Vpr抑制人大肠癌细胞株HT-29增殖及调节存活素表达的研究

Inhibition of the proliferation of HT-29 and the expression of survivin in HT-29 cells by Vpr

目的观察I型人类免疫缺陷病毒蛋白R(Viral protein R、Vpr)在体外对大肠癌细胞HT-29增殖抑制及探索Vpr对HT-29细胞存活素(Survivin)表达的调节作用。方法应用腺病毒转染系统将Vpr基因以不同感染复数转染至HT-29细胞。MTT法检测Vpr对HT-29细胞的增殖抑制作用;流式细胞仪检测细胞周期及细胞凋亡变化。逆转录-聚合酶链反应(RT-PCR)和免疫印迹(western blot)法检测mRNA水平和蛋白水平上Survivin的表达。结果实验组可见Vpr蛋白的表达;实验组大肠癌细胞HT-29增殖受到抑制(MOI=200),从转染72h开始,实验组MTT值与对照组及空载体组比较差异均具有有统计学意义(P<0.05),随着时间的延长,抑制逐渐增强;转染72h后(MOI=200),实验组细胞凋亡率与对照组及空载体组比较均差异具有显著统计学意义(P<0.01);实验组处于G2/M期细胞比例与对照组及空载体组比较均差异具有统计学意义(P<0.05);转染72h后,实验组(MOI=50)Survivin基因的mRNA水平及蛋白表达水平与对照组及空载体组(MOI=200)比较均差异具有统计学意义(P<0.05或P<0.01),并且survivin的表达随着MOI的增加而减弱。结论 Vpr体外可以诱导细胞HT-29G2/M期阻滞及凋亡从而有效抑制细胞HT-29增殖,其机制可能与Vpr下调Survivin基因的表达有关。Vpr在大肠癌的治疗中具有潜在的应用前景。

Objective To investigate the effect of Vpr on proliferation of HT-29 and expression of Survivin gene.Methods HT-29 cells were treated with Ad-Vpr or Adv for five days,The cell proliferation was measured by MTT assay everyday.72h post treatment,Flow cytometry（FCM） was employed to detect the cell cycle distribution and apoptosis.The expression of Vpr protein was detected by western blot;The rnRNA and protein expression levels of Survivin was determined by RT-PCR or Western blot.Results MTT showed that Vpr significantly inhibited the proliferation of HT-29 cells（72h post treatment,MOI=200 P0.05 vs.control or Adv group） in a time dependent manner.The results of FCM showed that Vpr markedly increased the ratio of apoptosis（72 h post treatment,MOI=200,P0.01 vs.control or Adv group） and cells in G2/M cell cycle（72 h post treatment,MOI=200,P0.05 vs.control or Adv group）.RT-PCR and Western blot showed that Vpr significant1ly downregulated the expression levels of Survivin（72 h post treatment,MOI：50,P0.05 or P0.01 vs.control or Adv（MOI=200） group）.Conclusion Vpr inhibits the proliferation of HT-29 in vitro by inducing cell cycle arrest and apoptosis.and Vpr significant1ly downregulated the expression of survivin.Vpr is a potential anticancer agent for colorectal cancer.