体外培养条件下人骨髓间充质干细胞碱性成纤维细胞生长因子基因的转染与鉴定

Identification of human bone marrow mesenchymal stem cells transfected with basic fibroblast growth factor gene in vitro

背景:骨髓间充质干细胞的定向分化需要合适生长因子的调控,利用细胞因子基因转染促进其生长、定向分化和加速骨缺损修复是近年来研究热点。目的:探讨在体外培养条件下转染碱性成纤维细胞生长因子基因对人骨髓间充质干细胞生物学特性的影响。方法:将含人pcDNA3.1-bFGF真核表达载体DH-5α菌扩增,用EndoFree质粒抽提纯化试剂盒抽提质粒,并对所提取的pcDNA3.1-bFGF重组表达质粒进行酶切鉴定和测序。利用脂质体将pcDNA3.1-bFGF质粒转染到生长良好的P3代骨髓间充质干细胞,G418筛选获得抗性克隆。采用荧光定量PCR、免疫组化、免疫荧光、Western-blot检测转染后骨髓间充质干细胞碱性成纤维细胞生长因子基因及其产物的表达,流式细胞仪检测细胞增殖周期。结果与结论:脂质体介导pcDNA3.1-bFGF重组表达质粒转染骨髓间充质干细胞后,转染细胞确实表达碱性成纤维细胞生长因子,且表达部位主要位于胞浆。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)。提示采用脂质体转染法能将pcDNA3.1-bFGF成功导入体外培养的骨髓间充质干细胞,碱性成纤维细胞生长因子基因转染后可改善骨髓间充质干细胞的生存状态,促进其增殖。

BACKGROUND：Directional differentiation of bone marrow mesenchymal stem cells（BMSCs） requires regulation of appropriate growth factors.Increasing attention has been paid to transfect cells with cytokine gene for promoting BMSCs to proliferate and differentiate and to accelerate repair process of bone defects.OBJECTIVE：To investigate the biological characteristics of BMSCs as seed cells of tissue engineering after transfected with basic fibroblast growth factor（bFGF） gene in vitro.METHODS：The phagemid expression vector with human pcDNA3.1-bFGF was amplified in E.coli.The plasmid was extracted,purified by EndoFree Plasmid Maxi Kit.The plasmid of pcDNA3.1-bFGF was analyzed and identified by enzymes digestion and sequencing analysis.pcDNA3.1-bFGF gene was transfected into P3 BMSCs using Lipofectamine 2000.Positive clones of BMSCs transfected bFGF gene were selected with G418.The expression of bFGF mRNA and its productions in the transfected BMSCs were detected by real time PCR,immunohistochemistry,immunofluorescence and Western-blot.The proliferative cycle of transfected BMSCs were examined by flow cytometry analysis.RESULTS AND CONCLUSION：BMSCs expressing bFGF gene were obtained by transfection with pcDNA3.1-bFGF gene via Lipofectamine.The cell lineage was confirmed that the expressed position of bFGF was mainly in cytoplasm after transfection with bFGF gene.Higher proportion of cells in proliferation cycle was shown in transfected BMSCs compared with non-transfected BMSCs（P 0.05）.Results show that pcDNA3.1-bFGF can be transfected into BMSCs cultured in vitro via Lipofectamine.Genetic modification of BMSCs with bFGF may promote the proliferation of BMSCs.