人端粒酶逆转录酶基因转染人髓核细胞的安全性研究

摘要

Objective To explore the effects of gene transfection of human telomerase reverse transcriptase (hTERT) on the safety of human nucleus pulposus (HNP) cells. Methods The second generation of HNP cells cultured in monolayer culture were transfected with recombinant adeno-associated virus vector-mediated hTERT gene (rAAV-hTERT) at multiplicity of infection (MOI) 105 v·g per cell. Three groups were designed for the experiment, rAAV-hTERT transfected group. AAV transfected group. No viral transfected group, The expression of the hTERT gene was determined by RT-PCR and Western-blot. The genetic phenotype of HNP cells was detected by karyotype analysis at 120 d after culture in vitro. The cells from the 3 groups (100 μX, 3 × 107/mL) were injected into nude mouse armpit to assess the oncogenic potentials and Hela cells were taken as positive control. Results The expressions of the rAAV-hTERT were successfully detected by RT-PCR. The cloning of the structural chromosomal abnormality was not seen through Giemsa-band karyotypic analysis. There was no tumor growth in all the nude mice. Conclusion The rAAV-hTERT can infect HNP cells and express in the transfected cells. It is safe for the rAAV-hTERT HNP cells in the limited time (120 d) in vitro. The study may provide a good safety reference for in-vivo study on the effect of rAAV-hTERT on HNP cells.%目的 评价人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因转染对人椎间盘髓核细胞“安全性”的影响.方法 将构建好的重组腺相关病毒人端粒酶逆转录酶基因( recombinant adeno-associated virus vector-mediated hTERT gene,rAAV-hTERT)按感染复数(multiplicity of infection,MOI) 105 v.g每细胞转染体外培养二代髓核细胞.设立rAAV-hTERT转染组,AAV空病毒转染组及未转染组;利用RT-PCR及Western-blot检测转染后hTERT基因的表达;对体外培养120 d的细胞进行染色体核型分析;将3组细胞(100 μL,3×107/mL)分别注入裸鼠腋下检测其成瘤性,以人Hela细胞为阳性成瘤实验对照.结果 成功检测出rAAV-hTERT转染髓核细胞后hTERT基因的表达;G-带核型分析未见染色体结构异常克隆;3组细胞均未观察到裸鼠体内肿瘤形成.结论 rAAV-hTERT能成功转染人椎间盘髓核细胞并正确表达,rAAV-hTERT转染髓核细胞在有限的体外培养过程中是安全的,可为进一步的在体研究提供安全性证据.