摘要
白血病(Leukemia)是造血干／祖细胞水平转化的一类克隆性恶性血液病。白血病细胞因分化障碍、增殖过度和凋亡受抑等机制而停滞在细胞发育的不同阶段并大量积聚,浸润多种组织器官,使正常造血细胞减少。临床上以贫血、出血、感染、浸润和高代谢为特点。白血病是造血系统的恶性肿瘤,病情凶险,死亡率高,在儿童及35岁以下成人中居第1位,严重威胁着人类的生命健康。近年来,随着对急性白血病的深入研究,联合化疗、骨髓干细胞移植以及分子生物学靶向治疗等方法的开展,使白血病患者的缓解率明显提高。缓解期、生存期延长,相当一部分患者长期存活,甚至治愈。但化疗强烈的毒副作用、化疗的多药耐药、化疗后支持治疗的昂贵费用以及有限的骨髓供体、高昂的移植费用、移植带来的严重并发症等致使白血病的疗效受到了很大的限制。80年代以来,国内外一些研究者以中医理论为基础,结合现代科学技术,运用一些中药提取物、中成药治疗急性白血病取得了明显的疗效。如我国首先研究报道的从中药砒霜中提取的三氧化二砷或亚砷酸对M3型白血病的惊人疗效。从三尖杉植物中提取出的三尖杉酯碱和高三尖杉酯碱,已广泛用于急性非淋巴细胞白血病的治疗,并获得了较好的疗效。以及青黛、喜树、蟾酥、葛根等众多中药成分均有不同程度的抗白血病作用。青蒿琥酯作为植物青蒿的提取衍生物,临床抗疟应用已久。从2004年开始,在导师杨洪涌教授的指导下,本专业研究生团队运用青蒿琥酯先后对K562、HL-60细胞株,以及部分急性白血病患者原代细胞做了体外抑制试验。结果均表明,青蒿琥酯对白血病细胞具有明显的抑制作用。
本研究所用药物青蒿琥酯是中药青蒿的提取衍生物。药理学研究证实,青蒿素及其衍生物除具有抗疟疾效应外,还有抑制白细胞增殖效应。药代动力学研究证实青蒿素类药物在体内吸收快、分布广、排泄快。毒理学研究证实其毒副作用轻微,毒性主要靶器官为骨髓,可抑制骨髓造血,对白血病细胞有较强的靶向性。体内外实验表明：青蒿素及其衍生物可选择性杀伤肿瘤细胞,且与传统化疗药物无交叉耐药,并能逆转肿瘤细胞的多重耐药现象。本研究是在既往研究成果的基础上,通过建立急性白血病患者骨髓原代细胞培养体系,以经典化疗药物柔红霉素为对照药物,同时设立1640培养液空白对照组,观察不同剂量青蒿琥酯对人急性白血病原代细胞增殖的影响。从细胞增殖周期、细胞凋亡与胀亡、细胞凋亡相关蛋白、白血病相关基因等方面进行研究,为青蒿琥酯治疗急性白血病提供细胞形态学、分子生物学及细胞遗传学方面的初步依据。
本课题通过提取11例急性白血病患者(初发组7例,复发组4例)的骨髓液进行原代细胞培养,建立原代细胞体外培养体系,模拟白血病细胞体内生长环境,观察原代白血病细胞自然生长趋势及特点,以对数生长期细胞为干预点,分别设立青蒿琥酯高、中、低治疗组,柔红霉素对照组及空白对照组。取对数生长期细胞,调细胞浓度为1×106/L,分别接种于96孔板、12孔板或6孔板中,将终浓度分别为50μg/ml、25μg/ml、12.5μg/m的青蒿琥酯,终浓度为25μg/ml的柔红霉素以及相同剂量1640培养液作用于细胞,作用一定时间后,分别采用MTT(四甲基偶氮唑盐)显示法,检测青蒿琥酯对人AL原代细胞增殖的影响；运用透射电子显微镜观察青蒿琥酯作用后细胞的形态变化；运用蛋白印迹法定量检测青蒿琥酯作用后白血病细胞相关凋亡蛋白含量变化情况；运用基因芯片技术检测青蒿琥酯作用后白血病相关基因表达情况,从而探讨青蒿琥酯抗白血病作用机制及作用靶点。
1.人AL骨髓原代细胞体外自然生长趋势观察结果显示：人AL骨髓原代细胞的生长大致经历延滞期、对数生长期、平台期、缓慢凋亡期、快速死亡期五期。细胞从培养24h开始进入对数生长期,48h到72h基本处于稳定的平台期,72h之后先经历一个缓慢凋亡期,然后迅速进入快速死亡期。所以我们选择对数生长期细胞为干预点对细胞进行干预研究。
2.四甲基偶氮唑盐(MTT)法检测青蒿琥酯对人AL原代细胞增殖作用的结果显示：与空白对照组比较,浓度为50μg/ml、25μg/ml、12.5μg/ml的青蒿琥酯各组作用48h时对AL骨髓原代细胞抑制率分别为(41.55±0.73)%、(31.48±1.32)%、(25.14±0.89)%,差异具有统计学意义(P<0.01)；作用72h时对AL骨髓原代细胞抑制率分别为(54.88±2.47)%、(49.39±5.30)%、(36.85±1.08)%,差异具有统计学意义(P<0.01)；各药物浓度组相比,差异具有统计学意义(P<0.01)；48h与72h各组比较差异也具有统计学意义(P<0.01)。提示青蒿琥酯对人AL骨髓原代培养细胞的增殖有抑制作用,且抑制率与药物剂量、作用时间呈依赖关系。
3.透射电子显微镜观察青蒿琥酯作用后细胞形态结果显示：青蒿琥酯高剂量组细胞大部分死亡,且呈现两种不同的死亡形态学改变。少部分细胞出现细胞体积缩小染色质固缩,或裂解形成一个或数个大小不一的致密凋亡小体；大部分细胞出现细胞体积明显增大,胞浆呈空泡样改变,细胞膜部分完整,线粒体肿胀,细胞核多被挤向细胞一侧,染色质凝集在核膜、核仁周围。青蒿琥酯中剂量组细胞部分死亡,也呈现两种不同的死亡形态学改变。大部分细胞可见细胞体积变小,染色质固缩,或裂解形成凋亡小体；少数细胞可见细胞体积缩小,染色质固缩,但伴见胞浆肿胀或呈空泡样；仅个别部分细胞细胞体积明显增大,胞浆呈空泡样改变,细胞膜部分完整,线粒体肿胀,细胞核多被挤向细胞一侧,染色质凝集在核膜、核仁周围。青蒿琥酯低剂量组电镜下细胞形态改变基本同青蒿琥酯中剂量组,部分细胞可见Auer小体。柔红霉素组细胞几乎全部溶解,细胞膜破裂,细胞器消失,细胞核溶解。空白对照组可见部分细胞细胞膜、细胞器及细胞核完整,细胞浆分布均匀的白血病细胞；个别细胞体积缩小,染色质固缩。提示青蒿琥酯具有诱导人AL骨髓原代细胞凋亡和胀亡的作用,由于各浓度组细胞病理学变化相类似,说明青蒿琥酯诱导人AL骨髓原代细胞凋亡和胀亡的形态学改变不具有剂量依赖性。
