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  • 纪琼琼,李明华,王培军.不同方法诱导小鼠胚胎成纤维细胞直接转化为神经干细胞效率的比较[J].同济大学学报(医学版),2019,30(3):268-273.    [点击复制]
  • JI Qiong-qiong,LI Ming-hua,WANG Pei-jun.Comparison of different methods for induction of mouse embryonic fibroblasts into neural stem cells[J].同济大学学报(医学版),2019,30(3):268-273.   [点击复制]
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不同方法诱导小鼠胚胎成纤维细胞直接转化为神经干细胞效率的比较
纪琼琼,李明华,王培军
0
(同济大学附属同济医院医学影像科,上海 200065)
摘要:
目的 比较不同方法诱导小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs)直接转化为诱导型神经干细胞(induced neural stem cells, iNSCs)的效率,以进一步优化诱导方法。方法 构建慢病毒表达载体pLenti6.3-Sox2-IRES2-EGFP,分别采用单转录因子Sox2、小分子物质、转录因子Sox2联合小分子物质共同作用三种方法将MEFs直接转化为iNSCs。qPCR检测神经干细胞的标志基因和多潜能标志基因的表达。免疫荧光检测神经干细胞的标记物nestin,计算iNSCs的转染效率。iNSCs在分化培养基中贴壁培养7~14d,免疫荧光分别检测神经元、星形胶质细胞和少突胶质细胞的细胞标记物MAP2、GFAP和Olig2的表达。结果 诱导后4~6d,MEFs的形态发生明显改变。免疫荧光显微镜下的结果显示诱导因子Sox2加小分子的诱导方法较单独使用Sox2和单独使用小分子物质进行诱导的效率高,差异具有统计学意义(P<0.05)。qPCR的结果证明,3组iNSCs多种神经干细胞的标志基因(Sox2、nestin、Blbp、Pax6)的表达均较MEFs明显增高,3组间差异无统计学意义(P>0.05);共聚焦显微镜的结果证明3种方法诱导的iNSCs具有分化为神经元、星形胶质细胞、少突胶质细胞的能力,3组间差异无统计学意义(P>0.05)。结论 转录因子Sox2联合小分子共同作用可显著提高MEFs转化为iNSCs的效率且并不改变iNSCs的细胞功能特点。3组的iNSCs均具有神经干细胞自我增殖和多向分化的能力。
关键词:  Sox2  小分子  小鼠胚胎成纤维干细胞  诱导型神经干细胞
DOI:10.16118/j.1008-0392.2019.03.002
投稿时间:2018-12-11
基金项目:国家自然科学基金(81571655)
Comparison of different methods for induction of mouse embryonic fibroblasts into neural stem cells
JI Qiong-qiong,LI Ming-hua,WANG Pei-jun
(Dept. of Medical Imaging, Tongji Hospital, Tongji University, Shanghai 200065, China)
Abstract:
Objective To compare different methods for induction of mouse embryonic fibroblasts (MEFs) into induced neural stem cells (iNSCs). Methods The lentiviral vector pLenti6.3-Sox2-IRES2-EGFP was constructed and the mouse MEFs were directly transformed into iNSCs by using the transcription factor Sox2 (group S), the small molecule substance (group M) or transcription factor Sox2 with small molecular substances (group S+M). The efficiency of the transfection was calculated by immunofluorescence. The expressions of marker genes of neural stem cells and pluripotency were detected by qPCR. The iNSCs were cultured in differentiation medium for 7-14 days, immunofluorescence was performed to detect the differentiative capacity of neural stem cells. Results The morphology of MEFs changed significantly 4-6d after transduction. The results of immunofluorescence microscopy showed that the transduction efficiency as shown by nestin positive colonies in S+M group was significantly higher than that in S group and M group (P<0.05). Quantitative real-time RT-PCR confirmed that nestin, Blbp and Pax6 were expressed in iNSCs in all three groups, and there was no significant difference in expression levels among the three groups (P>0.05). Immunofluorescence demonstrated that iNSCs induced with three methods had the ability to differentiate into neurons, astrocytes and oligodendrocytes. Conclusion The combination of Sox2 and small molecules can significantly improve the transformation efficiency of MEFs into iNSCs, but the function of iNSCs induced by different methods is not different.
Key words:  Sox2  small molecule  mouse embryonic fibroblasts  induced neural stem cells

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