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胶质源性神经生长因子在人胚胎神经干细胞中的表达调控

, PP. 868-875

Keywords: 神经干细胞,胶质源性神经营养因子,慢病毒载体,四环素调控

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Abstract:

胶质源性神经生长因子(glialcellderivedneurotrophicfactor,GDNF)对帕金森病等神经退行性疾病的治疗有广泛的应用前景,而人胚胎神经干细胞已被证实是一种将GDNF安全而有效地携带到受损脑区的良好细胞载体.本研究利用含有GDNF基因的可调控慢病毒载体转染人胚胎神经干细胞,实现了GDNF在人胚胎神经干细胞在体外培养条件下的表达调控.实验中首先利用含有GDNF基因的慢病毒载体成功转染人胚胎神经干细胞,其中GDNF基因受四环素操纵子7(tetracyclineoperator7,tetO7)调控,由延长因子1(elongationfactor1,EF1-a)驱动;转染后的神经干细胞经过潮霉素筛选后同时转入对下游基因起抑制作用的慢病毒载体TTS(tetra-cyclinerepressorfusionprotein,TTS),TTS蛋白表达后可与tetO7相互作用抑制GDNF在人神经干细胞中的表达.当加入强力霉素(doxycycline,DOX)后,TTS与tetO7解离,GDNF表达可恢复到未调控前水平.转染GDNF后的人胚胎神经干细胞仍然表达神经干细胞特异性抗原nestin,Sox2;放线菌素D诱导凋亡后,转染GDNF组较正常对照组凋亡细胞比例有显著降低;同时,转染GDNF的神经干细胞分化成神经元的比例有显著提高.利用可调控的慢病毒调控GDNF在人胚胎神经干细胞中的表达为GDNF应用于神经退行性疾病的治疗提供了实验数据.

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