本文選題：人胚胎干細(xì)胞　切入點(diǎn)：擬胚體　出處：《山東大學(xué)》2006年碩士論文

【摘要】：第一部分：人自體基因胚胎干細(xì)胞體外定向誘導(dǎo)分化神經(jīng)元樣細(xì)胞 目的：初步建立人自體基因胚胎干細(xì)胞向神經(jīng)元樣細(xì)胞分化的培養(yǎng)體系，為神經(jīng)系統(tǒng)細(xì)胞替代治療提供一種含有人自體基因、無免疫排斥反應(yīng)、具有高分化潛能的全新種子細(xì)胞。 方法：將人自體基因胚胎干細(xì)胞制備的擬胚體(EBs)分別懸浮培養(yǎng)4、6、8、10天后，使其貼壁，加入N_2培養(yǎng)液和20ng／mL bFGF誘導(dǎo)其分化形成神經(jīng)干細(xì)胞，分離神經(jīng)干細(xì)胞進(jìn)行懸浮培養(yǎng)，檢測其神經(jīng)干細(xì)胞標(biāo)記物巢蛋白(nestin)，體外擴(kuò)增，將神經(jīng)干細(xì)胞貼壁后加入N_2培養(yǎng)液和10ng／mL BDGF誘導(dǎo)其分化為神經(jīng)元樣細(xì)胞；并通過比較：①不同培養(yǎng)方法形成EB的效率。②不同直徑大小的EBs分化為神經(jīng)干細(xì)胞的效率。③培養(yǎng)不同時(shí)間的EBs貼壁后出現(xiàn)神經(jīng)分化的比例。④不同誘導(dǎo)方法獲得神經(jīng)元樣細(xì)胞的效率，以初步探討影響人自體基因胚胎干細(xì)胞向神經(jīng)元樣細(xì)胞分化的體外培養(yǎng)因素。 結(jié)果：①胚胎干細(xì)胞經(jīng)序貫法誘導(dǎo)分化為神經(jīng)元樣細(xì)胞，，表現(xiàn)為：細(xì)胞形成短的突起，有的相互形成突觸狀的連接，且免疫細(xì)胞化學(xué)染色顯示神經(jīng)元標(biāo)記物MAP_2，β-tubulin，NSE染色陽性。②經(jīng)低粘附六孔板懸浮培養(yǎng)法形成的EBs數(shù)多于懸滴法和細(xì)菌培養(yǎng)皿懸浮培養(yǎng)法。③小EBs的分化效率較低，中等EBs的分化效率最高，大EBs的分化效率介于兩者之間。④培養(yǎng)6、8天的EBs誘導(dǎo)分化得到的神經(jīng)干細(xì)胞多于培養(yǎng)4、10天的EBs。⑤采用序貫誘導(dǎo)法所得神經(jīng)元樣細(xì)胞的比例明顯高于RA組和自發(fā)分化組。 結(jié)論：①初步建立了人自體基因胚胎干細(xì)胞向神經(jīng)元樣細(xì)胞分化的誘導(dǎo)體系：序貫誘導(dǎo)法得到14.2％的神經(jīng)元樣細(xì)胞。②低粘附六孔板懸浮培養(yǎng)法比懸滴和細(xì)菌培養(yǎng)皿懸浮培養(yǎng)法更利于EBs的形成。③中等大小的EBs向神經(jīng)分化的效率最高。④EBs培養(yǎng)6-8天是其向神經(jīng)分化的最佳時(shí)段⑤序貫誘導(dǎo)法比RA法產(chǎn)
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 袁淑蘭,黃光琦,黃韌敏,宋毅;丹參酮體外誘導(dǎo)分化作用及其機(jī)制的研究[J];癌癥;1996年06期

2 賈太蓮,朱風(fēng)蘭,王崢;復(fù)方丹參注射液治療新生兒缺氧缺血性腦病100例[J];實(shí)用兒科臨床雜志;2004年08期

3 何麗娜,何素冰,楊軍,王靜,劉超;丹參酮對原代大鼠神經(jīng)細(xì)胞缺血性損傷的保護(hù)作用[J];中國藥理學(xué)通報(bào);2001年02期

4 劉相名,楊立業(yè),苗宏生,孫兵,栗超躍,惠國楨,郭禮和;脂肪組織來源的多能干細(xì)胞培養(yǎng)和外源基因的表達(dá)[J];中華實(shí)驗(yàn)外科雜志;2003年02期

5 夏文杰,張秀明,李艷,李樹濃,陳振光,張麗蓉,項(xiàng)鵬;隱丹參酮誘導(dǎo)骨髓間質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞的實(shí)驗(yàn)研究[J];中國中西醫(yī)結(jié)合雜志;2002年12期

本文編號：1697805