热休克培养对bFGF转染骨髓间充质干细胞FGFRs及凋亡相关蛋白的影响

Expressions of Fibroblast Growth Factor Receptors and Apoptosisrelated Proteins in Basic Fibroblast Growth Factor-transfected Bone Marrow Mesenchymal Stem Cells Under Heat Shock Cultured

目的探讨热休克培养对碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)转染骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)后成纤维细胞生长因子受体(fibroblast growth factor receptors,FGFRs)及Bcl-2、Bax、Caspase-3等凋亡相关蛋白的影响。方法 4周龄雄性SD大鼠处死后获取BMSCs,24h热休克培养,分别转染b FGF基因过表达腺病毒载体Ad-b FGF和空载体Ad-GFP,并设立阴性对照组,q RT-PCR和Western blot法分别鉴定3组细胞b FGF基因和蛋白的表达情况,Western blot法测定FGFRs、Bcl-2、Bax、Caspase-3的表达情况。结果转染后48h,RT-PCR和Western blot结果显示,转染Ad-b FGF组细胞有b FGF基因和蛋白表达,而阴性对照组和空载体Ad-GFP组未检测;Western blot结果表明,与阴性对照组和空载体Ad-GFP组相比,转染Ad-b FGF组FGFRs、Bcl-2表达增高,而Bax、Caspase-3表达下降,Bax/Bcl-2比值下降(P<0.05),阴性对照组和空载体Ad-GFP组差异无统计学意义(P>0.05)。结论腺病毒介导的基因转染技术能实现b FGF转染到BMSCs中,借助于热休克培养能提高FGFRs表达水平,并增强细胞抗凋亡能力,有望成为基因治疗的理想载体。

Objective To observe the effects of basic fibroblast growth factor（b FGF） transfection on fibroblast growth factor receptors（FGFRs）and apoptosis-related proteins including Bcl-2,Bax,Caspase-3 of bone marrow mesenchymal stem cells（BMSCs） cultured under heat shock conditions.Methods BMSCs were obtained from the killed male SD rats of 4 weeks of age,and cultured for 24 h under heat shock conditions,then were transfected by b FGF gene overexpression adenovirus vector Ad-b FGF and empty vector Ad-GFP respectively,meanwhile negative control group was set up.The expression of b FGF gene and protein were identified by q RT-PCR and Western blot respectively,while the expression of FGFRs,Bcl-2,Bax,Caspase-3 in these three groups of cells were evaluated by Western blot respectively.Results The results of RT-PCR and Western blot showed that the transfected Ad-b FGF group cells had b FGF gene and protein expression,while the negative control and empty vector Ad-GFP groups did not detect the gene and protein,after 48 h transfection.Moreover,compared with the negative control and empty vector Ad-GFP groups,the expressions of FGFRs and Bcl-2 increased,while the expressions of Bax and Caspase-3 and the ratio of Bax/Bcl-2 decreased in the transfected Ad-b FGF group（P 〈0.05）.There was no significant difference between the negative control and empty vector Ad-GFP groups（P 〉0.05）.Conclusion Adenovirus mediated gene transfection can achieve b FGF transfection into BMSCs,supported by heat shock culture,and the expression of FGFRs and anti apoptosis ability of cells can be enhanced.Therefore,it is expected to be an ideal carrier for gene therapy.