新生鼠体外神经干细胞培养、分化及鉴定

中国儿童保健杂志 ›› 2014, Vol. 22 ›› Issue (1): 31-34.

新生鼠体外神经干细胞培养、分化及鉴定

王钰^{1, 2}, 尹晓娟^{1, 2}

1 北京军区总医院附属八一儿童医院, 北京 100700;
2 安徽医科大学研究生学院, 安徽合肥 230001

收稿日期:2013-06-18 发布日期:2014-01-10 出版日期:2014-01-10
通讯作者: 尹晓娟, E-mail:yyinxiaojuan@163.com
作者简介:王钰(1986-), 女, 硕士学位, 研究方向为新生儿脑损伤的临床与基础研究。

Cultivation, differentiation and identification in vitro of the neural stem cells from neonatal rat.

WANG Yu^{1, 2}, YIN Xiao-juan^{1, 2}.

1 Bayi Children's Hospital Affiliated to General Hospital of Beijing Military Command of People's Liberation Army, Beijing 100700, China; 2 Graduate School of Anhui Medical University, Hefei, Anhui 230001, China

Received:2013-06-18 Online:2014-01-10 Published:2014-01-10
Contact: YIN Xiao-juan, E-mail:yyinxiaojuan@163.com

摘要/Abstract

摘要： 目的探索新生SD大鼠神经干细胞体外分离培养基鉴定的方法。方法分离新生SD大鼠海马组织, 无血清培养技术培养神经干细胞, 免疫组织化学技术检测其巢蛋白的表达;并用溴脱氧尿嘧啶核苷掺入试验, 免疫荧光双标技术观测神经干细胞的增殖状况;诱导神经干细胞分化, 分别用神经元特异性烯醇化酶抗体和胶质纤维酸性蛋白抗体进行免疫组织化学技术鉴定分化细胞。结果获得大量悬浮生长的单克隆神经球, 并可以分化为神经元和星形胶质细胞。结论成功培养的新生SD大鼠神经干细胞具有增殖、自我更新和多分化潜能, 为体外基础和临床研究奠定基础。

关键词: 新生鼠, 神经干细胞, 细胞培养, 免疫细胞化学

Abstract: Objective To investigate the cultivation, identification and identification cultivation of the neural stem cells (NSCs) from neonatal rats. Methods Hippocampus was isolated from neonatal SD rats and NSCs were cultured in serum free medium.Nestin expression in NSCs was detected by immunohistochemical fluorescence.The proliferation of NSCs was assessed by both of 5-bromodeoxyuridine (5-BrdU) incorporation assay and double-marked immunofluorescence.The differentiation of NSCs was induced.To identify the differentiated NSCs, antibodies of neuron-specific enolase (NSE) and antibody of glial fibrillary acidic protein (GFAP) were employed respectively with immunohistochemical fluorescence staining. Results A mass of neurospheres were obtained.Those NSCs could differentiate into neurons and astrocytes. Conclusions The fetal rat NSCs are successfully obtained, which have the capacities of proliferation, self-renew and pluripotentiality.They could differentiate into neurons and astrocytes, lay the foundation for the basic and clinical research in vitro.

Key words: neonatal rat, neural stem cells, cell culture, immunohistochemistry

中图分类号:

Q426

王钰, 尹晓娟. 新生鼠体外神经干细胞培养、分化及鉴定[J]. 中国儿童保健杂志, 2014, 22(1): 31-34.

WANG Yu, YIN Xiao-juan.. Cultivation, differentiation and identification in vitro of the neural stem cells from neonatal rat.[J]. journal1, 2014, 22(1): 31-34.

参考文献

[1] Lawn JE, Cousens S, Zupan J.4 million neonatal deaths:when?Where?Why?[J].Lancet, 2005, 365(9462):891-900.
[2] Volpe JJ, Hypoxic-ischemic encephalopathy[M]//Neurology of the Newborn.Saunders WB, Philadelphia PA.2001:245-480.
[3] Azzopardi DV, Strohm B, Edwards AD, et al.Moderate hypothermia to treat perinatal asphyxial encephalopathy[J].N Engl J Med, 2009, 361:1349-1358.
[4] Zhu C, Kang W, Xu F, et al.Erythropoietin improved neurologic outcomes in newbornswith hypoxic-ischemic encephalopathy[J].Pediatrics, 2009, 124:e218-226.
[5] 邵肖梅, 叶鸿瑁, 丘小汕.实用新生儿学[M].4版.2011:700-705.
[6] Doetsch F, Caille I, Lim DA, et al.Subventrcular zone astrocytes are neural stem cells in the adult mamMalian brain[J].Cell, 1999, 97(6):703-716.
[7] Gage FH, Coates PW, Palmer TD, et al.Servival and differentiation of adult neuronal progenitor cells transplanted to the adult brain[J].Proc Natl Acad Sci USA, 1995, 92(11):11879-11883.
[8] Weiss S, Dunne C, Hewson J, et al.Multipotent CNS stem cells are present in the adult mamMalian spinal cord and ventricular neuroaxis[J].J Neurosci, 1996, 16(23):7599-7609.
[9] Tropepe V, Coles BLK, Chiasson BJ, et al.Retinal stem cells in the adult mamMalian eye[J].Science, 2000, 287(5460):2032-2036.
[10] 历俊华, 郑淑荣, 万虹, 等.血清预培养促进NSCs的增殖[J].中国康复理论与实践, 2005, 11(5):339 -340.
[11] Tsymbaliuk VI.Human nerve stem cells in vitro[J].Tsitol Genet, 2012, 46:71-80.
[12] Svendsen CN, Caldwell MA, Shen J, et al.Long-term survival of human central nervous system Progenitor cells transplanted into a rat model of Parkinson's disease[J].Exp Neurol, 1997, 148:135-146.
[13] Zhang CP.Characteristics of neural stem cells expanded in lowered oxygen and the potential role of hypoxia-inducible factor-1[J].Alha Neurosignals, 2006, 15(5):259-265.
[14] Milosevie J, Storeh A, Sehwarz J.Spontaneous apoptosis inmurine free-floating neurosPheres[J].Experimental Cell Researeh, 2004, 294:19-27.
[15] Miehalezyk K, Ziman M.Nestin strueture and predieted function in cellular cytoskeletal organization[J].Histol HistoPathol, 2005, 20(2):665-667.
[16] De Paula S, Greggio S, DaCosta JC.Use of stem cells in perinatal as-phyxia:From bench to bedside[J].J Pediatr ( Rio J), 2010, 86(6):451-464.
[17] Lee IS, Jung K, Kim M, et al.Neural stem cells:properties and thera-peutic potentials for hypoxic-ischemic brain injury in newborn infants[J].Pediatr Int, 2010, 52(6):855-865.
[18] Vill A, Bueno C, Frcuso S, et al.Long-term molecular and cellular stability of human neural stem cell lines[J].Experi-mental Cell Research, 2004, 294:559-566.