上调表达Smad7显著抑制TGF-β介导的大鼠腹膜间皮细胞Smad2的表达

目的： 探讨外源性转入并上调表达抑制性信号蛋白Smad7对TGF-β₁作用下大鼠腹膜间皮细胞Smad2表达的影响。 方法： 通过脂质体介导的方法，将表达Smad7的重组质粒（PCDNA3-Smad7）转染培养的大鼠腹膜间皮细胞，分别培养于不同浓度TGF-β₁培养液(0、1.25、2.5和10 μg/L)，用RT-PCR及Western blotting的方法检测不同时间（0、5、15、30、60和120 min）Smad2、Smad7表达的水平。 结果： 正常间皮细胞Smad2 mRNA和蛋白在TGF-β₁刺激后5 min开始表达，呈时间依赖性，在30 min达到高峰，而后逐渐减弱；Smad7 mRNA和蛋白也在TGF-β₁刺激后5 min可见表达，但此后逐渐减弱，30 min达到最低，60 min又开始逐渐增强。Smad2 和Smad7 mRNA和蛋白，随TGF-β₁浓度的增高而表达增强。转染后大鼠腹膜间皮细胞可见Smad7 mRNA和蛋白表达显著上调，并可持续高表达。转染后的腹膜间皮细胞在TGF-β₁刺激下，Smad2 mRNA及蛋白的表达均低下，刺激0、5、15、30、60 和120 min后Smad2 mRNA表达分别低33%、56%、67%、71%、63%和57%（P<0.05）。Smad2蛋白表达分别低78%、89%、89%、88%和76%（P<0.05）。 结论： 上调表达抑制性信号蛋白Smad7可显著抑制腹膜间皮细胞中受体调控信号蛋白Smad2的表达和活性，提示Smad7可能对TGF-β₁起反向调控作用。

Overexpression of Smad7 inhibits TGF- β- induced Smad2 mRNA and protein expression in peritoneal mesothelial cells

AIM: To investigate the role of Smad7 in the Smad2 expression induced by transforming growth factor-β₁ (TGF-β₁) in rat peritoneal mesothelial cells (PMCs).METHODS: Rat PMCs were cultured at different doses of TGF-β₁ (0，1.25，2.5，10 μg/L) for different time (0，5，15，30，60，120 min).PCDNA3-Smad7 was then transfected into cultured rat PMCs by lipofectamine, and the cells were stimulated like the above.Endogenous Smad2 and Smad7 expression was evaluated by RT-PCR and Western blotting.RESULTS: TGF-β₁ induced increase in Smad2 mRNA and protein expression at 5 min, peaked at 30 min, and declined to baseline levels at 120 min, which was in a time-dependent manner.TGF-β₁ also induced Smad7 mRNA expression at 5 min, and then declined, down to the lowest at 30 min, but at 60 min it increased again.Smad2, Smad7 mRNA and protein expression induced by TGF-β₁ were also dose-dependent.After transfection, overexpressions of Smad7 mRNA and protein in rat PMCs were observed, which did not decline with time.The expression of Smad2 mRNA significantly decreased by 33%, 56%, 67%, 71%, 63% and 57% (P<0.05), the expression of Smad2 protein declined by 78%，89%，89%，88% and 76% (P<0.05) respectively at 0, 5, 15, 30, 60 and 120 min.CONCLUSION: Overexpression of Smad7 inhibits Smad2 gene and protein expression in peritoneal mesothelial cells.Smad7 may be a negative regulator of TGF-β₁ signaling.