DFO、DFO联合AS_2O_3对HL-60细胞移植瘤裸鼠的治疗作用及对NF-κBp65表达影响
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摘要
研究目的
采用铁螫合剂去铁胺(DFO)、去铁胺联合三氧化二砷(AS2O3)对高致瘤性HL-60白血病细胞移植瘤裸鼠进行治疗,观察上述药物对移植瘤裸鼠的治疗作用及对移植瘤组织NF-κBp65表达水平的影响,探讨其作用机制,为临床采用铁螫合剂治疗或辅助治疗白血病提供实验依据。
研究方法
1.采用皮下接种的方法,构建高致瘤性HL-60白血病细胞裸鼠移植瘤模型,将荷瘤裸鼠随机分为4组：50mg/kg DFO单药组、联合用药组(50mg/kg DFO+1.5 mg/kg As2O3)、3 mg/kg As2O3单药组、生理盐水对照组；于接种的当天开始给药,连续给药10天,全部采用腹腔注射,实验协议通过郑州大学第一临床学院伦理委员会批准。
2.给药期间密切观察裸鼠的一般状况,裸鼠接种后的成瘤时间及移植瘤的生长特点,同时观察荷瘤裸鼠对药物的不良反应。用游标卡尺每天测量移植瘤块的最大长径(a)和短径(b),依据公式：体积=ab2/2,计算瘤体积,绘制肿瘤生长曲线。
3.荷瘤裸鼠在全部用药结束后24小时处死,计算移植瘤的瘤重量、抑瘤率
4.荷瘤裸鼠在处死后,取其移植瘤组织做常规病理学检查,观察移植瘤组织的形态学改变；取荷瘤裸鼠骨髓,观察有无骨髓抑制和白血病细胞浸润；取荷瘤裸鼠的心、肝、脾、肺、肾组织,观察有无病理改变及瘤转移。
5.采用免疫组织化学即用型二步法(非生物素)检测各组移植瘤组织NF-κBp65的表达水平变化,运用专业图像分析系统Biosens Digital Imaging System (vl.6)进行积分光密度测定,用阳性区平均积分光密度值来表示NF-κBp65免疫组化染色阳性的表达程度。
6.统计分析：采用SPSS 16.0 for Windows软件包进行统计学处理；样本数据采用均数±标准差(X±S)表示；治疗组与生理盐水对照组的移植瘤重量比较采用t检验,4组移植瘤组织免疫组化NF-κBp65表达水平比较采用单因素方差分析；统计检验均为双侧概率检验(α=0.05), P<0.05为差异有统计学意义。
结果
1.裸鼠接种后的成瘤情况及移植瘤的生长特点：用体外培养的高致瘤性HL-60细胞(约1×106个细胞／只)接种,于接种后第7～8天30只裸鼠全部成瘤,出现肉眼可见的瘤块,直径约0.5cm～1.0cm大小,成瘤率为100%。移植瘤生长早期仅在裸鼠接种局部皮下生长,生长迅速,呈向外膨胀性生长的特性,表现为圆形或椭圆形,随着瘤体增长逐渐成为不规则形,表面呈多个结节融合状。从外部观察这些移植瘤边界较清楚,肿瘤与皮肤无粘连,活动度可,似有包膜,表皮血管丰富,但随着移植瘤体积进行性增大,肿瘤活动度逐渐下降。当移植瘤直径长至2.0cm及其以上时,在移植瘤表皮中央出现褐色的边缘不规则的坏死灶,坏死面积随着时间延长持续扩大。该移植瘤组织较易剥离,离体后可见瘤组织有一层完整的包膜、包膜上血供丰富、切面似鱼肉状、组织松脆易碎。
2.给药期间裸鼠的一般状况：给药第1天至给药第7天各组荷瘤裸鼠精神状态均良好,活动正常,反应敏捷,进食、进水正常,皮肤颜色如常,大便正常,未见明显异常反应。从给药第8天开始,随着移植瘤体积的增大,荷瘤裸鼠正常活动逐渐受到影响,行动较前迟缓,进食、进水减少。当移植瘤直径长至2.0cm及其以上时,荷瘤裸鼠逐渐呈现恶病质状态,肤色逐渐由粉色转为淡青色,且随着移植瘤的增大体重逐渐增长。给药期间,观察到As2O3单药组有2只裸鼠出现腹水,对照组有1只裸鼠出现眼部转移瘤,除此之外,各治疗组荷瘤裸鼠一般状况与对照组相比无明显差异。
3.不同用药组移植瘤体积增长情况：随着时间的延长,荷瘤裸鼠移植瘤体积越来越大。各治疗组移植瘤体积增长速度均较对照组慢,其中以DFO联合As2O3组移植瘤体积增长最慢,其次为As2O3单药组和DFO单药组。给药第1天至给药第5天,所有裸鼠无移植瘤生长,从给药第6天开始,各治疗组的移植瘤平均体积均小于生理盐水对照组,且随着给药时间的延长,各治疗组与对照组移植瘤平均体积的差值进一步扩大。到第11天时,生理盐水对照组移植瘤平均体积最大,其次是DFO单药组,再次是As2O3单药组,而DFO联合As2O3用药组的移植瘤平均体积最小,说明不同用药组对高致瘤HL-60白血病细胞裸鼠异种移植瘤体积随时间变化的趋势有影响。
