小鼠来源诱导多能干细胞定向分化为神经元的体外实验研究

发布时间：2018-08-25 07:59

【摘要】：目的:探讨源于小鼠的诱导多能干细胞(induced pluripotent stem cells,iPSCs)体外定向分化为神经元的方法,为大量稳定地获得神经元及提供种子细胞治疗脊髓损伤奠定体外实验基础。方法:源于小鼠的iPSCs在不同的诱导培养基中经悬浮、贴壁、再悬浮和再贴壁培养。免疫荧光检测iPSCs多能性标志物、神经干细胞及其分化细胞标志物的表达。RT-PCR检测神经元及胶质细胞基因表达并行神经元基因测序鉴定,流式细胞术检测iPSCs定向分化为神经干细胞及进一步分化为神经元的比例。结果:源于小鼠的iPSCs表达干细胞多能性标志物Sox2、Oct4和SSEA1。悬浮培养可以形成类球形拟胚体;经诱导及机械分离后的细胞可形成神经球,神经球经诱导后可分化为神经元。免疫荧光显示iPSCs可诱导出表达nestin的神经球,贴壁培养的神经球可分化为分别表达微管相关蛋白2(MAP-2)、胶质细胞原纤维酸性蛋白(GFAP)及髓磷脂碱性蛋白(MBP)的神经元、星形胶质细胞和少突胶质细胞。RT-PCR及基因鉴定结果显示形成小鼠神经元;流式细胞术检测结果显示分化细胞中神经干细胞和神经元的比例分别为63.93%±1.47%和21.40%±1.70%。结论:源于小鼠的诱导多能干细胞能够稳定地在体外定向分化为神经元,为脊髓损伤的治疗提供了一个可靠的细胞来源。
[Abstract]:Aim: to investigate the method of inducing pluripotent stem cells (induced pluripotent stem cells,iPSCs) derived from mice to differentiate into neurons in vitro, so as to provide in vitro experimental basis for the stable acquisition of neurons and the provision of seed cells for the treatment of spinal cord injury (sci). Methods: iPSCs derived from mice was cultured in different culture medium, such as suspension, adhesion, resuspension and reattachment. The expression of multipotent markers of iPSCs, neural stem cells and differentiated cell markers were detected by immunofluorescence. RT-PCR was used to detect the gene expression of neurons and glial cells. The proportion of iPSCs differentiated into neural stem cells and further differentiated into neurons was detected by flow cytometry. Results: iPSCs derived from mice expressed stem cell pluripotent markers Sox2,Oct4 and SSEA1.. Suspension culture can form globular embryoid body, and the cells after induction and mechanical separation can form neuronal spheres, which can be differentiated into neurons after induction. Immunofluorescence showed that iPSCs could induce the expression of nestin, and the adherent neurons could differentiate into neurons expressing microtubule-associated protein 2 (MAP-2), glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP), respectively. The results of RT-PCR and gene identification of astrocytes and oligodendrocytes showed that mouse neurons were formed, and the percentages of neural stem cells and neurons in differentiated cells were 63.93% 卤1.47% and 21.40% 卤1.70%, respectively. Conclusion: the induced pluripotent stem cells derived from mice can stably differentiate into neurons in vitro and provide a reliable cell source for the treatment of spinal cord injury.
【作者单位】：中山大学附属第三医院脊柱外科;
【基金】：国家自然科学基金资助项目(No.31170947) 广东省自然科学基金资助项目(No.S2012020011099;No.S2013010016413) 广州市科技计划(No.2013J4100062)
【分类号】：Q813;R318

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