increase the survival time of hu-Balb/c-nu-KG1a leukemic mice.
Innovation of our research
Our research firstly discovered the fact that NNAVC combined with activated immune cells could be used to treat acute myeloid leukemia, and NNAVC had no side effect on activated immune cells when they killed leukemic cells.
Values of our research
Our research demonstrated NNAVC combined with activated immune cells could kill the KGla cells more effectively and treat hu-Balb/c-nu-KGla leukemic mice,whose survival times were increased.The research provided a new insight for the the therapy of AML and long
引文
[1]Murray JA, Khanim FL, Hayden RE, et al.Combined bezafibrate and medroxyprogesterone acetate have efficacy without haematological toxicity in elderly and relapsed acute myeloid leukaemia (AML). Br J Haematol.2010 Jan 13. [Epub ahead of print]
[2]Lu C, Hassan HT. Human stem cell factor-antibody [anti-SCF] enhances chemotherapy cytotoxicity in human CD34+ resistant myeloid leukaemia cells. Leuk Res,2006,30:296-302.
[3]Krause DS, Van Etten RA. Right on target:Eradicating leukemic stem cells. Trends Mol Med,2007,13:470-481.
[4]Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukemia after transplantation into SCID mice. Nature,1994,367:645-648.
[5]Blagosklonny MV.. Target for cancer therapy:proliferating cells or stem cells.[J].Leukemia,2006, (20):385-391.
[6]Connelly-Smith L, Pattinson J, Grundy M,et al. P-glycoprotein is downregulated in KGla-primitive leukemia cells by LDL cholesterol deprivation and by HMG-CoA reductase inhibitors. Exp Hematol.2007 Dec;35(12):1793-800.
[7]Didier C, Cavelier C, Quaranta M,et al. G2/M checkpoint stringency is a key parameter in the sensitivity of AML cells to genotoxic stress. Oncogene.2008 Jun 19;27(27):3811-20.
[8]Aversa SM, Trentin C, Soraru M, et al. Acute promyelocytic leukemia after Stanford V plus radiotherapy for advanced Hodgkin lymphoma. Leuk Lymphoma.2009;50(7):1214-6.
[9]Li YF, Liu X, Liu DS, et al.The effect of homoharringtonine in patients with chronic myeloid leukemia who have failed or responded suboptimally to imatinib therapy. Leuk Lymphoma.2009;50(11):1889-91.
[10]Jin Y, Lu Z, Cao K, Zhu Y, et al. The antitumor activity of homoharringtonine against human mast cells harboring the KIT D816V mutation. Mol Cancer Ther.2010 Jan;9(1):211-23.
[11]Duan XF, Wu YL, Xu HZ,et al. Synergistic mitosis-arresting effects of arsenic trioxide and paclitaxel on human malignant lymphocytes. Chem Biol Interact. 2010;183(1):222-30.
[12]Thomadaki H, Floros KV, Scorilas A. Molecular response of HL-60 cells to mitotic inhibitors vincristine and taxol visualized with apoptosis-related gene expressions, including the new member BCL2L12. Ann N Y Acad Sci. 2009;1171:276-83.
[13]Masami Ishibashi, Takasbi Obtsuki. Studies on search for bioactive natural products targeting TRAIL signaling leading to tumor cell apoptosis[J]. Med Res Rev.2008;28(5):688-714.
[14]El-Refael MF, Sarkar NH. Snake venom inhibits the growth of mouse mammary tumor cells in vitro and in vivo. Toxicon.2009;54(1):33-41.
[15]Zouari-Kessentini R, Luis J, Karray A,et al. Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration. Toxicon.2009;53(4):444-53.
[16]Rodrigues RS, Izidoro LF, de Oliveira RJ Jr,et al. Snake venom phospholipases A2:a new class of antitumor agents. Protein Pept Lett. 2009;16(8):894-8.
[17]Gebrim LC, Marcussi S, Menaldo DL,et al. Antitumor effects of snake venom chemically modified Lys49 phospholipase A2-like BthTX-I and a synthetic peptide derived from its C-terminal region. Biologicals.2009;37(4):222-9.
[18]Alves RM, Antonucci GA, Paiva HH,et al. Evidence of caspase-mediated apoptosis induced by 1-amino acid oxidase isolated from Bothrops atrox snake venom. Comp Biochem Physiol A Mol Integr Physiol.2008;151(4):542-50.
[19]Park MH, Son DJ, Kwak DH, et al. Snake venom toxin inhibits cell growth through induction of apoptosis in neuroblastoma cells. Arch Pharm Res.2009 Nov;32(11):1545-54.
[20]Brown MC, Staniszewska I, Del Valle L,et al. Angiostatic activity of obtustatin as alpha 1 beta 1 integrin inhibitor in experimental melanoma growth. Int J Cancer.2008 Nov 1;123(9):2195-203.
[21]Xie Q, Wan R, Lin X,et al. Effects of snake venom cystatin gene on gene expression profiles in mouse melanoma cell line B16F1.Ai Zheng.2008 Jul;27(7):716-22.
[22]Zhang L, Cui L. A cytotoxin isolated from Agkistrodon acutus snake venom induces apoptosis via Fas pathway in A549 cells. Toxicol In Vitro.2007 Sep;21(6):1095-103.
[23]Kim DS, Jang YJ, Jeon OH,et al. Saxatilin, a snake venom disintegrin, suppresses TNF-alpha-induced ovarian cancer cell invasion. J Biochem Mol Biol.2007 Mar 31;40(2):290-4.
[24]Dong Ju Son, Mi Hee Park, Sang Jin Chae, et al. Inhibitory effect of snake venom toxin from Vipera lebetina turanica on hormone- refractory human prostate cancer cell growth:induction of apoptosis through inactivation of nuclear factorKB. Mol Cancer Ther 2007;6(2):675-683.
