Fas siRNA真核表达载体构建及其抑制Jurkat细胞凋亡的作用

 目的 构建Fas（CD95）特异性siRNA真核表达载体psilencircle-FasSi，转染Fas-FasL凋亡敏感细胞株Jurkat，研究其抗凋亡作用。方法 通过聚合酶链式反应（PCR）制备siRNA表达框架(SECs)，转染Jurkat细胞，实时荧光定量PCR检测Fas mRNA抑制率，筛选出高效抑制Fas mRNA表达的siRNA；把筛选出的siRNA序列插入siRNA真核表达质粒psilencircle，构建psilencircle-FasSi；psilecircle FasSi转染Jurkat细胞，实时荧光定量PCR检测Fas mRNA、Western blot检测Fas蛋白；anti-Fas mAb刺激Jurkat细胞凋亡，流式细胞术检测Jurkat细胞凋亡率。结果 酶切、测序证明成功构建了psilecircle-FasSi，实时荧光定量PCR及Western blot表明psilencircle-FasSi可抑制Fas mRNA和蛋白表达，流式细胞术检测证实psilencircle-FasSi可抑制Jurkat凋亡率。结论 我们成功构建了Fas特异性siRNA真核表达载体psilencircle-FasSi，它不仅可以下调Jurkat细胞的Fas表达而且可以显著抑制Fas-FasL途径诱导的凋亡。

Construction of Fas Targeting siRNA-expressing Vector and Its Apoptotic Inhibitory Effect

Objective To construct Fas-targeting siRNA expressing vector,to transfect Fas-FasL sensitive Jurkat cell line and to study its anti-apoptotic effect. Methods The siRNA expression cassettes(SECs) are generated by PCR, then Jurkat cells were transfected by Fas targeting SECs. The efficient SEC was selected by comparison of Fas mRNA level using real time fluorescent quantitative PCR(FQ-PCR) and construct the psilecircle FasSi SEC. Fas expression of Jurkat cells transfected by psilencircle-FasSi(Jurkat-FasSi) was analyzed by FQ-PCR and Western blot. Jurkat-FasSi was treated by anti-Fas mAb to induce apoptosis and apoptotic rate was detected by flow cytometry. ResultsRestriction enzyme digestion analysis and the sequence analysis confirmed that the psilecircle-FasSi was successfully constructed. FQ-PCR and Western blot results showed that Fas expression in Jurkat FasSi cells was down regulated. Flow cytometry analysis using Annexin/PI staining indicated that apoptosis induced by anti-Fas antibody in Jurkat FasSi cells was significantly inhibited. Conclusion The Fas siRNA eukaryotic expression plasmid(psilencircle FasSi) was successfully constructed. It can down regulate Fas expression by transfected it into Jurkat cell line and inhibited Fas-FasL pathway induced apoptosis significantly through RNAi.