过表达Sox2基因可增强人骨髓干细胞向神经元分化（英文）
|
摘要
目的:研究Sox2对于人骨髓间充质干细胞(hBMSCs)向神经元跨分化能力的影响。方法:利用Western Blot检测hBMSCs中Sox2、Oct4和c-Myc的表达,将携带Sox2和rhGFP编码序列的Tet-On慢病毒感染hBMSCs,通过多西环素(Dox)处理10 d诱导Sox2和rhGFP表达;利用神经诱导培养基诱导感染慢病毒的hBMSCs分化,通过免疫荧光染色技术检测神经元标记物Tuj1的表达;利用Western Blot技术检测Smad 2/3信号通路蛋白在hBMSCs向神经元分化过程中的表达水平。结果:Western Blot结果显示hBMSCs不仅表达Sox2和Oct4,同时还表达c-Myc;光镜下发现Sox2的过表达促进hBMSCs的形态向神经元样细胞转化,同时通过免疫荧光染色发现Tuj1的表达水平增加; Western Blot证实Sox2基因过表达引起hBMSCs中磷酸化Smad 2/3的表达水平降低。结论:Sox2可通过Smad 2/3信号通路增强人骨髓间充质干细胞向神经元分化。
Objective: To study the effects of sex determining region Y box containing gene 2( Sox2) on human bone marrow mesenchymal stem cells( hBMSCs) trans-differentiation into neurons. Methods: The expression of Sox2,Oct,and c-Myc was tested using Western Blot. Tet-On lentivirus carrying Sox2 and rhGFP were infected into hBMSCs and its expression level was regulated by doxycycline( Dox). Then hBMSCs were cultured in the neuronal induction medium for 10 days. The expression of neuronal marker Tuj1 was detected by immunofluorescence staining. The expression level of p Smad 2/3 protein of hBMSCs was detected by Western Blot. Results: Western Blot results confirmed the expression of Sox2,Oct4,and c-Myc in hBMSCs. Over-expression of Sox2 promoted the ability of hBMSCs tans-differentiation into neuron-like cells. Immunofluorescence staining showed that the expression level of Tuj1 increased. Western Blot results also showed that the expression level of p Smad 2/3 decreased in hBMSCs when over-expression of Sox2. Conclusion:Sox2 could enhance neuronal differentiation of hBMSCs through the Smad 2/3 signaling pathway.
Objective: To study the effects of sex determining region Y box containing gene 2( Sox2) on human bone marrow mesenchymal stem cells( hBMSCs) trans-differentiation into neurons. Methods: The expression of Sox2,Oct,and c-Myc was tested using Western Blot. Tet-On lentivirus carrying Sox2 and rhGFP were infected into hBMSCs and its expression level was regulated by doxycycline( Dox). Then hBMSCs were cultured in the neuronal induction medium for 10 days. The expression of neuronal marker Tuj1 was detected by immunofluorescence staining. The expression level of p Smad 2/3 protein of hBMSCs was detected by Western Blot. Results: Western Blot results confirmed the expression of Sox2,Oct4,and c-Myc in hBMSCs. Over-expression of Sox2 promoted the ability of hBMSCs tans-differentiation into neuron-like cells. Immunofluorescence staining showed that the expression level of Tuj1 increased. Western Blot results also showed that the expression level of p Smad 2/3 decreased in hBMSCs when over-expression of Sox2. Conclusion:Sox2 could enhance neuronal differentiation of hBMSCs through the Smad 2/3 signaling pathway.
引文
[1]George PM,Steinberg GK.Novel stroke therapeutics:unraveling stroke pathophysiology and its impact on clinical treatments[J].Neuron,2015,87(2):297-309.DOI:10.1016/j.neuron.2015.05.041.
[2]Meybodi AT,Kim H,Nelson J,et al.Surgical treatment vs nonsurgical treatment for brain arteriovenous malformations in patients with hereditary hemorrhagic telangiectasia:a retrospective multicenter consortium study[J].Neurosurgery,2018,82(1):35-47.DOI:10.1093/neuros/nyx168.
