E1A激活基因阻遏子促进人血管平滑肌细胞分化和迁移  

The Cellular Repressor of E1A-stimulated Genes Induces Differentiation and Migration of Human Vascular Smooth Muscle Cells

作　　者：韩雅玲[1] 胡叶[1] 刘海伟[1] 康建[1] 闫承慧[1] 李少华 

机构地区：[1]沈阳军区总医院全军心血管病研究所心内科 [2]Department of Pathology,Robert Wood Johnson Medical School,New Jersy 08854,USA

出　　处：《生物化学与生物物理进展》2005年第6期517-522,共6页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30070280)~~

摘　　要：为探讨E1A激活基因阻遏子(cellularrepressorofE1A-stimulatedgenes,CREG)蛋白在人血管平滑肌细胞株HITASY分化和迁移中的调控作用,构建了重组逆转录病毒载体pLNCX2(+)/CREG和pLXSN(-)/CREG.以带绿色荧光蛋白(greenfluorescentprotein,GFP)的空载体pLNCX(+)/GFP和正常HITASY为对照,用磷酸钙共沉淀法将重组逆转录病毒载体转染293细胞,包装出完整的逆转录病毒后,感染HITASY.经G418筛选,获得稳定感染的细胞克隆.应用免疫荧光染色、蛋白质印迹等方法检测CREG和平滑肌分化标志蛋白α-肌动蛋白(SMα-actin)表达,同时通过刮伤实验、慢速显微摄像技术检测细胞迁移能力以及用明胶酶谱方法分析细胞基质金属蛋白酶(matrixmetalloproteinase,MMPs)的活性.结果表明:pLNCX2(+)/CREG稳定感染的HITASY中CREG和SMα-actin蛋白表达上调,MMP-2和MMP-9活性升高,细胞迁移速度加快;pLXSN(-)/CREG稳定感染的HITASY中CREG和SMα-actin蛋白表达下调,MMP-2和MMP-9活性降低,细胞迁移速度减慢.上述研究提示CREG在诱导血管平滑肌细胞分化的同时促进细胞迁移.In order to study the effect of the cellular repressor of E1A-stimulated genes (CREG) on differentiation and migration of human vascular smooth muscle cells (VSMCs)-HlTASY, the full length human sense and antisense-CREG cDNA retroviral vectors, pLNCX(2)(+)/CREG and pLXSN(-)/CREG, were constructed. Western blot and immunoflourescence analysis showed that the expression of CREG and SM alpha -actin increased in HITASY after infection with pLNCX(2)(+) /CREG. The migration of HITASY infected with pLNCX(2)(+)/CREG obviously enhanced compared with that of normal HITASY and pLXSN(-)/CREG cells showed by scrape-wounding and time-lapse analysis. Moreover, CREG over-expression increased the secretion of MMPs in HITASY tested by Western blot. Gelatin SDS-PAGE zymography analysis revealed that the activities of MMPs also increased in HITASY infected with pLNCX2(+)/CREG. On the other hand, the opposite effects were observed when CREG expression decreased by using antisense pLXSN(-)/CREG. These results suggest that CREG may be able to induce the VSMCs differetiation and promote the VSMCs migration in the meantime.

关 键 词：阻遏子 腺病毒蛋白E1A 分化 迁移 逆转录病毒载体 平滑肌 

分 类 号：Q555.7[生物学—生物化学]