摘要
目的利用短发夹RNA（shRNA）在肝癌HepG-2细胞内诱导RNA干扰（RNAi），抑制Bcl—xL基因表达，探讨对肝癌细胞HepG-2生长和凋亡的影响。方法构建Bcl—xL靶向的shRNA，用脂质体Lipofectamine^TM2000转染入肝癌细胞HepG-2。经RT—PCR和流式细胞术分别检测shRNA对Bcl—xL mRNA和蛋白水平的抑制效应，采用MTT，TUNEL等技术检测shRNA处理前后细胞生物学行为的变化。结果与未转染组相比，shRNA能够有效的抑制Bcl—xL基因表达，mRNA和蛋白表达明显降低，抑制率分别为86．6％和70．2％；肝癌细胞生长明显减慢，转染对照组（HepG-2hk）和未转染组（HepG-2-w）细胞的生长曲线较为接近，说明转染非特异性shRNA后的细胞增殖特性未受到影响（P〉0．05）；而转染组的细胞（HepG-2-si）增殖曲线位于前两者的下方，细胞增殖特性发生明显改变，差异有统计学意艾（P〈0．05）；凋亡明显增加，HepG-2-si组细胞每视野凋亡数为15．0±1．1，HepG-2-w，HepG-2hk组细胞凋亡数分别为1．7±1．0和3．2±1．2。HepG-2-si与HepG-2-w组间细胞凋亡数差异具有统计学显著性意义（P〈0．01），而HepG-2hk组与HepG-2-w组间细胞凋亡数差异无统计学显著性意义（P〉0．05）。结论RNAi在体外明显抑制了肝癌细胞中Bcl—xL基因的表达和肿瘤细胞增殖，这种抑制作用是通过细胞凋亡增加实现的，为开辟Bcl—xL靶向的RNA干扰治疗肝癌提供实验基础。
Objective To investigate the inhibitory action of short hairpin RNA （shRNA） targeting at the Bcl-xL gene expression in HepG-2 cells and to determine the effect of shRNA on the change of cell growth and cell apoptosis. Methods Constructed a small interference RNA homologous to Bcl-xL gene,transfected it into hepatocarcinoma cell HepG-2,then detected Bcl-xL gene expression on mRNA and proteins level ,respectively. MTT detected the change of cell growth and TUNEL detected the change of cell apoptosis. Results The expression of Bcl-xL was obviously inhibited by RNAi. The inhibition rates were 86.6% and 70. 2% respectively. The cell growth became significantly slow （P〈0. 05）,and apoptosis promoting simultaneously （P〈 0. 01）. Conclusion shRNA-Bcl-xL effectively inhibits Bcl-xL gene expression and cell growth,promotes apoptosis.
出处
《现代检验医学杂志》
CAS
2010年第3期98-101,共4页
Journal of Modern Laboratory Medicine
基金
辽宁省教育厅2008年度科研计划项目（2008161）.