4.DFO单药组和As2O3单药组对高致瘤性HL-60细胞裸鼠异种移植瘤的抑瘤率分别为2.56%和10.67%,移植瘤重量分别为(2.55±0.82)g和(2.34±0.79)g,与生理盐水对照组相比较,差异均无统计学意义(分别为F=1.290,P=0.866 >0.05和F=1.714,P=0.474>0.05):DFO联合As2O3用药组对高致瘤HL-60细胞裸鼠异种移植瘤的抑瘤率为25.49%,移植瘤重量为(1.95±0.39)g,与生理盐水对照组相比较,明显低于生理盐水对照组,差异有统计学意义(F=0.028,P=0.019<0.05),提示DFO联合As2O3用药对HL-60高致瘤细胞裸鼠异种移植瘤的生长具有抑制作用,DFO联合As2O3在抑制高致瘤HL-60细胞裸鼠异种移植瘤的生长上具有协同作用。
5.荷瘤裸鼠在处死后,肉眼观察各组移植瘤组织,可见出血坏死区,有的瘤块表面可见黑色痂样改变。光镜下可见,对照组瘤细胞分布密集,瘤细胞圆形或椭圆形,细胞胞体较大,大小基本一致,明显异型,核大而深染,胞浆量少,呈嗜碱性,可见个别凋亡细胞及凋亡小体,内有散在的局灶性细胞坏死区：各治疗组移植瘤组织中瘤细胞数目明显较生理盐水对照组减少,细胞体积变小,核深染,有的细胞固缩,有的仅存胞核,可见不规则坏死、出血,呈片状的嗜伊红结构,凋亡细胞和凋亡小体小片状分布,其中以DFO联合As2O3用药组较DFO单药组和As2O3单药组显著。
6.荷瘤裸鼠心、肝、脾、肺、肾病理组织学检查结果显示：在显微镜下观察可见,每只荷瘤裸鼠的心、肝、脾、肺、肾组织,结构完整,除充血程度不同外,均无明显的病理改变,未见HL-60白血病细胞转移。
7.荷瘤裸鼠骨髓病理组织学检查结果显示：4组均可见有核细胞增生活跃,粒红比例正常,末见明显的骨髓抑制和HL-60白血病细胞浸润。
8.免疫组织化学PowerVisionTM二步法检测移植瘤组织NF-κBp65的表达,统计学结果显示：DFO单药组,As2O3单药组与DFO联合As2O3用药组3个治疗组的平均积分光密度值均低于生理盐水对照组,与生理盐水对照组比较均有统计学差异(P<0.05)；且NF-κBp65表达的积分光密度值：生理盐水对照组>As2O3单药组>DFO单药组>DFO联合As2O3用药组。4组两两相互比较差异均有统计学意义(P<0.05)。提示DFO和As2O3都可降低高致瘤HL-60细胞裸鼠异种移植瘤组织的NF-κBp65表达,二者联合降低移植瘤组织NF-κBp65表达的作用更强。
结论
1.采用高致瘤性HL-60白血病细胞(1×106个细胞／只)在裸鼠皮下接种,可成功建立移植瘤动物模型,接种后7～8天均能形成直径约0.5cm～1.0cm大小的移植瘤,成瘤率达100%,且成瘤后瘤体生长迅速。
2.DFO和As2O3单药应用均可抑制高致瘤HL-60白血病细胞裸鼠异种移植瘤的生长,二者联合作用显著。
3.DFO联合As2O3应用荷瘤裸鼠能较好耐受,DFO可降低As2O3用量,促进其杀伤肿瘤作用,且可降低As2O3毒副作用。
4.DFO和As2O3均可降低高致瘤HL-60细胞裸鼠异种移植瘤组织的NF-κBp65的表达水平,二者联合降低移植瘤组织NF-κBp65表达的作用更明显。
Objective
To observe the therapeutic effect and the influence on NF-KBp65 expression level of transplanted tumors of deferoxamine and deferoxamine(DFO) in combination with arsenic trioxide (As2O3)by establishing a model of implanted tumor of human high tumorigenic leukemia cell line HL-60 in nude mice,and investigate the mechanism,so as to provide the experimental evidence for treatment or auxiliary treatment clinically for leukemia of iron chelators.