[25]Hong SY, Koh YS, Chung KH, Kim DS. Snake venom disintegrin, saxatilin, inhibits platelet aggregation, human umbilical vein endothelial cell proliferation, and smooth muscle cell migration. Thromb Res. 2002;105:79-86.
[26]Minn AJ, Velez P, Schendel SL, et al. Bcl-x forms anion channel in synthetic lipid membranes [J]. Science,1997,385:353-357.
[27]Robert A. Eagle, John Trowsdale. Promiscuity and the single receptor: NKG2D [J]. Nature Reviews Immunology.2007; 7:737-744.
[28]Nausch N, Cerwenka A.NKG2D ligands in tumor immunity. Oncogene.2008 Oct 6;27(45):5944-58.
[29]Guilloton F, de ThonelA, Jean C, et al. TNFalpha stimulates NKG2D mediated lytic activity of acute myeloid leukemic cells [J]. Leukemia,2005,19:2206-2214.
[30]Sutherland C, Rabinovich B, ChalupnyN, et al. ULBPs, human ligands of the NKG2D receptor, stimulate tumor immunity with enhancement by IL-15. Blood,2006,108:1313-1319.
[31]Decot V, Voillard L, Latger-Cannard V,et al. Natural-killer cell amplification for adoptive leukemia relapse immunotherapy:Comparison of three cytokines, IL-2, IL-15, or IL-7 and impact on NKG2D, KIR2DL1, and KIR2DL2 expression. Exp Hematol.2010 Feb 19. [Epub ahead of print]
[1]Rygaard J, Povlsen CO. Heterotransplantation of a human malignant tumour to "Nude" mice. Acta Pathol Microbiol Scand.1969;77(4):758-60.
[2]Gazdar AF, Carney DN, Sims HL,et al. Heterotransplantation of small-cell carcinoma of the lung into nude mice:comparison of intracranial and subcutaneous routes. Int J Cancer.1981;28(6):777-83.
[3]Giovanella BC, Stehlin JS Jr, Shepard RC, et al. Correlation between response to chemotherapy of human tumors in patients and in nude mice. Cancer. 1983;52(7):1146-52.
[4]Fodstad O, Aass N, Pihl A.Response to chemotherapy of human, malignant melanoma xenografts in athymic, nude mice. Int J Cancer.1980;25(4):453-8.
[5]Petrich T, Korkmaz Z, Krull D,et al. In vitro experimental (211)At-anti-CD33 antibody therapy of leukaemia cells overcomes cellular resistance seen in vivo against gemtuzumab ozogamicin. Eur J Nucl Med Mol Imaging.2010 Jan 27. [Epub ahead of print]
[6]HP Koeffler, R Billing, AJ Lusis, et al. An undifferentiated variant derived from the human acute myelogenous leukemia cell line (KG-1).Blood.1980; 56:265-273.
[1]Estey E, Dohner H. Acute myeloid leukaemia.Lancet.2006;368:1894-1907.
[2]Kell J.Treatment of relapsed acute myeloid leukaemia. Rev Recent Clin Trials. 2006;1(2):103-111.
[3]Oliansky DM, Antin JH, Bennett JM, et al. The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of myelodysplastic syndromes:an evidence-based review. Biol Blood Marrow Transplant. 2009;15(2):137-172.
[4]McLornan DP, McMullin MF, Johnston P, et al. Molecular mechanisms of drug resistance in acute myeloid leukaemia. Expert Opin Drug Metab Toxiclo.2007;3:363-377.
[5]Mak DH, Schober WD, Chen W,et al.Triptolide induces cell death independent of cellular responses to imatinib in blast crisis chronic myelogenous leukemia cells including quiescent CD34+ primitive progenitor cells. Mol Cancer Ther. 2009;8(9):2509-16.
[6]Chui CH, Lau FY, Tang JC, et al.Activities of fresh juice of Scutellaria barbata and warmed water extract of Radix Sophorae Tonkinensis on anti-proliferation and apoptosis of human cancer cell lines. Int J Mol Med.2005;16(2):337-41.
[7]Cecchinato V, Chiaramonte R, Nizzardo M, et al. Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells. Biochem Pharmacol.2007;74(11):1568-74.
[8]Goergens A, Frericks M, Esser C. The arylhydrocarbon receptor is only marginally involved in the antileukemic effects of its ligand curcumin. Anticancer Res.2009 Nov;29(11):4657-64.
[9]Kok SH, Chui CH, Lam WS, et al. Mechanistic insight into a novel synthetic cantharidin analogue in a leukaemia model. Int J Mol Med.2006;18(2):375-9.
[10]Zhu L, Yuan C, Chen Z, et al. Expression, purification and characterization of recombinant Jerdonitin, a P-Ⅱ class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. Toxicon.2010;55(2-3): 375-80.
[11]de Figueiredo-Pontes LL, Pintao MC, Oliveira LC, et al. Determination of P-glycoprotein, MDR-related protein 1, breast cancer resistance protein, and lung-resistance protein expression in leukemic stem cells of acute myeloid leukemia. Cytometry B Clin Cytom.2008;74(3):163-168.
[12]Estrov Z.The leukemia stem cell. Cancer Treat Res.2009;145:1-17.
[13]Didier C, Cavelier C, Quaranta M, et al. G2/M checkpoint stringency is a key parameter in the sensitivity of AML cells to genotoxic stress[J].Oncogene,2008, 27(27):3811-3820.
[14]Rainey MD, Black EJ, Zachos G, et al.Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase[J]. Oncogene,2008,27(7):896-906.
[1]Wang XD. The expanding role of mitochondria in apoptosis. Genes Dev.2001, 15(11):2922-2933.
[2]Green DR, Reed JC. Mitochondria and apoptosis. Science.1998; 281(5381):1309-12.
[3]Xiao L, Caino MC, von Burstin VA,et al. Phorbol ester-induced apoptosis and senescence in cancer cell models. Methods Enzymol.2008;446:123-39.
