小鼠原代肝细胞中PTEN基因沉默所需siRNA的筛选

Screening of siRNA for PTEN gene silencing in mouse primary hepatocytes

目的筛选出小鼠原代肝细胞中PTEN基因沉默所需的高效siRNA。方法采用两步胶原酶灌注法分离小鼠原代肝细胞,放入25 cm^2胶原包被瓶中(1×106个/瓶),均分为5组:对照组(C组)、阴性对照组(NC组)、siR-996组、siR-1381组、siR-1519组。采用Hi PerFect转染试剂将各组对应的siRNA序列转入小鼠原代肝细胞,24 h后提取RNA和蛋白,通过Real-time PCR检测各组PTEN mRNA表达,通过Western blot检测各组PTEN蛋白含量。结果siR-996组、siR-1381组和siR-1519组的PTEN蛋白含量低于NC组,差异有统计学意义(P<0.01),siR-996组、siR-1381组和siR-1519组的PTEN mRNA水平低于NC组,差异有统计学意义(P<0.01)。siR-996组的PTEN蛋白含量及PTEN mRNA水平最低,其PTEN蛋白含量及PTEN m RNA水平的沉默效率高于siR-1381组和siR-1519组。结论 siR-996是小鼠原代肝细胞中PTEN基因沉默所需的高效siRNA。

Objective To screen siRNA for PTEN gene silencing in mouse primary hepatocytes. Methods Primary mouse hepatocytes were isolated by two-step collagenase perfusion, the hepatocytes were plated in collagen-coated 25 cm^2 flasks at a density of 1×10^6 cells/flask. The hepatocytes were divided into five groups on average: control group (C group), negative control group (NC group), siR-996 group, siR-1381 group, siR-1519 group. The siRNA of each group were transferred into mouse primary hepatocytes by HiPerFect transfection reagent, extracted RNA and protein after 24 h. The expression of PTEN mRNA was detected by Real-time PCR and the content of PTEN protein was detected by Western blot. Results The PTEN protein content of the siR-996 group, siR-1381 group and siR-1519 group were lower than that of the NC group, the differences were statistically significant (P<0.01). The PTEN mRNA level of the siR-996 group, siR-1381 group and siR-1519 group were lower than that of the NC group, the differences were statistically significant (P<0.01). The PTEN protein content and PTEN mRNA level of siR-996 group were the lowest, the silencing efficiency of PTEN protein content and PTEN mRNA level was higher than that of siR-1381 group and siR-1519 group. Conclusion SiR-996 is a highly efficient siRNA for PTEN gene silencing in mouse primary hepatocytes.