Methods
1.Xenograft tumor model of human high tumorigenic leukemia cell line HL-60 in nude mice was established by means of inoculating subcutaneously,then the tumor-bearing mice were randomly divided into four groups:50mg/kg DFO group, combination group(50mg/kg DFO+1.5mg/kg As2O3),3mg/kg As2O3 group, normal saline control group. The tumor-bearing mice were given drugs from that day of inoculating subcutaneously,and drug administration continued for 10 days continuously through intraperitoneal injection.The experimental protocol was approved by the ethical committee of First Clinical Medical College of Zhengzhou University.
2.Observing closely general conditions and adverse drug effects of the tumor-bearing mice,time of forming tumor, growth characteristic of transplanted tumor during administration of drugs.The maximum longitudinal(a) and transverse(b) diameters of transplanted tumor were measured with slide callipers every day. Tumor volumes were calculated according to the equation(volume=ab2/2),and then transplanted tumor growth curves were drawn.
3.The next day The tumor-bearing mice were executed after administration of drugs.Then tumor weights and tumor inhibitory rate of transplanted tumors were calculated.
4.After the tumor-bearing mice were executed, the specimens of transplanted tumors were stained with HE.Routine pathological examination were performed on transplanted tumors to observe the changes of tissue morphology. Then the changes of each specimen in different experimental groups were analyzed. The bone marrows of the tumor-bearing mice were observed to determine whether bone marrow depression and infiltration of leukemic cells existed or not. The tissues of heart,liver,spleen,lung and kidney of the tumor-bearing mice were observed to determine whether pathological changes and tumor metastasis existed or not.
5.NF-κBp65 expression levels of transplanted tumors were detected by two-step Immunohistochemistry detection(PowerVisionTM),and then integrate optical density(IOD) was detected by using professional software Biosens Digital Imaging System (v1.6).Mean integrate optical density value of the positive area was used for representing the expression levels of NF-κBp65.
6.The results of experiment were analyzed by SPSS16.0 for Windows.Sample averages were expressed by X±S.The differences of the weights of transplanted tumors between treatment groups and control group were test by t test.The differences of the NF-κBp65 expression levels of transplanted tumors between four experimental groups were test by one-way ANOVA.Statistic test were two-sided test of probability(α=0.05).Probability of less than 0.05 were considered significant.
Results
1.General conditions of forming tumor, growth characteristic of transplanted tumor after inoculating:Inoculating human high tumorigenic leukemia cell line HL-60 (about 1×106 cells/each mouse) culturing in vitro,tumor grew in 100% of animals.From the seventh day to the eighth day, macroscopic tumors were observed 0.5cm～1.0cm in diameter. Tumors which were orbicular or elliptic grew only at the sites of cells inoculation and expansive grew outward with rapid growth at the time of early growth.After that, with the enlargement of tumor size, tumors became irregular gradually and multinodular confluent on the surface.From the outside watching, clear boundary, no adhesion with the skin, good mobility, having peplos apparently and epidermis rich in blood vessels were observed,but the mobility of tumor decreased gradually with the progressive increase of tumor volume. When the tumors were bigger than two centimeters in diameter, brown necrotic foci which had irregular margins were observed on the central Epidermis of tumors,and necrotic area continued to expand with time.The transplanted tumor tissues were easily stripped with layer of intact peplos rich in blood vessels.The section of tumors looks like fish.The tissues were crispy and easily broken.
2.General conditions of the tumor-bearing mice during administration course:A good mental state, normal activities,agile response,eating and drinking normally, normal skin color, normal stool and no abnormal situation were observed in all the tumor-bearing mice from the first day to the seventh day of administration of drugs.With the enlargement of tumor size, normal activities of the tumor-bearing mice were influenced gradually from the eighth day of administration of drugs,for example,a slowing of movement and reduced eating and drinking.When the tumors were bigger than two centimeters in diameter, cachexia was observed in the tumor-bearing mice.Skin color gradually changed from pink to light cyan,and the weight of the tumor-bearing mice increased gradually with the enlargement of tumor size from the eighth day. Ascites were observed in two tumor-bearing mice of 3mg/kg As2O3 group,and metastatic tumor were observed in a tumor-bearing mice of control group. Apart from this,there was no significant difference between treatment groups and control group in general conditions of the tumor-bearing mice.