4.免疫印迹法对细胞凋亡相关蛋白(Bcl-2、Bax、细胞色素c)的测定结果显示：浓度为50μg/ml、25μg/ml、12.5μg/ml的青蒿琥酯各组作用48h后人AL骨髓原代细胞凋亡抑制蛋白Bc1-2表达变化分别为0.62±0.06、0.72±0.11、0.81±0.16；凋亡蛋白Bax表达变化分别为0.95±0.05、0.87±0.04、0.82±0.05；凋亡蛋白细胞色素C表达变化分别为1.05±0.10、0.95±0.98、0.91±0.13。与空白对照组、柔红霉素组比较,各药物浓度组均具有统计学意义(P<0.05)；各药物浓度组组间相比,差异无统计学意义(P>0.05)。提示青蒿琥酯作用48小时后各浓度青蒿琥酯均可下调凋亡抑制蛋白Bcl-2含量,同时促进凋亡蛋白Bax、细胞色素c的释放,但对于细胞Bcl-2、Bax、细胞色素c含量的影响与青蒿琥酯浓度无明显相关性,该结果提示线粒体途径可能是青蒿琥酯诱导白血病细胞凋亡的主要途径。
5.基因芯片技术检测青蒿琥酯对AL原代细胞基因表达谱的影响结果初步分析显示：青蒿琥酯能上调AL骨髓原代细胞的Bax表达水平,下调Bcl-2的表达,提示青蒿琥酯可能通过抑制](?)ax-Bcl-2异源二聚体的形成,促进Bax同源二聚体的形成而达到诱导细胞凋亡的作用。青蒿琥酯能明显上调CACNA2D3基因表达水平,该基因属于电压依赖性钙离子通道基因家族,其作用能使胞浆内钙离子浓度升高,与细胞色素C结合,激活caspase非依赖性细胞死亡途径从而诱导细胞凋亡。青蒿琥酯能上调AL骨髓原代细胞细胞周期相关基因如CDK8、CDKL4、CCND2等基因的表达水平,提示青蒿琥酯可能通过细胞周期调控机制使AL原代细胞周期阻滞于某一期,从而抑制其增殖,诱导细胞凋亡。综上所述,青蒿琥酯具有确切的抗白血病作用。其诱导细胞凋亡可能通过影响细胞凋亡信号传导通路中的某些相关基因而达到抗白血病作用。其中某些基因可能也参与了原代细胞胀亡的过程,但尚需进一步验证。
广州中医药大学第一临床医学院血证研究室杨洪涌教授根据急性白血病相关文献研究及中药青蒿琥酯抗白血病相关实验研究,同时结合自己的临床经验,提出急性白血病发病初期起病急骤,病势凶险,多有高热、出血、感染、发斑等表现,类似温热病中感受温热毒邪引起的急性热病。急性白血病之中期,极易发生出血、感染等合并症。其出血之症不外古人之“热伤阳络则衄血,热伤阴络则便血”所述。临床观察阴虚内热期出血,多以鼻衄、肌衄、齿衄、便血为多。急性白血病之初期和中期都易发生感染而危及生命,此乃温毒湿热之邪侵及人体,邪毒炽盛,正气无力抗邪所致。本研究所用药物青蒿琥酯是中药青蒿的提取衍生物,其味苦、性辛、寒,具有清虚热,除骨蒸,解暑,截疟的功效,临床用治温邪伤阴、余热未清、夜热早凉或热病后低热不退。还用治阴虚发热、劳热骨蒸及感受暑热之邪,发热口渴等。这与急性白血病之初期感受温热毒邪之发热,中后期气阴两虚之内热的病机相类似。从2004年开始,在导师杨洪涌教授的指导下,本专业研究生团队运用青蒿琥酯先后对白血病细胞株,以及急性白血病患者原代细胞做了一系列体外抑制试验,结果显示,青蒿琥酯可通过升高细胞内钙离子浓度而致细胞死亡；可能通过降低其细胞内线粒体膜电位致细胞死亡；也可通过对癌基因与抑癌基因表达的影响达到抗肿瘤的作用。本研究通过青蒿琥酯作用后急性白血病骨髓原代细胞增殖明显受抑；在电子显微镜下同时存在凋亡和胀亡两种细胞形态；其对细胞凋亡相关蛋白含量的影响以及对白血病相关基因表达的影响,结合既往研究结果,我们进一步肯定了青蒿琥酯抗白血病的作用。其诱导细胞凋亡可能通过影响细胞凋亡信号传导通路中的某些相关基因而达到抗白血病作用的。其中某些基因可能也参与了青蒿琥酯诱导人急性白血病骨髓原代细胞胀亡的过程,但尚需进一步验证。据此结合既往研究结果推断,线粒体凋亡途径的激活可能是其抗白血病及逆转多药耐药的主要分子机制。
Background:Acute leukemia is a malignant tumor in hematopoietic system. The mortality rate is high. In recent years, with the further study, the chemotherapy, bone marrow transplant and molecular biology treatment method for the leukaemia had improved remission rate obviously, even be cured. But the strong poisonous effect, multidrug resistance and the expensive cost, limited marrow providing bodys, and transplant bring serious neurological complications limited therapy of modern medicine.Since the 1980s, some researchers of TCM used traditional chinese medicine extracts to treat acute leukemia. Had gain a noticeable effection. Art as an plants can anti-malarial in clinical application. In 2007, under the guidance of professor Yang Hongyong, we have used Art to experimentalize on K562, HL-60 cell line and 16 cases of acute leukemia patients.The results all showed that:Art can inhibit the growth of human acute leukemia primary cells.But its the mechanism and the target are up for exploring further.
