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目的探讨ERK1/2途径是否参与Pkd2基因低表达诱导主动脉血管平滑肌细胞(VSMCs)表型转化及可能分子机制。方法原代培养小鼠主动脉VSMCs,转染Pkd2+/-突变载体,构建Pkd2基因低表达细胞模型。实验共分为空白对照的Control组、Pkd2+/-组、空病毒组、Ets1组(Pkd2促进剂)、PD98059组(ERK抑制剂)、EGF组(ERK激动剂)。Western blot检测多囊蛋白2(PC2,Pkd2编码蛋白)、a-SMA(VSMCs收缩型标志蛋白)、骨桥蛋白(OPN,VSMCs增殖型标志蛋白)、ERK1/2、ERK磷酸化蛋白(P-ERK1/2)表达水平;RT-PCR检测PC2、a-SMA、OPN、ERK1、ERK2 mRNA表达水平;MTT法检测细胞增殖。结果 (1)Pkd2+/-组中PC2蛋白及mRNA表达明显下调,Pkd2基因低表达模型构建成功。(2)Pkd2+/-组中a-SMA表达下调、OPN表达升高,VSMCs细胞增殖明显,细胞发生了表型转化;加入Ets1后被明显抑制(P<0.05)。(3)Pkd2+/-组中ERK1/2及P-ERK1/2表达明显升高,加入PD98059后表达被抑制,但PC2表达无变化(P<0.05)。(4)Pkd2+/-组中加入PD98059后,a-SMA表达升高、OPN表达下调,VSMCs细胞增殖被抑制,细胞表型转化被抑制。结论 Pkd2基因低表达可以诱导小鼠主动脉VSMCs表型转化,这一过程可能与ERK1/2异常活化有关。
Objective To investigate whether ERK1 / 2 pathway is involved in phenotypic transformation of aortic vascular smooth muscle cells (VSMCs) induced by low expression of Pkd2 gene and its possible molecular mechanism. Methods Primary cultured mouse aorta VSMCs were transfected with Pkd2 +/- mutant vector to construct a low expression Pkd2 cell model. The experiment was divided into blank control group, Pkd2 +/- group, empty virus group, Ets1 group (Pkd2 promoter), PD98059 group (ERK inhibitor) and EGF group (ERK agonist). Western blot was used to detect the expression of PC2 and Pkd2 protein, a-SMA (VSMCs contractile marker), osteopontin (OPN), ERK1 / 2 and ERK phosphorylated protein (P- ERK1 / 2). The mRNA expressions of PC2, a-SMA, OPN, ERK1 and ERK2 were detected by RT-PCR. Cell proliferation was detected by MTT assay. Results (1) PC2 protein and mRNA expression were significantly down-regulated in Pkd2 +/- group, and Pkd2 low expression model was successfully constructed. (2) The expression of a-SMA in Pkd2 +/- group was down-regulated and the expression of OPN was up-regulated. The proliferation of VSMCs was obvious and the phenotype of cells was transformed. Ets1 was significantly inhibited (P <0.05). (3) The expressions of ERK1 / 2 and P-ERK1 / 2 were significantly increased in Pkd2 +/- group. The expression of PDK802 was inhibited but the expression of PC2 was unchanged (P <0.05). (4) After adding PD98059 to Pkd2 +/- group, the expression of a-SMA increased and the expression of OPN decreased. The proliferation of VSMCs was inhibited and the phenotype of cells was inhibited. Conclusion The low expression of Pkd2 can induce the phenotype of VSMCs in aorta of mice. This process may be related to the abnormal activation of ERK1 / 2.