[4]Blagosklonny MV. Target for cancer therapy:proliferating cells or stem cells. Leukemia,2006,20:385
[5]陈纯,陈崇宏.眼镜蛇毒细胞毒素CTX-d诱导NB4细胞凋亡及其机制研究.中草药,2007,38(12)：1838
[6]吴穗晶,杜欣,蒋文玲,等.中华眼镜蛇毒对K562和难治性白血病细胞体外作用的研究.现代肿瘤医学,2008,16(3)：428
[7]LalierL, Cartron PF, Juin P, et al. Bax activation and mitochondrial insertion during apoptosis. Apoptosis,2007,12(5):887-896.
[8]Del Poeta G, Ammatuna E, Lavorgna S,et al. The genotype nucleophosmin mutated and FLT3-ITD negative is characterized by high bax/bcl-2 ratio and favourable outcome in acute myeloid leukaemia. Br J Haematol.2010 Feb 8. [Epub ahead of print]
[9]Farrow S N, White J H M, Martinou I, et al. Cloning of a bcl-2 homologue by interaction with adenovirus E1B-19K. Nature,1995,374(6524):731-733.
[10]Wong WW, Puthalakath H.Bcl-2 family proteins:the sentinels of the mitochondrial apoptosis pathway.IUBMB Life.2008 Jun;60(6):390-7.
[11]Kuwana T, Newmeyer DD. Bcl-2 family proteins and the role of mitochondria in apoptosis. Curr Opin Cell Biol,2003,15:691-699.
[12]Klee M, Pallauf K, Alcala S, et al. Mitochondrial apoptosis induced by BH3-only molecules in the exclusive presence of endoplasmic reticular Bak..EMBO,2009,28(12):1757-1768.
[13]Cavalcanti GB Jr, Scheiner MA, Simoes Magluta EP,et al. p53 flow cytometry evaluation in leukemias:Correlation to factors affecting clinical outcome. ytometry B Clin Cytom.2010 Mar 2. [Epub ahead of print]
[14]Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, Green DR.Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosisScience.2004;303(5660):1010-4.
[15]Egger L, Madden DT, Rheme C, et al. Endoplasmic reticulum stress-induced cell death mediated by the proteasome. Cell Death Differ,2007,14:1172-1180.
[16]Qiu Q, Wang Z, Jiang JM, et al. Effect of lidamycin on mitochontria initiated apoptotic pathway in human cancer cells. Acta Pharm Sin,2007,42:132-138.
[17]E Szegezdi, S Cahill, M Meyer, et al. TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt. Br J Cancer,2006,94(3):398-406.
[18]Hongkui Jin, Renhui Yang, Jed Ross, et al. Cooperation of the Agonistic DR5 Antibody Apomab with Chemotherapy to Inhibit Orthotopic Lung Tumor Growth and Improve Survival[J]. Clinical Cancer Research,2008, 14:7733-7740.
[19]Hofmanova, J., Vaculova, A., Hyzd'Alova, M., et al. Response of normal and colon cancer epithelial cells to TNF-family apoptotic inducers[J]. Oncology Reports,2008,19 (2):567-573.
[1]Biron CA, Nguyen KB, Pien GC, et al. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol. 1999;17:189-220.
[2]Trinchieri G. Biology of natural killer cells. Adv Immunol.1989;47:187-376.
[3]Chen XM, Xu XQ, Sun K, et al. NKG2D ligands expression and NKG2D-mediated cytotoxicity in human laryngeal squamous carcinoma cells[J]. Scand Imm unol,2008,67 (5):441-447.
[4]Gasser S, Raulet DH. Activation and self2tolerance of natural killer cells[J]. Immunol Rev,2006,214:130-142.
[5]Brenner CD, King S, Przewoznik M, et al. Requirements for control of B-cell lymphoma by NK cells. Eur J Immunol.2010;40(2):494-504.
[6]Barber A, Rynda A, Sentman CL. Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment. J Immunol.2009;183(11):6939-47.
[7]Koebel CM, Vermi W, Swann JB,et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature.2007 Dec 6;450(7171):903-7.
[8]Decot V, Voillard L, Latger-Cannard V,et al. Natural-killer cell amplification for adoptive leukemia relapse immunotherapy:Comparison of three cytokines, IL-2, IL-15, or IL-7 and impact on NKG2D, KIR2DL1, and KIR2DL2 expression. Exp Hematol.2010 Feb 19. [Epub ahead of print]
[9]郭坤元,梅家转,姚开泰,尹晓林.NK细胞免疫编辑后的人鼻咽癌细胞株CNE2 HLA I类分子和MICA/MICB的表达及其对NK细胞杀伤活性的影响.第四军医大学学报；2007(11)：1005-1007.
[10]Poggi A, Massaro AM, Negrini S, et al. Tumor-induced apoptosis of human IL-2 activated NK cells:role of natural cytotoxicity receptors. J Immunol.2005; 174(5):2653-2660.
[11]Barber A, Rynda A, Sentman CL. Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment. J Immunol.2009;183(11):6939-47.
[12]Armeanu S, Krusch M, Baltz KM, et al. Direct and natural killer cell-mediated antitumor effects of low-dose bortezomib in hepatocellular carcinoma. Clin Cancer Res.2008; 14(11):3520-3528.
[13]Leslie Elsner, Vijayakumar Muppala, Mathias Gehrmann,et al. The Heat Shock Protein HSP70 Promotes Mouse NK Cell Activity against Tumors That Express Inducible NKG2D Ligands. The Journal of Immunology.2007; 179: 5523-5533.
[14]Kato N, Tanaka J, Sugita J, et al. Regulation of the expression of MHC class I related chain A, B (MICA, MICB) via chromatin remodeling and its impact on the susceptibility of leukemic cells to the cytotoxicity of NKG2D-expressing cells. Leukemia.2007; 21(10):2103-2108.
[15]Zhang C, Wang Y, Zhou Z, et al. Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis. Cancer Immunol Immunother.2009;8(58): 1275-1285.
