诱导人脐血间充质干细胞分化为多巴胺能神经元样细胞的研究

中国生物工程杂志

2009, Vol. 29

Issue (03): 1-8

研究报告

诱导人脐血间充质干细胞分化为多巴胺能神经元样细胞的研究

林燕¹,孙海梅¹,杨慧²,曾晓蓓¹,季凤清¹

1. 首都医科大学组织胚胎学教研室
2. 首都医科大学北京神经科学研究所

The study of CB-MSCs differentiating to become dopaminergic neuron-like cells

全文: PDF(1130 KB) HTML

摘要：

目的：探讨人羊膜上皮细胞条件培养液(ACM)及SHH，FGF8诱导人脐血间充质干细胞（CB-MSCs）分化为多巴胺( dopamine,DA )能神经元样细胞的作用及机制。方法：利用沉降红细胞、密度梯度离心和贴壁筛选法纯化CB-MSCs，加入ACM及SHH，FGF8分为CON，ACM，SHH+FGF8，ACM+SHH+FGF8四组诱导48h后，免疫细胞化学染色鉴定分化细胞的DA神经元表型。应用高压液相色谱技术测定细胞培养上清中DA含量，并应用Realtime PCR方法观察DA能神经元发育过程中的相关基因En-1，Foxa2，Lmx1b，Gli-1，Pitx3，Nurr1以及Ngn2的表达。结果：加诱导剂诱导48h后，各诱导组的TH和DAT的表达量均比对照组高。早期的转录因子En1，Foxa2 在各组中均有表达；而Pitx3，Lmx1b表达在各诱导组中；Lmx1b在ACM+SHH+FGF8组中表达最高；Pitx3在各诱导组中均有表达且无明显差异。Nurr1，Ngn2在SHH+FGF8组中表达很强。结论：ACM及SHH，FGF8可诱导脐血间充质干细胞分化为DA能神经元样细胞，其作用机制与多巴胺能神经元发育过程中的各种基因En-1，Foxa2，Lmx1b，Gli-1，Pitx3，Nurr1以及Ngn2相关。

关键词： CB-MSCs;DA能神经元样细胞;ACM;SHH+FGF8;诱导

Abstract:

Abstract : Objective :To detect the dopaminergic neural cells differentiated pathway, the related gene in dopaminergic neural cells development process were investigated. The role of Human amnion epithelial conditional media (ACM) and SHH, FGF8 was focused on in cultivation process of human umbilical cord blood mesenchymal stem cells (CB-MSCs). Method: Preparation of the mononuclear cell fraction was performed by the Ficoll gradient technique according to the manufacturer’s instructions . ACM and SHH, FGF8 were combined to induce CB-MSCs, and there were four groups CON, ACM, SHH+FGF8, ACM+SHH+FGF8. After 48 hours of co-cultivation , Immunophenotypes of CB-MSCs were observed by immunocytochemistry and the related gene in dopaminergic neural cells development process En-1、Foxa2、Lmx1b、Gli-1、Pitx3，Nurr1 and Ngn2 were detected by RT-PCR. Results: After 48 hours of co-cultivation ，the positive cells numer of TH and DAT were higher in induction groups than in control group. By RT-PCR, En-1，Foxa2，Pitx3, Lmx1b were higher in ACM, SHH+FGF8, ACM+SHH+FGF8 groups than in the control group(p<0.01). Nurr1,Ngn2 were strongly higher in SHH+FGF8. Conclusion:The related gene in dopaminergic neural cells development process，En-1、Foxa2、Lmx1b、Gli-1、Pitx3，Nurr1 and Ngn2 play a role in the differentiated pathway form CB-MSCs to dopaminergic neural cells.

Key words: CB-MSCs;dopaminergic neuron-like cells;ACM;SHH+FGF8;induction

收稿日期: 2008-10-30 出版日期: 2009-03-31

ZTFLH:

Q812

基金资助:

国家基础科学研究项目(2006CB500706);北京自然科学基金(7042008);其它项目基金

通讯作者: 季凤清

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	林燕孙海梅杨慧曾晓蓓季凤清

引用本文:

林燕,孙海梅,杨慧,曾晓蓓,季凤清. 诱导人脐血间充质干细胞分化为多巴胺能神经元样细胞的研究[J]. 中国生物工程杂志, 2009, 29(03): 1-8.

LIN Yan- Sun-Hai-Mei- Yang-Hui- Ceng-Xiao-Bei- Ji-Feng-Qing. The study of CB-MSCs differentiating to become dopaminergic neuron-like cells. China Biotechnology, 2009, 29(03): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I03/1

［1］ SanchezRamos J R, Song S, Kamath S G, et al. Expression of neural markers in human umbilical cord blood. Exp Neurol,2001,171:109~115

［2］ Buzanska L, Machaj E K, Zablocka B, et al. Human cord bloodderived cells attain neuronal and glial features in vitro. J Cell Sci,2002,115:2131~2138

［3］ McGuckin C P, Forraz N, Allouard Q, et al. Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro. Exp Cell Res,2004,295:350~359

［4］ Uchida S, Inanaga Y, Kobayashi M, et al. Neurotrophic function of conditioned medium from human amniotic epithelial cells. J Neurosci Res,2000,62:585~590

［5］ Chen L W, Zhang J P, KwokYan S D, et al. Localization of nerve growth factor, neurotrophin3, and glial cell linederived neurotrophic factor in nestinexpressing reactive astrocytes in the caudateputamen of 1methyl4phenyl1,2,3,6tetrahydropyridinetreated C57/Bl mice. J Comp Neurol,2006,497:898~909

