摘要
目的 研究 si RNA(sm all interfering RNA)对 MCF- 7细胞株 Bcl- x L 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 Bcl- x L 基因的 si RNA;荧光标记法标记 Bcl- x Lsi RNA;脂质体法将 si RNA转入 MCF- 7细胞株 ;流式细胞术检测 si RNA的转染效率 ;半定量 RT- PCR法检测si RNA对 Bcl- x L 基因表达的抑制作用 ;MTT法检测 si RNA对细胞生长增殖抑制作用 ;荧光染色法观察 si RNA诱导细胞凋亡的作用。结果 si RNA的转染效率在一定剂量范围内随浓度的增加而增加 ,si RNA转染组 Bcl- x Lm RNA表达水平可下调约 72 % ,在对照组内 m RNA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 ,si RNA5 0、10 0、2 0 0 nm ol/L 转染组出现典型的凋亡形态学变化。结论 脂质体法对 si RNA的瞬时转染效率较高 ,体外转录合成的 si RNA能特异有效下调 Bcl- x L 基因的表达 ,瞬时转染 Bcl- x L si RNA能有效抑制乳腺癌细胞增殖并诱导其凋亡。低剂量 Bcl- x L si
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.
出处
《实用肿瘤杂志》
CAS
2005年第1期25-29,共5页
Journal of Practical Oncology
基金
广东省自然科学基金资助项目 (3196 2)
