二氯化钴诱导大鼠肝癌细胞缺氧及凋亡及增殖探究.docVIP

二氯化钴诱导大鼠肝癌细胞缺氧及凋亡及增殖探究 　【摘要】 　　目的：了解大鼠肝癌细胞系CBRH7919在缺氧状态下凋亡和增殖的关系，探讨低氧诱导因子（hypoxia induced factor, HIF-1α）在此诱导过程中的作用。方法：二氯化钴（CoCl2）作为缺氧诱导剂。利用RT-PCR和免疫细胞化学法检测CBRH7919细胞HIF-1α在转录水平和翻译水平的表达，利用流式细胞仪检测CBRH7919细胞在不同缺氧程度下细胞凋亡率、细胞周期和线粒体膜电位的变化。结果：（1）随缺氧程度的加重，HIF-1α在转录和翻译水平的表达逐渐增加；（2）流式细胞仪结果显示经300 μM CoCl2分别处理0、4、24小时后细胞凋亡率分别为（2.31±1.1）%、（5.62±2.4）%、（21.2±7.5）%。对照组和4小时处理组细胞周期改变不显著，24小时处理组出现显著G0/G1期阻滞。处理组细胞线粒体膜电位较对照组有不同程度的降低。结论：大鼠肝癌细胞随着缺氧程度的加重，细胞凋亡明显增加，伴随细胞周期进入G0/G1阻滞及线粒体膜电位降低；HIF-1α的表达增加在此过程中发挥重要作用。 【关键词】 肝癌细胞 缺氧 凋亡 低氧诱导因子 　　[Abstract] Objective: To investigate the apoptosis and proliferation of hepatocellular carcinoma cells induced by hypoxia, and to study the role of HIF-1α in the induction course. Methods: CBRH7919 cells were treated with 300 μM CoCl2 for 0, 4 and 24 hours. The expressions of HIF-1α at the levels of transcription and translation were detected by RT-PCR and immunocytochemistry. The change of cell cycle, membrane potential of mitochondria and apoptotic ratio were detected by flow cytometry. Results: HIF-1α was upregulated both at the transcriptional and translational levels under hypoxic condition. Apoptotic ratios of CBRH7919 cells were (2.31±1.1)%, (5.62±2.4)% and (21.2±7.5)% respectively. CBRH7919 cells appeared notable cell arrest at G0/G1 phase under severely hypoxia. There was also a decrease in the mitochondria membrane potential. Conclusion: Cobalt dichloride can induce apoptosis of rat hepatocellular cell line CBRH7919, and HIF-1α plays an important role in this hypoxia-induced apoptosis. 　　[Key words] Hepatocellular carcinoma cell; Hypoxia; Apoptosis, Hypoxia inducible factor 　　氧稳态的维持是细胞生命活动的基本前提。低氧诱导因子（hypoxia inducible factor，HIF）在缺氧的情况下被激活，是最重要的低氧信号转录调节因子[1，2]。本实验从缺氧诱导大鼠肝癌细胞凋亡和增殖的角度，初步探讨HIF-1α在缺氧环境下的表达及其与细胞凋亡和增殖的关系。 　　1 材料和方法 　　1.1 材料 大鼠肝癌细胞系CBRH7919购自中科院上海细胞生物学研究所, 二氯化钴（CoCl2）购自汕头化学试剂公司（分子量237.93），SP免疫组化试剂盒购自北京中山试剂公司，单克隆抗体HIF-1α购自Neomarkers，Trizol Rna购自Invitrogen， RT-PCR 试剂盒购自TaKaRa，Annexin V-FITC 试剂盒购自Beckman Coulter。余试剂及仪器均由天津市肿瘤研究所中心实验室提供。