3.There was an increase of the transplanted tumor's volume as time went on. The rate of tumor volume increase in control group was the fastest, followed by 50mg/kg DFO group and 3mg/kg As2O3 group.And the rate in combination group(50mg/kg DFO+1.5mg/kg As2O3)was the lowest.No tumour grew from the first day to the fifth day of administration of drugs. From the sixth day mean volumes of tumors in control group were higher than them in treatment groups.And the differences of mean volumes of tumors between treatment groups and control group were further expanded as time went on.On the eleventh day the tumor volume of control group was the largest, followed by 50mg/kg DFO group, and 3mg/kg As2O3 group is next. The tumor volume of combination group(50mg/kg DFO+1.5mg/kg As2O3) was the smallest.So we can see that different drugs affected the trend of transplanted tumor's volume over time.
4.The tumor inhibitory ratioes of 50mg/kg DFO group and 3mg/kg As2O3 group were 2.56% and 10.67% respectively.The transplanted tumor weights of 50mg/kg DFO group and 3mg/kg As2O3 group were(2.55±0.82)g and(2.34±0.79)g respectively.There was no significant difference between DFO group and the control group(F=1.290,P=0.866>0.05),and there was no significant difference between As2O3 group and the control group (F=1.714, P=0.474>0.05).The tumor inhibitory ratio of combination group(50mg/kg DFO +1.5mg/kg As2O3)was 25.49%, The transplanted tumor weight of combination group was(1.95±0.39)g,and significantly lower than that of control group(F=0.028,P=0.019<0.05).It indicated that DFO combined with As2O3 had the synergistic anti-tumor effect.
5.After the tumor-bearing mice were executed, hemorrhage, necrosis and black scab were observed on the tumor tissue through macroscopic observation.By light microscope the tumor cells in control group were large, orbicular or elliptic, heterogeneous and basophilic,with high nucleocytoplasmic ratio,and distributed compactly. A few of apoptosis cells and apoptotic bodies were found in control group,in which areas of focal necrosis scattered. The number of tumor cells in tissue of treatment groups were smaller than that of normal saline control group.Small tumor cells with hyperchromatic nuclei,pycnosis, Some remaining nucleus,irregular necrosis,hemorrhage, flaky eosinophilic Structure,apoptosis cells and apoptotic bodies with a small flaky distribution were found in treatment groups.especially in combination group.
6.By light microscope,Complete structure, no obvious pathological changes except of congestion in different degrees and no metastases were found in the tissues of heart,liver,spleen,lung and kidney of the tumor-bearing mice.
7.After administration completed, bone marrow depression and infiltration of leukemic cells HL-60 were not observed in the bone marrows of the tumor-bearing mice,and the active proliferation of bone marrow karyocyte and normal myeloid erythroid ratio were observed in bone marrows.
8.NF-κBp65 expression levels of transplanted tumors were detected by two-step Immunohistochemistry detection(PowerVisionTM),and then the statistical results showed that there was significant difference in average value of integrate optic density(IOD) among experimental groups(P<0.05).The average value of IOD of control group was the largest, followed by 3mg/kg As2O3 group, and 50mg/kg DFO group is next. The average value of IOD of combination group(50mg/kg DFO+1.5mg/kg As2O3) was the smallest. So both DFO and As2O3 could lower NF-icBp65 expression levels of transplanted tumors, and especially DFO combined with As2O3 had a stronger effect.
Conclusion
1.The xenograft model of the human leukemia cell line HL-60 in nude mice was successfully established by Inoculating human high tumorigenic leukemia cell line HL-60 (about 1×106 cells/each mouse).Transplanted tumor grew in 100% of animals,and grew rapidly after forming tumours.From the seventh day to the eighth day after inoculating, macroscopic tumors were observed 0.5cm～1.0cm in diameter.
2.Both DFO and As2O3 could inhibit the growth of transplanted tumors,and DFO combined with As2O3 had a obvious and significant effect on inhibiting the growth of transplanted tumors.
3.When DFO combined with As2O3,it was well tolerated and there were no significant adverse effects.DFO not only lowered the dosage of As2O3,but also increased the killing effect of As2O3 on tumor.
