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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 effects of transient expression of TRPC6 on the expression of nephrin, desmin and caspase9 in mouse renal podocytes and the effects of TGF-β1 on the apoptosis. Methods The mouse podocytes cultured in vitro were transfected with the mouse pCRP-TRPC6 eukaryotic expression vector pEX-3-TRPC6 by lipofectamine 2000. The expression of TRPC6 protein was detected by Western blot 48 h after transfection. The podocytes were divided into 4 groups: normal control group, TGF-β1 intervention group, TGF-β1 + p EX-3-NC group (empty vector group), TGF-β1 + p EX-3-TRPC6 group The expression of caspase9, desmin, nephrin protein and mRNA were detected by western blot and real time PCR after 48 hours intervention. The apoptosis rate of each group was detected by flow cytometry. The morphological changes of apoptotic nuclei were observed by DAPI staining. Results TRPC6 protein expression in TRPC6 high expression group was significantly higher than that in normal control group 48 hours after transfection (P <0.01), TRPC6 protein expression in empty vector group had no significant change; 48 hours after TGF-β1 intervention caspase9, desmin protein and mRNA (P <0.05). The expression of nephrin protein and mRNA was significantly decreased (P <0.01), and the above changes were more obvious after high expression of TRPC6 (P <0.05). The apoptosis of podocytes increased after TGF-β1 intervention The apoptosis rate of podocyte in TGF-β1 -treated group was (12.30 ± 0.81)%, and that of TRPC6-overexpressed group was (21.26 ± 1.16)%. The difference between the two groups was statistically significant (P <0.01). There was no significant difference between the apoptosis rate of empty vector group and TGF-β1 intervention group. Conclusion TRPC6 plays an important role in podocyte injury induced by TGF-β1. One of the mechanisms may be through the mitochondrial apoptosis pathway that induces podocyte apoptosis, decreases nephrin expression and increases desmin expression.