支架蛋白JLP对人肾小管上皮细胞转分化的影响

Role of JLP on the epithelial to mesenchymal transition in renal tubular epithelial cells

目的 观察支架蛋白JLP对人肾小管上皮细胞(HK-2)间充质转分化的影响,探讨p38 MAPK信号通路在其中的作用.方法 构建敲除jlp基因的细胞模型,细胞被分为阴性对照(Ctrl-shRNA)组、jlp基因敲除(jlp-shRNA)组、阴性对照+成纤维细胞生长因子(FGF-2)组和jlp基因敲除+成纤维细胞生长因子(jlp-shRNA+ FGF-2)组.Western印迹法检测各组细胞JLP、E钙黏蛋白(E-cadherin)、转化生长因子(TGF)β1、α-平滑肌肌动蛋白(α-SMA)、p38丝裂原活化蛋白激酶(MAPK)的表达;免疫荧光法检测FGF-2诱导24 h后,α-SMA、Ⅰ型胶原(COL-Ⅰ)、纤维连接蛋白(FN)的表达.结果 与Ctrl-shRNA组比较,FGF-2组JLP蛋白表达量明显下调(P＜0.05);与FGF-2组比较,jlp-shRNA+ FGF-2组TGF-β1、α-SMA、p38 MAPK蛋白表达量明显上调,E-cadherin蛋白表达量明显下调(P＜0.05);与FGF-2组比较,jlp-shRNA+FGF-2组α-SMA、COL-Ⅰ、FN的荧光表达明显上调.结论 支架蛋白JLP在促纤维化环境形成中可抑制HK-2细胞间充质转分化,其机制可能与JLP负调控p38 MAPK信号通路有关.

Objective To observe the effect of JLP on transdifferentiation of human renal proximal tubular epithelial cells (HK-2),and to investigate the role of p38 MAPK signaling pathway in this process.Methods The knock-down plasmids of JLP were constructed.HK-2 cells were randomly divided into four groups:negative control cells (Ctrl-shRNA group),knock-down jlp cells (jlpshRNA group),negative control cells with FGF-2 treatment (FGF-2 group) and knock-down jlp cells with FGF-2 treatment(jlp-shRNA +FGF-2 group).The expressions of JLP,E-cadherin,TGF-β1,α-SMA,p-p38 MAPK protein were detected by Western blotting.After the induction of FGF-2 for 24 hours,the expressions of α-SMA,COL-Ⅰ,FN were detected by immunocytochemistry.Results Compared with Ctrl-shRNA group,the expression of JLP protein was significantly down-regulated in FGF-2 group.Compared with FGF-2 group,the expressions of TGF-β1,α-SMA,p-p38 MAPK protein were significantly up-regulated,while E-cadherin protein was significantly down-regulated (P ＜ 0.05).Compared with FGF-2 group,the expressions of α-SMA,COL-Ⅰ,FN immunostaining increased markedly in jlp-shRNA+FGF-2 group.Conclusion Scaffolding protein JLP is critical in preventing EMT in the course of fibrosis through the inhibition of p-p38 activation in HK-2 cells.