骨形态发生蛋白4慢病毒载体构建及对鼠骨髓间充质干细胞成牙功能影响

目的:构建骨形态发生蛋白4(BMP4)慢病毒载体,检测其对鼠骨髓间充质干细胞成牙功能的影响,以期为组织工程牙筛选种子细胞。方法:以成熟人胎盘组织为材料来源,克隆人BMP4基因的全长cDNA,转克隆至plenti6/v5-D-TOPO表达载体,构建出BMP4-plenti6/v5-D-TOPO重组慢病毒表达载体;转染SD大鼠骨髓间充质干细胞,用MTT法检测转染前后细胞的增殖活性,实时荧光定量PCR检测Ⅰ型胶原蛋白、成釉蛋白、牙本质基质蛋白1、同源异型盒基因1成牙相关基因的mRNA水平相对表达量的变化。结果:BMP4基因转染后,细胞体外增殖活性增强,成釉蛋白、牙本质基质蛋白1、同源异型盒基因1 mRNA水平表达量增多,差异有统计学意义(P<0.05),Ⅰ型胶原蛋白 mRNA水平差异无显著性意义。结论:BMP4可提高骨髓间充质干细胞体外增殖活性和牙向分化能力。

Construction and Odontogenic Effects on Rat Bone Marrow Mesenchymal Stem Cells of Bone Morphogenetic Protein4 Lentiviral Vector.

Objective: To construct lentiviral vector carrying bone morphogenetic protein4, and to detect the odontogenic effects on rat bone marrow mesenchymal stem cells for tissue- engineered tooth seed cells. Methods: hBMP4 cDNA was gained by RT-PCR from the mRNA ,which was extracted from mature human placenta. Then it was cloned into plenti6/v5-D-TOPO vector. Lentiviral vectors carrying BMP4 were constructed to transfect rat bone marrow mesenchymal stem cells(BMSCs). The RNA was extracted using real-time quantitative PCR to detect the changes mRNA levels of collagen I, enamel protein,dentin matrix protein 1, and homeobox gene 1. And the proliferation ability of BMSCs was detected with MTT method before and after transfection. Results: After transfection, mRNA levels of collagen I, enamel protein and dentin matrix protein 1 were higher, and the difference was statistically significant (P<0.05),but the mRNA levels of homeobox gene 1 were not significant different, and the proliferation ability was increased. Conclusion: BMP4 is able to improve the proliferation and odontogenic differentiation potential of BMSCs in vitro.