GFAP启动子与骨髓基质干细胞神经胶质分化的筛选(.docVIP

GFAP启动子与骨髓基质干细胞神经胶质分化的筛选? ?作者单位 100044 北京大学人民医院创伤骨科 通讯作者 姜保国 本课题受国家863计划（2002AA205071），国家973重大基础研究项目（2001cca01300）基金资助 张培训 姜保国 何湘君 赵富强 魏光如 张殿英 傅中国 张宏波 摘要 目的 研究骨髓基质干细胞体外诱导分化，结合特异性GFAP启动子控制的EGFP的表达来筛选诱导的骨髓基质干细胞。方法 构建报告载体pGFAP-EGFP, 原代培养的骨髓基质干细胞，在β-ME, RA, forskolin, bFGF, PDGF-AA, HRG-β等复合物的诱导7天后，pGFAP-EGFP载体转染，G418抗生素筛选。荧光显微镜下观察存活细胞体内的EGFP表达情况，流式细胞仪测定荧光细胞的比率。 结果 报告载体pGFAP-EGFP构建成功，转染骨髓基质干细胞后部分细胞存活，流式细胞仪测定82.74%的细胞表达EGFP。筛选出的细胞GFAP免疫细胞化学鉴定阳性。体外扩增后可以得到大量的目的细胞。 结论 特异性GFAP 启动子控制的EGFP的表达可以在体外有效的进行骨髓基质干细胞的筛选和扩增。筛选后的细胞具有胶质细胞表型，可提供足够的胶质细胞进行神经系统的细胞替代治疗。 关键词 骨髓基质干细胞; GFAP启动子; 增强型绿色荧光蛋白（EGFP）; 细胞标记 The differentiation selection of bone marrow stromal cells into neuralglial cells with GFAP promoter Zhang peixun, Jiang baoguo, He xiangjun, et al. Traumatic orthopaedics of People Hospital, Peking University, Abstract Objective To study the bone marrow stromal cells differentiation in vitro and to select the differentiated bone marrow stromal cells with the GFAP promoter-controled EGFP expression. Methods Reconstruct the report vector pGFAP-EGFP, culture bone marrow stromal cells primarily and induce with composite of β-ME, RA, forskolin, bFGF, PDGF-AA, HRG-β for 7 days. . Transfected bone marrow stromal cells with pGFAP-EGFP and selected with G418 antibiotic. The survived bone marrow stromal cells were observed under fluorescence microscopy and flow cytometry. Results The report vector pGFAP-EGFP was reconstructed successfully. Transfected bone marrow stromal cells surviced and 82.74% expressed EGFP. Survived cells were positive to GFAP immunocytochemistry staining. The survived bone marrow stromal cells can be enriched in vitro. Conclusions Bone marrow stromal cells can be selected and enriched in vitro under the GFAP-controled EGFP expression. The selected cells possessed the phenotypes of neuralglial cells and can provide plenty of cells quantities for cell substitution therapy. Key Words: Bone marrow stromal cells; GFAP promoter; EGFP; cell labeling