4.Both DFO and As2O3 could lower NF-κBp65 expression levels of transplanted tumors, and especially DFO combinated with As2O3 had an obvious effect.
采用铁螫合剂去铁胺(DFO)、去铁胺联合三氧化二砷(AS2O3)对高致瘤性HL-60白血病细胞移植瘤裸鼠进行治疗,观察上述药物对移植瘤裸鼠的治疗作用及对移植瘤组织NF-κBp65表达水平的影响,探讨其作用机制,为临床采用铁螫合剂治疗或辅助治疗白血病提供实验依据。
研究方法
1.采用皮下接种的方法,构建高致瘤性HL-60白血病细胞裸鼠移植瘤模型,将荷瘤裸鼠随机分为4组：50mg/kg DFO单药组、联合用药组(50mg/kg DFO+1.5 mg/kg As2O3)、3 mg/kg As2O3单药组、生理盐水对照组；于接种的当天开始给药,连续给药10天,全部采用腹腔注射,实验协议通过郑州大学第一临床学院伦理委员会批准。
2.给药期间密切观察裸鼠的一般状况,裸鼠接种后的成瘤时间及移植瘤的生长特点,同时观察荷瘤裸鼠对药物的不良反应。用游标卡尺每天测量移植瘤块的最大长径(a)和短径(b),依据公式：体积=ab2/2,计算瘤体积,绘制肿瘤生长曲线。
3.荷瘤裸鼠在全部用药结束后24小时处死,计算移植瘤的瘤重量、抑瘤率
4.荷瘤裸鼠在处死后,取其移植瘤组织做常规病理学检查,观察移植瘤组织的形态学改变；取荷瘤裸鼠骨髓,观察有无骨髓抑制和白血病细胞浸润；取荷瘤裸鼠的心、肝、脾、肺、肾组织,观察有无病理改变及瘤转移。
5.采用免疫组织化学即用型二步法(非生物素)检测各组移植瘤组织NF-κBp65的表达水平变化,运用专业图像分析系统Biosens Digital Imaging System (vl.6)进行积分光密度测定,用阳性区平均积分光密度值来表示NF-κBp65免疫组化染色阳性的表达程度。
6.统计分析：采用SPSS 16.0 for Windows软件包进行统计学处理；样本数据采用均数±标准差(X±S)表示；治疗组与生理盐水对照组的移植瘤重量比较采用t检验,4组移植瘤组织免疫组化NF-κBp65表达水平比较采用单因素方差分析；统计检验均为双侧概率检验(α=0.05), P<0.05为差异有统计学意义。
结果
1.裸鼠接种后的成瘤情况及移植瘤的生长特点：用体外培养的高致瘤性HL-60细胞(约1×106个细胞／只)接种,于接种后第7～8天30只裸鼠全部成瘤,出现肉眼可见的瘤块,直径约0.5cm～1.0cm大小,成瘤率为100%。移植瘤生长早期仅在裸鼠接种局部皮下生长,生长迅速,呈向外膨胀性生长的特性,表现为圆形或椭圆形,随着瘤体增长逐渐成为不规则形,表面呈多个结节融合状。从外部观察这些移植瘤边界较清楚,肿瘤与皮肤无粘连,活动度可,似有包膜,表皮血管丰富,但随着移植瘤体积进行性增大,肿瘤活动度逐渐下降。当移植瘤直径长至2.0cm及其以上时,在移植瘤表皮中央出现褐色的边缘不规则的坏死灶,坏死面积随着时间延长持续扩大。该移植瘤组织较易剥离,离体后可见瘤组织有一层完整的包膜、包膜上血供丰富、切面似鱼肉状、组织松脆易碎。
2.给药期间裸鼠的一般状况：给药第1天至给药第7天各组荷瘤裸鼠精神状态均良好,活动正常,反应敏捷,进食、进水正常,皮肤颜色如常,大便正常,未见明显异常反应。从给药第8天开始,随着移植瘤体积的增大,荷瘤裸鼠正常活动逐渐受到影响,行动较前迟缓,进食、进水减少。当移植瘤直径长至2.0cm及其以上时,荷瘤裸鼠逐渐呈现恶病质状态,肤色逐渐由粉色转为淡青色,且随着移植瘤的增大体重逐渐增长。给药期间,观察到As2O3单药组有2只裸鼠出现腹水,对照组有1只裸鼠出现眼部转移瘤,除此之外,各治疗组荷瘤裸鼠一般状况与对照组相比无明显差异。
3.不同用药组移植瘤体积增长情况：随着时间的延长,荷瘤裸鼠移植瘤体积越来越大。各治疗组移植瘤体积增长速度均较对照组慢,其中以DFO联合As2O3组移植瘤体积增长最慢,其次为As2O3单药组和DFO单药组。给药第1天至给药第5天,所有裸鼠无移植瘤生长,从给药第6天开始,各治疗组的移植瘤平均体积均小于生理盐水对照组,且随着给药时间的延长,各治疗组与对照组移植瘤平均体积的差值进一步扩大。到第11天时,生理盐水对照组移植瘤平均体积最大,其次是DFO单药组,再次是As2O3单药组,而DFO联合As2O3用药组的移植瘤平均体积最小,说明不同用药组对高致瘤HL-60白血病细胞裸鼠异种移植瘤体积随时间变化的趋势有影响。
