氯化钴诱导低氧环境对人肝癌HepG2细胞株的生长影响  

Effects of hypoxia induced by the cobalt chloride on the growth and apoptosis in hepatic cancer cell HepG2

作　　者：陈国凤[1] 张培建[1] 刘歆农[1] 刘霞[1] 朱以佳[1] 朱世春[1] 

机构地区：[1]扬州市第一人民医院普外科,扬州225000

出　　处：《国际外科学杂志》2016年第1期17-23,F0003,共8页International Journal of Surgery

基　　金：江苏省扬州市科技攻关项目（YZ2009051,YZ2010087）

摘　　要：目的 建立人肝癌HepG2细胞在氯化钴诱导的低氧环境下的生长模型,观察不同浓度氯化钴（CoCl2）诱导的低氧环境对人肝癌HepG2细胞的增殖、细胞的周期与凋亡即细胞的生长状况影响,探讨氯化钴诱导的低氧对人肝癌HepG2细胞生长的作用机制.方法 将对数期人肝癌细胞株HepG2细胞,分为加入不同浓度（50 μm/L、100 μm/L、150 μm/L、200 μm/L）氯化钴的实验组和加入等体积培养基的对照组,（1）通过MTT法检测HepG2细胞增殖情况计算细胞的抑制率并绘制HepG2细胞生长抑制曲线;（2）划痕实验观察HepG2细胞的迁移能力;（3）通过流式细胞仪的Annexin-VFITC/PI双染法和PI单染法检测细胞的凋亡和周期变化;（4）通过Western Blot法检测HepG2细胞的Bcl-2蛋白的表达.结果 （1）通过MTT法检测结果示在一定的时间和浓度范围内氯化钴抑制人肝癌HepG2细胞的增殖,并呈时间-剂量依赖性关系.（2）划痕损伤实验说明：氯化钴抑制HepG2细胞的迁移及损伤修复能力,呈浓度依赖性关系.（3）流式细胞仪的检测结果显示：对照组、不同浓度（50 μm/L、100 μm/L、200 μm/L、300 μm/L、500 μm/L）的实验组的细胞凋亡率（％）分别是：3.42、7.74、13.07、20.56、28.53、44.45（P ＜0.05）,与对照组相比实验组的凋亡率明显增加,并呈浓度依赖性.细胞的周期结果显示：随着浓度增加,氯化钴能明显抑制HepG2细胞周期的变化,阻滞细胞周期于G1期,从而抑制细胞的增殖.（4） Western Blot法检测：与对照组相比,经过不同浓度氯化钴处理后的实验组Bcl-2蛋白的表达明显减少.结论 在一定范围内,氯化钻诱导的低氧环境可以抑制人肝癌HepG2细胞的增殖以及愈合迁移的能力,诱导细胞凋亡并呈时间-剂量的依赖性关系,其作用机制可能与Bcl-2蛋白的表达减少有关.Objective To establish a human HepG2 cell growth model under the low oxygen environment induced by cobalt chloride in order to observe the impacts of human HepG2 cell proliferation,cellular cycles and apoptosis,namely cellular growth conditions,under the low oxygen environment induced by cobalt chloride with different concentrations and to study the HepG2 cell growth mechanism under the low oxygen environment induced by cobalt chloride.Methods The human HepG2 cells in the logarithmic phase were randomized grouping as control group and CoCl2 experimental group with different concentrations （50 μm/L,100 μm/L,150 μm/L and 200 μm/L）.① HepG2 cell proliferation was tested by MTT assay to calculate cell＇s suppression rate and draw HepG2 cell growth inhibition curves.② The move ability of HepG2 cells was observed by scratch test.③ The cellular apoptosis and periodic changes were detected using the flow cytometer Annexin-V FITC/PI double-staining and PI single staining methods.④HepG2 cell＇s Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and Bcl-2 protein expression were detected by Western Blot.Results ① The test results obtained via MTT assay showed that CoCl2 suppressed the human HepG2 cell proliferation within a certain amount of time and concentration and presented a time-dose dependent relation.② Scratch damage trial suggested that the cobalt chloride suppressed the HepG2 cell migration and wound repair capacity and presented a concentration dependent relation.③ Flow cytometer＇ s test results revealed that the apoptosis rates （%） in control group and experimental group with different concentrations （50 μm/L,100 μm/L,150 μm/L and 200 μm/L） were 3.42,7.74,13.07,20.56,28.53 and 44.45 （P 〈0.05）,respectively.The apoptosis rate of the experimental group was significantly increased compared with the control group,as well as showing a concentration dependency.The results of cellular cycles revealed that the cobalt chloride significantly suppressed the HepG2 cell＇s periodi

关 键 词：肝肿瘤 细胞低氧 生长 代谢 

分 类 号：R737.14[医药卫生—肿瘤]