2种诱导iPSC向神经干细胞分化方法的比较

发布时间：2018-01-19 08:19

本文关键词： 诱导性多能干细胞分化神经干细胞　出处：《中国病理生理杂志》2015年01期 　论文类型：期刊论文

【摘要】：目的:整体比较2种促进诱导性多能干细胞(induced pluripotent stem cells,i PSC)向神经干细胞(neural stem cells,NSC)分化的方法,确定一种稳定、高效的获得NSC的方法,并对NSC进行系统鉴定。方法:方法A:SB431542和drosomophorin的浓度均为5μmmol/L,诱导初始密度100%;方法 B:SB431542的浓度为5 mmol/L,drosomophorin的浓度为1 mmol/L,诱导初始密度为40%。比较及鉴定方法:镜下观察诱导获得NSC的状态;realtime PCR比较神经干细胞相关基因Pax6、nestin、Sox1、Sox2等表达量;流式细胞术分析诱导第16天Pax6阳性率;免疫荧光定性分析神经干细胞相关蛋白的表达及其自发分化的能力。结果:方法 A获得的NSC悬起后成球趋势明显,圆形,透明;方法 B诱导获得NSC形状不规则,色灰暗。Real-time PCR结果证明方法 A诱导获得的细胞神经干细胞相关基因的表达量高于方法 B。流式细胞术分析证明第16天,PAX6的阳性率,方法 A高于方法 B。经鉴定,方法 A获得的神经干细胞高表达Pax6、nestin、Sox2等基因自发分化30 d,形成明显的神经纤维束,表达TUJ-1、MAP2及GFAP等神经元和胶质细胞的特异性标志物。结论:方法 A整体优于方法 B,我们推荐方法 A作为诱导i PSC向神经干细胞分化的方法。
[Abstract]:Objective: to compare two kinds of induced pluripotent stem cells for promoting inducible pluripotent stem cells. The method of differentiation into neural stem cells of neural stem cells was used to determine a stable and efficient method for obtaining NSC. Methods: the concentrations of drosomophorin and A: SB431542 were 5 渭 mol / L, and the initial induction density was 100 渭 mol / L; Methods the concentration of B: SB431542 was 5 mmol / L drosomophorin for 1 mmol/L. The initial density of induction was 40. Comparison and identification methods: the state of NSC was observed under microscope. Realtime PCR was used to compare the expression of neural stem cell related gene Pax6nestinine, Sox1, Sox2 and so on. The positive rate of Pax6 was analyzed by flow cytometry on the 16th day after induction. Results: after suspension of NSC obtained by A, the globular tendency was obvious, round and transparent. Methods the shape of NSC was irregular induced by B. The results of gray and dark. Real-time PCR showed that the expression of neural stem cell related genes induced by method A was higher than that by B. flow cytometry on the 16th day. The positive rate of PAX6 was higher in method A than that in method B. the neural stem cells obtained by method A were highly expressed in Pax6netin Sox2 gene for 30 days. The specific markers of neurons and glial cells, such as TUJ-1MAP2 and GFAP, were expressed in the formation of obvious nerve fiber bundles. Conclusion: method A is superior to method B. We recommend method A as a method to induce I PSC to differentiate into neural stem cells.
【作者单位】：山东省医学科学院基础医学研究所;济南大学-山东省医学科学院医学与生命科学学院;山东大学附属济南市中心医院中心实验室;山东省移植与组织工程研究中心;
【基金】：973计划资助项目(No.2012CB966503;No.2012CB966504) 国家青年科学基金资助项目(No.81200950)
【分类号】：R329
【正文快照】： 神经干细胞(neural stem cells,NSC)可用于建立神经系统疾病的细胞模型,因此在神经系统疾病的机制及治疗的研究中日趋重要[1]。神经干细胞获得有以下几个途径:(1)从流产的胎儿脑组织中分离[2],体外扩增,但该来源有限,且受到伦理约束;(2)人胚胎干细胞(human embryonic stem cel

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