人端粒酶反转录酶转染大鼠许旺细胞的生物学特性

Human telomerase reverse transcriptase gene-transfected effects on biological characteristics of Schwann cells

背景:研究表明,基因修饰许旺细胞可使许旺细胞在体内存活时间延长,促进神经再生和功能的恢复。目的:以反转录病毒PLXSN为载体,将hT ERT基因转染入体外培养的大鼠许旺细胞,检测许旺细胞端粒酶活性及细胞生物学特性。方法:体外培养Wistar大鼠许旺细胞,经反转录病毒PLXSN为载体介导人端粒酶反转录酶基因转染,在同等条件下进行空载病毒转染,以正常培养的许旺细胞为对照组。采用RT-PCR,Western blot检测许旺细胞人端粒酶反转录酶基因和蛋白的表达,流式细胞仪测定细胞周期分布的变化。以细胞生长曲线、MTT比色法观察细胞生长的优化作用。结果与结论:人端粒酶反转录酶基因转染许旺细胞48 h后,检测到人端粒酶反转录酶mR NA和蛋白水平表达明显。与对照组和空载病毒组比较,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)。结果表明通过反转录病毒PLXSN为载体介导人端粒酶反转录酶基因转染使许旺细胞端粒酶活性明显升高,能够促进体外培养的大鼠许旺细胞增殖。

BACKGROUND：Studies have shown that genetical y modified Schwann cel s can survive for a longer time in vivo, and promote nerve regeneration and functional recovery. OBJECTIVE：To transfect human telomerase reverse transcriptase （hTERT） gene into rat Schwann cel s cultured in vitro via PLXSN vector, and to detect the telomerase activity and biological characteristics of Schwann cel s. METHODS：Schwann cel s from Wistar rats were cultured in vitro and transfected by PLXSN vector with （hTERT group） or without hTERT （empty vector group）. Normal Schwann cel s were selected as control group. RT-PCR and western blot methods were used to detect the hTERT protein and mRNA levels in Schwann cel s, and flow cytometry was used to measure the cel cycle distribution. Cel growth was observed by cel growth curve and MTT colorimetric method. RESULTS AND CONCLUSION：At 48 hours after transfection, the mRNA and protein expressions of hTERT were remarkably seen in Schwann cel s. Compared with the control and empty vector groups, the cel s grew faster, the number of cel s at G 0/G 1 Schwann cel s cultured in vitro. phase was reduced, but the number of S phase cel s was increased in the hTERT group （P〈0.05）. These findings indicate that PLXSN vector-mediated hTERT transfection of Schwann cel s can significantly improve the activity of telomerase in Schwann cel s as wel as promote the proliferation of Schwann cells cultured in vitro.