Objective:This study is on the basis of research results in the past, by establishing the training system with human acute leukemia primary cells, in contrast with DUR and the saline water in the blank, Observe the effects of different concentration of Art on the proliferation of the primary cells, Provide the preliminary support for Art to treat leukemia from cell cycle、apoptosis and oncosis、molecular biology gene and cell genetics.
Methods:11 patients who were all diagnosed as acute leukemia were recruited into research. Establishing the training system in vitro with their bone marrow primary cells. Cells were cultured in logarithmic growth phase were seeded in 96、12 and 6 well plates Respectively.To a final concentration of 50 u g/ml、25μg/ml、12.5μg/ml of Art, concentration of 25μg/ml DNR and the same dosage of saline solution for a certain time. Using the method of MTT to detect the effect of the proliferation cells; Using electron microscope to observe the forms of apoptosis and onconsis; Using Western Blotting testive technology to detect the protein of the cell apoptosis; Using Gene chip testive technology to detect the perform expression of AL related genes. To explore the mechanism and target for Art on human acute leukemia.
1. MTT detective results showed that:Art can inhibit the growth of human acute leukemia primary cells. And the inhibition ratio is dependent on the dose of Art and the functionary time.
2. Electron microscopic observational results showed that:Art can cause the different degrees of the apoptosis and and oncosis of leukemia primary cells after 48 hours. And the degree of apoptosis and and oncosis is dependent on the dose of Art.
3. Western Blotting testive results showed that:Art can down-regulate protein Bcl-2;at the same time up-regulate protein Bax and cyc-c.
4. Gene chip testive results showed that:Art can perform expression of AL related genes.
Conclusion:Art can inhibit the growth of human acute leukemia primary cells definitivly. Mitochondrial channe may be the main way of Antileukemic function and reversion of multidrug resistance. So Art is worth to be developed to the new drug of Antileukemia.
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