CASK下调表达对人血管内皮细胞ECV-304增殖能力的影响  

Effects of CASK down-regulation on the proliferation of ECV-304 cells

作　　者：陈建[1] 苏踊跃[1] 梁光萍[1] 陈渝[1] 罗向东[1] 杨宗城[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所,创伤,烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2009年第12期1144-1146,共3页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划)(2005CB522601);国家自然科学基金重点项目(30730093)~~

摘　　要：目的建立CASK下调表达细胞株,研究钙/钙调蛋白依赖性丝氨酸蛋白激酶(calcium,calmodulin-associated serine/threonine kinase,CASK)下调表达对ECV-304细胞增殖能力的影响。方法将包含针对CASK基因干扰siRNA片段的pGenesil-1-hCASK重组质粒转染人ECV-304细胞株,经G418加压筛选,成功筛选出CASK下调表达的细胞株。用蛋白免疫印迹法鉴定转染细胞中CASK基因的表达水平。采用流式细胞技术、MTT法观察CASK下调表达对ECV-304细胞增殖能力的变化。结果成功筛选出CASK下调表达ECV-304细胞株(siCASK细胞株),这些细胞在传代过程中绿色荧光蛋白能维持表达,细胞株中转染阳性率在90%以上;经蛋白免疫印迹法检测CASK表达明显降低;siCASK细胞株中G0/G1细胞比率下降,G2M期细胞比率明显上升,增殖指数明显提高,生长曲线左移。结论成功建立了CASK下调表达的ECV-304细胞株,CASK下调表达可导致细胞增殖能力增强。Objective To establish calcium, calmodulin-associated sefine/threonine kinase （CASK） down-regulation ECV-304 cell line and to study the effects of CASK down-regulation on the proliferation of ECV-304 cells. Methods The recombinant pGenesil-l-hCASK plasmids containing siRNA targeting CASK gene were transfected into ECV-304 cells. The stable cell strain was screened by adding G418 into cell culture medium. The protein expression levels of CASK gene were detected by Western Blotting. Flow cytometry and M3T method were used to investigate the effects of CASK down-regulation on the proliferation of the transfected cells. Results CASK down-regulation cell strain, siCASK cell line, was successfully screened. These cells could maintain over 90% green fluorescent protein expression. The decreased expression of CASK could lower but enhance the cell population at G0/G1 phase but G2 M phase and lead to left-shift of the cell growth curve. Conclusion CASK down-regulation cell strain was successfully constructed and CASK down-regulation could enhance the cell proliferation.

关 键 词：RNA干扰 钙/钙调蛋白依赖性丝氨酸蛋白激酶 真核细胞转染 细胞增殖 

分 类 号：R322.12[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]