转染bFGF基因对人骨髓间充质干细胞增殖特性的影响

目的 探讨作为组织工程种子细胞的人骨髓间充质干细胞,在体外培养条件下转染bFGF基因对其增殖特性的影响.方法 利用脂质体将含人pcDNA3.1-bFGF质粒转染到生长良好的P3代人BMSCs,G418筛选获得抗性克隆.采用荧光定量PCR和免疫荧光检测转染bFGF的骨髓间充质干细胞bFGF基因及其产物的表达,MTY法检测和流式细胞仪检测细胞的增殖情况和细胞增殖周期,并将转染和非转染BMSCs分别成骨诱导分化,对其碱性磷酸酶活性进行测定.结果 脂质体介导pcDNA3.1-bFGF重组表达质粒转染BMSCs,经荧光定量PCR和免疫荧光检测.证实转染细胞表达bFGF.转染细胞增殖活力加强,处于增殖周期的细胞比例更高（P〈0.05）.碱性磷酸酶活性检测结果表明转染细胞的碱性磷酸酶活性高于非转染细胞（P〈0.05）.结论 用脂质体转染法能将pcDNA3.1-bFGF成功导入体外培养的hBMSCs,bFGF基因改良的BMSCs可以改善其生存状态、促进其增殖,并可促进向成骨细胞分化.

Impact of transfection of bFGF gene on the proliferative characteristics of human BMSCs

Objective To investigate the proliferative characteristics of hBMSCs as seed cells of tissue engineering after being transfected with bFGF gene in vitro.Methods The plasmid containing human pcDNA3.1-bFGF was transfected into P3 population of hBMSCs by using lipofectamine 2000. Positive clones of BMSCs with bFGF gene were selected with G418. The expression of bFGF mRNA and its productions in the transfected BMSCs were detected by real-time PCR and immunofluorescence. The proliferation and cell cycle of transfected BMSCs were examined by MTT colorimetric assay and flow cytometry. The transfected BMSCs and non-transfected BMSCs were induced to differentiate into osteo-blasts and the ALP activity was detected. Results BMSCs expressing bFGF gene were obtained by transfection with pcDNA3.1-bFGF gene via lipofectamine. The cells were demonstrated to express bFGF mRNA and bFGF protein by real-time PCR and immunofluorescence test. MTT colorimetric assay revealed that more proliferative activity of transfected BMSCs was shown than that of non-transfected cells; and a higher proportion of cells in proliferation cycle and higher ALP activity were also shown in transfected BMSCs (P<0.05). Conclusions Via lipofectamine, pcDNA3.1-bFGF can be transfected into BMSCscultured in vitro. Genetic modification of hBMSCs with bFGF may improve the viability of BMSCs, promote the proliferation of the cells, and make them into osteoblasts.