中文摘要：

目的：研究腺病毒载体介导人内皮型一氧化氮合酶（human endothelial nitric oxide synthase，heNOS）和增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）在体外培养的大鼠骨髓间充质干细胞（mesenchymal stem cells，MSCs）的表达。方法：体外培养、扩增大鼠骨髓间充质干细胞，并对培养的细胞进行成骨、成脂肪诱导分化和细胞免疫表型鉴定；用PmeI线性化后去磷酸化的pShuttle—heNOS—EGFP转化含腺病毒骨架质粒pAdEasy--1的感受态BJ 5183，使其在细菌内发生同源重组，获得阳性重组质粒pAdEasy—heNOS—EGFP。重组质粒经PCR、酶切和测序鉴定正确后经Pac I酶切，转人AD 293细胞，包装成重组腺病毒Ad—heNOS—EGFP。用重组腺病毒Ad—heNOS—EGFP对MSCs进行转染，并用荧光显微镜及免疫荧光染色法观察转染细胞内EGFP和heNOS蛋白质的表达。结果：pShuttle—heNOS—EGFP与pAdeasy--1在BJ 5183中成功地发生了同源重组，得到了重组腺病毒质粒pAdEasy—heNOS—EGFP。重组质粒在AD 293细胞中包装成重组腺病毒Ad—heNOS—EGFP。经基因转染的MSCs中高表达heNOS和EGFP。结论：采用腺病毒载体可以将外源性的heNOS基因转染至MSCs中，并得到有效表达，且携带的EGFP标记可以直观地检测靶细胞感染和外源基因的表达情况，这将为勃起功能障碍的细胞治疗提供一条新的途径。

英文摘要：

Objective To investigate the expression of human endothelial nitric oxide synthase（heNOS） and enhanced green fluorescent protein （EGFP） in rat mesenchymal stem cells cultured in vitro. Methods Mesenchymal stem cells （MSCs） were isolated from bone marrow of new - born SD rats and expanded in vitro. MSCs were identified with their abilities to differentiate into adipogenic and osteogenic lineages and their immunophenotype of CD44, CIM5 and CD90. The vector pShut- tle - heNOS - EGFP was linearlzed with Pme I and transformed into ultracompetent BJ 5183 bacteria containing pAdEasy - 1. The positive clone of homologous recombination （pAdEasy- heNOS- EGFP） was identified by PCR, endonuclease digestion, and DNA sequencing. The positive recombinant adenoviral plasmid was digested with Pac I and transfected AD 293 cells with Lipofectamine^TM 2000 to package recombinant adenovirus particles. Thereafter, the recombinant adenovirus infected MSCs. The expression of heNOS and EGFP in MSCs were detected by immunofluorescent staining and fluorescent microscopy, respectively. The non - infected MSCs served as control. Results Homologous recombination occurred between pShuttle - heNOS - EGFP and pAdEasy - 1 within BJ 5183 to generate pAdEasy - heNOS - EGFP. The recombinant adenovirus Ad - heNOS - EGFP was confirmed to be successfully packaged within AD 293 cells with PCR, and efficientlyinfected MSCs. The green and red fluorescence were seen in MSCs infected with Ad - heNOS - EGFP under fluorescent microscope, which demonstrated the expression of EGFP and heNOS in MSCs. Gonclusion Exogenous heNOS cDNA could be efficiently transduced into MSCs mediated by adenoviral vector and overexpressed within MSCs, which would provide a new strategy to cell - based therapy of erectile dysfunction. Moreover, EGFP expression is a useful tool for directly monitoring the infection of target cells and the etexpression of gene of interest.