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目的研究稳定表达端粒酶催化亚基的人骨髓间充质干细胞(hBMSCs)生物学特性。方法用含有人端粒酶返转录酶(hTERT)基因的反转录病毒质粒pBabepuro-hTERT感染hBMSCs,经嘌呤霉素(puro)筛选出转基因细胞,我们将其命名为hTERT-hBMSCs。采用TRAP-ELISA法检测hBMSCs细胞转染前后端粒酶活性的变化;采用四甲基偶氮唑蓝(MTT)法,染色体核型分析,测量平板克隆形成率,成骨诱导等方法对hTERT-hBMSCs细胞生物学特性进行分析。结果TRAP-ELISA法检测转染hTERT的hBMSCs的端粒酶为阳性;hTERT-hBMSCs细胞表面抗原CD44表达阳性,CD34阴性;hTERT-hBMSCs比hBMSCs增殖活跃,目前已传了11代,尚未见衰老迹象,其染色体数目和结构正常,未见畸变;克隆形成率较低,为正常细胞;并保持干细胞成骨分化潜能。结论将外源性hTERT基因转入hBMSCs后,不影响其生物学特性和分化功能。
Objective To study the biological characteristics of human bone marrow mesenchymal stem cells (hBMSCs) stably expressing telomerase catalytic subunit. Methods hBMSCs were infected with the retroviral plasmid pBabepuro-hTERT containing the human telomerase reverse transcriptase (hTERT) gene and the transgenic cells were screened by puro. We named hTERT-hBMSCs. The changes of telomerase activity before and after transfection of hBMSCs were detected by TRAP-ELISA. MTT assay, chromosome karyotype analysis, plate formation rate and osteogenic induction were used to detect the changes of hTERT- hBMSCs cell biological characteristics were analyzed. Results hTERT-hBMSCs transfected hTERT hTERT positive for telomerase; hTERT-hBMSCs positive for cell surface antigen CD44, CD34 negative; hTERT-hBMSCs than hBMSCs proliferation active, has passed 11 generations, has not seen signs of aging , Its chromosome number and structure of normal, no distortion; clone formation rate is low, normal cells; and maintain the potential of osteogenic differentiation of stem cells. Conclusion The exogenous hTERT gene transferred into hBMSCs does not affect its biological characteristics and differentiation.