4.DFO单药组和As2O3单药组对高致瘤性HL-60细胞裸鼠异种移植瘤的抑瘤率分别为2.56%和10.67%,移植瘤重量分别为(2.55±0.82)g和(2.34±0.79)g,与生理盐水对照组相比较,差异均无统计学意义(分别为F=1.290,P=0.866 >0.05和F=1.714,P=0.474>0.05):DFO联合As2O3用药组对高致瘤HL-60细胞裸鼠异种移植瘤的抑瘤率为25.49%,移植瘤重量为(1.95±0.39)g,与生理盐水对照组相比较,明显低于生理盐水对照组,差异有统计学意义(F=0.028,P=0.019<0.05),提示DFO联合As2O3用药对HL-60高致瘤细胞裸鼠异种移植瘤的生长具有抑制作用,DFO联合As2O3在抑制高致瘤HL-60细胞裸鼠异种移植瘤的生长上具有协同作用。
5.荷瘤裸鼠在处死后,肉眼观察各组移植瘤组织,可见出血坏死区,有的瘤块表面可见黑色痂样改变。光镜下可见,对照组瘤细胞分布密集,瘤细胞圆形或椭圆形,细胞胞体较大,大小基本一致,明显异型,核大而深染,胞浆量少,呈嗜碱性,可见个别凋亡细胞及凋亡小体,内有散在的局灶性细胞坏死区：各治疗组移植瘤组织中瘤细胞数目明显较生理盐水对照组减少,细胞体积变小,核深染,有的细胞固缩,有的仅存胞核,可见不规则坏死、出血,呈片状的嗜伊红结构,凋亡细胞和凋亡小体小片状分布,其中以DFO联合As2O3用药组较DFO单药组和As2O3单药组显著。
6.荷瘤裸鼠心、肝、脾、肺、肾病理组织学检查结果显示：在显微镜下观察可见,每只荷瘤裸鼠的心、肝、脾、肺、肾组织,结构完整,除充血程度不同外,均无明显的病理改变,未见HL-60白血病细胞转移。
7.荷瘤裸鼠骨髓病理组织学检查结果显示：4组均可见有核细胞增生活跃,粒红比例正常,末见明显的骨髓抑制和HL-60白血病细胞浸润。
8.免疫组织化学PowerVisionTM二步法检测移植瘤组织NF-κBp65的表达,统计学结果显示：DFO单药组,As2O3单药组与DFO联合As2O3用药组3个治疗组的平均积分光密度值均低于生理盐水对照组,与生理盐水对照组比较均有统计学差异(P<0.05)；且NF-κBp65表达的积分光密度值：生理盐水对照组>As2O3单药组>DFO单药组>DFO联合As2O3用药组。4组两两相互比较差异均有统计学意义(P<0.05)。提示DFO和As2O3都可降低高致瘤HL-60细胞裸鼠异种移植瘤组织的NF-κBp65表达,二者联合降低移植瘤组织NF-κBp65表达的作用更强。
结论
1.采用高致瘤性HL-60白血病细胞(1×106个细胞／只)在裸鼠皮下接种,可成功建立移植瘤动物模型,接种后7～8天均能形成直径约0.5cm～1.0cm大小的移植瘤,成瘤率达100%,且成瘤后瘤体生长迅速。
2.DFO和As2O3单药应用均可抑制高致瘤HL-60白血病细胞裸鼠异种移植瘤的生长,二者联合作用显著。
3.DFO联合As2O3应用荷瘤裸鼠能较好耐受,DFO可降低As2O3用量,促进其杀伤肿瘤作用,且可降低As2O3毒副作用。
4.DFO和As2O3均可降低高致瘤HL-60细胞裸鼠异种移植瘤组织的NF-κBp65的表达水平,二者联合降低移植瘤组织NF-κBp65表达的作用更明显。
Objective
To observe the therapeutic effect and the influence on NF-KBp65 expression level of transplanted tumors of deferoxamine and deferoxamine(DFO) in combination with arsenic trioxide (As2O3)by establishing a model of implanted tumor of human high tumorigenic leukemia cell line HL-60 in nude mice,and investigate the mechanism,so as to provide the experimental evidence for treatment or auxiliary treatment clinically for leukemia of iron chelators.
