转染人骨形成蛋白2共培养条件下NIH3T3细胞生物学性状的观察

The biological changes of NIH3T3 cells co-cultured with human bone morphogenetic protein-2 gene transfecting cells

目的利用细胞共培养系统研究转基因诱导表达的分泌型人骨形成蛋白2(hBMP-2),对NIH3T3细胞超微结构和碱性磷酸酶(ALP)活性的影响。方法用阳离子聚合物转染试剂将hBMP-2真核表达载体pcDNA3·1-B2导入NIH3T3细胞,G418筛选获得阳性细胞克隆。免疫组化和酶联免疫吸附试验检测BMP-2胞内和胞外的表达,并利用细胞共培养系统Transwell,将转基因细胞与NIH3T3细胞共培养。结果转染hBMP-2基因的NIH3T3细胞胞内和胞外都有BMP-2的表达。与转基因细胞共培养的NIH3T3细胞在共培养后超微结构的变化和ALP的高表达,都提示其向成骨样细胞分化的趋势。结论经转基因诱导表达的分泌型BMP-2具有诱导成纤维细胞向成骨样细胞分化的作用。

Objective To investigate the ultrastructure and the alkaline phosphatase （ALP） activity changes of NIH3T3 cells incubated with secretive human bone morphagenetic protein-2 （hBMP-2） that is induced by gene transfection through transwell system. Methods Eukaryonic expression vector （pcDNA3.1-B2） was transduced into NIH3T3 cells by Sofast^TM, a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracelluler expression of BMP-2 in the NIH3T3 cells were determined by immunohistochemical and enzyme-linked immunosorbent assay （ELISA）. NIH3T3 cells were co-cultured with hBMP-2 gene transfecting ceils through transwell system, and the ultrastructure and ALP activity （the markers of osteogenetic differentiation） changes were observed. Results There were cytoplasmic and extracelluler expression of BMP-2 in transfecting NIH3T3 cells. The ultrastructure changes and the high expression of ALP suggested the osteogenetic differentiation tendency of NIH3T3 cells cocultured with transfecting NIH3T3 cells. Conclusions Secretive BMP-2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.