天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (6): 517-522.

• 实验研究 • 上一篇    下一篇

扶肾方调节各种细胞因子干预腹膜间皮细胞 EMT的实验研究

杨波 1，王孟孟 2，李洁 1，乔延恒 1，杨洪涛 1△
  

  1. 1天津中医药大学第一附属医院肾病科（邮编300381）；2安徽省阜阳市第四人民医院内分泌科

  • 收稿日期:2019-11-22 修回日期:2020-03-16 出版日期:2020-06-15 发布日期:2020-06-15
  • 通讯作者: 杨波 E-mail:yb8203@126.com
  • 基金资助:
    基于Smurf2串话TGF-β/Smads通路探讨扶肾方防治腹膜间皮细胞EMT的研究;基于Dll4/Notch信号通路探讨扶肾方抑制腹膜血管新生及miRNA调控机制的研究

Experimental study of Fushen recipe regulating various cytokines on intervention of EMT of peritoneal mesothelial cells

YANG Bo1, WANG Meng-meng2, LI Jie1, QIAO Yan-heng1, YANG Hong-tao1△ #br#   

  1. 1 Department of Nephrology, the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin
    300381, China; 2 Department of Endocrinology, the Fourth People    ’s Hospital, Fuyang City, Anhui Province
  • Received:2019-11-22 Revised:2020-03-16 Published:2020-06-15 Online:2020-06-15

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摘要： 摘要：目的 探讨扶肾方对大鼠腹膜间皮细胞转化生长因子-β受体Ⅰ（TGF-βRⅠ）、TGF-βRⅡ、Smad泛素化相 关因子2（Smurf2）、E-钙黏蛋白（E-cadherin）、紧密连接蛋白-1（ZO-1）的影响及抑制上皮-间充质转分化（EMT）的作 用机制。方法 原代培养大鼠腹膜间皮细胞，传至第2代并鉴定后，分为空白对照（C）组，含药血清（B）组，模型（T） 组（40 μg/L TGF-β1），模型+含药血清（T+B）组，模型+MG132（T+M）组。培养24 h后收集细胞和培养液上清液，采用 蛋白印迹法检测Smurf2蛋白水平，qPCR检测各组TGF-βRⅠ、TGF-βRⅡ、E-cadherin、ZO-1的mRNA转录水平。结果 与C组比较，T组E-cadherin、ZO-1、TGF-β1RⅡ mRNA、Smurf2蛋白表达明显上调（P＜0.05）；与T组比较，B组 E-cadherin、ZO-1 mRNA 表达均上调，T+B 组E-cadherin、ZO-1、TGF-β1RⅡ mRNA 表达均上调，Smurf2蛋白表达下 调，T+M组E-cadherin、ZO-1、TGF-β1RⅠ mRNA、Smurf2蛋白表达均下调，TGF-β1RⅡ mRNA表达上调（P＜0.05）；与 B组比较，T+B组和T+M组E-cadherin mRNA表达下调，T+M组ZO-1、TGF-β1RⅠ mRNA表达下调（P＜0.05）；与T+B 组比较，T+M组E-cadherin、ZO-1、TGF-β1R，TGF-β1RⅡ mRNA表达均下调（P＜0.05）。结论 扶肾方抑制大鼠腹膜 间皮细胞EMT可能与上调TGF-βRⅡ mRNA转录，下调Smurf2蛋白含量，促进E-cadherin、ZO-1 mRNA的转录相关。

关键词: 上皮-间质转化, 受体, 转化生长因子β, 紧密连接蛋白质类, E-钙黏蛋白, Smad泛素化相关因子2, 腹膜间皮细胞, 扶肾方

Abstract: Abstract: Objective To investigate the effect of Fushen recipe on transforming growth factor-β receptor Ⅰ (TGF-βR Ⅰ), transforming growth factor-β receptor Ⅱ (TGF-βRⅡ), smad ubiquitination related factor 2 (Smurf2) and E-cadherin and tight junction protein-1 (ZO-1) in peritoneal mesothelial cells of rats, and the mechanism of its inhibiting epithelialmesenchymal transition (EMT). Methods The rat peritoneal mesothelial cells were primary cultured. After passing to the second generation and identification, cells were divided into blank control group (C), medicated serum group (B), model group (T, 40 μg/L TGF-β1), model + medicated serum intervention group (T+B) and model + MG132 intervention group (T+ M). Cells and the culture supernatant were collected after 24-h incubation. Western blot assay was used to detect the Smurf2 protein level. mRNA transcription levels of TGF- βR Ⅰ , TGF- βR Ⅱ , E-cadherin and ZO-1 were detected by qPCR. Results Compared with group C, the expressions of E-cadherin, ZO-1, TGF- β1R Ⅱ mRNA and Smurf2 protein were increased significantly in group T (P＜0.05). Compared with group T, the expressions of E-cadherin and ZO-1 mRNA were up-regulated in group B. The expressions of E-cadherin, ZO-1 and TGF- β1R Ⅱ mRNA were up-regulated and the expression of Smurf2 protein was down-regulated, in group T+B. The expressions of E-cadherin, ZO-1, TGF-β1RI mRNA and Smurf2 protein were down-regulated, and the expression of TGF-β1RⅡ mRNA was up-regulated, in group T+M (P＜ 0.05). Compared with group B, the expressions of E-cadherin mRNA were down-regulated in group T+B and group T+M, while the expressions of ZO-1 and TGF-β1RⅠ mRNA were down-regulated in group T+M (P＜0.05). Compared with T+B group, E-cadherin, ZO-1, TGF-β1R and TGF-β1RⅡ mRNA expression were all down-regulated in T+M group (P＜0.05). Conclusion The inhibition of EMT in rat peritoneal mesothelial cells by Fushen recipe may be related to the up-regulation of TGF- βR Ⅱ mRNA transcription, the down-regulation of Smurf2 protein and the promotion of E-cadherin and ZO-1 mRNA transcription.

Key words: pepithelial-mesenchymal transition, receptors, transforming growth factor beta, tight junction proteins,
E-cadherin, smad ubiquitination correlation factor 2, peritoneal mesothelial cells, fushen recipe