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目的证实脐血中含有间充质干细胞,探讨其体外分离、培养条件,并进行生物学特性和表面抗原的鉴定,初步探索间充质干细胞作为基因治疗的载体携带目的基因的可能性及方法。方法无菌条件下取正常足月剖宫产的脐血分离、培养和纯化,获得间充质干细胞;对获得的间充质干细胞进行形态学观察;绘制生长曲线,分析间充质干细胞的增殖情况;用流式细胞仪检测间充质干细胞表面抗原的表达情况。采用脂质体转染法将pDNA316-IRES-EGFP质粒转入间充质干细胞中,观察绿色荧光的表达以及转染后细胞生长情况。结果自脐血中成功分离获得间充质干细胞,可传代培养,经表面标志检测,表达CD29、CD44、CD105,不表达CD45和HLA-DR。质粒pDNA316-IRES-EGFP通过脂质体介导转染间充质干细胞后,荧光显微镜下观察到报告基因EGFP的表达。转染后的细胞生长受到一定影响。结论人脐血来源的间充质干细胞能在体外分离、培养、扩增,并且具有和骨髓源间充质干细胞类似的生物形态和抗原表型。EGFP基因可被成功转入间充质干细胞中并获表达。转基因间充质干细胞用于基因治疗的持久性和有效性仍需进一步研究。
Objective To confirm that mesenchymal stem cells are contained in umbilical cord blood, to explore its isolation and culture conditions in vitro, and to identify biological characteristics and surface antigens in order to explore the possibility and method of carrying mesenchymal stem cells as a vector for gene therapy. Methods Umbilical cord blood from normal term cesarean section was isolated, cultured and purified under sterile conditions to obtain mesenchymal stem cells. Morphological observation was performed on the obtained mesenchymal stem cells. Growth curves were drawn to analyze the proliferation of mesenchymal stem cells The expression of mesenchymal stem cell surface antigen was detected by flow cytometry. The plasmid pDNA316-IRES-EGFP was transfected into mesenchymal stem cells by lipofection method. The expression of green fluorescence and the growth of cells after transfection were observed. Results Mesenchymal stem cells (MSCs) were successfully isolated from umbilical cord blood and subcultured. The surface markers of CD29, CD44 and CD105 were expressed and CD45 and HLA-DR were not expressed. After plasmid pDNA316-IRES-EGFP was transfected into mesenchymal stem cells by liposome, EGFP expression was observed under a fluorescence microscope. The cell growth after transfection is affected to some extent. Conclusion Human umbilical cord blood-derived mesenchymal stem cells can be isolated, cultured and expanded in vitro with similar biological morphology and antigenic phenotype as bone marrow-derived mesenchymal stem cells. EGFP gene can be successfully transferred into mesenchymal stem cells and expressed. The persistence and effectiveness of genetically modified mesenchymal stem cells for gene therapy still need further study.