RNA 干扰技术建立人CASK表达缺陷细胞株 - 第三军医大学学报.DOCVIP

CASK下调表达对人血管内皮细胞ECV-304增殖能力的影响 陈建　苏踊跃　梁光萍 陈愉　罗向东 杨宗城 (第三军医大学西南医院烧伤研究所,创伤、烧伤与复合伤国家重点实验室，重庆 400038) 　　 摘 要:目的　建立CASK下调表达细胞株, 研究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片断的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。 结论　CASK下调表达可促进ECV-304细胞增殖，CASK下调表达细胞株的建立为CASK基因功能研究提供了有效手段。 关键词:　RNA 干扰； CASK；　真核细胞转染 ；细胞增殖 The effect of CASK down-regulation on the proliferation of ECV-304 cells CHEN Jian, SU Yong-yue, LIANG Guang-ping，CHEN Yu， LUO Xiang-dong，YANG Zong-cheng(State key laboratory of trauma, burns and combined injury, Institute of Burn Research，Southwest Hospital，Third Military Medical University，Chongqing 400038, China) Abstract: Objective 　To establish calcium,calmodulin-associated serine/threonine kinase（CASK) down-regulation ECV-304 cell line and to study the effect of CASK down-regulation on the proliferation of ECV-304 cells. Methods 　Transfected ECV-304 cells with the recombinant pGenesil-1-hCASK plasmids containing shRNA targeting CASK gene. 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 cell counting were used to measure the change of the proliferation of transfected cells. Results CASK down-regulation cell strain was successfully screened out. The decreased expression of CASK could lower the G0/G1 phase and elevate G2M phase of the cell population. Conclusion 　The successe in constructing CASK down-regulation cell strain offered an effective way to further study the function of CASK gene. The decreased expression of CASK could promote the proliferation of ECV-304 cells. Key words: RNA interference , CASK , eukaryotic transfection ,cell proliferation 　　 烧伤后多种因素可导致血管内皮细胞的激活与损伤[1]，损伤的血管内皮细胞使毛细血管通透性增加、组织脏器微循环障碍，是烧伤后多脏器功能不全综合征( multiple organ dysfunction syndrome, MODS)的重要发病机制[2]。 微循环功