［6］ CasteloBranco G,Sousa K M,Bryja V, et al. Ventral midbrain glia express regionspecific transcription factors and regulate dopaminergic neurogenesis through Wnt5a secretion.Mol Cell Neurosci,2006,31:251~262

［7］ Simon H H, Scholz C,O′Leary D D.Engrailed genes control developmental fate of serotonergic and noradrenergic neurons in mid and hindbrain in a gene dosedependent manner. Mol Cell Neurosci,2005,28:96~105

［8］ Smidt M P, Asbreuk C H, Cox J J, et al. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci,2000,3:337~341

［9］ Smits S M, Smidt M P.The role of Pitx3 in survival of midbrain dopaminergic neurons. J Neural Transm Suppl,2006:57~60

［10］Gang E J, Hong S H, Jeong J A,et al. In vitro mesengenic potential of human umbilical cord bloodderived mesenchymal stem cells. Biochem Biophys Res Commun, 2004,321:102~108

［11］Uchida S, Inanaga Y, Kobayashi K, et al. Neurotrophic function of conditioned medium from human amniotic ep ithelial cells.Neurosci Res, 2000, 62 (4): 585~590

［12］Kgler G,Sensken S,Airey J A, et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential.J Exp Med, 2004,200:123~135

［13］FallahiSichani M,Soleimani M，Kiani J,et al.In vitro differentiation of cord blood unrestricted somatic stem cells expressing dopamineassociated genes into neuronlike cells. Cell Biology International,2007,31:299~303

［14］Filippa M,Sancho M I,Gasull E.Encapsulation of methyl and ethyl salicylates by βcyclodextrin HPLC, UVvis and molecular modeling studies. Journal of Pharmaceutical and Biomedical Analysis,2008,48: 969~973

［15］Fiszman M L, Zuddas A, Masana M I,et al.Dopamine synthesis precedes dopamine uptake in embryonic rat mesencephalic neurons. J Neurochem,1991,56:392~399

［16］Fu Y S, Cheng Y C, Lin M Y, et al.Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism. Stem Cells,2006,24:115~124

［17］Simon H H, Bhatt L, Gherbassi D, et al. Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors. Ann N Y Acad Sci,2003,991:36~47

［18］Abeliovich A,Hammond R. Midbrain dopamine neuron differentiation: Factors and fates. Developmental Biology,2007,304: 447~454

［19］Ye W, Shimamura K, Rubenstein J L, et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell,1998,93:755~766

［20］Martinat C, Bacci J J, Leete T, et al. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype. Proc Natl Acad Sci U S A,2006,103:2874~2879

[1]	万方, 张斌, 陈民良, 陈进聪, 陈雪岚. *比较pta及ack敲除对钝齿棒杆菌产L-精氨酸生理代谢的影响*[J]. 中国生物工程杂志, 2015, 35(9): 28-34.
[2]	吴梦, 刘作华, 林保忠, 兰国成, 邹贤刚, 葛良鹏. 转基因猪研究进展[J]. 中国生物工程杂志, 2015, 35(3): 92-98.
[3]	万方, 陈民良, 张斌, 陈进聪, 陈雪岚. 代谢工程改造微生物高产氨基酸的策略[J]. 中国生物工程杂志, 2015, 35(3): 99-103.
[4]	王鹏, 郑兆鑫, 刘明秋. 猪Mx1和牛Mx1蛋白在PK-15细胞中的表达及其对伪狂犬病病毒的抑制[J]. 中国生物工程杂志, 2015, 35(3): 1-7.
[5]	高文, 高向东, 陆小冬, 徐晨. 蛋白质层析柱复性及工艺评价[J]. 中国生物工程杂志, 2015, 35(3): 84-91.
[6]	杨江科, 毛玲, 周文静, 陈江山, 胡臣. 基于基因的重新设计与高通量筛选策略选育解脂耶氏酵母脂肪酶高产菌株[J]. 中国生物工程杂志, 2014, 34(8): 54-60.
[7]	刘玉雪, 张祎昕, 王磊, 林心萍, 朱志伟, 赵宗保. 重组酿酒酵母催化二氢大豆苷元生产雌马酚[J]. 中国生物工程杂志, 2014, 34(4): 41-45.
[8]	张斌, 陈进聪, 万方, 陈民良, 杨慧林, 陈雪岚. L-精氨酸产生菌的分子育种[J]. 中国生物工程杂志, 2014, 34(4): 127-132.
[9]	田晋红, 刘琦, 战丽萍, 周泽扬. 可视化突变建模、定点突变和表达超耐热菌D-乳酸脱氢酶[J]. 中国生物工程杂志, 2012, 32(05): 73-78.
[10]	金慧, 栾雨时. 转录因子在植物抗病基因工程中的研究进展[J]. 中国生物工程杂志, 2010, 30(10): 94-99.
[11]	廖冰,吴宁,韩凤桐,林秀坤. 牛性别决定新基因Fgf9的克隆及生物信息学分析[J]. 中国生物工程杂志, 2009, 29(08): 45-50.
[12]	曲立娟,黄英. ΦC31整合酶与转基因动物研制[J]. 中国生物工程杂志, 2009, 29(02): 103-107.
[13]	张雨良,张智俊,杨峰山,Mahesh Kulye,袁辉,罗淑萍. 新疆盐生植物车前PmNHX1基因的克隆及生物信息学分析[J]. 中国生物工程杂志, 2009, 29(01): 27-33.

Viewed

Full text

Abstract

Cited

Shared

Discussed