【摘要】 目的:将外源性人端粒酶反转录酶基因转入人骨髓间充质干细胞,以建立永生化细胞株。方法:实验于2006-01/2007-01在石河子大学新疆地方与民族高发病省部共建教育部重点实验室完成。①实验材料:骨髓标本采自因意外事故引产死亡的8个月正常胎儿,家属均签署知情同意书。大肠杆菌DH5α分子克隆的受体菌由本室保存。质粒pBabepuro-hTERT由美国Weinberg博士惠赠。PA317细胞和Hela细胞由郭志儒博士惠赠。NIH3T3细胞由黄瑾博士惠赠。②实验方法:无菌条件下将骨块置于α-MEM培养液中,分离及扩增培养人骨髓间充质干细胞。提取并纯化质粒pBabepuro-hTERT,转染包装骨髓间充质干细胞,经嘌呤霉素筛选后获得抗性克隆,选取病毒滴度最高的细胞克隆感染生长状态良好的第3代骨髓间充质干细胞。③实验评估:采用端粒酶重复序列扩增法-酶链免疫吸附法检测人骨髓间充质干细胞转染前后端粒酶活性的变化,样本的△A值高于0.2视为端粒酶阳性。结果:①骨髓间充质干细胞的形态观察:第3代人骨髓间充质干细胞形态不规则,体积较大的细胞逐渐死亡,以梭形为主。②基因转染包装细胞和克隆筛选结果:嘌呤霉素筛选7d后,转染的细胞出现抗性克隆。待细胞克隆长至肉眼可见时将细胞克隆消化后扩增培养,挑选出4个抗性克隆,分别命名为克隆1,2,3,4。③病毒滴度的测定:1～4号细胞克隆的病毒滴度分别为3×106,7×105,4×105,9×106cfu/L。④端粒酶活性变化的检测:未转染的第3代骨髓间充质干细胞△A值为0.121±0.043,端粒酶活性为阴性。第3代人端粒酶反转录酶-骨髓间充质干细胞的△A值为1.741±0.103,端粒酶活性为阳性。结论:导入外源性人端粒酶反转录酶基因后可激活骨髓间充质干细胞的端粒酶活性。更多还原

【Abstract】 AIM:Human bone mesenchymal stem cells(hMSCs)were stably transduced by a retroviral vector containing the telomerase reverse transcriptase gene(hTERT)to construct immortalized human bone mesenchymal stem cells.METHODS:The experiment was conducted in Xinjiang Endemic and Ethical Laboratory of Shihezi University from January 2006 to January 2007.①The marrow specimen was provided by a 8-month embryo who was induced labor because of the misadventure with the informed consents of parturients.The Escherichia coli DH5α was conserved in this Laboratory,and the plasmid pBabepuro-hTERT was presented by Dr.Weinberg in American.PA317 cells and Hela cells were presented by Dr.Guo.NIH3T3 cells were presented by Dr.Huang.②The bone of embryo was separated and cultured to get hMSCs in the α-MEM culture medium under sterile condition.The pBabepuro-hTERT that was extracted and purified was transduced into HMSCs,and then to acquire resistance clones by puromycin screening.The 3rd generation hMSCs were infected by the highest of virus titer of cell clone.③The telomerase activity of hTERT-MSCs cells was detected by TRAP(PCR)-enzyme labeled immunosorbent assay(ELISA).If △A of sample exceeded 0.2,it was positive.RESULTS:①Observation of hMSCs morphous:The morphous of the 3rd generation hMSCs was irregular.And the bigger cells were died gradually.There were mostly spindle-shaped.②Assay of gene transfection incasing cells and clone screening:After transduction cells were screened 7 days by puromycin,resistance clones were gained.After being seen by naked eye,the resistance clones,named clone 1,2,3,4,were digested and cultured in another pan.③Determination of virus titer:The virus titer of the 4 cell clones was 3×106,7×105,4×105,9×106 cfu/L,respectively.④Detection of telomerase activation:The△A of the 3rd generation hMSCs was 0.121±0.043,thus telomerase activation of hMSCs was negative.The △A of the 3rd generation hTERT-hMSCs was 1.741±0.103,thus telomerase activation of hTERT-hMSCs was positive.CONCLUSION:The telomerase of hMSCs can be activated by the hTERT gene.更多还原