转导人端粒酶逆转录酶基因致人成骨细胞永生化的研究

Immortalization of human osteoblast by transferring hTERT gene

目的 建立永生化的人成骨细胞系供骨科临床和基础研究使用。方法 将人骨髓基质干细胞定向诱导分化为成骨细胞 ,以逆转录病毒为载体 ,导入人端粒酶逆转录酶基因 (hTERT)全长cDNA ,经筛选得到阳性克隆 ,体外连续传代 ,检测hTERT基因的整合情况及其表达 ,并对不同代次细胞的成骨特性进行了检测。结果 成功地将hTERT基因转入人成骨细胞 ,得到的转化细胞端粒酶表达阳性 ,增殖旺盛 ,至今已传至 6 2代 ,对第 2 5、第 5 5代转化细胞分别进行碱性磷酸酶、Ⅰ型胶原、骨桥素的检测 ,证明此永生化细胞保持了良好的成骨特性。结论 转导外源性的hTERT基因可以导致人成骨细胞永生化且可保持其正常的成骨特性。

Objective To establish an immortalized osteoblast cell line used for basal and clinical research of orthopaedics. Methods Human marrow stromal cells (hMSCs) were inducted into osteoblasts directionally, and human telomerase reverse transcriptase(hTERT )cDNA was transferred into the osteoblasts by retroviral vector pLPChTERT .The resultant stable clones reproduced successively and the expression of hTERT as well as the osteogenesis characteristics of different eras were identified. Result hTERT gene has been transferred into human osteoblasts successfully .The transformed cells expressed telomerase activity and divided vigorously. P62 has been obtained so far. The expression of bone specific alkaline phosphatase, Collagen type Ⅰand osteopontin of p25,p55 were checked and it is proved that the immortalized cell line preserved typical osteogenetic characteristics. Conclusion Osteoblasts can be immortalized by transferring exogenous hTERT gene to reconstitute telomerase activity, and the immortalized cell reserve their normal osteogenetic characteristics.