p27Kip1基因shRNA表达质粒的构建鉴定及功能研究  

Construction and identification of p27Kip1 shRNA expression plasmid and its functional investigation

作　　者：黄渝侃[1] 张明昌[1] 王勇[2] 张进祥[3] 范可顺[1] 张光红[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院眼科,武汉430022 [2]武汉爱尔眼科医院,武汉430030 [3]华中科技大学同济医学院附属协和医院急诊科,武汉430022

出　　处：《第三军医大学学报》2007年第22期2148-2151,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30500487);湖北省自然科学基金(2004ABA250)~~

摘　　要：目的构建细胞周期性激酶抑制剂p27Kip1的小发夹RNA(short hairpin RNA,shRNA)表达质粒,并观察其对牛角膜内皮细胞增殖能力的影响。方法设计有发夹状结构的3条p27Kip1-shRNA对应模板DNA序列,并构建无关序列HK-shRNA作为阴性对照。转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的pGenSil-1质粒,构建重组质粒pGenSil-1/p27Kip1-1、pGenSil-1/p27Kip1-2、pGenSil-1/p27Kip1-3、pGenSil-1/HK。转化DH5α菌株,提取质粒行酶切及测序鉴定。以脂质体法将4个重组质粒分别导入牛角膜内皮细胞。转染后48h以Western blot法检测p27Kip1蛋白水平及MTT法检测各实验组和对照组细胞增殖情况。结果酶切及测序证实4个重组质粒均构建成功。3个实验组p27Kip1蛋白水平分别下降了66.41%、61.51%、71.32%,shRNA可显著促进牛角膜内皮细胞增殖。结论成功构建了针对p27Kip1的RNA干扰表达载体。Objective To construct a plasmid expressing a short hairpin RNA （shRNA） targeting a cyclin-dependent kinase inhibitor （CKI）, p27Kipl, and evaluate its effect on the proliferation of bovine corneal endothelial cells （bCEC）. Methods Three p27Kipl-shRNA template DNA sequences were designed and synthesized as experimental groups, a plasmid expressing irrelevant shRNA with a random combination was used as negative control. The products were inserted into the pGenSil-1 plasmid containing U6 promoter and EGFP. The recombinant plasmids of pGenSil-1/p27Kipl-1, pGenSil-1/p27Kipl-2, pGenSil-1/p27Kipl-3 and pGenSil-1/ HK were constructed respectively. Then the recombinant plasmids were expanded in DHSot cells and identified by restriction enzyme and sequence analysis. The 4 plasmids were transfected into bCEC respectively by Lipo fectamine 2000TM. The level of p27Kipl protein was detected by Western blotting and the proliferation of bCEC were detected by MTF 48 h after transfection. Results The 4 recombinant plasmids was confirmed to be con- structed successfully. The inhibitory rates of p27Kipl protein were 66.41%, 61.51% and 71.32% in the 3 experimental groups respectively, and the proliferation of bCEC was promoted NA- p27Kipl RNAi system has been constructed successfully. It facilitates the tion of bCEC by the method of RNAi. significantly, Conclusion shRexperimental study of the promotion of bCEC by the method of RNAi.

关 键 词：RNA干扰 P27KIP1 质粒 

分 类 号：R322.91[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]