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
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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,i NSCs)的效率,以进一步优化诱导方法。方法构建慢病毒表达载体p Lenti6. 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i NSCs。q PCR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i NSCs的转染效率。i NSCs在分化培养基中贴壁培养7～14 d,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4～6 d,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)。q PCR的结果证明,3组i NSCs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i NSCs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i NSCs的效率且并不改变i NSCs的细胞功能特点。3组的i NSCs均具有神经干细胞自我增殖和多向分化的能力。
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 4-6 d 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.
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 4-6 d 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.
引文
[1]SABELSTROM H,STENUDD M,FRISEN J.Neural stem cells in the adult spinal cord[J].Exp Neurol,2014,260:44-49.
[2]ZHANG W,GU G J,ZHANG Q,et al.NSCs promote hippocampal neurogenesis,metabolic changes and synaptogenesis in APP/PS1 transgenic mice[J].Hippocampus,2017,27(12):1250-1263.
[3]PACHERNIK J,ESNER M,BRYJA V,et al.Neural differentiation of mouse embryonic stem cells grown in monolayer[J].Reprod Nutr Dev,2002,42(4):317-326.
[4]TAKAHASHI K,YAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126(4):663-676.
[5]HAN D W,TAPIA N,HERMANN A,et al.Direct reprogramming of fibroblasts into neural stem cells by defined factors[J].Cell Stem Cell,2012,10(4):465-472.
[6]RING K L,TONG L M,BALESTRA M E,et al.Direct reprogramming of mouse and human fibroblasts into multipotent neural stem cells with a single factor[J].Cell Stem Cell,2012,11(1):100-109.
[7]HAN Y C,LIM Y,DUFFIELDL M D,et al.Direct reprogramming of mouse fibroblasts to neural stem cells by small molecules[J].Stem Cells Int,2016,2016:4304916.
[8]胡馨予,高正良,徐俊,等.2-DG对神经干细胞增殖和分化影响的研究[J].同济大学学报(医学版),2016,37(6):35-40.
[9]KIM S M,KIM J W,KWAK T H,et al.Generation of integration-free induced neural stem cells from mouse fibroblasts[J].J Biol Chem,2016,291(27):14199-14212.
[10]THIER M,WORSDORFER P,LAKES Y B,et al.Direct conversion of fibroblasts into stably expandable neural stem cells[J].Cell Stem Cell,2012,10(4):473-479.
[11]HUANGFU D W,OSAFUNE K,MAEHR R,et al.Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2[J].Nat Biotechnol,2008,26(11):1269-1275.
[12]SHI Y,DO J T,DESPONTS C,et al.A combined chemical and genetic approach for the generation of induced pluripotent stem cells[J].Cell Stem Cell,2008,2(6):525-528.
[13]LEE J P,JEYAKUMAR M,GONZALEZ R,et al.Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease[J].Nat Med,2007,13(4):439-447.
[14]YANG W F,WEI W,SHI C,et al.Pluripotin combined with leukemia inhibitory factor greatly promotes the derivation of embryonic stem cell lines from refractory strains[J].Stem Cells,2009,27(2):383-389.
[15]TOJO M,HAMASHIMA Y,HANYU A,et al.The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelialto-mesenchymal transition by transforming growth factor-beta[J].Cancer Sci,2005,96(11):791-800.
[16]ESTEBAN M A,WANG T,QIN B M,et al.Vitamin C enhances the generation of mouse and human induced pluripotent stem cells[J].Cell Stem Cell,2010,6(1):71-79.
[2]ZHANG W,GU G J,ZHANG Q,et al.NSCs promote hippocampal neurogenesis,metabolic changes and synaptogenesis in APP/PS1 transgenic mice[J].Hippocampus,2017,27(12):1250-1263.
[3]PACHERNIK J,ESNER M,BRYJA V,et al.Neural differentiation of mouse embryonic stem cells grown in monolayer[J].Reprod Nutr Dev,2002,42(4):317-326.
[4]TAKAHASHI K,YAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126(4):663-676.
[5]HAN D W,TAPIA N,HERMANN A,et al.Direct reprogramming of fibroblasts into neural stem cells by defined factors[J].Cell Stem Cell,2012,10(4):465-472.
[6]RING K L,TONG L M,BALESTRA M E,et al.Direct reprogramming of mouse and human fibroblasts into multipotent neural stem cells with a single factor[J].Cell Stem Cell,2012,11(1):100-109.
[7]HAN Y C,LIM Y,DUFFIELDL M D,et al.Direct reprogramming of mouse fibroblasts to neural stem cells by small molecules[J].Stem Cells Int,2016,2016:4304916.
[8]胡馨予,高正良,徐俊,等.2-DG对神经干细胞增殖和分化影响的研究[J].同济大学学报(医学版),2016,37(6):35-40.
[9]KIM S M,KIM J W,KWAK T H,et al.Generation of integration-free induced neural stem cells from mouse fibroblasts[J].J Biol Chem,2016,291(27):14199-14212.
[10]THIER M,WORSDORFER P,LAKES Y B,et al.Direct conversion of fibroblasts into stably expandable neural stem cells[J].Cell Stem Cell,2012,10(4):473-479.
[11]HUANGFU D W,OSAFUNE K,MAEHR R,et al.Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2[J].Nat Biotechnol,2008,26(11):1269-1275.
[12]SHI Y,DO J T,DESPONTS C,et al.A combined chemical and genetic approach for the generation of induced pluripotent stem cells[J].Cell Stem Cell,2008,2(6):525-528.
[13]LEE J P,JEYAKUMAR M,GONZALEZ R,et al.Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease[J].Nat Med,2007,13(4):439-447.
[14]YANG W F,WEI W,SHI C,et al.Pluripotin combined with leukemia inhibitory factor greatly promotes the derivation of embryonic stem cell lines from refractory strains[J].Stem Cells,2009,27(2):383-389.
[15]TOJO M,HAMASHIMA Y,HANYU A,et al.The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelialto-mesenchymal transition by transforming growth factor-beta[J].Cancer Sci,2005,96(11):791-800.
[16]ESTEBAN M A,WANG T,QIN B M,et al.Vitamin C enhances the generation of mouse and human induced pluripotent stem cells[J].Cell Stem Cell,2010,6(1):71-79.