论文部分内容阅读
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 d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。
Objective To study the differentiation of bone marrow stromal stem cells in vitro and to screen the induced bone marrow stromal stem cells in combination with the expression of EGFP under the control of specific GFAP promoter. Methods The reporter vector pGFAP-EGFP and primary cultured bone marrow stromal cells were constructed and transfected with pGFAP-EGFP vector after 7 d induction of β-ME, RA, forskolin, bFGF, PDGF-AA and HRG- G418 antibiotic screening. The expression of EGFP in surviving cells was observed under a fluorescence microscope, and the ratio of fluorescent cells was measured by flow cytometry. Results The pGFAP-EGFP vector was successfully constructed. Some cells survived after transfection of bone marrow stromal cells, and EGFP was detected in 82.74% of the cells by flow cytometry. The selected cells GFAP immunocytochemistry identification positive. In vitro amplification can be a large number of target cells. Conclusion The expression of EGFP under the control of specific GFAP promoter can effectively screen and amplify bone marrow stromal stem cells in vitro. After screening cells with glial phenotype, can provide enough glial cells in the nervous system cell replacement therapy.