去甲斑蝥素靶向抑制PP2Ac抗肾间质纤维化的分子机制研究

发布时间：2018-07-09 14:30

  本文选题：肾间质纤维化 + 蛋白磷酸酶2A　； 参考：《中南大学》2014年硕士论文

【摘要】：目的 通过TGF-β1体外刺激人肾小管上皮细胞(HK-2细胞)建立肾间质纤维化细胞模型,探讨：(1)NCTD是否通过靶向抑制PP2Ac发挥抗肾间质纤维化作用；(2)NCTD抗肾间质纤维化是否与其抑制PP2Ac介导Smad3-L区去磷酸化有关。 方法 1、常规培养HK-2细胞,分别通过过表达PP2Ac和小干扰RNA敲低PP2Ac表达后,应用TGF-β1(5ng/ml)刺激和NCTD(2.5μg/ml)进行干预24h。采用RT-PCR和Western印迹法检测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,应用NCTD (2.5μg/ml)进行预干预24h和TGF-β1(5ng/ml)刺激1h。采用间接细胞免疫荧光法检测pSmad3-L(Ser204)、pSmad3-L(Ser208)在细胞的分布,Western印迹法检测总蛋白中PP2Ac和核蛋白中pSmad3-L(Ser204)、pSmad3-L(Ser208)的表达。 结果 1、TGF-β1刺激HK-2细胞24h致PP2Ac表达上调的同时,FN、 Col-I和α-SMA表达上调,E-cadherin表达下调,HK-2细胞纤维化加重;而转染PP2Ac过表达质粒后再用TGF-β1刺激,则PP2Ac表达上调更为明显,HK-2细胞纤维化加重更显著；NCTD干预使PP2Ac表达下调,同时FN、Col-I和α-SMA表达下调,E-cadherin表达上调,HK-2细胞纤维化缓解； 2、小干扰RNA敲低PP2Ac表达后,FN、Col-I和α-SMA表达下调,E-cadherin表达上调,HK-2细胞纤维化缓解；而NCTD与小干扰RNA共同抑制PP2Ac表达后,对HK-2细胞纤维化的缓解程度同单纯PP2Ac小干扰RNA干预组比较无明显差异； 3、免疫荧光结果显示,空白对照组中pSmad3-L(Ser204)、 pSmad3-L(Ser208)基本无表达,TGF-β1刺激HK-2细胞1h后pSmad3-L(Ser204)、pSmad3-L(Ser208)在细胞核内表达明显增多,NCTD干预使pSmad3-L(Ser204)、pSmad3-L(Ser208)在细胞核内表达进一步增多。Western印迹结果显示,TGF-β1刺激1h使得PP2Ac蛋白表达上调的同时,细胞核内pSmad3-L(Ser204)、pSmad3-L(Ser208)蛋白表达均增多,NCTD干预使PP2Ac表达下调的同时,细胞核内pSmad3-L(Ser204)、pSmad3-L(Ser208)蛋白表达进一步增多。 结论 1.NCTD靶向抑制PP2Ac发挥抗肾间质纤维化作用； 2.NCTD可能通过抑制PP2Ac介导Smad3-L区去磷酸化,即促进Smad3-L区磷酸化发挥抗肾间质纤维化作用。
[Abstract]:Objective to establish a renal interstitial fibrosis cell model by stimulating human renal tubular epithelial cells (HK-2 cells) with TGF- 尾 1 in vitro, and to explore whether NCTD can inhibit renal interstitial fibrosis by targeting PP2Ac; (2) whether NCTD inhibits renal interstitial fibrosis by inhibiting PP2Ac mediated Smad3-L dephosphorylation. Methods 1. HK-2 cells were treated with TGF- 尾 1 (5ng/ml) stimulation and NCTD (2.5 渭 g/ml) for 24 h after overexpression of PP2Ac and small interfering RNA knockout PP2Ac. The mRNA and protein expressions of PP2ActFN, Col-I, 伪 -SMA and E-cadherin were detected by RT-PCR and Western blot. HK-2 cells were cultured routinely. NCTD (2.5 渭 g/ml) was pretreated for 24 h and stimulated by TGF- 尾 1 (5ng/ml) for 1 h. The expression of pSmad3-L (Ser204) pSmad3-L (Ser208) in the total protein and pSmad3-L (Ser204) pSmad3-L (Ser208) in the total protein was detected by Western blot. Results 1 the expression of PP2Ac was up-regulated in HK-2 cells induced by TGF- 尾 1 for 24 h, while the expression of FN, Col-I and 伪 -SMA was down-regulated and the fibrosis of HK-2 cells was aggravated after transfection of PP2Ac overexpression plasmid, and then stimulated by TGF- 尾 1. The expression of PP2Ac was up-regulated and the fibrosis of HK-2 cells was aggravated significantly. The expression of PP2Ac was down-regulated by NCTD, and the expression of E-cadherin was up-regulated by FN-1 Col-I and 伪 -SMA, and the fibrosis of HK-2 cells was alleviated. 2After the expression of PP2Ac was reduced by small interfering RNA knockout, the expression of PP2Ac was down-regulated and the expression of E-cadherin was up-regulated, while the expression of PP2Ac was inhibited by NCTD and small interfering RNA, and the expression of PP2Ac was inhibited by NCTD and small interfering RNA. The remission degree of HK-2 cells fibrosis was not significantly different from that of PP2Ac small interfering RNA intervention group. The expression of pSmad3-L (Ser204), pSmad3-L (Ser208) and pSmad3-L (Ser208) in the nucleus of HK-2 cells was significantly increased 1 h after stimulation of pSmad3-L (Ser204) and pSmad3-L (Ser208). Western blot showed that the expression of pSmad3-L (Ser204) pSmad3-L (Ser208) increased in the nucleus of HK-2 cells. Western blotting showed that the expression of PP2Ac protein was up-regulated by TGF- 尾 1 stimulation for 1 h, while the expression of pSmad3-L (Ser204) pSmad3-L (Ser208) increased significantly. The expression of pSmad3-L (Ser204) pSmad3-L (Ser208) in the nucleus was increased. The expression of pSmad3-L (Ser204) pSmad3-L (Ser208) in the nucleus was further increased while the expression of PP2Ac was down-regulated by NCTD. 2. NCTD could inhibit the dephosphorylation of Smad3-L region by inhibiting PP2Ac phosphorylation, that is, promote the phosphorylation of Smad3-L region and play an anti-interstitial role in renal interstitial fibrosis. 2.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692

