大鼠EPO真核表达载体的构建及其在骨髓间充质干细胞中的表达.docVIP

大鼠EPO真核表达载体的构建及其在骨髓间充质干细胞中的表达 王文益王文益，男,（1981.3-），四川资阳人，汉族，硕士，医师，工作单位为四川省三台县人民医院儿科。E-mail： HYPERLINK mailto:xuehaitianshu@163.com 王文益，男,（1981.3-），四川资阳人，汉族，硕士，医师，工作单位为四川省三台县人民医院儿科。E-mail： HYPERLINK mailto:xuehaitianshu@163.com xuehaitianshu@163.com 手通信作者 赵聪敏 教授，第三军医大学第二附属医院儿科 主任医师 硕士研究生导师。电话：(023E-mail： HYPERLINK mailto:zhao_54@163.com zhao_54@163.com 基金项目：重庆市科委项目（CSCT,2007BB5057）；重庆市计生委项目（2009C175） 摘 要:目的　构建表达大鼠促红细胞生成素(erythropoietin, EPO)的真核表达质粒pEGFP-N1/EPO,并检测其在体外的表达,为治疗缺氧缺血性脑病引起的脑损伤提供实验基础。方法　从大鼠肾脏获得EPO基因片段并用RT-PCR方法扩增,将其连接于真核表达质粒pEGFP-N1,测序鉴定后,用脂质体包裹转染大鼠骨髓间充质干细胞（mesenchymal stem cells, MSCs）,采用免疫组化和RT-PCR检测EPO的表达。结果　经酶切和测序鉴定证实本实验构建的重组表达质粒正确,该质粒在体外转染大鼠MSCs细胞后可表达EPO蛋白。结论　成功构建的pEGFP-N1/EPO重组质粒能在体外表达EPO蛋白。 关键词：促红细胞生成素；真核表达载体；骨髓间充质干细胞；缺氧缺血性脑病 Construction of eukaryotic expression plasmid vector pEGFPN1-EPO and its expression vitro Wang Wen-Yi, Zhao Cong-Min，Liao Wei (Department of pediatrics,Xinqiao Hospital, Third Military Medical University,Chongqing,40037) Abstract: Objective　To construct eukaryotic expression plasmid vector pEGFP-N1/EPO,and to detect the expression of the plasmid in vitro, which is the basis for further study on the treatment of HIBD(hypoxic-ischemic brain damage). Methods　we gain EPO cDNA fragment of the rat’s kidney, amplified by RT-PCR, and inserted into pEGFP-N1 (-) vector, then identified by restriction endonuclease digestion and nucleotide sequencing. MSCs cells were transfected with the plasmid using Lipofectamine 2000. The expression of EPO protein was detected by immunohistochemistry techniques and RT-PCR. Results　The constructed recombinant plasmid contained the sequence of rat’s EPO gene. After transfection with the plasmid, EPO protein could be expressed in MSCs cells. Conclusion　The constructed eukaryotic expression plasmid vector pEGFP-N1/EPO can express EPO in vitro. Key words: EPO;erythropoietin;eukaryotic expression plasmid;MSCs;HIBD EPO是是一种耐热的含唾液酸的酸性糖蛋白,能促进造血祖细胞的增殖分化。近年来发现其具有阻断兴奋性氨基酸的毒性、抗神经细胞凋亡、抑制炎症反应、