siRNA介导的Bcl-xL基因沉默诱导人乳腺癌细胞凋亡的研究

目的研究siRNA(small interfering RNA)对MCF-7细胞株Bel—xL基因表达的抑制作用及对癌细胞增殖抑制和凋亡诱导作用。方法体外转录法合成特异针对人Bel—xL基因的siRNA；荧光标记法标记Bel—xL siRNA；脂质体法将siRNA转入MCF-7细胞株；流式细胞术检测siRNA的转染效率；半定量RT—PCR法检测siRNA对Bcl—xL基因表达的抑制作用；MTT法检测siRNA对细胞生长增殖抑制作用；荧光染色法观察siRNA诱导细胞凋亡的作用。结果siRNA的转染效率在一定剂量范围内随浓度的增加而增加，siRNA转染组Bcl—xL mRNA表达水平可下调约72％，在对照组内mRNA表达水平无明显改变。细胞生长增殖抑制率在一定范围内具有剂量依赖性和时间依赖性。荧光显微镜下观察肿瘤细胞发现，siRNA50、100、200nmol／L转染组出现典型的凋亡形态学变化。结论脂质体法对siRNA的瞬时转染效率较高，体外转录合成的siRNA能特异有效下调Bel—xL基因的表达，瞬时转染Bel—xL siRNA能有效抑制乳腺癌细胞增殖并诱导其凋亡。低剂量Bcl—xL siRNA对瘤细胞显示了显著的抗增殖作用。

Inhibition of Bcl-xL gene expression by siRNA induces apoptosis in MCF-7 cell line

Objective To study the inhibition of Bcl -xL gene expression induced by siRNA in MCF-7 cell line, and the related pr oliferation inhibition and apoptotic induction of MCF-7 cells. Methods siRNA targeting Bcl-xL gene were synthesized using web design software provided by Ambion and the Silencer siRNA construction kit; Fluorescein-lab eled siRNA was done by FAM-Silencer TMsiRNA Labeling Kit; siRNA was t ransfected into MCF-7 cells by Lipofectamine TM2000 reagent; siRNA transf ection efficiencies were analyzed by flow cytometry; The expression of Bcl-xL w as detected by RT-PCR; MTT assay was used to assess the cell growth; Apoptosis of MCF-7 cells was analyzed by fluorescent microscope. Results The expression of Bcl-xL mRNA decreased by 72% in the siRNA transfected MCF- 7, while no significant changes were found in control groups. The inhibitory rat e of cell proliferation depended on time and concentrations to some extent. The typical morphological changes in apoptosis were found by fluorescence staining i n siRNA groups. Conclusions The synthesized siRNA in vitro ca n down-regulate the expression of Bcl-xL, which is associated with growth inhi bition and apoptosis of MCF-7 cells.