逆转录病毒介导hBMP_7基因转染兔骨髓间充质干细胞的表达

HUMAN BONE MORPHOGENETIC PROTEIN 7 EXPRESSION IN RABBIT BONE MARROW STEM CELL TRANSFERRED BY RETROVIRAL VECTOR MEDIATED hBMP_7 GENE

采用粘 粘端连接方法构建hBMP7逆转录病毒载体 ,重组质粒转染包装细胞PT6 7后 ,制备含目的基因的重组逆转录病毒液感染兔骨髓间充质干细胞。限制性内切酶酶切分析筛选插入方向正确的重组质粒并进行基因测序。结果显示 ,hBMP7逆转录病毒载体中外源基因插入方向正确、无碱基错误和缺失。原位杂交和免疫组织化学检测表明 ,感染后 2天经基因转染的BMSc原位杂交和免疫组化检测结果呈阳性 ,未经转染的细胞检测结果呈阴性。转染的BMSc经G4 18筛选至 4周仍有外源BMP蛋白的表达。提示采用逆转录病毒介导方法转染的BMSc中可有外源性BMP7mRNA和蛋白的表达。

The human bone morphogenetic protein 7 (hBMP 7) gene was reconstructed in retroviral vector and transferred into incasing cells PT67 by liposome mediated method.The clones of the cells transferred with BMP 7 were selected by G418, and targeted rabbit bone marrow stem cells were infected with the virus granules which secreted from PT67 cells and also selected by G418. The mRNA and protein of BMP 7 gene in transferred cells were analyzed with hybridization in situ and immunohistochemistry. BMP 7 retrovirus vetor was successfully reconstructed. Cells transferred by PLNCX 2 hBMP 7 expressed abundant human BMP 7 mRNA and protein in the cytoplasm. However positive findings were not found in those cells that were not transferred. It may be used to increase the osteogenic capability of BMSc in the study of bone tissue engineering.