[16]牛新清,胡亮杉,郭坤元,宋朝阳,涂三芳,梅家转.AS203对CD34+白血病细胞NKG2D配体表达及NK杀伤活性的影响[J].中国免疫学杂志.2008；24(6)：509-511.
[1]Wang XD. The expanding role of mitochondria in apoptosis[J]. Genes Dev,2001,15(11):2922-2933.
[2]Galluzzi L, Zamzami N, de La Motte Rouge T, Lemaire C, Brenner C, Kroemer G. Methods for the assessment of mitochondrial membrane permeabilization in apoptosis. Apoptosis.2007 May;12(5):803-13.
[3]Wieckowski MR. Postepy Biochem. The role of mitochondrial permeability transition pore in physiology and pathology of the cell.2008;54(1):71-81.
[4]喻爱喜,周志刚,张促宁等.TRAIL诱导骨肉瘤HOS-8606细胞凋亡与线粒体跨膜电位关系的研究.现代肿瘤医学,2005,13(5)586-588.
[5]Belosludtsev KN, Belosludtseva NV, Mironova GD. Possible mechanism for formation and regulation of the palmitate-induced cyclosporin A-insensitive mitochondrial pore.Biochemistry (Mosc).2005 Jul; 70(7):815-21.
[6]Orrenius S.Reactive oxygen species in mitochondria-mediated cell death. Drug Metab Rev.2007;39(2-3):443-55.
[7]Leu J I, Dumont P, Hafey M, et al.Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcll complex [J]. Nat Cell Biol,2004,6(5):443-450.
[8]Cheng E H, Wei M C, Weiler S, et al. BCL-2, BCL-X(L) sequester BH3 domainonly molecules preventing BAX- and BAK-mediated mitochondrial apoptosis [J]. Mol Cell,2001,8(3):705-711.
[9]Wong WW, Puthalakath H.Bcl-2 family proteins:the sentinels of the mitochondrial apoptosis pathway.IUBMB Life.2008 Jun; 60(6):390-7.
[10]Green DR, Reed JC.Mitochondria and apoptosis.Science.1998 Aug 28;281(5381):1309-12.
[11]Gogvadze V, Robertson JD, Zhivotovsky B, Orrenius S. Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax.J Biol Chem.2001 Jun 1;276(22):19066-71. Epub 2001 Mar 22.
[12]Masami Ishibashi, Takasbi Obtsuki. Studies on search for bioactive natural products targeting TRAIL signaling leading to tumor cell apoptosis[J].Med Res Rev. Feb 2008, [Epub ahead of print].
[13]Dong Ju Son, Mi Hee Park, Sang Jin Chae, et al. Inhibitory effect of snake venom toxin from Vipera lebetina turanica on hormone- refractory human prostate cancer cell growth:induction of apoptosis through inactivation of nuclear factor KB. Mol Cancer Ther 2007;6(2):675-683.
[14]Minn AJ, Velez P, Schendel SL, et al. Bcl-x forms anion channel in synthetic lipid membranes [J]. Science,1997,385:353-357.
[15]Torii S,Yamane K,Mashima T,et al.Molecular cloning and functional analysis of apoxin Ⅰ,a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity [J]. Biochemistry,2000,39 (12):3197-3205.
[16]张若燕.中药斑蝥及复方斑蝥制剂在治疗恶性肿瘤中的作用[J].内科,2006,1(1)：73-74.
[17]何太平,莫丽儿,梁念慈.斑蝥素诱导高转移卵巢癌细胞HO-8910PM凋亡的 研究[J].中国药科大学学报,2005,36(2)：164-167.
[18]Thomas Efferth, Rolf Rauh, Stefan Kahl, et al. Molecular modes of action of cantharidin in tumor cells [J]. Biochemical Pharmacology,2005, 69(5):811-818.
[19]Jeong-Eun Huh, Kyung-Sun Kang, Chanhee Chae, et al.Roles of p38 and JNK mitogen-activated protein kinase pathways during cantharidin-induced apoptosis in U937 cells [J]. Biochemical Pharmacology,2004, 67(10):1811-1818.
[20]Xianxi Wang. Ranyia Matta. Gang Shen.Leif D. Nelin. Dehua Pei. Yusen Liu. Mechanism of triptolide-induced apoptosis:effect on caspase activation and Bid cleavage and essentiality of the hydroxyl group of triptolide [J]. Mol Med (2006)84:405-415
[21]Bing Z. Carter, Duncan H. Mak, Wendy D. Schober, Teresa McQueen, David Harris, Zeev Estrov, Randall L. Evans, Michael Andreeff.Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells[J].Blood 2006,108:630-637
[22]Guang-Biao Zhou, Hui Kang, Lan Wang, et al. Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21) leukemia in vitro and in vivo[J]. Blood.2007;109(8):3441-3450.
[23]Simone Fulda, Klaus-Michael Debatin. Resveratrol modulation of signal transduction in apoptosis and cell survival:A mini-review [J].Cancer Detection and Prevention.2006;30:217-223.
[24]Prasad KN, Kumar B, Yan XD, et al. Alpha - tocopheryl succinate, the most effective form of vitamine E for adjuvant cancer treatment:a review [J]. J Am Coll Nutr,2003,22 (2):108-117.
[25]Ripoll EA, Rama BN, Webber MM. Vitamine E enhances the chemotherapeutic effectsof adriamycinon human prostatic carcinoma cells in vitro[J].J Urol,1986, 136 (2):529-531.
[26]Weber T,Dalen H,AnderaL, et al. Mitochondrial playa central role in apoptosis induced by alpha - tocopheryl succinate, an agent with antineoplastic activity: comparisionwith receptor- mediated proapoptotic signaling [J]. Biochemistry, 2003,42(14):4277-4291.
[27]Efferth T, BenakisA, RomeroM R, et al. Enhancement of cytotoxicity of artemisinins toward cancer cells by ferrous iron [J]. Free Radic Biol Med, 2004,37 (7):998-1009.