[3]Gao YY,Zhang ZH,Zhuang Z,et al.Recombinant milk fat globuleEGF factor-8 reduces apoptosis via integrin hata3/FAK/PI3K/AKTsignaling pathway in rats after traumatic brain injury[J].Cell Death Dis,2018,9(9):845.DOI:10.1038/s41419-018-0939-5.
[4]Green NS,Bhatia M,Griffith EY,et al.Enhanced long-term brain magnetic resonance imaging evaluation of children with sickle cell disease after hematopoietic cell transplantation[J].Biol Blood Marrow Transplant,2017,23(4):670-676.DOI:10.1016/j.bbmt.2017.01.007.
[5]Wuebben EL,Rizzino A.The dark side of SOX2:cancer-a comprehensive overview[J].Oncotarget,2017,8(27):44917-44943.DOI:10.18632/oncotarget.16570.
[6]Sheng C,Jungverdorben J,Wiethoff H,et al.A stably self-renewing adult blood-derived induced neural stem cell exhibiting patternability and epigenetic rejuvenation[J].Nat Commun,2018,9(1):4047.DOI:10.1038/s41467-018-06398-5.
[7]Yang WH,Yang C,Xue YQ,et al.Regulated expression of lentivirus-mediated GDNF in human bone marrow-derived mesenchymal stem cells and its neuroprotection on dopaminergic cells in vitro[J].PLo S One,2013,8(5):e64389.DOI:10.1371/journal.pone.0064389.
[8]Erdlenbruch F,Rohwedel J,Ralfs P,et al.Generation of induced pluripotent stem cells(i PSCs)from human foreskin fibroblasts[J].Stem Cell Res,2018,33(4):79-82.DOI:10.1016/j.scr.2018.10.010.
[9]Bani-Yaghoub M,Tremblay RG,Lei JX,et al.Role of Sox2 in the development of the mouse neocortex[J].Dev Biol,2006,295(1):52-66.DOI:10.1016/j.ydbio.2006.03.007.
[10]Ellis P,Fagan BM,Magness ST,et al.SOX2,a persistent marker for multipotential neural stem cells derived from embryonic stem cells,the embryo or the adult[J].Dev Neurosci,2004,26(2-4):148-165.DOI:10.1159/000082134.
[11]Ben-David U,Nissenbaum J,Benvenisty N.New balance in pluripotency:reprogramming with lineage specifiers[J].Cell,2013,153(5):939-940.DOI:10.1016/j.cell.2013.04.051.
[12]Niu W,Zang T,Zou Y,et al.In vivo reprogramming of astrocytes to neuroblasts in the adult brain[J].Nat Cell Biol,2013,15(10):1164-1175.DOI:10.1038/ncb2843.
[13]Stenudd M,Sabelstrom H,Frisen J.Role of endogenous neural stem cells in spinal cord injury and repair[J].JAMA Neurol,2015,15(10):235-237.DOI:10.1001/jamaneurol.2014.2927.
[14]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.DOI:10.1016/j.cell.2006.07.024.
[15]Carbone LD,BukováP,Fink HA,et al.Association of plasma SDF-1 with bone mineral density,body composition,and hip fractures in older adults:the cardiovascular health study[J].Calcif Tissue Int,2017,100(6):569-608.DOI:10.1007/s00223-017-0245-8.
[16]Lu Y,Futtner C,Rock JR,et al.Evidence that SOX2 overexpression is oncogenic in the lung[J].PLo S One,2010,5(6):e11022.DOI:10.1371/journal.pone.0011022.
[17]Ichida JK,Blanchard J,Lam K,et al.A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog[J].Cell Stem Cell,2009,5(5):491-503.DOI:10.1016/j.stem.2009.09.012.
