背景：研究表明，感染人端粒酶反转录酶基因后的干细胞具有稳定表达高水平端粒酶活性，且能使细胞增殖旺盛，这为建立基因工程的永生化干细胞系奠定了基础。目的：探讨人端粒酶反转录酶基因感染对体外培养鼠胎肝干细胞增殖及细胞周期的影响。方法：体外培养鼠胎肝干细胞，经重组腺相关病毒作为载体介导人端粒酶反转录酶基因感染，用 RT-PCR、Western blot检测鼠胎肝干细胞人端粒酶反转录酶基因和蛋白的表达，CCK-8法、细胞生长曲线检测细胞生长增殖情况，流式细胞仪测定细胞周期分布的变化。结果与结论：与对照组、空载病毒组相比，基因感染组人端粒酶反转录酶基因和蛋白水平均有表达，细胞的生长速度明显增快，G0/G1期细胞减少，S期细胞数增多。结果表明以重组腺相关病毒作为载体介导人端粒酶反转录酶基因感染能促进体外培养的鼠胎肝干细胞增殖，对其培养有优化作用。%BACKGROUND:Studies have shown that stem cels transfected with human telomerase reverse transcriptase (hTERT) gene can stably express high-level telomerase activity and strengthen cel proliferation, which lays the foundation to establish geneticaly engineered immortalized stem cel lines. OBJECTIVE:To explore the effects of hTERT transfection on proliferation and cel cycle of rat fetal liver stem cels in vitro. METHODS:Rat fetal liver stem cels culturedin vitro were transfected by recombinant adeno-associated virus carrying hTERT genes. RT-PCR and western blot assay were used to detect the expression of hTERT mRNA and protein, respectively. Cel Counting Kit-8 method and cel growth curve were used to detect cel growth and proliferation. Changes in cel cycle distribution were determined by flow cytometry. RESULTS AND CONCLUSION:Compared with the control group and empty virus group, in the hTERT infection group, hTERT expressed at gene and protein levels, the growth rate of the cels increased, and the number of cels at G0/G1 phase and S phase was decreased and increased, respectively. The results show that recombinant adeno-associated virus as a vector of hTERT gene used for gene transfection can promote the in vitro proliferation of rat fetal liver stem cels and play an optimal role in cel culture.
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