[28]Efferth T, GiaisiM, MerlingA, et al. Artesunate induces ROS2mediated apop tosis in doxorubicin2resistant T leukemia cells [J]. PLoSONE,2007,2 (1) e693.
[29]Yao K, Ye P, Zhang L, et al. Epigallocatechin gallate protects against oxidative stress-induced mitochondria-dependent apoptosis in human lensepithelial cells [J].Mol Vis,2008,14:217-223.
[2]Lu C, Hassan HT. Human stem cell factor-antibody [anti-SCF] enhances chemotherapy cytotoxicity in human CD34+ resistant myeloid leukaemia cells. Leuk Res,2006,30:296-302.
[3]Krause DS, Van Etten RA. Right on target:Eradicating leukemic stem cells. Trends Mol Med,2007,13:470-481.
[4]Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukemia after transplantation into SCID mice. Nature,1994,367:645-648.
[5]Blagosklonny MV.. Target for cancer therapy:proliferating cells or stem cells.[J].Leukemia,2006, (20):385-391.
[6]Connelly-Smith L, Pattinson J, Grundy M,et al. P-glycoprotein is downregulated in KGla-primitive leukemia cells by LDL cholesterol deprivation and by HMG-CoA reductase inhibitors. Exp Hematol.2007 Dec;35(12):1793-800.
[7]Didier C, Cavelier C, Quaranta M,et al. G2/M checkpoint stringency is a key parameter in the sensitivity of AML cells to genotoxic stress. Oncogene.2008 Jun 19;27(27):3811-20.
[8]Aversa SM, Trentin C, Soraru M, et al. Acute promyelocytic leukemia after Stanford V plus radiotherapy for advanced Hodgkin lymphoma. Leuk Lymphoma.2009;50(7):1214-6.
[9]Li YF, Liu X, Liu DS, et al.The effect of homoharringtonine in patients with chronic myeloid leukemia who have failed or responded suboptimally to imatinib therapy. Leuk Lymphoma.2009;50(11):1889-91.
[10]Jin Y, Lu Z, Cao K, Zhu Y, et al. The antitumor activity of homoharringtonine against human mast cells harboring the KIT D816V mutation. Mol Cancer Ther.2010 Jan;9(1):211-23.
[11]Duan XF, Wu YL, Xu HZ,et al. Synergistic mitosis-arresting effects of arsenic trioxide and paclitaxel on human malignant lymphocytes. Chem Biol Interact. 2010;183(1):222-30.
[12]Thomadaki H, Floros KV, Scorilas A. Molecular response of HL-60 cells to mitotic inhibitors vincristine and taxol visualized with apoptosis-related gene expressions, including the new member BCL2L12. Ann N Y Acad Sci. 2009;1171:276-83.
[13]Masami Ishibashi, Takasbi Obtsuki. Studies on search for bioactive natural products targeting TRAIL signaling leading to tumor cell apoptosis[J]. Med Res Rev.2008;28(5):688-714.
[14]El-Refael MF, Sarkar NH. Snake venom inhibits the growth of mouse mammary tumor cells in vitro and in vivo. Toxicon.2009;54(1):33-41.
[15]Zouari-Kessentini R, Luis J, Karray A,et al. Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration. Toxicon.2009;53(4):444-53.
[16]Rodrigues RS, Izidoro LF, de Oliveira RJ Jr,et al. Snake venom phospholipases A2:a new class of antitumor agents. Protein Pept Lett. 2009;16(8):894-8.
[17]Gebrim LC, Marcussi S, Menaldo DL,et al. Antitumor effects of snake venom chemically modified Lys49 phospholipase A2-like BthTX-I and a synthetic peptide derived from its C-terminal region. Biologicals.2009;37(4):222-9.
[18]Alves RM, Antonucci GA, Paiva HH,et al. Evidence of caspase-mediated apoptosis induced by 1-amino acid oxidase isolated from Bothrops atrox snake venom. Comp Biochem Physiol A Mol Integr Physiol.2008;151(4):542-50.
[19]Park MH, Son DJ, Kwak DH, et al. Snake venom toxin inhibits cell growth through induction of apoptosis in neuroblastoma cells. Arch Pharm Res.2009 Nov;32(11):1545-54.
[20]Brown MC, Staniszewska I, Del Valle L,et al. Angiostatic activity of obtustatin as alpha 1 beta 1 integrin inhibitor in experimental melanoma growth. Int J Cancer.2008 Nov 1;123(9):2195-203.
[21]Xie Q, Wan R, Lin X,et al. Effects of snake venom cystatin gene on gene expression profiles in mouse melanoma cell line B16F1.Ai Zheng.2008 Jul;27(7):716-22.
[22]Zhang L, Cui L. A cytotoxin isolated from Agkistrodon acutus snake venom induces apoptosis via Fas pathway in A549 cells. Toxicol In Vitro.2007 Sep;21(6):1095-103.
[23]Kim DS, Jang YJ, Jeon OH,et al. Saxatilin, a snake venom disintegrin, suppresses TNF-alpha-induced ovarian cancer cell invasion. J Biochem Mol Biol.2007 Mar 31;40(2):290-4.
[24]Dong Ju Son, Mi Hee Park, Sang Jin Chae, et al. Inhibitory effect of snake venom toxin from Vipera lebetina turanica on hormone- refractory human prostate cancer cell growth:induction of apoptosis through inactivation of nuclear factorKB. Mol Cancer Ther 2007;6(2):675-683.
[25]Hong SY, Koh YS, Chung KH, Kim DS. Snake venom disintegrin, saxatilin, inhibits platelet aggregation, human umbilical vein endothelial cell proliferation, and smooth muscle cell migration. Thromb Res. 2002;105:79-86.
[26]Minn AJ, Velez P, Schendel SL, et al. Bcl-x forms anion channel in synthetic lipid membranes [J]. Science,1997,385:353-357.
[27]Robert A. Eagle, John Trowsdale. Promiscuity and the single receptor: NKG2D [J]. Nature Reviews Immunology.2007; 7:737-744.