【参考文献】

相关期刊论文 前5条

1 陈琼;李瑛;罗俊辉;杨阳;李军;孙林;肖力;许向青;彭佑铭;刘伏友;;去甲斑蝥素对高糖刺激的HK-2细胞FN,Col Ⅳ和TGF β1 mRNA及蛋白表达的影响[J];中南大学学报(医学版);2012年03期

2 刘伏友;孙岩;孙林;凌光辉;李瑛;刘健;袁曙光;刘映红;张东山;夏运成;;去甲斑蝥素对TGF-β_1诱导的人近端肾小管细胞上皮间质转分化以及转录因子Snail1表达的影响[J];中国中西医结合肾病杂志;2009年11期

3 李瑛;陈琼;刘伏友;彭佑铭;李军;孙林;罗俊辉;杨阳;段绍斌;凌光辉;刘虹;刘映红;;去甲斑蝥素不依赖钙调蛋白磷酸酶信号通路抑制高糖刺激肾小管上皮细胞细胞外基质的表达[J];肾脏病与透析肾移植杂志;2010年06期

4 孙岩;李瑛;孙林;刘健;凌光辉;刘映红;周循;刘伏友;;去甲斑蝥素抑制单侧输尿管梗阻大鼠肾小管上皮-间充质转分化的实验研究[J];肾脏病与透析肾移植杂志;2012年01期

5 高振梅,万鲲,王芮,王世岭;斑蝥素抑制NIH/3T3细胞增殖对防治器官组织纤维化的作用[J];中国临床康复;2004年02期

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