拓扑替康诱导人肝癌细胞株HepG2凋亡的初步研究

Preliminary Study of Apoptosis of Human Hepatocarcinoma Cell LineHepG2 Induced by Topotecan

背景与目的:拓扑替康(topotecan,TPT)是新一代水溶性、半合成喜树碱类衍生物,系拓扑异构酶I抑制剂、细胞周期特异性抗肿瘤药物;肝癌为我国发病率居第三位的恶性肿瘤,现有治疗方法的临床疗效均不理想。本文研究TPT诱导肝癌细胞株HepG2凋亡的作用,及其细胞毒作用。方法:运用MTT、HE染色、透射电镜、流式细胞仪、免疫组化技术等实验方法,对TPT杀伤HepG2细胞的程度进行了测定并观察了其诱导凋亡的过程。结果:TPT能通过诱导HepG2细胞凋亡的方式,杀伤HepG2细胞,TPT对HepG2细胞具有较强的体外杀伤作用,并存在明显的剂量、时间依赖关系,IC50约为95μg/L。TPT将HepG2细胞阻断在S期,并进一步诱导其凋亡。凋亡形态特征表现为典型的间期凋亡:细胞核染色质固缩,呈新月形聚集于核膜周缘,胞质浓缩,出现空泡。在TPT诱导HepG2细胞凋亡的过程中,不影响bcl-2基因蛋白正常表达(P>0.05)。结论:TPT能诱导HepG2细胞凋亡,细胞毒性较强。

Background & Objective: Topotecan, a semisynthetic water soluble derivative of camptothecin, is a potent inhibitor of DNA topoisomeraseⅠ and cell cycle specific antitumor agent; The incidence rate of hepatocarcinoma ranks the third in all types of malignant tumor in China. The clinical curative effect of the present treatments and drugs for hepatocarcinoma are not so satisfactory. The inducing effect of topotecan on apoptosis of hepatocarcinoma cell line HepG2 and its cytotoxicity on HepG2 were studied in this paper. Methods: Microculture tetrazolium assay (MTT), HE staining, transmission electron microscope (TEM), flow cytometer (FCM) and immunohistochemistry were employed to determine and observe the apoptosis of HepG2 induced by topotecan and its lethal action on HepG2. Results:Topotecan killed HepG2 cell by inducing apoptosis. Topotecan had strong anti HepG2 activity in vitro, suggesting distinct dose and time dependent relationship. The drug IC50 was about 95μg/L. Topotecan block HepG2 in S phase, then further initiate apoptosis program. The morphologic characteristic of apoptosis showed that it is a typical interphase apoptosis process: the nuclei showed chromatin pycnosis, and clustered on the inner border of karyotheca, cytoplasm condensed, many vacuoles occurred in it. The expression of bcl 2 were not affected in the course of HepG2 apoptosis induced by topotecon (P >0.05). Conclusion: Topotecan had significant cytotoxicity on HepG2 by inducing apoptosis.