血管内皮生长因子抑制肾小管上皮间充质转化及其与结缔组织生长因子和PI3K-Akt信号通路的关系  

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1 - induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

作　　者：连耀国[1] 何春梅[1] 郑法雷[1] 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院肾内科,北京100730

出　　处：《中华肾脏病杂志》2009年第6期445-451,共7页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30570854）

摘　　要：目的探讨血管内皮生长因子（VEGF）对转化生长因子β1（TGF—β1）诱导的肾小管上皮间充质转化（EMT）的作用，及其与结缔组织生长因子（CTGF）、P13K—Akt信号通路的关系。方法（1）将体外培养的HK2细胞分为正常对照组、TGF-β1（5μg／L，下同）组、VEGF组（100μg／L，下同）、TGF—β1＋VEGF组。HK2细胞体外培养48h，用免疫组化双染方法检测肾小管上皮细胞α平滑肌肌动蛋白（α-SMA）和E钙黏蛋白的表达。（2）将体外培养的HK2细胞分为正常对照组、TGF-β1组、VEGF组、TGF—β1＋VEGF组、PI3K—Akt信号通路阻断剂LY294002组（25txmol／L，下同）、TGF-β1＋LY294002组、VEGF＋LY294002组、TGF-β1＋VEGF＋LY294002组。HK2细胞体外培养48h，用Western印迹和RTPCR方法检测α-SMA和CTGF的表达；用ELISA方法检测培养上清中纤连蛋白（FN）和Ⅰ型胶原（ColⅠ）的表达。结果免疫组化结果显示，TGF—β1组α-SMA表达比正常对照组增强，而E钙黏蛋白表达减弱；TGF-β1＋VEGF组-αSMA表达比TGF—β1组显著减弱，而E钙黏蛋白表达增强。TGF—β1组α—SMA、CTGF蛋白和mRNA及FN、ColⅠ表达比正常对照组显著增强（均P〈0．05）；TGF-β1＋VEGF组α-SMA、CTGF蛋F1和mRNA及FN、ColI表达比TGF—β1组显著减弱（均P〈0．05）；TGF-β1＋VEGF＋LY294002组α-SMA、CTGF蛋白和mRNA及FN、ColI表达比TGF—β1＋VEGF组显著增强（均P〈0．05）。结论VEGF能抑制TGF—β1诱导的体外培养的HK2细胞发生EMT，其机制可能与VEGF下调HK2细胞CTGF表达及减少细胞外FN、ColⅠ合成有关。VEGF的这种作用可能部分通过PI3K-Akt信号转导通路实现，其确切机制有待进一步研究。Objective To examine the relationship of the inhibitory effect of vascular endothelial growth faetor（VEGF） on epithelial-mesenchymal transition （EMT） induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor （CTGF） and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups： normal control group, TGF-β1 （5 μg/L） group, VEGF （100μg/L） group, TGF-β1 plus VEGF group. LY294002 （25μmol/L）, the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, a-smooth muscle actin （α-SMA） and E-eadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin （FN） and collagen Ⅰ （Col Ⅰ ） in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control （P〈0.05）, while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone （P〈0.05, respectively）. The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF α-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment （P 〈0.05）. Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 cells in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ , which may be partly dependent on PI3K-Akt pathway.

关 键 词：血管内皮生长因子类 转化生长因子Β1 上皮细胞 结缔组织生长因子 上皮间充质转化 

分 类 号：R692.9[医药卫生—泌尿科学]