Pkd2基因低表达调控ERK1/2途径诱导血管平滑肌细胞表型转化

Low expression of Pkd2 gene induces phenotype transformation of vascular smooth muscle cells by ERK1/2 pathway

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 study whether the ERK1/2 pathway is involved in aortic vascular smooth muscle cells（VSMCs）phenotype transformation induced by low expression of Pkd2 gene and to explore its possible molecular mechanisms.Methods Mouse aortic VSMCs were primarily cultured,Pkd2＋/-mutant vectors were transfected,and low expression cell model of Pkd2 gene was established.The experiment included 6 groups：the control group,Pkd2＋/-group,blank vector group,Ets1（Pkd2 agonist） group,PD98059（ERK inhibition） group and EGF（ERK agonist） group.Western blot was used to detect the expression levels of polycystin 2（PC2,Pkd2 encoding protein）,a-SMA（VSMCs contraction marker）,osteopontin（OPN,VSMCs proliferation marker）,ERK1/2 and the phosphorylation of ERK protein（P-ERK1/2）.The expression levels of PC2,a-SMA,OPN,ERK1 and ERK2 mRNAs were measured by real-time PCR.MTT assay was used to detect cell proliferation.Results The expression of PC2 protein and mRNA in Pkd2＋/-group was significantly down-regulated,and the low expression model of Pkd2 gene was successfully constructed.The expression of a-SMA in Pkd2＋/-group was down-regulated,while the expression of OPN was elevated.The proliferation of VSMCs was obvious,and cellular phenotype transformation occurred.The cell proliferation was significantly inhibited（P 0.05） after Ets1 was added.The expression of ERK1/2 and P-ERK1/2 in Pkd2＋/-group was significantly elevated,but was inhibited after PD98059 was added,while the expression of PC2 remained unchanged（P 0.05）.After PD98059 was added in Pkd2＋/-group,the expression of a-SMA was elevated,while the expression of OPN was down-regulated.The proliferation of VSMCs and cellular phenotype transformation were both inhibited.Conclusions The low expression of Pkd2 gene can induce the phenotype transformation of aortic VSMCs in mice,and this process may be related to the abnormal activation of ERK1/2.