小干扰RNA干扰PC12细胞内源性Kir6.2基因后对鱼藤酮诱导的细胞损伤及信号通路研究

摘要

目的 通过小干扰RNA(siRNA)干扰内源性Kir6.2基因表达来研究Kir6.2亚基缺失对鱼藤酮诱导的帕金森病细胞模型的作用及可能的信号通路.方法 以PC12细胞为研究对象,采用RT-PCR检测5组细胞(正常对照组,阴性对照组,siRNA干扰1组,siRNA干扰2组和siRNA干扰3组)Kir6.2干扰情况,Western blot方法检测2组细胞(阴性对照组,siRNA干扰1组)Kir6.2干扰情况,水溶性四氮唑检测4组细胞(阴性无药组、干扰组无药组、阴性加药组、干扰加药组)在鱼藤酮处理24 h后的细胞活力;Western blot方法检测4组细胞(阴性对照组、干扰组无药组、鱼藤酮组、PKC抑制剂组)鱼藤酮处理前后细胞内PKC及磷酸化PKC表达变化.结果 RT-PCR结果显示,siRNA干扰1组,siRNA干扰2组干扰成功.Western blot结果显示,siRNA干扰1组内源性Kir6.2表达较阴性对照组明显降低(0.55±0.07 vs 0.89±0.09,P＜0.05).四氮唑结果显示,Kir6.2干扰组较阴性对照组PC12细胞活力显著减低(P＜0.05).Western blot结果提示PKC抑制剂组PKC和磷酸化PKC水平较阴性对照组、siRNA干扰组、鱼藤酮组明显降低(P＜0.05).结论 内源性Kir6.2能保护PC12细胞抵抗鱼藤酮的毒性作用,Kir6.2可能通过激活磷酸化PKC在调节鱼藤酮诱导的PC12细胞活力中发挥重要作用.%Objective To study the effect of siRNA-interfered endogenous Kir6.2 gene deletion on rotenone-induced cell damage and signal pathway according to the expression of siRNA-interfered endogenous Kir6.2 gene.Methods The expression of endogenous Kir6.2 gene in PC12 cells was detected by RT-PCR and Western bolt.The viability of PC12 cells was tested using WST-1.The expression of PKC and phosphorylated PKC in PC12 cells was detected by Western blot in negative control group,siRNA/Kir6.2 group,siRNA/Kir6.2 + rotenone group,siRNA/Kir6.2+ rotenone+PKC inhibitor group before and afer treatment with rotenone.Results RT-PCR showed that the interference was successful in siRNA/Kir6.2/group 1 and siRNA/Kir6.2/ group 2.Western blot displayed that the expression level of endogenous Kir 6.2 gene was significantly lower in siRNA/Kir6.2/ group 1 than in negative control group (0.55±0.07 vs 0.89±0.09,P＜ 0.05).WST-1 revealed that the viability of PC12 cells was significantly lower in siRNA/Kir6.2 group than in negative control group (P＜0.05).Western blot demonstrated that the expression of PKC and phosphorylated PKC was significantly lower in siRNA/Kir6.2+-rotenone+PKC inhibitor group than in the other three group (P＜0.05).Conclusion Endogenous Kir6.2 can protect PC12 cells against the toxicity of rotenone and plays an important role in regulating the rotenone-induced viability of PC12 cells by activating the phosphorylated PKC.