【摘要】 背景:目前小鼠胚胎干细胞体外向神经方向诱导分化的研究报道诸多,但其分化的过程很难控制,很多实验的操作方法复杂,分化比率也很低。目的:建立一种稳定、高效、简便的实验方法,将小鼠胚胎干细胞诱导分化为神经细胞。设计、时间及地点:对比观察实验,于2006-08/2008-01在中国医科大学发育生物学教研室完成。材料:小鼠胚胎干细胞系AB2.1,由美国南加州大学医学院惠赠。方法:在饲养层细胞上培养小鼠胚胎干细胞,采用"序贯诱导法",优化实验条件,将小鼠胚胎干细胞向神经方向诱导,对诱导后细胞进行成熟神经细胞标志物鉴定,并用流式细胞仪测定诱导分化比率。主要观察指标:①倒置荧光显微镜下观察小鼠胚胎干细胞形态特征。②优化序贯诱导法的3个实验条件。③实验条件优化后诱导至第10天分化细胞的形态特征。④诱导后第10天细胞免疫荧光法和蛋白免疫印迹检测神经细胞特异性标志物的表达。⑤蛋白免疫印迹检测诱导分化第4天和第10天细胞的神经细胞β-微管蛋白J1的表达。⑥流式细胞仪检测神经元特异性核蛋白阳性细胞百分比。结果:①生长于饲养层上的未分化鼠胚胎干细胞聚集呈圆形或椭圆形集落样生长,细胞排列紧密,集落边界清楚。②优化后的"序贯诱导法"在诱导时首次接种所用培养液的血清浓度为12.5%,小鼠胚胎干细胞首次接种最佳密度为1.0×108L-1,完全更换为无血清培养液的时间为第5天。③诱导至第10天分化细胞出现的突起可达胞体长度的10倍以上。也有小部分分化细胞胞体呈多边形,有多个短突起,仅有一个细长突起,出现突触样结构和生长端样结构,类似神经元。④诱导后第10天细胞免疫荧光法检测神经元特异性标志物巢蛋白、神经细胞β-微管蛋白J1、神经元特异性核蛋白、神经细胞黏附分子1表达阳性,神经胶质细胞标志物胶质细胞纤维酸性蛋白表达阴性。⑤与未分化鼠胚胎干细胞相比,诱导分化第4天和第10天的细胞蛋白免疫印迹检测神经细胞β-微管蛋白J1表达升高(P<0.05)。⑥流式细胞仪检测神经元特异性核蛋白阳性细胞百分比,计算出诱导第10天胚胎干细胞分化比率为(89.91±2.03)%。结论:应用优化后的"序贯诱导法",可稳定、简便的诱导小鼠胚胎干细胞高比率分化为神经元样细胞。更多还原

【Abstract】 BACKGROUND:Nowadays,there are plenty of the investigations of embryonic stem cells,especially of neural induced differentiation.But the differentiation in vitro is poorly controlled,the methods are complicated,and the proportions of differentiation are not very high.OBJECTIVE:To establish a method characterizing stabilization,high efficacy,and convenience to induce mouse embryonic stem cells(mESCs) to differentiate into neural cells.DESIGN,TIME AND SETTING:A contrast study was performed at Department of Developmental Biology,China Medical University from August 2006 to January 2008.MATERIALS:mESCs AB2.1 was provided by Medical College of University of Southern California,USA.METHODS:mESCs were cultured on PMEF feeder cells.The conditions of sequential neural differentiation were optimized,neural specific markers were determined,and the differentiation proportion was detected using flow cytometry.MAIN OUTCOME MEASURES:① Morphology of mESCs was observed under inverted fluorescence microscope;② three conditions of sequential method;③ morphology of mESCs at 10 days after differentiation;④ specific marker expression was detected using immumofluorescence method and Western blot method at 10 days after induction;⑤ neurocyte β-microtubulin J1 expression was detected using Western blot method at day 4 and 10 after differentiation;⑥ neuron specific nuclear protein-positive cell percentage was detected using flow cytometry.RESULTS:① Undifferentiated mESCs were aggregated like round or oval-shape colony,tightly arrayed with clear boundary.② The concentration of culture solution for the first time was 12.5% using optimized sequential method.The best density of mESCs for the first time was 1.0×108 /L,and on the fifth day,the culture solution was replaced by serum-free culture media.③ The process observed at day 10 after differentiation was 10 times as cell body.A few of differentiated cells were polygon with some short processes but one long process.Synapse-like structure and growing end-like structure were observed like neurons.④ Expressions of neuron-specific Nestin,neurocyte β-microtubulin J1,neuron specific nuclear protein,and neural cell adhesion molecule-1 were positive,but glia fiber acidic protein expression was negative.⑤ Western blot method demonstrated that neurocyte β-microtubulin J1 expression of differentiated cells was increased compared to that of undifferentiated cells at day 4 and 10 after differentiation(P < 0.05).⑥ Neuron specific nuclear protein positive cell percentage was detected using flow cytometery to calculate differentiation ratio of mESCs at day 10,which was(89.91±2.03)%.CONCLUSION:The optimized sequential neural induction can induce mESCs into neuron-like cells effectively and stably.更多还原