Pyk2/PI3K/NF-κB在心脏成纤维细胞表型转化及迁移中的作用  

Pyk2/PI3K/NF-κB improves phenotypic transition and migration of cardiac fibroblasts in vitro

作　　者：郝嘉[1] 游凯[1] 陈林[1] 肖颖彬[1] 

机构地区：[1]第三军医大学新桥医院全军心血管外科中心,重庆400037

出　　处：《第三军医大学学报》2011年第11期1110-1114,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30700822)~~

摘　　要：目的用短发夹RNA干扰技术选择性下调大鼠心脏成纤维细胞(cardiac fibroblasts,CFs)中富含脯氨酸的酪氨酸激酶2(prolein-rich tyrosine kinase 2,Pyk2)的表达,检测Pyk2/PI3K/NF-κB在细胞表型转化及迁移的作用。方法利用Pyk2特异短发夹RNA质粒载体,转染血管紧张素Ⅱ刺激的原代培养大鼠心脏成纤维细胞。使用Transwell小室模型、体外划痕法检测细胞迁移变化,并结合免疫荧光观察α-平滑肌肌动蛋白表达变化。蛋白印迹法检测Pyk2和磷酸化磷酸肌醇3激酶(phosphoinositide-3-kinase,PI3K)p85亚基的蛋白水平,EMSA检测核转录因子κB(NF-κB)活性。结果 Transwell实验显示CFs跨膜细胞数随培养时间延长而上升,但Pyk2 shRNA转染组在不同时相点跨膜细胞数较对照组均明显减少,在6 h分别为[(19.1±6.1)和(30.8±8.6),P<0.05],在12 h分别为[(37.7±12.4)和(53.6±11.3),P<0.05],而在24 h分别为[(78.9±18.5)和(102.3±21.7),P<0.01]。转染Pyk2 shRNA后α-平滑肌肌动蛋白表达明显受抑制,绿色阳性染色细胞的比例下降,荧光强度减弱。体外划痕法显示Pyk2 shRNA转染组细胞迁移距离明显下降,在24 h分别为0.838 mm和1.213 mm(P<0.05),在48 h分别为1.626 mm和2.049 mm(P<0.01)。Pyk2 shRNA转染组Pyk2和PI3K p85表达的相对条带强度较对照组减弱(0.47 vs 0.61,P<0.05),磷酸化PI3K p85表达水平也较对照组有明显降低(0.31 vs 0.42,P<0.05)。NF-κB平均光密度分别为2.17和3.59(P<0.05)。结论 Pyk2/PI3K/NF-κB可能是促进CFs向肌成纤维细胞表型转化和介导细胞迁移的重要信号途径,Pyk2可能成为防治心脏纤维化的新靶点。Objective To down-regulate the expression of prolein-rich tyrosine kinase 2（Pyk2） in cultured adult rat cardiac fibroblasts（CFs） with short hairpin RNA interference,and evaluate the role of Pyk2/PI3K/NF-κB in cell phenotypic transition and migration.Methods The Pavu6＋27-Pyk2 shRNA expression vector was constructed by gene recombination,then transfected into cultured adult rat CFs stimulated by angiotension Ⅱ.Untransfected cells（control）,cells transfected with Pavu6＋27 vector（empty vector） and those transfected with negative shRNA vector（negative control） were used as control.Transwell chamber test and in vitro scratching assay were adopted to evaluate the cell migration.Change of α-smooth muscle actin（α-SMA） in CFs was analyzed in transwell chamber by immunofluorescence.The protein expressions of Pyk2 and phosphate phosphoinositide-3-kinase protein（PI3K） p85 subunit in CFs were determined by Western blotting.Electrophoretic mobility shift assay was employed to determine the activation of NF-κB.Results After Pyk2 shRNA transfection,transwell assay showed the number of migrated CFs was increased with the prolongation of culture.However,comparing to control group,the number of migrated CFs in Pyk2 shRNA transfection group was deceased significantly after transfection（19.1±6.1 vs 30.8±8.6 at 6 h,37.7±12.4 vs 53.6±11.3 at 12 h,78.9±18.5 vs 102.3±21.7 at 24 h,P〈0.05,0.01）.But no difference was found among these control,empty vector and negative control.Immunofluorescence staining showed that α-actin expression and migration in CFs was inhibited after Pyk2 shRNA transfection.In vitro scratching assay revealed there were significant difference between the migration distance of CFs with Pyk2 shRNA transfection and control（0.838 vs 1.213 mm at 24 h,1.626 vs 2.049 mm at 48 h,P〈0.05,0.01）.The protein expression of Pyk2（0.47 vs 0.61） and phosphated PI3K p85（0.31 vs 0.42） were declined after Pyk2 shRNA transfection（P〈0.05）.Electrophoretic mobility shift ass

关 键 词：富含脯氨酸的酪氨酸激酶2 心脏成纤维细胞 表型 迁移 

分 类 号：R322.11[医药卫生—人体解剖和组织胚胎学] R394.33[医药卫生—基础医学]