重组慢病毒载体转染羊骨髓间充质干细胞及成骨基因表达量的变化

Recombinant lentiviral vector transfected sheep bone marrow mesenchymal stem cells and osteogenic gene expression changes

背景:碱性成纤维细胞生长因子具有促进骨髓基质细胞分裂增殖作用,而骨形态发生蛋白2在诱导新骨形成方面有重要的研究意义。目的:分析骨形态发生蛋白2和碱性成纤维细胞生长因子基因单独和联合转染对体外培养的骨髓间充质干细胞的增殖和骨向分化的影响,比较Ⅰ型胶原蛋白、骨钙蛋白、骨桥蛋白等非特异性成骨基因于转染前后骨髓间充质干细胞相对表达量的差别,为构建组织工程骨的种子细胞做理论依据。方法:构建碱性成纤维细胞生长因子、骨形态发生蛋白2慢病毒载体,分别转染羊骨髓间充质干细胞,得到碱性成纤维细胞生长因子组、骨形态发生蛋白2组、联合组、对照组细胞,提取RNA后采用实时定量PCR的方法检测Ⅰ型胶原蛋白、骨钙蛋白、骨桥蛋白等非特异性成骨基因的mRNA水平相对表达量的变化。结果与结论:对照组、碱性成纤维细胞生长因子、骨形态发生蛋白2及联合组4组间非特异性成骨基因表达量存在明显差异(P<0.05),且碱性成纤维细胞生长因子和骨形态发生蛋白2具有交互作用(P<0.05),碱性成纤维细胞生长因子、骨形态发生蛋白2联合组的Ⅰ型胶原蛋白和骨钙蛋白基因表达量明显高于其他3组,且差异有显著性意义(P<0.05);但是骨桥蛋白基因中差异无显著性意义(P>0.05)。体外实验结果显示联合转染组中的Ⅰ型胶原蛋白、骨钙蛋白、骨桥蛋白等非特异性成骨基因mRNA水平相对表达量较多,该组细胞的成骨功能最强,适合作为组织工程骨的种子细胞。

METHODS：Lentiviral vectors carrying bFGF and BMP-2 were constructed to transfect sheep bone marrow mesenchymal stem cells. cells were divided into four groups：bFGF group, BMP-2 group, co-transfection group＆amp;nbsp;BACKGROUND：Basic fibroblast growth factor （bFGF） can promote the proliferation of bone marrow stromal cells, and bone morphogenetic protein-2 （BMP-2） has an important significance in the induction of new bone formation. OBJECTIVE：To analyze the effects of bFGF, BMP-2 and their co-transfection on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and to compare the relative expressions of col agen I, osteocalcin and osteopontin before and after celltransfection, thereby providing theoretical implications for seed cells in the construction of tissue-engineered bone. and control group. The RNA was extracted using real-time quantitative PCR to detect mRNA levels of col agen I, osteocalcin, and osteopontin. RESULTS AND CONCLUSION：Significant difference in non-specific osteogenic gene expressions was found among the four groups （P〈0.05）. bFGF and BMP-2 showed an interaction （P〈0.05）. Expressions of col agen I and osteocalcin in the co-tranfection group were higher than those in the other three groups （P〈0.05）, but osteopontin expression exhibited no difference （P〉0.05）. In vitro experiments showed that the relative expression of col agen I, osteocalcin and osteopontin were higher in the co-transfection group, indicating the cells from the co-transfection group have strongest osteogenic capacity that are suitable for seed cells for bone tissue engineering.