TRPC6高表达对血管紧张素Ⅱ诱导的小鼠足细胞凋亡的影响

Effects of overexpression of TRPC6 on angiotensin Ⅱ-induced apoptosis of mouse podocytes

目的观察瞬间受体电位阳离子通道蛋白6（TRPC6）高表达对血管紧张素Ⅱ（AngⅡ）诱导的小鼠足细胞凋亡的影响并探讨其作用机制。方法用脂质体将针对小鼠TRPC6的基因真核表达载体pEGFP—N1-mTRPC6转染体外培养的永生化小鼠足细胞系。24h后用荧光显微镜观察增强绿色荧光蛋白（EGFP）的表达。Western印迹法检测转染后TRPC6蛋白表达的变化。将足细胞分组，应用不同浓度AngⅡ处理足细胞，Fluo-3AM结合激光共聚焦显微镜检测各组足细胞胞质内钙离子的浓度。RT—PCR及Western印迹法检测Bax、Bcl-2mRNA及蛋白的水平。流式细胞仪及Hoechst染色法检测足细胞凋亡。结果pEGFP—N1-mTRPC6转染足细胞后，约35％细胞出现绿色荧光；足细胞TRPC6蛋白表达明显上调（P〈0．01）。TRPC6高表达可以明显促进AngⅡ诱导的足细胞钙离子内流（P〈0．01），在蛋白及基因水平均上调Bax的表达而下调Bcl-2的表达（P〈0．01，P〈0．05）。在低剂量AngⅡ（10^-10mol／L）作用下，足细胞凋亡率为（2．50±0．72）％，转染TRPC6以后凋亡率为（4．33±0．45）％，差异有统计学意义（P〈0．05）。在高剂量AngⅡ（10^-6 mol／L）作用下，足细胞凋亡率为（15．46±1．40）％，转染TRPC6以后凋亡率为（18．33±0.87）％，差异有统计学意义（P〈0．01）。结论TRPC6在AngⅡ诱导的足细胞凋亡中发挥重要作用。TRPC6可能通过增加钙离子内流，进而启动其下游的凋亡调节成分参与凋亡过程。

Objective To study the effect of overexpression of TRPC6 on Ang Ⅱ- induced apoptosis of mouse podocytes in vitro and to explore the possible mechanisms. Methods Mouse TRPC6 cDNA eukaryotic expression vector pEGFP-NI-mTRPC6 was transfected to conditionally immortalized murine podocyte cell line by liposome. The fluorescent microscopy was used to examine the expression of EGFP after 24 hours. The change of TRPC6 protein expression was observed by Western-blot. Podocytes were treated by different concentrations of AngⅡ . The podocyte intracellular calcium concentration was measured with laser-scanning confocal microscope. The expression of Bax and Bcl-2 mRNA was assessed by RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot. The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst staining. Results About 35% of the cells expressed EGFP. An up-regulation of protein expression of TRPC6 was detected in podocytes when transfected with pEGFP-NI-mTRPC6 （P〈0.01）. The overexpression of TRPC6 promoted the Ang Ⅱ-induced influx of extracellular calcium and elevated the expression of Bax but decreased the expression of Bcl-2 （P〈0.01, P〈O.05）. The apoptotic ratio of podocyte was （2.50±0.72）% when treated by low-dose Ang Ⅱ （10-10 mol/L）, and it was increased to （4.33±0.45）% when transfected with pEGFP-N1- mTRPC6 （P 〈0.05 ）. Transfection with pEGFP-NI-mTRPC6 increased apoptosis rate from （15.46± 1.40）% to （18.33±0.87）%（P〈0.01） by high-dose AngⅡ （10.6 mol/L）. Conclusion TRPC6 plays an important role in the Ang Ⅱ-induced apoptosis of podocytes by promoting the influx of extracellular calcium, which leads to the apoptosis cascade initiation.