上调Smad7表达对大鼠腹膜纤维化模型腹膜间皮细胞转分化的影响

目的 探讨上调Smad7表达对腹膜纤维化大鼠模型腹膜间皮细胞转分化的影响，为进一步研究防止腹膜纤维化的措施提供依据。 方法 将24只体重180~200 g SD雄性大鼠随机分为4组:A组(6只):正常对照组；B组(6只):腹膜纤维化模型组，每日按100 ml/kg腹腔注射4.25%透析液，并于造模后第8、10、12、22、24、26天按0.6 mg/kg腹腔注射脂多糖(LPS)；C组(6只):空白载体组，在建立模型的第1、14天转染空白载体和pEF purop-Tet-on质粒；D组(6只):Smad7转染组，在建立模型第1、14天转染Smad7和pEF purop-Tet-on质粒。各组均在每日饮水中加入强力霉素(200 mg/L)诱导基因表达。所有动物于实验第28天杀检，取脏层腹膜组织行光镜及电镜检查。用RT-PCR、间接免疫荧光和Western印迹方法检测α-SMA、E-钙黏蛋白(cadherin)、Smad7、磷酸化(P)-Smad2/3 mRNA和蛋白表达。 结果 与正常对照组比较，腹膜纤维化模型组和空白载体组p-Smad2/3蛋白水平显著升高，α-SMA mRNA和蛋白表达上调，但E-cadherin mRNA和蛋白水平明显下调。电镜结果显示，腹膜纤维化模型组和空白载体组腹膜间皮细胞微绒毛脱落，基底膜断裂，并向间皮下组织迁徙，胞浆中有密体、密斑和肌丝出现。与上述2组比较，Smad7基因转染组Smad7蛋白表达明显增加，p-Smad2/3蛋白水平明显下调，α-SMA mRNA和蛋白水平也明显降低，而E-cadherin mRNA和蛋白水平无明显变化。电镜结果显示，Smad7基因转染组腹膜间皮细胞微绒毛明显增多，细胞连接和基底膜趋于完整。 结论 基因转染上调Smad7表达可通过部分抑制TGF-β受体调控信号通路(Smad2/3)，抑制腹膜间皮细胞向肌成纤维细胞的转分化。

Smad7 overexpression inhibits epithelial-mesenchymal transition in peritoneal fibrosis rat model

Objective To investigate the role of overexpression of Smad7， the inhibitory factor of TGF-β/Smads signaling，in epithelial-mesenchymal transition(EMT) of peritoneal mesothelial cells. Methods Peritoneal fibrosis rat model was built by daily intraperitoneal injection with 4.25% Dineal (100 ml/kg) and lipopolysaccharide(LPS)(0.6 mg/kg) at day 8， 10， 12， 22， 24， 26. Smad7 or control empty vectors was transferred at day 0，14 and was induced by doxycline in the daily drinking water(200 mg/L). Rats were sacrificed on day 28 and the expression of TGF-beta/Smads， α-SMA and E-cadherin was examined. Results Compared with normal rats， empty vector rats showed higher expression of phosphorylated Smad2/3. α-SMA expression was elevated but E-cadherin was reduced. Under electron microscope，the mesothelial cells removed to submesothelial zone and showed large bundles of actin microfilaments and dense bodies within the cytoplasm. Basement membrane was broken. After induction of Smad7 in peritoneal fibrosis rats， the morphology of mesothelial cells normalized partly， phosphorylated Smad2/3 was reduced. Moreover， expression of E-cadherin was increased， expression of α-SMA was dramatically reduced. Conclusion Inhibition of TGF-β/Smad signaling by Smad7 overexpression may inhibit the epithelial-mesenchymal transition of mesothelial cell， which may provide a new therapeutic method for peritoneal fibrosis by overexpression of Smad7.