脂质体介导TGF-β1 shRNA转染的方法

The Method of TGF-β1 shRNA Transfection Efficiency Mediated by Liposomes

目的探讨脂质体介导的转化生长因子β1(TGF-β1)短发夹RNA(shRNA)在人肿瘤细胞株的转入效率,为研究TGF-β1信号转导在肿瘤微环境中的作用机制提供方法依据。方法化学合成TGF-β1小干扰RNA(siRNA),与脂质体转染介质混合制备转染复合物(Lip-shRNA),处理细胞24h后用流式细胞术(FCM)及人工计数法测定RD细胞的转入率;免疫荧光技术检测细胞TGF-β1蛋白表达水平。结果荧光显微镜观察硫代磷酸化修饰的寡核苷酸转入4h后,shRNA主要定位在细胞浆,8h后细胞核中有较多的shRNA;FCM测得转入细胞转入率与Lip-shRNA复合物呈剂量依赖关系,人工测得shRNA的转入率与转入复合物在低剂量呈剂量依赖关系,但在高剂量时增高不明显。转入6d后各组细胞TGF-β1蛋白表达量较转入3d时明显减少,与对照组比较,差异有统计学意义(P<0.05)。结论脂质体介导的TGF-β1shRNA在常见肿瘤细胞株中的转入效率与寡核苷酸的稳定性、Lip-shRNA复合物剂量、转入时间密切相关,该转染技术可以作为RNA干扰手段研究TGF-β1信号转导的病理学机制。

Objective Study on transfer efficiency in human tumor cell lines by liposome mediated TGF-β1shRNA and provides a possible research method of RNA interference to explore pathogenic role of TGF-β1 signaling pathway in tumor microenvironment. Methods Chemically synthetic siRAN against the TGF-β1 protein, combined with LipofectamineTM2000 to prepare Lip-shRNA complexes. After exposure to the different dose of complexes for 24 hours, the fluorescence indexes of RD cells were analyzed by flow cytometry. The transfer rates were assessed by manual counting under fluorescent microscope. The TGF-β1 protein expression of cells transferred for different time was inspected by immunofluorescent. Results The fluorescent microscope showed that the fluorescent phosphorothioated oligonucleotide located in cytoplasm of RD cells after transferred for 4 h, and the shRNA shifted to nucleus of RD cells after 8 h, The transfer rate examined by FCM showed compellent dependent correlation with the doses of Lip-shRNA complexes. The transfer rate by manual counting were dependently correlated with the lower doses of Lip-shRNA complexes, but didn＇t increase obviously with the high dose of complexes. The TGF-β1 protein expression of RD cells transferred for 6 days is decreased more than that of RD cells transferred for 3 days, and there were significant difference （P〈0.05） compare to the control group. Conclusion The transfer efficiency of TGF-β1 shRNA mediated by liposome in tumor cells depends on stability of oligonucleotide, dose of Lip-shRNA complexes, and transfer time,this technology may used as a way of research to explore pathogenic role of TGF-β1 signaling pathway in tumor microenviron- ment.