[2]Meybodi AT,Kim H,Nelson J,et al.Surgical treatment vs nonsurgical treatment for brain arteriovenous malformations in patients with hereditary hemorrhagic telangiectasia:a retrospective multicenter consortium study[J].Neurosurgery,2018,82(1):35-47.DOI:10.1093/neuros/nyx168.
[3]Gao YY,Zhang ZH,Zhuang Z,et al.Recombinant milk fat globuleEGF factor-8 reduces apoptosis via integrin hata3/FAK/PI3K/AKTsignaling pathway in rats after traumatic brain injury[J].Cell Death Dis,2018,9(9):845.DOI:10.1038/s41419-018-0939-5.
[4]Green NS,Bhatia M,Griffith EY,et al.Enhanced long-term brain magnetic resonance imaging evaluation of children with sickle cell disease after hematopoietic cell transplantation[J].Biol Blood Marrow Transplant,2017,23(4):670-676.DOI:10.1016/j.bbmt.2017.01.007.
[5]Wuebben EL,Rizzino A.The dark side of SOX2:cancer-a comprehensive overview[J].Oncotarget,2017,8(27):44917-44943.DOI:10.18632/oncotarget.16570.
[6]Sheng C,Jungverdorben J,Wiethoff H,et al.A stably self-renewing adult blood-derived induced neural stem cell exhibiting patternability and epigenetic rejuvenation[J].Nat Commun,2018,9(1):4047.DOI:10.1038/s41467-018-06398-5.
[7]Yang WH,Yang C,Xue YQ,et al.Regulated expression of lentivirus-mediated GDNF in human bone marrow-derived mesenchymal stem cells and its neuroprotection on dopaminergic cells in vitro[J].PLo S One,2013,8(5):e64389.DOI:10.1371/journal.pone.0064389.
[8]Erdlenbruch F,Rohwedel J,Ralfs P,et al.Generation of induced pluripotent stem cells(i PSCs)from human foreskin fibroblasts[J].Stem Cell Res,2018,33(4):79-82.DOI:10.1016/j.scr.2018.10.010.
[9]Bani-Yaghoub M,Tremblay RG,Lei JX,et al.Role of Sox2 in the development of the mouse neocortex[J].Dev Biol,2006,295(1):52-66.DOI:10.1016/j.ydbio.2006.03.007.
[10]Ellis P,Fagan BM,Magness ST,et al.SOX2,a persistent marker for multipotential neural stem cells derived from embryonic stem cells,the embryo or the adult[J].Dev Neurosci,2004,26(2-4):148-165.DOI:10.1159/000082134.
[11]Ben-David U,Nissenbaum J,Benvenisty N.New balance in pluripotency:reprogramming with lineage specifiers[J].Cell,2013,153(5):939-940.DOI:10.1016/j.cell.2013.04.051.
[12]Niu W,Zang T,Zou Y,et al.In vivo reprogramming of astrocytes to neuroblasts in the adult brain[J].Nat Cell Biol,2013,15(10):1164-1175.DOI:10.1038/ncb2843.
[13]Stenudd M,Sabelstrom H,Frisen J.Role of endogenous neural stem cells in spinal cord injury and repair[J].JAMA Neurol,2015,15(10):235-237.DOI:10.1001/jamaneurol.2014.2927.
[14]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.DOI:10.1016/j.cell.2006.07.024.
[15]Carbone LD,BukováP,Fink HA,et al.Association of plasma SDF-1 with bone mineral density,body composition,and hip fractures in older adults:the cardiovascular health study[J].Calcif Tissue Int,2017,100(6):569-608.DOI:10.1007/s00223-017-0245-8.
[16]Lu Y,Futtner C,Rock JR,et al.Evidence that SOX2 overexpression is oncogenic in the lung[J].PLo S One,2010,5(6):e11022.DOI:10.1371/journal.pone.0011022.
[17]Ichida JK,Blanchard J,Lam K,et al.A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog[J].Cell Stem Cell,2009,5(5):491-503.DOI:10.1016/j.stem.2009.09.012.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |