碱性成纤维细胞生长因子重组腺病毒转染兔骨髓间充质干细胞的表型变化

Rabbit bone marrow mesenchymal stem cells transfected with recombinant adenovirus vectors carrying basic fibroblast growth factor: variation of cell phenotypes

背景:外源性碱性成纤维细胞生长因子在韧带组织的愈合过程中发挥着重要作用,采用转基因方法将外源性基因转入细胞内能促进碱性成纤维细胞生长因子分泌。目的:观察碱性成纤维细胞生长因子重组腺病毒载体转染兔骨髓间充质干细胞后表型变化及碱性成纤维细胞生长因子蛋白的表达。方法:取P2代骨髓间充质干细胞,分为3组,正常培养组为对照组,其他2组分别转染重组腺病毒(Ad.bFGF-eGFP)及空病毒(Ad.eGFP)。相差显微镜观察各组细胞形态变化,MTT法测定细胞增殖曲线,ELISA法分析各组细胞上清液中碱性成纤维细胞生长因子蛋白质量浓度。结果与结论:转染后细胞呈现均一的成纤维细胞表型;碱性成纤维细胞生长因子重组腺病毒组细胞增殖活性高于空病毒组及对照组;ELISA结果提示碱性成纤维细胞生长因子重组腺病毒组在7 d内上清液中碱性成纤维细胞生长因子蛋白质量浓度较空病毒组及对照组增加,差异有显著性意义(P<0.05)。提示碱性成纤维细胞生长因子重组腺病毒转染骨髓间充质干细胞可促进其向成纤维细胞分化,增加增殖活力,促进其碱性成纤维细胞生长因子蛋白的表达。

BACKGROUND：Exogenous basic fibroblast growth factor （bFGF） plays an important role in the ligament tissue healing process, and the use of transgenic methods to transfect exogenous genes into cells can promote the secretion of bFGF. OBJECTIVE：To observe phenotypic changes and the bFGF protein expression after bFGF recombinant adenovirus was used to transfect rabbit bone marrow mesenchymal stem cells （BMSCs）. METHODS：Passage 2 BMSCs were divided into three groups：Ad.bFGF-eGFP group, Ad.eGFP group and control group. Under a phase contrast microscope we observed the changes in cellmorphology. The expression of bFGF protein in BMSCs was determined by enzyme-linked immunosorbent assay （ELISA）. The proliferative curve was detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT）. RESULTS AND CONCLUSION：The transfected cells showed a uniform phenotype of fibroblasts. MTT colorimetric assay revealed that more proliferative activity of transfected BMSCs was shown in the Ad.bFGF-eGFP group than in the Ad.eGFP group and control group. ELISA results showed that expression of bFGF protein was higher in the Ad.bFGF-eGFP group than in the Ad.eGFP group and control group （P〈0.05）. BFGF recombinant adenovirus can induce the differentiation of BMSCs into fibroblasts, increase proliferative ability and promote the expression of bFGF protein.