Transfection of hTERT into newborn rat cochlear basilar membrane cells
Transfection of hTERT into newborn rat cochlear basilar membrane cells
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发表时间：
2012
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通讯作者：
X. Min
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作者：
X. Min
目的通过基因转染的方法获得新生大鼠耳蜗基底膜细胞永生化细胞系。方法原代培养大鼠耳蜗基底膜细胞，将pCI-neo-hTERT质粒转染入细胞，用G418筛选稳定转染细胞系，RT-PCR、端粒酶活性检测、流式细胞仪检测转染细胞系的端粒酶活性，并进行流式细胞仪检测转染细胞系的端粒酶活性。结果RT-PCR法可检测到人端粒酶逆转录酶（human telomerase reverse transcriptase，hTERT）基因的表达;转染后72h进行PCR扩增，G418筛选获得稳定转染的细胞株;流式细胞仪检测hTERT转染细胞的端粒酶活性，结果显示hTERT能轻微提高细胞活力，结论hTERT基因转染新生大鼠耳蜗基底膜细胞可减少细胞凋亡提高细胞传代能力，为耳蜗细胞实验提供了充足的细胞来源。
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.