[28]Nausch N, Cerwenka A.NKG2D ligands in tumor immunity. Oncogene.2008 Oct 6;27(45):5944-58.
[29]Guilloton F, de ThonelA, Jean C, et al. TNFalpha stimulates NKG2D mediated lytic activity of acute myeloid leukemic cells [J]. Leukemia,2005,19:2206-2214.
[30]Sutherland C, Rabinovich B, ChalupnyN, et al. ULBPs, human ligands of the NKG2D receptor, stimulate tumor immunity with enhancement by IL-15. Blood,2006,108:1313-1319.
[31]Decot V, Voillard L, Latger-Cannard V,et al. Natural-killer cell amplification for adoptive leukemia relapse immunotherapy:Comparison of three cytokines, IL-2, IL-15, or IL-7 and impact on NKG2D, KIR2DL1, and KIR2DL2 expression. Exp Hematol.2010 Feb 19. [Epub ahead of print]
[1]Rygaard J, Povlsen CO. Heterotransplantation of a human malignant tumour to "Nude" mice. Acta Pathol Microbiol Scand.1969;77(4):758-60.
[2]Gazdar AF, Carney DN, Sims HL,et al. Heterotransplantation of small-cell carcinoma of the lung into nude mice:comparison of intracranial and subcutaneous routes. Int J Cancer.1981;28(6):777-83.
[3]Giovanella BC, Stehlin JS Jr, Shepard RC, et al. Correlation between response to chemotherapy of human tumors in patients and in nude mice. Cancer. 1983;52(7):1146-52.
[4]Fodstad O, Aass N, Pihl A.Response to chemotherapy of human, malignant melanoma xenografts in athymic, nude mice. Int J Cancer.1980;25(4):453-8.
[5]Petrich T, Korkmaz Z, Krull D,et al. In vitro experimental (211)At-anti-CD33 antibody therapy of leukaemia cells overcomes cellular resistance seen in vivo against gemtuzumab ozogamicin. Eur J Nucl Med Mol Imaging.2010 Jan 27. [Epub ahead of print]
[6]HP Koeffler, R Billing, AJ Lusis, et al. An undifferentiated variant derived from the human acute myelogenous leukemia cell line (KG-1).Blood.1980; 56:265-273.
[1]Estey E, Dohner H. Acute myeloid leukaemia.Lancet.2006;368:1894-1907.
[2]Kell J.Treatment of relapsed acute myeloid leukaemia. Rev Recent Clin Trials. 2006;1(2):103-111.
[3]Oliansky DM, Antin JH, Bennett JM, et al. The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of myelodysplastic syndromes:an evidence-based review. Biol Blood Marrow Transplant. 2009;15(2):137-172.
[4]McLornan DP, McMullin MF, Johnston P, et al. Molecular mechanisms of drug resistance in acute myeloid leukaemia. Expert Opin Drug Metab Toxiclo.2007;3:363-377.
[5]Mak DH, Schober WD, Chen W,et al.Triptolide induces cell death independent of cellular responses to imatinib in blast crisis chronic myelogenous leukemia cells including quiescent CD34+ primitive progenitor cells. Mol Cancer Ther. 2009;8(9):2509-16.
[6]Chui CH, Lau FY, Tang JC, et al.Activities of fresh juice of Scutellaria barbata and warmed water extract of Radix Sophorae Tonkinensis on anti-proliferation and apoptosis of human cancer cell lines. Int J Mol Med.2005;16(2):337-41.
[7]Cecchinato V, Chiaramonte R, Nizzardo M, et al. Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells. Biochem Pharmacol.2007;74(11):1568-74.
[8]Goergens A, Frericks M, Esser C. The arylhydrocarbon receptor is only marginally involved in the antileukemic effects of its ligand curcumin. Anticancer Res.2009 Nov;29(11):4657-64.
[9]Kok SH, Chui CH, Lam WS, et al. Mechanistic insight into a novel synthetic cantharidin analogue in a leukaemia model. Int J Mol Med.2006;18(2):375-9.
[10]Zhu L, Yuan C, Chen Z, et al. Expression, purification and characterization of recombinant Jerdonitin, a P-Ⅱ class snake venom metalloproteinase comprising metalloproteinase and disintegrin domains. Toxicon.2010;55(2-3): 375-80.
[11]de Figueiredo-Pontes LL, Pintao MC, Oliveira LC, et al. Determination of P-glycoprotein, MDR-related protein 1, breast cancer resistance protein, and lung-resistance protein expression in leukemic stem cells of acute myeloid leukemia. Cytometry B Clin Cytom.2008;74(3):163-168.
[12]Estrov Z.The leukemia stem cell. Cancer Treat Res.2009;145:1-17.
[13]Didier C, Cavelier C, Quaranta M, et al. G2/M checkpoint stringency is a key parameter in the sensitivity of AML cells to genotoxic stress[J].Oncogene,2008, 27(27):3811-3820.
[14]Rainey MD, Black EJ, Zachos G, et al.Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase[J]. Oncogene,2008,27(7):896-906.
[1]Wang XD. The expanding role of mitochondria in apoptosis. Genes Dev.2001, 15(11):2922-2933.
[2]Green DR, Reed JC. Mitochondria and apoptosis. Science.1998; 281(5381):1309-12.
[3]Xiao L, Caino MC, von Burstin VA,et al. Phorbol ester-induced apoptosis and senescence in cancer cell models. Methods Enzymol.2008;446:123-39.
[4]Blagosklonny MV. Target for cancer therapy:proliferating cells or stem cells. Leukemia,2006,20:385
[5]陈纯,陈崇宏.眼镜蛇毒细胞毒素CTX-d诱导NB4细胞凋亡及其机制研究.中草药,2007,38(12)：1838
[6]吴穗晶,杜欣,蒋文玲,等.中华眼镜蛇毒对K562和难治性白血病细胞体外作用的研究.现代肿瘤医学,2008,16(3)：428
[7]LalierL, Cartron PF, Juin P, et al. Bax activation and mitochondrial insertion during apoptosis. Apoptosis,2007,12(5):887-896.
