PTEN基因沉默对小鼠原代肝细胞Akt/GSK-3β信号通路的影响

Effect of PTEN gene silencing on Akt/GSK-3β signaling pathway in primary mouse hepatocytes

目的探讨第10号染色体缺失性磷酸酶-张力蛋白同源物蛋白(PTEN)对小鼠原代肝细胞蛋白激酶B/糖原合酶激酶-3β(Akt/GSK-3β)信号通路的影响。方法采用两步胶原酶灌注法分离小鼠原代肝细胞,均分为4组:阴性对照组(NC-C组)、阴性对照白细胞介素-6(IL-6)组(NC-IL组)、siR-996对照组(996-C组)、siR-996与IL-6组(996-IL组)。采用HiPerFect转染试剂将siRNA-996转入小鼠原代肝细胞,24h后提取蛋白,通过蛋白质印迹法(Western blot)检测各组Akt、磷酸化Akt(p-Akt)、GSK-3β、磷酸化GSK-3β(pGSK-3β)、PTEN蛋白水平。结果在PTEN蛋白水平上,与NC-C组相比,NC-IL组升高(P<0.05),996-C组和996-IL组降低(P<0.01)。在p-Akt/Akt上,与NC-C组相比,NC-IL组降低(P<0.05),996-C组和996-IL组升高(P<0.05)。在p-GSK-3β/GSK-3β上,与NC-C组相比,NC-IL组降低(P<0.05),996-C组和996-IL组升高(P<0.01)。结论 PTEN基因沉默可逆转IL-6对小鼠原代肝细胞Akt/GSK-3β信号通路的抑制,增强小鼠原代肝细胞Akt/GSK-3β信号通路的表达。

Objective To investigate the effect of phosphatase and tensin homologue deleted on chromosome 10(PTEN)on protein kinase B/glycogen synthase kinase-3β(Akt/GSK-3β)signaling pathway in mouse primary hepatocytes.Methods The mouse primary hepatocytes were isolated by two-step collagenase perfusion and divided into four groups on average:the negative control group(the NC-C group),the negative control IL-6 group(the NC-IL group),the siR-996 control group(the 996-C group)and the siR-996 and IL-6 group(the 996-IL group).siRNA-996 was transfected into mouse primary hepatocytes by HiPerFect transfection reagent.After 24 h culture,the protein was extracted,and the protein levels of Akt,p-Akt,GSK-3β,p-GSK-3βand PTEN in each group were detected by Western blot.Results Compared with the NC-C group,the level of PTEN protein increased in the NC-IL group(P<0.05),while decreased significantly in the 996-C group and the 996-IL group(P<0.01).Compared with the NC-C group,p-Akt/Akt was decreased in the NC-IL group(P<0.05),while it was increased in the 996-C group and the 996-IL group(P<0.05).Compared with the NC-C group,p-GSK-3β/GSK-3βwas decreased in the NC-IL group(P<0.05),while it was increased significantly in the 996-C group and the 996-IL group(P<0.01).Conclusion PTEN gene silencing can reverse the inhibition of IL-6 on Akt/GSK-3βsignaling pathway in mouse primary hepatocytes,and enhance the expression of Akt/GSK-3βsignaling pathway in mouse primary hepatocytes.