hTERT转染神经干细胞端粒酶活性及hTERT mRNA检测

目的以逆转录病毒PLXSN为载体,将hTERT基因转染入体外培养的人神经干细胞,检测神经干细胞端粒酶活性及hTERT mRNA表达情况,为建立永生化神经干细胞系提供一种新的思路.方法体外扩增人神经干细胞及基因转染后,应用PCR-ELISA法检测神经干细胞端粒酶活性,荧光定量PCR法检测hTERT mRNA表达情况.结果通过逆转录病毒介导的hTERT基因转染人神经干细胞后,端粒酶活性明显升高,持续表达,hTERT mRNA也早期呈高水平表达,但继之下降,维持在一个较低水平.结论转染hTERT基因后的神经干细胞具有表达高水平端粒酶活性,并稳定表达,这为建立基因工程的永生化神经干细胞系奠定了基础.而hTERT mRNA则逐渐下降,并维持在一个较低水平,不能作为检测神经干细胞增殖和分化能力的指标.

Detection of telomerase activity and hTERT mRNA in hTERT transfected human neural stem cells

Objective To detect the telomerase activity and the expression of hTERT mRNA in hTERT transfected human neural stem cells(NSCs) and explore to establish immortalized NSCs line.Methods Human NSCs cultured in vitro were transfected by retroviral vector carrying hTERT gene.Telomerase activity(TA) was detected by a quantitative telomerase PCR-ELISA,and hTERT mRNA expression was quantified by online RT-PCR.Results High levels of telomerase activity and hTERT expression were detected in transfected NSCs in early stage.The high level of telomerase activity lasted,but the expression of hTERT mRNA decreased gradually,then lasted in a low level.Conclusion hTERT transfected human NSCs held a high-level,stable telomerase activity,paving the way to establish immortalized NSCs line.hTERT mRNA could not be used as a molecular target of NSCs for describing their proliferation potential and differentiation status.