[8]Del Poeta G, Ammatuna E, Lavorgna S,et al. The genotype nucleophosmin mutated and FLT3-ITD negative is characterized by high bax/bcl-2 ratio and favourable outcome in acute myeloid leukaemia. Br J Haematol.2010 Feb 8. [Epub ahead of print]
[9]Farrow S N, White J H M, Martinou I, et al. Cloning of a bcl-2 homologue by interaction with adenovirus E1B-19K. Nature,1995,374(6524):731-733.
[10]Wong WW, Puthalakath H.Bcl-2 family proteins:the sentinels of the mitochondrial apoptosis pathway.IUBMB Life.2008 Jun;60(6):390-7.
[11]Kuwana T, Newmeyer DD. Bcl-2 family proteins and the role of mitochondria in apoptosis. Curr Opin Cell Biol,2003,15:691-699.
[12]Klee M, Pallauf K, Alcala S, et al. Mitochondrial apoptosis induced by BH3-only molecules in the exclusive presence of endoplasmic reticular Bak..EMBO,2009,28(12):1757-1768.
[13]Cavalcanti GB Jr, Scheiner MA, Simoes Magluta EP,et al. p53 flow cytometry evaluation in leukemias:Correlation to factors affecting clinical outcome. ytometry B Clin Cytom.2010 Mar 2. [Epub ahead of print]
[14]Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, Green DR.Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosisScience.2004;303(5660):1010-4.
[15]Egger L, Madden DT, Rheme C, et al. Endoplasmic reticulum stress-induced cell death mediated by the proteasome. Cell Death Differ,2007,14:1172-1180.
[16]Qiu Q, Wang Z, Jiang JM, et al. Effect of lidamycin on mitochontria initiated apoptotic pathway in human cancer cells. Acta Pharm Sin,2007,42:132-138.
[17]E Szegezdi, S Cahill, M Meyer, et al. TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt. Br J Cancer,2006,94(3):398-406.
[18]Hongkui Jin, Renhui Yang, Jed Ross, et al. Cooperation of the Agonistic DR5 Antibody Apomab with Chemotherapy to Inhibit Orthotopic Lung Tumor Growth and Improve Survival[J]. Clinical Cancer Research,2008, 14:7733-7740.
[19]Hofmanova, J., Vaculova, A., Hyzd'Alova, M., et al. Response of normal and colon cancer epithelial cells to TNF-family apoptotic inducers[J]. Oncology Reports,2008,19 (2):567-573.
[1]Biron CA, Nguyen KB, Pien GC, et al. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol. 1999;17:189-220.
[2]Trinchieri G. Biology of natural killer cells. Adv Immunol.1989;47:187-376.
[3]Chen XM, Xu XQ, Sun K, et al. NKG2D ligands expression and NKG2D-mediated cytotoxicity in human laryngeal squamous carcinoma cells[J]. Scand Imm unol,2008,67 (5):441-447.
[4]Gasser S, Raulet DH. Activation and self2tolerance of natural killer cells[J]. Immunol Rev,2006,214:130-142.
[5]Brenner CD, King S, Przewoznik M, et al. Requirements for control of B-cell lymphoma by NK cells. Eur J Immunol.2010;40(2):494-504.
[6]Barber A, Rynda A, Sentman CL. Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment. J Immunol.2009;183(11):6939-47.
[7]Koebel CM, Vermi W, Swann JB,et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature.2007 Dec 6;450(7171):903-7.
[8]Decot V, Voillard L, Latger-Cannard V,et al. Natural-killer cell amplification for adoptive leukemia relapse immunotherapy:Comparison of three cytokines, IL-2, IL-15, or IL-7 and impact on NKG2D, KIR2DL1, and KIR2DL2 expression. Exp Hematol.2010 Feb 19. [Epub ahead of print]
[9]郭坤元,梅家转,姚开泰,尹晓林.NK细胞免疫编辑后的人鼻咽癌细胞株CNE2 HLA I类分子和MICA/MICB的表达及其对NK细胞杀伤活性的影响.第四军医大学学报；2007(11)：1005-1007.
[10]Poggi A, Massaro AM, Negrini S, et al. Tumor-induced apoptosis of human IL-2 activated NK cells:role of natural cytotoxicity receptors. J Immunol.2005; 174(5):2653-2660.
[11]Barber A, Rynda A, Sentman CL. Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment. J Immunol.2009;183(11):6939-47.
[12]Armeanu S, Krusch M, Baltz KM, et al. Direct and natural killer cell-mediated antitumor effects of low-dose bortezomib in hepatocellular carcinoma. Clin Cancer Res.2008; 14(11):3520-3528.
[13]Leslie Elsner, Vijayakumar Muppala, Mathias Gehrmann,et al. The Heat Shock Protein HSP70 Promotes Mouse NK Cell Activity against Tumors That Express Inducible NKG2D Ligands. The Journal of Immunology.2007; 179: 5523-5533.
[14]Kato N, Tanaka J, Sugita J, et al. Regulation of the expression of MHC class I related chain A, B (MICA, MICB) via chromatin remodeling and its impact on the susceptibility of leukemic cells to the cytotoxicity of NKG2D-expressing cells. Leukemia.2007; 21(10):2103-2108.
[15]Zhang C, Wang Y, Zhou Z, et al. Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis. Cancer Immunol Immunother.2009;8(58): 1275-1285.
[16]牛新清,胡亮杉,郭坤元,宋朝阳,涂三芳,梅家转.AS203对CD34+白血病细胞NKG2D配体表达及NK杀伤活性的影响[J].中国免疫学杂志.2008；24(6)：509-511.
[1]Wang XD. The expanding role of mitochondria in apoptosis[J]. Genes Dev,2001,15(11):2922-2933.
