细胞增殖抑制基因对大鼠肝星状细胞HSC-T6增殖与凋亡的影响

Effect of HSG gene on proliferation and apoptosis of rat hepatic stellate cell line HSC-T6

目的 观察细胞增殖抑制基因（HSG）对大鼠肝星状细胞系HSC-T6增殖与凋亡的影响.方法 制备携带HSG基因的重组腺病毒载体,瞬时转染至大鼠肝星状细胞HSC-T6,逆转录-聚合酶链反应（RT-PCR）和Western blot分别检测细胞中HSG信使核糖核酸（mRNA）及蛋白的表达水平,细胞计数试剂盒（CCK-8）法检测细胞增殖活性,流式细胞术分析HSG对细胞周期和凋亡状态的影响,Western blot检测HSG对细胞周期蛋白激酶抑制剂p21、p27及细胞增殖核抗原（PCNA）的作用.结果 与空白对照组和阴性对照组比较,转染组HSG的mRNA（14.93 ±1.10比1.03±0.33,14.93±1.10比1.00 ±0.15）和蛋白表达量明显增加（P＜0.05）;转染后细胞生长减缓,细胞增殖活性明显受到抑制（P＜0.05）;转染组G0/G1期细胞比例和凋亡率分别为[（66.57±1.29）％、（23.88 ±0.96）％],与空白对照组[（49.32±2.86）％、（3.38±0.16）％]和阴性对照组[（52.46±3.17）％、（3.54±0.24）％]比较明显增高（P＜0.05）;转染后细胞周期调控蛋白p21、p27表达升高,PCNA表达降低,差异均有统计学意义（P＜0.05）.结论 HSG基因过表达可有效的抑制大鼠肝星状细胞系HSC-T6的增殖,使其阻滞于G0/G1期并诱导其凋亡,并可能是通过参与细胞周期的调节发挥作用.

Objective To discuss the effects of HSG gene on proliferation and apoptosis of rat hepatic stellate cell line HSC-T6.Methods To construct the recombinant retroviral vector carrying rat HSG gene,transient transfection into rat hepatic stellate cell line HSC-T6,real-time quantitative polymerase chain reaction （RT-PCR） and Western blotting were used to detect the mRNA expression and protein levels of HSG respectively.Cell counting kit-8 （CCK-8） assay was used to investigate cell proliferation activity.Flow cytometry was used to measure the cell cycle progression and cell apoptosis.Effect of HSG on HSC-T6 cells proliferation were investigated by Western blotting for the expression of cyclin dependent kinase inhibitors p21,p27 and proliferating cell nuclear antigen （PCNA）.Results Compared with blank control group and negative control group,the protein and mRNA expression （14.93 ± 1.10 vs.1.03 ± 0.33,14.93 ± 1.10 vs.1.00 ± 0.15） of HSG was significantly increased in transfection group （all P 〈 0.05）.Meahwhile,the cell proliferation activity remarkably decreased in transfection group （both P 〈 0.05）.Furthermore,cell cycle arrested in the G0/G1 phase with the increasing apoptosis rate [（23.88 ± 0.96） % vs.（3.38±0.16）%,（23.88±0.96）% vs.（3.54 ±0.24）%] as well as the expression of p21 and p27,and the reducing expression of PCNA （all P 〈 0.05）.Conclusion Overexpression of HSG could inhibit the proliferation of rat hepatic stellate cell line HSC-T6,the cell cycle arrest in the G0/G1 phase and induce its apoptosis.HSG may play a role by participating in the regulation of cell cycle.