Methods
1.Xenograft tumor model of human high tumorigenic leukemia cell line HL-60 in nude mice was established by means of inoculating subcutaneously,then the tumor-bearing mice were randomly divided into four groups:50mg/kg DFO group, combination group(50mg/kg DFO+1.5mg/kg As2O3),3mg/kg As2O3 group, normal saline control group. The tumor-bearing mice were given drugs from that day of inoculating subcutaneously,and drug administration continued for 10 days continuously through intraperitoneal injection.The experimental protocol was approved by the ethical committee of First Clinical Medical College of Zhengzhou University.
2.Observing closely general conditions and adverse drug effects of the tumor-bearing mice,time of forming tumor, growth characteristic of transplanted tumor during administration of drugs.The maximum longitudinal(a) and transverse(b) diameters of transplanted tumor were measured with slide callipers every day. Tumor volumes were calculated according to the equation(volume=ab2/2),and then transplanted tumor growth curves were drawn.
3.The next day The tumor-bearing mice were executed after administration of drugs.Then tumor weights and tumor inhibitory rate of transplanted tumors were calculated.
4.After the tumor-bearing mice were executed, the specimens of transplanted tumors were stained with HE.Routine pathological examination were performed on transplanted tumors to observe the changes of tissue morphology. Then the changes of each specimen in different experimental groups were analyzed. The bone marrows of the tumor-bearing mice were observed to determine whether bone marrow depression and infiltration of leukemic cells existed or not. The tissues of heart,liver,spleen,lung and kidney of the tumor-bearing mice were observed to determine whether pathological changes and tumor metastasis existed or not.
5.NF-κBp65 expression levels of transplanted tumors were detected by two-step Immunohistochemistry detection(PowerVisionTM),and then integrate optical density(IOD) was detected by using professional software Biosens Digital Imaging System (v1.6).Mean integrate optical density value of the positive area was used for representing the expression levels of NF-κBp65.
6.The results of experiment were analyzed by SPSS16.0 for Windows.Sample averages were expressed by X±S.The differences of the weights of transplanted tumors between treatment groups and control group were test by t test.The differences of the NF-κBp65 expression levels of transplanted tumors between four experimental groups were test by one-way ANOVA.Statistic test were two-sided test of probability(α=0.05).Probability of less than 0.05 were considered significant.
Results
1.General conditions of forming tumor, growth characteristic of transplanted tumor after inoculating:Inoculating human high tumorigenic leukemia cell line HL-60 (about 1×106 cells/each mouse) culturing in vitro,tumor grew in 100% of animals.From the seventh day to the eighth day, macroscopic tumors were observed 0.5cm～1.0cm in diameter. Tumors which were orbicular or elliptic grew only at the sites of cells inoculation and expansive grew outward with rapid growth at the time of early growth.After that, with the enlargement of tumor size, tumors became irregular gradually and multinodular confluent on the surface.From the outside watching, clear boundary, no adhesion with the skin, good mobility, having peplos apparently and epidermis rich in blood vessels were observed,but the mobility of tumor decreased gradually with the progressive increase of tumor volume. When the tumors were bigger than two centimeters in diameter, brown necrotic foci which had irregular margins were observed on the central Epidermis of tumors,and necrotic area continued to expand with time.The transplanted tumor tissues were easily stripped with layer of intact peplos rich in blood vessels.The section of tumors looks like fish.The tissues were crispy and easily broken.
2.General conditions of the tumor-bearing mice during administration course:A good mental state, normal activities,agile response,eating and drinking normally, normal skin color, normal stool and no abnormal situation were observed in all the tumor-bearing mice from the first day to the seventh day of administration of drugs.With the enlargement of tumor size, normal activities of the tumor-bearing mice were influenced gradually from the eighth day of administration of drugs,for example,a slowing of movement and reduced eating and drinking.When the tumors were bigger than two centimeters in diameter, cachexia was observed in the tumor-bearing mice.Skin color gradually changed from pink to light cyan,and the weight of the tumor-bearing mice increased gradually with the enlargement of tumor size from the eighth day. Ascites were observed in two tumor-bearing mice of 3mg/kg As2O3 group,and metastatic tumor were observed in a tumor-bearing mice of control group. Apart from this,there was no significant difference between treatment groups and control group in general conditions of the tumor-bearing mice.
