PP2Ac介导Smad3中间区去磷酸化促肾间质纤维化研究
发布时间：2018-11-19 08:22
【摘要】：目的 通过TGF-β1体外刺激人肾小管上皮细胞(HK-2)建立肾间质纤维化细胞模型,拟(1)通过PP2Ac过表达及小干扰RNA质粒转染HK-2细胞研究PP2Ac在肾小管间质纤维化中作用；(2)探讨PP2Ac促肾小管间质纤维化是否与其介导Smad3中间连接区去磷酸化有关。 方法 1、常规培养HK-2细胞,实验分组：空白对照组、TGF-β1刺激组(模型组)、质粒转染+TGF-β1组(PP2Ac过表达和小干扰RNA质粒转染HK-2细胞后,予5ng/ml TGF-β1刺激干预24h)。采用RT-PCR和Western blot法检测PP2Ac、FN、Col-Ⅰ、α-SMA和E-cadherin mRNA及蛋白表达水平。 2、常规培养HK-2细胞同上,实验分组：空白对照组、TGF-β1刺激组(模型组)、质粒转染+TGF-β1组(PP2Ac过表达和小干扰质粒转染HK-2细胞后,TGF-β1刺激1h)。分别提取总蛋白和核蛋白,采用Western blot法检测Smad3、Smad3中间连接区pSmad3-L (Ser204)、pSmad3-L (Ser208)蛋白表达情况。 结果 1.RT-PCR和Western blot结果均显示：TGF-β1刺激HK-2细胞24h使得PP2Ac表达上调的同时,FN、Col-Ⅰ和a-SMA表达增加,E-cadherin表达下降,纤维化加重；PP2Ac过表达质粒转染HK-2细胞再予TGF-β1刺激24h后,PP2Ac mRNA及蛋白水平表达均较TGF-β1刺激组升高,同时FN、Col-Ⅰ和a-SMA表达明显上调,E-cadherin表达下调,纤维化进一步加重；PP2Ac小干扰RNA转染HK-2细胞再予TGF-β1刺激24h后,较TGF-β1刺激组,PP2Ac的mRNA及蛋白表达均降低,FN、Col-Ⅰ和a-SMA表达减少,E-cadherin表达增高,纤维化缓解。差异均有统计学意义(P0.05)。 2.予TGF-β1刺激HK-2细胞后以Western blot检测不同时间点PP2Ac、pSmad3-L (Ser204)、pSmad3-L (Ser208)和Smad3的变化。实验结果显示在TGF-β1刺激HK-2细胞1h时可使PP2Ac的表达上调达到高峰,同时pSmad3-L (Ser204)在TGF-β1刺激1h左右表达最高,pSmad3-L (Ser208)在1/2h处达高峰,但1h处仍表达明显,Smad3在各时间点的表达基本无变化。故在下面的实验中选用TGF-β1刺激HK-2细胞1h研究PP2Ac促肾间质纤维化是否与其介导Smad3中间连接区去磷酸化有关。 3.Western blot结果显示：TGF-β1刺激HK-2细胞1h使得PP2Ac蛋白表达上调的同时,总蛋白和核蛋白中pSmad3-L (Ser204)和pSmad3-L (Ser208)表达均增加；HK-2细胞转染PP2Ac过表达质粒再予TGF-β1刺激1h后,较TGF-β1刺激组PP2Ac表达进一步增高,总蛋白和核蛋白中pSmad3-L (Ser204)、pSmad3-L (Ser208)表达均下降；PP2Ac小干扰RNA转染HK-2细胞再予TGF-β1刺激1h后,PP2Ac较TGF-β1刺激组表达减少,同时总蛋白和核蛋白中pSmad3-L (Ser204)、pSmad3-L (Ser208)蛋白表达均增加。差异均有统计学意义(P0.05)。 结论 1.PP2Ac的表达同FN、Col-I和a-SMA表达正相关,与E-cadherin表达负相关,提示PP2Ac促进细胞外基质的生成及肾小管EMT； 2.PP2Ac通过介导Smad3中间连接区去磷酸化,即抑制其磷酸化促进肾间质纤维化。
[Abstract]:Objective to establish a renal interstitial fibrosis cell model by stimulating human renal tubular epithelial cells (HK-2) with TGF- 尾 1 in vitro. (1) to study the role of PP2Ac in renal tubulointerstitial fibrosis by overexpression of PP2Ac and transfection of small interfering RNA plasmid into HK-2 cells. (2) to investigate whether PP2Ac promotes renal tubulointerstitial fibrosis and whether it mediated Smad3 intermediate junction dephosphorylation. Methods 1.Conventionally cultured HK-2 cells were divided into blank control group, TGF- 尾 1-stimulated group (model group), plasmid transfected TGF- 尾 1 group (PP2Ac overexpression and small interfering RNA plasmid transfected HK-2 cells). 5ng/ml TGF- 尾 1 was given for 24 h). The expression levels of PP2Ac,FN,Col- 鈪，
本文编号：2341682
[Abstract]:Objective to establish a renal interstitial fibrosis cell model by stimulating human renal tubular epithelial cells (HK-2) with TGF- 尾 1 in vitro. (1) to study the role of PP2Ac in renal tubulointerstitial fibrosis by overexpression of PP2Ac and transfection of small interfering RNA plasmid into HK-2 cells. (2) to investigate whether PP2Ac promotes renal tubulointerstitial fibrosis and whether it mediated Smad3 intermediate junction dephosphorylation. Methods 1.Conventionally cultured HK-2 cells were divided into blank control group, TGF- 尾 1-stimulated group (model group), plasmid transfected TGF- 尾 1 group (PP2Ac overexpression and small interfering RNA plasmid transfected HK-2 cells). 5ng/ml TGF- 尾 1 was given for 24 h). The expression levels of PP2Ac,FN,Col- 鈪，
本文编号：2341682
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