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胶质源性神经生长因子(glial cell derived neurotrophic factor,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(tetracy cline operator7,tetO7)调控,由延长因子1(elongation factor1,EF1-)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-cycline repressorfusi onprotein,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.
Glial cell derived neurotrophic factor (GDNF) has broad application prospects for the treatment of neurodegenerative diseases such as Parkinson’s disease, while human embryonic neural stem cells have been proved to be a safe and effective way to GDNF Carried to the damaged brain area of a good cell carrier.In this study, the use of GDNF gene containing regulatory lentiviral vector transfected human embryonic neural stem cells, to achieve GDNF in human embryonic neural stem cells cultured in vitro conditions regulation.In the experiment first Human embryonic neural stem cells were successfully transfected with lentiviral vector containing GDNF gene. The GDNF gene was regulated by tetracycline operator7 (tetO7) and driven by elongation factor 1 (EF1-). After transfection After being screened by hygromycin, the neural stem cells are transfected into the lentiviral vector TTS (tetrazine (TTS)), which inhibits the downstream genes. The expression of TTS protein can interact with tetO7 to inhibit the expression of GDNF in human neural stem cells After addition of doxycycline (DOX), TTS dissociated from tetO7 and GDNF expression returned to the unregulated level.Transfection of G After DNF, the human embryonic neural stem cells still expressed neural stem cell-specific antigen nestin, Sox2; after actinomycin D-induced apoptosis, the percentage of apoptotic cells in GDNF transfected group was significantly lower than that in normal control group; meanwhile, The percentage of neural stem cells differentiated into neurons is significantly increased.Controlling the expression of GDNF in human embryonic neural stem cells using a regulatable lentivirus provides experimental data for the application of GDNF in the treatment of neurodegenerative diseases.