干细胞体外定向诱导分化神经元样细胞的研究

发布时间：2018-04-01 23:30

本文选题：人胚胎干细胞　切入点：拟胚体　出处：《山东大学》2006年硕士论文

【摘要】：第一部分：人自体基因胚胎干细胞体外定向诱导分化神经元样细胞目的：初步建立人自体基因胚胎干细胞向神经元样细胞分化的培养体系，为神经系统细胞替代治疗提供一种含有人自体基因、无免疫排斥反应、具有高分化潜能的全新种子细胞。方法：将人自体基因胚胎干细胞制备的拟胚体(EBs)分别悬浮培养4、6、8、10天后，使其贴壁，加入N_2培养液和20ng／mL bFGF诱导其分化形成神经干细胞，分离神经干细胞进行悬浮培养，检测其神经干细胞标记物巢蛋白(nestin)，体外扩增，将神经干细胞贴壁后加入N_2培养液和10ng／mL BDGF诱导其分化为神经元样细胞；并通过比较：①不同培养方法形成EB的效率。②不同直径大小的EBs分化为神经干细胞的效率。③培养不同时间的EBs贴壁后出现神经分化的比例。④不同诱导方法获得神经元样细胞的效率，以初步探讨影响人自体基因胚胎干细胞向神经元样细胞分化的体外培养因素。结果：①胚胎干细胞经序贯法诱导分化为神经元样细胞，，表现为：细胞形成短的突起，有的相互形成突触状的连接，且免疫细胞化学染色显示神经元标记物MAP_2，β-tubulin，NSE染色阳性。②经低粘附六孔板悬浮培养法形成的EBs数多于悬滴法和细菌培养皿悬浮培养法。③小EBs的分化效率较低，中等EBs的分化效率最高，大EBs的分化效率介于两者之间。④培养6、8天的EBs诱导分化得到的神经干细胞多于培养4、10天的EBs。⑤采用序贯诱导法所得神经元样细胞的比例明显高于RA组和自发分化组。结论：①初步建立了人自体基因胚胎干细胞向神经元样细胞分化的诱导体系：序贯诱导法得到14.2％的神经元样细胞。②低粘附六孔板悬浮培养法比悬滴和细菌培养皿悬浮培养法更利于EBs的形成。③中等大小的EBs向神经分化的效率最高。④EBs培养6-8天是其向神经分化的最佳时段⑤序贯诱导法比RA法产
[Abstract]:Part I: in vitro directional induction of neuron-like cells by human autologous embryonic stem cells. Objective: to establish a culture system for the differentiation of human autologous embryonic stem cells into neuron-like cells, and to provide a kind of human autologous gene for neuronal cell replacement therapy without immune rejection. New seed cells with high differentiation potential. Methods: human embryonic stem cells derived from human autologous gene embryonic stem cells were cultured in vitro for 10 days. After suspension culture for 10 days, the embryonic stem cells were induced to differentiate into neural stem cells (NSCs) by adding 20ng/mL bFGF and N _ 2 medium to induce them to differentiate into neural stem cells (NSCs). The neural stem cells (NSCs) were isolated for suspension culture. The neural stem cell marker nestin was detected and expanded in vitro. The neural stem cells were induced to differentiate into neuron-like cells by adding NST2 culture medium and 10ng/mL BDGF. By comparing the efficiency of different culture methods to form EB. 2. The efficiency of differentiation of different diameter EBs into neural stem cells. 3. 3. 3. The proportion of neural differentiation after EBs adherent for different time. 4. 4 different induction methods to obtain nerve. The efficiency of meta-like cells, To explore the factors affecting the differentiation of human autologous embryonic stem cells into neuron-like cells in vitro. Results the stem cells were induced to differentiate into neuron-like cells by the sequential method, which showed that the cells formed short processes and some formed synaptic connections with each other. Immunocytochemical staining showed that the number of EBs formed by low adhesion six-well plate suspension culture method was higher than that by suspension culture method and the differentiation efficiency of 尾 -tubulin NSE positive staining was lower than that of suspension culture method and bacterial culture dish suspension culture method, and the differentiation efficiency of 尾 -tubulin NSE positive staining was lower than that of suspension culture method. The differentiation efficiency of medium EBs was the highest. The differentiation efficiency of large EBs was higher than that of EBs.5 cultured for 4 days. The percentage of neuron-like cells obtained by Sequential induction method was significantly higher than that in RA group and spontaneous differentiation group. Conclusion the induction system of differentiation of human autologous embryonic stem cells into neuron-like cells was preliminarily established by 1: 1: 14.2% of neuron-like cells were obtained by sequential induction method with low adhesion six-hole plate suspension culture method compared with suspension drop and culture dish suspension. Floatation culture method is more conducive to the formation of EBs. 3 medium size EBs has the highest efficiency of neural differentiation. 4 EBs culture for 6 to 8 days is the best time for nerve differentiation. 5 sequential induction method is more effective than RA method.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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