瞬时受体电位阳离子通道蛋白6高表达对转化生长因子-β1诱导体外培养小鼠肾足细胞损伤的作用

Effect of TRPC6 over expression on mice podocyte damage induced by TGF-β1

目的探讨瞬时受体电位阳离子通道蛋白6(TRPC6)高表达对转化生长因子-β1(TGF-β1)干预下体外培养小鼠肾足细胞nephrin、desmin、caspase9表达及细胞凋亡的影响。方法用脂质体Lip2000将针对小鼠TRPC6的基因真核表达载体p EX-3-TRPC6转染体外培养的小鼠足细胞,48小时后Western blot检测转染后TRPC6蛋白表达变化。将足细胞分为4组:正常对照组,TGF-β1干预组,TGF-β1+p EX-3-NC组(空载体组),TGF-β1+p EX-3-TRPC6组(TRPC6高表达组),干预48小时后用western blot和real time PCR检测caspase9、desmin、nephrin蛋白和mRNA表达水平,用流式细胞术检测各组细胞凋亡率,DAPI染色观察凋亡细胞核形态学变化。结果转染48小时后TRPC6高表达组TRPC6蛋白水平较正常对照组明显升高(P<0.01),空载体组TRPC6蛋白表达水平无明显变化;TGF-β1干预48小时后caspase9、desmin蛋白和mRNA表达水平显著升高(P<0.05),nephrin蛋白和mRNA表达水平明显下降(P<0.01),使TRPC6高表达后上述变化更明显(P<0.05);TGF-β1干预后足细胞凋亡增多并出现典型凋亡细胞核形态学改变,TGF-β1干预组足细胞凋亡率为(12.30±0.81)%,TRPC6高表达组凋亡率为(21.26±1.16)%,组间比较差异有统计学意义(P<0.01),空载体组细胞凋亡率与TGF-β1干预组比较无明显差异。结论 TRPC6在TGF-β1诱导足细胞损伤中发挥重要作用,其机制之一可能通过线粒体凋亡途径诱导足细胞凋亡,减少nephrin表达,增加desmin表达来实现。

Objective To investigate the effect of transient receptor potential cation channel 6 （TRPC6） over expression on the expression of nephrin,desmin,caspase9 and apoptosis on mice podocyte damage induced by transfonning growth factor beta 1 （TGF-β1） .Methods Mice TRPC6 cDNA eukaiyotic expression vector pEX-3-TRPC6 was transfected to culture in vitro murine podocyte cell by Lip 2000.The change of TRPC6 protein expression was detected by Western bolt after 48 hours. Podocytes were divided into the following four groups：control group,TGF-β1 treatment group,TGF-β1＋pEX-3-NC empty vec-tor group and TGF-β1＋pEX-3-TRPC6 high expression group.After inten^ening 48 hours,the expression of caspase9,desmin, nephrin protein and mRNA were measured by Western-blot and real time RT-PCR respectively, and the apoptotic ratio of podocytes was monitored by flow cytometiy, and morphological changes of apoptotic cells were obsen^ed by DAPI staining. Re-sults After transfecting 48 hours, protein expression level of TRPC6 in the TRPC6 high expression group significantly in-creased while comparing to the control group （P〈0.01） , while that in the empty vector group had no obvious change. After TGF-β1 being inten^ened for 48 hours,the caspase9, desmin protein and mRNA expression level significantly increased （P 〈 0.05） ,while the decrease of nephrin protein and mRNA expression level were also obvious （P〈0.01） ,and the above-men tioned changes were more obvious after the high expression of TRPC6（ P〈0.05） .Podocyte apoptosis increased and typical ap- optotic morphological changed alter inten^ention of TGF-β1,and the podocyte apoptosis rate in the TGF-β1 treatment group was （12.30±0.81）%,ancl that in the TRPC6 high expression group was （21.26±1.16）%,so difference was statistically significant（ P〈0.01） .No statistically significant difference was found in the podocyte apoptosis rate in TGF-β1＋pEX-3-NC empty vector group and TGF-β1 treatment group. Conclusion TRPC6 plays an important role in the TGF-β1 induced damage of podocytes, one of the mechanisms of which may realize by inducing podocyte apoptosis, decreasing the expression of nephrin and increasing the expression of desmin via mitochondria apoptosis pathway.