低密度脂蛋白和氧化低密度脂蛋白诱导人肾小管上皮细胞转分化的通路研究

目的探讨低密度脂蛋白（LDL）和氧化（OX）LDL是否可诱导人肾小管上皮细胞转分化及其可能机制。方法体外培养的第2代肾小管上皮细胞随机分为（1）阴性对照组；（2）LDL（50mg／ml）组；（3）oxLDL（50mg／ml）组；（4）LDL（50mg／m1）＋PD98059（5μmol／L）组；（5）oxLDL（50mg／ml）＋PD98059（5μmol／L）组。应用形态学、免疫荧光、Western印迹观察LDL和oxLDL刺激小管细胞角蛋白（cytokerafin）、E-钙粘糖蛋白（cadhefin）、α-SMA和波形蛋白（vimenfin）表达的改变、Ⅰ型胶原产生的改变，及其与ERK1／2MAPK通路活化的关系。结果OXLDL较LDL有更强的致小管上皮细胞转分化的作用，与对照组相比。细胞中上皮细胞标记cytokeratin和E-cadherin表达减少，间充质细胞标记α-SMA和vimentin明显增加，Ⅰ型胶原产生增多（P〈0．05）。LDL和oxLDL刺激组细胞中ERK1／2MAPK和GSK-3β磷酸化活化较对照组明显增强（P〈0．05）；β-catenin发生细胞核内转移。MAPK抑制剂PD98059可抑制GSK-3β的磷酸活化，并几乎完全阻断oxLDL诱导的肾小管上皮细胞转分化，但对LDL诱导的小管上皮细胞转分化只有部分阻断作用。结论本研究首次在体外观察到（1）oxLDL可诱导肾小管细胞上皮细胞转分化和Ⅰ型胶原的产生，作用明显强于LDL；（2）ERK1／2MAPK、GSK-3β和β-catenin组成一条信号通路调节LDL和oxLDL诱导的小管上皮细胞转分化。

Study of signaling pathway of LDL and oxLDL inducing kidney tubular epithelial cells transdifferentiation

Objective To explore whether LDL and oxLDL may induce kidney tubular epithelial-mesenchymal transition (EMT) and its mechanism. Methods The second generation human kidney tubular epithelial cells(TECs) were cultured for 24 hours in different conditions as (1)serum free as control， (2) treated with LDL(50 μg/ml)， (3) treated with oxLDL(50 μg/ml)， (4)treated with LDL(50 μg/ml) plus PD98059(5 μmlo/L)， (5) treated with oxLDL(50 μg/ml) plus PD98059(5 μmol/L). The expression of cytokeratin， E-cadherin， α-SMA and vimentin was assessed by immunofluorescence and Western-blot. Western-blot was also performed to test the expression of collagen I and phospho-ERK1/2MAPK and phospho-GSK-3β. Results oxLDL was more potently in inducing tubular EMT than LDL at 24 hours as demonstrated by de novo α-SMA expression， increased expression of vimentin， partial loss of cytokeratin and reduction of E-cadherin expression by TECs. The expression of collagen I and phospho-ERK1/2MAPK and phospho-GSK-3β was increased in TECs stimulated by LDL or oxLDL. MAPK inhibitor(PD98059) inhibited the phosphorylation of GSK-3β and almost completely blocked oxLDL-induced tubular EMT. However，PD98059 alone was able to inhibit LDL-induced tubular EMT partially. Conclusions oxLDL is more potently in inducing tubular EMT than LDL. The ERK1/2MAPK-GSK-3β signaling pathway mediates the LDL or oxLDL-induced tubular EMT.