人碱性成纤维细胞生长因子基因转染大鼠骨髓间充质干细胞的研究

Transfection and expression of basic fibroblast growth factor gene mediated by lentivirus vector into rat mesenchymal stem cells

目的 观察人碱性成纤维细胞生长因子（bFGF）基因体外转染对大鼠骨髓间充质干细胞（MSCs）bFGF表达的影响.方法 密度梯度离心、贴壁法培养分离SD雄性大鼠MSCs,体外扩增,流式细胞仪检测MSCs表面抗原表达.利用慢病毒载体系统介导将具有人源性bFGF基因转染至第2代MSCs,在倒置荧光显微镜下观察转染后细胞形态和生长的变化,应用逆转录-聚合酶链反应（RT-PCR）、Western blot法鉴定bFGF在MSCs中的表达.结果 密度梯度离心、贴壁法培养分离可获得MSCs,P3代大鼠细胞利用流式细胞仪检测CD11b/c阳性细胞表达率为（13.2±0.6）%,CD34阳性细胞表达率为（1.2±0.5）%,CD44阳性细胞表达率（97.8±0.9）%,CD90阳性细胞表达率（96.8±1.4）%.MSCs转染48 h后,绿色荧光蛋白的表达明显增强.RT-PCR证实转基因MSCs表达bFGF mRNA明显增强,Western blot检测证实转基因MSCs在49 KDr出现特异性条带,而空白和空载组的MSCs则未见阳性条带.结论 采用慢病毒介导的基因转染技术可以将bFGF基因转染至MSCs中,并有外源性bFGFmRNA和蛋白的有效表达,MSCs可作为bFGF基因治疗的载体.

Objective To observe the transfection and expression of human basic fibroblast growth factor （bFGF） gene into rat mesenchymal stem cells （MSCs） in vitro, and provide a potential method for treatment of hind limb ischemia. Methods MSCs were separated from SD rat bone marrow with density gradient centrifugation, wall sticking screening and amplified in vitro. The surface antigens of MSCs were evaluated with fluorecene activated cell sorter. The bFGF gene was transfected into MSCs by lentivirus vector. The mRNA and protein expression of bFGF gene in the transferred cells was analyzed by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting respectively. Results MSCs could bo separated from SD rat bone marrow and amplified in vitro. The expression of CD11b/c, CD34, CD44 and CD90 was （ 13.2 ±0.6）%, （1.2 ±0.5）%, （97.8 ±0.9）% and （96.8 ± 1.4）% in the culture cells respectively. After transfection for 48 h, the expression of green fluorescent protein （GFP） was significantly increased. The bFGF gene was successfully transferred into MSCs by lentivirus vector. RT-PCR and Western blotting revealed that there was bFGF mRNA and protein expression in the transferred cells, and a significant positive zone of hybridization was visible at 49 kDr. However, no positive band was found in the other groups. Conclusion Gene transfer technology mediated by lentivirus vector can transfect human bFGF gene into MSCs, which can stably express exogenous bFGF mRNA and protein. MSCs can be used as the vector for genetic therapy of bFGF.