重建端粒酶活性的正常人成纤维细胞的成骨潜能研究  

OSTEOGENIC POTENTIAL OF FIBROBLASTS WITH RECONSTITUTED TELOMERASE ACTIVITY

作　　者：汪铮[1] 李莹[2] 朱佑明[2] 邓廉夫 易静[1] 汤雪明[1] 

机构地区：[1]上海第二医科大学细胞生物学教研室,上海200025 [2]上海第二医科大学发育生物学实验室 [3]上海市骨伤科研究所

出　　处：《中国修复重建外科杂志》2002年第3期200-204,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：上海联合利华研究发展基金资助 (990 7)

摘　　要：目的　通过重建端粒酶活性延长人成纤维细胞寿命 ,并对其成骨潜能进行研究 ,为解决组织工程骨修复种子细胞老化问题提供实验依据。方法　将人端粒酶催化亚基 (h TERT)基因用电穿孔法导入正常人原代成纤维细胞 ,用 TRAP- PCR检测细胞端粒酶活性 ,用β-半乳糖苷酶活性测定评价细胞衰老情况。在此基础上用骨形成蛋白(BMP- 2 )和肿瘤坏死因子 -α(TNF-α)联合诱导已重建端粒酶活性的成纤维细胞在体外培养条件下成骨 ,用四环素活体标记和茜素红染色显示钙盐结节形成。结果　转染 h TERT的人成纤维细胞能稳定表达端粒酶活性 ,培养超过 5 0代后细胞仍保持β-半乳糖苷酶阴性。经 BMP- 2和 TNF-α诱导后 ,转染后传 80代的人成纤维细胞仍可形成四环素标记和茜素红染色均为阳性的钙盐结节。结论　重建端粒酶活性。Objective To prevent the senescence of ' seed cells ' for tissue engineering, the life span of human fibroblasts is extended by reconstitution of telomerase activity, and the osteogenic potential of these fibroblasts are tested. Methods The pGRN145 plasmids encoding human telomerase reverse transcriptase (hTERT) were introduced into the normal human primary fibroblasts by electroporation. Telomerase activity was analyzed by TRAP PCR assay. The β galactosidase stain was used to indicate the signs of cell senescence. The hTERT positive fibroblasts were then induced to form bone nodules. The bone nodules were stained by tetracycline and Alizarin Red S. Results Stable telomerase activity could be detected in the transfected fibroblasts and no signs of cell senescence were found in the fibroblasts cultured for more than 50 doublings. The hTERT positive fibroblasts could form bone nodules when they were cultured in vitro induced by bone morphogentic protein 2 and tumor necrosis factor α. Conclusion The fibroblasts with reconstituted telomerase activity reserve their osteogenic potential.

关 键 词：组织工程 端粒酶 成纤维细胞 成骨潜能 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]