目的 通过基因转染法获得新生大鼠耳蜗基底膜细胞永生化细胞系.方法 通过脂质体法将pCI-neo-hTERT质粒转染原代培养的新生大鼠耳蜗基底膜细胞,G418筛选获得稳定转染的细胞系,并行转染细胞RT-PCR、端粒酶活性、细胞周期、细胞凋亡等检测.结果 转染72h后RT-PCR检测到人端粒酶逆转录酶(hTERT)基因阳性表达,转染细胞通过G418筛选传代后,可检测到端粒酶活性,流式细胞术检测提示转染细胞增殖活力增强不明显,但细胞凋亡明显减少.结论 通过脂质体转染hTERT基因,可使新生大鼠耳蜗基底膜细胞凋亡减少,传代能力增强,给耳蜗细胞实验提供足够的细胞来源.%Objective To obtain immortalized cell lines of newborn rat cochlear basilar membrane cells through gene transfection method. Methods Rat cochlear basilar membrane cells were grown in primary culture. pCI-neo-hTERT plasmid was transfected into the cells. Stably transfected cell lines were screened with G418. RT-PCR, telomerase activity detection, cell cycle and apoptosis detection were used to evaluate the effects. Results Human telomerase reverse transcriptase (hTERT) gene expression could be detected by RT-PCR 72 hours after transfection. The stably transfected cell lines were obtained by screening with G418; the telomerase activity of hTERT-transfected cells could be detected. Flow cytometry detection showed that hTERT could slightly improve cell vitality, but significantly reduce cell apoptosis. Conclusion Transferring hTERT gene into newborn rat cochlear basilar membrane cells can reduce cell apoptosis and improve cell passage ability, which provides enough cell sources for cochlear cell experiments.
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