3.There was an increase of the transplanted tumor's volume as time went on. The rate of tumor volume increase in control group was the fastest, followed by 50mg/kg DFO group and 3mg/kg As2O3 group.And the rate in combination group(50mg/kg DFO+1.5mg/kg As2O3)was the lowest.No tumour grew from the first day to the fifth day of administration of drugs. From the sixth day mean volumes of tumors in control group were higher than them in treatment groups.And the differences of mean volumes of tumors between treatment groups and control group were further expanded as time went on.On the eleventh day the tumor volume of control group was the largest, followed by 50mg/kg DFO group, and 3mg/kg As2O3 group is next. The tumor volume of combination group(50mg/kg DFO+1.5mg/kg As2O3) was the smallest.So we can see that different drugs affected the trend of transplanted tumor's volume over time.
4.The tumor inhibitory ratioes of 50mg/kg DFO group and 3mg/kg As2O3 group were 2.56% and 10.67% respectively.The transplanted tumor weights of 50mg/kg DFO group and 3mg/kg As2O3 group were(2.55±0.82)g and(2.34±0.79)g respectively.There was no significant difference between DFO group and the control group(F=1.290,P=0.866>0.05),and there was no significant difference between As2O3 group and the control group (F=1.714, P=0.474>0.05).The tumor inhibitory ratio of combination group(50mg/kg DFO +1.5mg/kg As2O3)was 25.49%, The transplanted tumor weight of combination group was(1.95±0.39)g,and significantly lower than that of control group(F=0.028,P=0.019<0.05).It indicated that DFO combined with As2O3 had the synergistic anti-tumor effect.
5.After the tumor-bearing mice were executed, hemorrhage, necrosis and black scab were observed on the tumor tissue through macroscopic observation.By light microscope the tumor cells in control group were large, orbicular or elliptic, heterogeneous and basophilic,with high nucleocytoplasmic ratio,and distributed compactly. A few of apoptosis cells and apoptotic bodies were found in control group,in which areas of focal necrosis scattered. The number of tumor cells in tissue of treatment groups were smaller than that of normal saline control group.Small tumor cells with hyperchromatic nuclei,pycnosis, Some remaining nucleus,irregular necrosis,hemorrhage, flaky eosinophilic Structure,apoptosis cells and apoptotic bodies with a small flaky distribution were found in treatment groups.especially in combination group.
6.By light microscope,Complete structure, no obvious pathological changes except of congestion in different degrees and no metastases were found in the tissues of heart,liver,spleen,lung and kidney of the tumor-bearing mice.
7.After administration completed, bone marrow depression and infiltration of leukemic cells HL-60 were not observed in the bone marrows of the tumor-bearing mice,and the active proliferation of bone marrow karyocyte and normal myeloid erythroid ratio were observed in bone marrows.
8.NF-κBp65 expression levels of transplanted tumors were detected by two-step Immunohistochemistry detection(PowerVisionTM),and then the statistical results showed that there was significant difference in average value of integrate optic density(IOD) among experimental groups(P<0.05).The average value of IOD of control group was the largest, followed by 3mg/kg As2O3 group, and 50mg/kg DFO group is next. The average value of IOD of combination group(50mg/kg DFO+1.5mg/kg As2O3) was the smallest. So both DFO and As2O3 could lower NF-icBp65 expression levels of transplanted tumors, and especially DFO combined with As2O3 had a stronger effect.
Conclusion
1.The xenograft model of the human leukemia cell line HL-60 in nude mice was successfully established by Inoculating human high tumorigenic leukemia cell line HL-60 (about 1×106 cells/each mouse).Transplanted tumor grew in 100% of animals,and grew rapidly after forming tumours.From the seventh day to the eighth day after inoculating, macroscopic tumors were observed 0.5cm～1.0cm in diameter.
2.Both DFO and As2O3 could inhibit the growth of transplanted tumors,and DFO combined with As2O3 had a obvious and significant effect on inhibiting the growth of transplanted tumors.
3.When DFO combined with As2O3,it was well tolerated and there were no significant adverse effects.DFO not only lowered the dosage of As2O3,but also increased the killing effect of As2O3 on tumor.
4.Both DFO and As2O3 could lower NF-κBp65 expression levels of transplanted tumors, and especially DFO combinated with As2O3 had an obvious effect.
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