人端粒酶反转录酶基因电转染优化培养大鼠前软骨干细胞

Electrotransfection by human telomerase reverse transcriptase gene: optimization in precartilagious stem cell culture

背景:将人端粒酶反转录酶转染至目的细胞中,可以提高其端粒酶活性,保持端粒的长度,在调控增殖和分化方面有重要作用。目的:探讨人端粒酶反转录酶基因转染对体外培养大鼠前软骨干细胞端粒酶活性及生物学特性的影响。方法:体外培养Wistar大鼠前软骨干细胞,以反转录病毒PLXSN为载体介导人端粒酶反转录酶基因转染,同时设置对照组、阴性对照序列组、转染组。用TRAP-ELISA法检测前软骨干细胞端粒酶活性,RT-PCR、Western blot检测前软骨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:(1)人端粒酶反转录酶基因转染前软骨干细胞48 h,端粒酶活性明显升高;(2)与对照组、阴性对照序列组相比,转染组人端粒酶反转录酶基因和蛋白水平表达明显,细胞的生长速度明显增快,G0/G1期细胞数减少,S期细胞数增多,差异有显著性意义(P<0.05);(3)结果表明,以反转录病毒PLXSN为载体介导人端粒酶反转录酶基因转染使前软骨干细胞端粒酶活性明显升高,能够促进大鼠前软骨干细胞增殖。

BACKGROUND： The human telomerase reverse transcriptase gene（hT ERT） transfected into target cells can play an important role in target cell proliferation and differentiation by increasing telomerase activity and maintaining telomere length. OBJECTIVE： To explore the effect of hT ERT transfection on telomerase activity and biological characteristics of precartilage stem cells culured in vitro. METHODS： Precartilage stem cells cultured in vitro were subjected to h TERT gene transfection via a retrovirus vector p LXSN. Meanwhile, control and negative control groups were set up. After transfection, TRAP-ELISA assay was used to detect telomerase activity; RT-PCR and western blot employed to detect hT ERT mR NA and protein expressions; cell counting kit-8 used to detect cell proliferaiton based on cell growth curve; and flow cytometry adopted to detect cell cycle and distribution. RESULTS AND CONCLUSION： The telomerase activity was significantly increased at 48 hours after hT ERT gene was transfected into the precartilage stem cells. After transfection of h TERT, hT ERT mR NA and protein levels were significantly increased, the cell growth rate was significantly increased, the proportion of cells at G0/G1 phase was decreased, and the number of S-phased cells increased compared with the control group and negative control group. There were significant differences among the groups（P 〈0.05）. In conclusion, hT ERT transfection via retrovirus vector pL XSN can promote the proliferation of precartilage stem cells in rats by increasing the telomerase activity.