[2]Galluzzi L, Zamzami N, de La Motte Rouge T, Lemaire C, Brenner C, Kroemer G. Methods for the assessment of mitochondrial membrane permeabilization in apoptosis. Apoptosis.2007 May;12(5):803-13.
[3]Wieckowski MR. Postepy Biochem. The role of mitochondrial permeability transition pore in physiology and pathology of the cell.2008;54(1):71-81.
[4]喻爱喜,周志刚,张促宁等.TRAIL诱导骨肉瘤HOS-8606细胞凋亡与线粒体跨膜电位关系的研究.现代肿瘤医学,2005,13(5)586-588.
[5]Belosludtsev KN, Belosludtseva NV, Mironova GD. Possible mechanism for formation and regulation of the palmitate-induced cyclosporin A-insensitive mitochondrial pore.Biochemistry (Mosc).2005 Jul; 70(7):815-21.
[6]Orrenius S.Reactive oxygen species in mitochondria-mediated cell death. Drug Metab Rev.2007;39(2-3):443-55.
[7]Leu J I, Dumont P, Hafey M, et al.Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcll complex [J]. Nat Cell Biol,2004,6(5):443-450.
[8]Cheng E H, Wei M C, Weiler S, et al. BCL-2, BCL-X(L) sequester BH3 domainonly molecules preventing BAX- and BAK-mediated mitochondrial apoptosis [J]. Mol Cell,2001,8(3):705-711.
[9]Wong WW, Puthalakath H.Bcl-2 family proteins:the sentinels of the mitochondrial apoptosis pathway.IUBMB Life.2008 Jun; 60(6):390-7.
[10]Green DR, Reed JC.Mitochondria and apoptosis.Science.1998 Aug 28;281(5381):1309-12.
[11]Gogvadze V, Robertson JD, Zhivotovsky B, Orrenius S. Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax.J Biol Chem.2001 Jun 1;276(22):19066-71. Epub 2001 Mar 22.
[12]Masami Ishibashi, Takasbi Obtsuki. Studies on search for bioactive natural products targeting TRAIL signaling leading to tumor cell apoptosis[J].Med Res Rev. Feb 2008, [Epub ahead of print].
[13]Dong Ju Son, Mi Hee Park, Sang Jin Chae, et al. Inhibitory effect of snake venom toxin from Vipera lebetina turanica on hormone- refractory human prostate cancer cell growth:induction of apoptosis through inactivation of nuclear factor KB. Mol Cancer Ther 2007;6(2):675-683.
[14]Minn AJ, Velez P, Schendel SL, et al. Bcl-x forms anion channel in synthetic lipid membranes [J]. Science,1997,385:353-357.
[15]Torii S,Yamane K,Mashima T,et al.Molecular cloning and functional analysis of apoxin Ⅰ,a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity [J]. Biochemistry,2000,39 (12):3197-3205.
[16]张若燕.中药斑蝥及复方斑蝥制剂在治疗恶性肿瘤中的作用[J].内科,2006,1(1)：73-74.
[17]何太平,莫丽儿,梁念慈.斑蝥素诱导高转移卵巢癌细胞HO-8910PM凋亡的 研究[J].中国药科大学学报,2005,36(2)：164-167.
[18]Thomas Efferth, Rolf Rauh, Stefan Kahl, et al. Molecular modes of action of cantharidin in tumor cells [J]. Biochemical Pharmacology,2005, 69(5):811-818.
[19]Jeong-Eun Huh, Kyung-Sun Kang, Chanhee Chae, et al.Roles of p38 and JNK mitogen-activated protein kinase pathways during cantharidin-induced apoptosis in U937 cells [J]. Biochemical Pharmacology,2004, 67(10):1811-1818.
[20]Xianxi Wang. Ranyia Matta. Gang Shen.Leif D. Nelin. Dehua Pei. Yusen Liu. Mechanism of triptolide-induced apoptosis:effect on caspase activation and Bid cleavage and essentiality of the hydroxyl group of triptolide [J]. Mol Med (2006)84:405-415
[21]Bing Z. Carter, Duncan H. Mak, Wendy D. Schober, Teresa McQueen, David Harris, Zeev Estrov, Randall L. Evans, Michael Andreeff.Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells[J].Blood 2006,108:630-637
[22]Guang-Biao Zhou, Hui Kang, Lan Wang, et al. Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21) leukemia in vitro and in vivo[J]. Blood.2007;109(8):3441-3450.
[23]Simone Fulda, Klaus-Michael Debatin. Resveratrol modulation of signal transduction in apoptosis and cell survival:A mini-review [J].Cancer Detection and Prevention.2006;30:217-223.
[24]Prasad KN, Kumar B, Yan XD, et al. Alpha - tocopheryl succinate, the most effective form of vitamine E for adjuvant cancer treatment:a review [J]. J Am Coll Nutr,2003,22 (2):108-117.
[25]Ripoll EA, Rama BN, Webber MM. Vitamine E enhances the chemotherapeutic effectsof adriamycinon human prostatic carcinoma cells in vitro[J].J Urol,1986, 136 (2):529-531.
[26]Weber T,Dalen H,AnderaL, et al. Mitochondrial playa central role in apoptosis induced by alpha - tocopheryl succinate, an agent with antineoplastic activity: comparisionwith receptor- mediated proapoptotic signaling [J]. Biochemistry, 2003,42(14):4277-4291.
[27]Efferth T, BenakisA, RomeroM R, et al. Enhancement of cytotoxicity of artemisinins toward cancer cells by ferrous iron [J]. Free Radic Biol Med, 2004,37 (7):998-1009.
[28]Efferth T, GiaisiM, MerlingA, et al. Artesunate induces ROS2mediated apop tosis in doxorubicin2resistant T leukemia cells [J]. PLoSONE,2007,2 (1) e693.
[29]Yao K, Ye P, Zhang L, et al. Epigallocatechin gallate protects against oxidative stress-induced mitochondria-dependent apoptosis in human lensepithelial cells [J].Mol Vis,2008,14:217-223.