【摘要】 目的建立永生化的人骨髓基质细胞(hMSC)系,以供神经外科临床和基础研究使用。方法原代培养人骨髓基质细胞,采用含有人端粒酶逆转录酶(hTERT)基因的逆转录病毒载体上清感染hMSC,经筛选得到阳性克隆,在体外进行连续培养。应用PCR、逆转录PCR(RT-PCR)和免疫细胞化学方法检测hTERT基因的整合及表达情况,并用10%β-巯基乙醇(β-ME)、反式维甲酸(RA)、Forskolin和碱性成纤维细胞生长因子(bFGF)对转化后的hMSC-hTERT细胞进行诱导,使其向神经元样细胞分化。结果外源性hTERT基因在DNA、RNA和蛋白质水平均有表达,诱导后的hMSC-hTERT细胞表达神经元特异性标志物神经核蛋白(NeuN)和βⅢ微管蛋白(Tubulin-βⅢ)。hMSC-hTERT细胞至今已传至82代。结论外源hTERT基因在hMSC细胞中稳定整合并表达,永生化的hMSC细胞系成功建立;在一定诱导条件下,永生化的hMSC细胞能够在体外分化成神经元样细胞。更多还原

【Abstract】 Objective To establish an immortalized marrow stromal cell line for basic and clinical research of neuroscience use. Methods Human telomerase reverse transcriptase (hTERT) cDNA was transferred into human marrow stromal cells (hMSCs) by retroviral vector pLPChTERT. PCR, RT-PCR, and immunocytochemical methods were used to detect integration and expression of ectopic hTERT gene. Subcultured hMSC-hTERT cells were induced to differentiate into neuron-like cells usingβ-mercaptoethanol, trans-retinoic acid, forskolin, and basic fibroblast growth factor (bFGF). Results Expression of hTERT gene can be detected on DNA, RNA, and at protein level. hMSC-hTERT cells expressed neuron-specific makers NeuN and tubulin β-Ⅲ after induction. The 82nd passage of hMSC-hTERT cells has been obtained so far. Conclusion Ectopic hTERT gene has been stably integrated and expressed in hMSCs, therefore, immortalized hMSC-hTERT cell line is successfully established. Under certain conditions, hMSC-hTERT cells can be differentiated into neuron-like cells.更多还原