人EPO基因真核表达载体构建及其在人脐血间充质干细胞中表达.docVIP

人EPO基因真核表达载体构建及其在人脐血间充质干细胞中表达 　 作者：夏桂枝，张国成，陈新民，洪新如 【摘要】 　　目的： 构建人EPO基因真核表达载体并转染人脐血间充质干细胞，获得稳定表达人EPO的人脐血间充质干细胞. 方法： 从人胎肝细胞中提取总RNA，经过RTPCR方法扩增人EPO全长cDNA，应用基因重组技术将EPO cDNA基因亚克隆至pcDNA3真核表达载体内，以酶切和测序方法鉴定重组质粒pcDNA3-EPO构建的正确性，再将pcDNA3-EPO经脂质体介导转染人脐血间充质干细胞，经G418筛选后获得稳定转染EPO基因的人脐血间充质干细胞，用RT-PCR，Western Blot和细胞免疫化学方法鉴定外源人EPO基因在人脐血间充质干细胞中的表达. 结果： 酶切和测序结果均证实pcDNA3-EPO构建正确，RT-PCR，Western blot和细胞免疫化学结果显示转染人EPO基因的人脐血间充质干细胞体外能表达EPO. 结论： 构建成功人EPO基因真核表达载体，转染人脐血间充质干细胞后在体外可获得稳定表达. 【关键词】 细胞生成素 胎血 间质干细胞 转染 　　【Abstract】 AIM: To construct the eukaryotic expression vector of human EPO gene and explore its expression in mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB). METHODS: The total RNA was extracted from human fetal liver cells. EPO cDNA was obtained by RT-PCR amplication and subcloned into pcDNA3 vector to construct recombinant pcDNA3-EPO plasmid. After identified by restriction enzyme analysis and sequencing,the pcDNA3-EPO was introduced into the human UCB-derived MSCs mediated by lipofectamine and the expression of exogenous EPO was examined by RT-PCR,Western blot and immunocytochemistry. RESULTS: The recombinant pcDNA3-EPO was shown to meet the design by restriction enzyme analysis and sequencing. The results of RT-PCR,Western blot and immunocytochemical analyses indicated that the exogenous EPO successfully expressed in human UCB-derived MSCs. CONCLUSION: The recombinant pcDNA3-EPO with full coding sequence of human EPO cDNA is successfully constructed and human UCB-derived MSCs with stable expression of exogenous EPO are established. 　　【Keywords】 erythropoietin；fetal blood；mesenchymal stem cells; transfection 　　0 引言 　　近年研究发现促红细胞生成素（erythropoietin，EPO）具有神经营养、神经保护和促进神经发育的作用，在脑组织损伤尤其是新生儿缺氧缺血性脑损伤中发挥着重要作用［1］. 间充质干细胞（mesenchymal stem cells，MSCs）是近年发现的一种具有多向分化潜能的成体干细胞［2］，而且是外源基因良好的细胞载体［3-4］. 我们构建人EPO基因真核表达载体，将其转染人脐血MSCs，观察EPO在人脐血MSCs中的表达情况，以探索EPO基因修饰的人脐血MSCs用于移植治疗新生儿缺氧缺血性脑病（hypoxic-ischemic encephalopathy，HIE）的可行性. 　　1 材料和方法 　　1.1 材料 人胎肝细胞系L02，质粒pcDNA3由福州总