摘要
目的:分离培养大鼠胚胎脑源性神经干细胞,并进行增殖分化鉴定。方法:实验于2005-06/12在中国科学技术大学生命科学学院周江宁研究室进行。①自孕14dWistar大鼠胚胎分离大脑皮质及皮质下组织,经机械吹打分离成单细胞后,利用无血清原代及传代培养方法,在添加重组人碱性成纤维细胞生长因子、重组人表皮生长因子和B27添加剂的DMEM/F12(1∶1)培养基中进行悬浮培养,获得具有克隆能力的细胞群;用有限稀释法,得到来源于同一细胞的亚细胞系克隆。②从培养基中撤除生长因子和B27添加剂,换成含体积分数为0.1的胎牛血清的DMEM/F12培养基,将培养的细胞球接种于预先铺有多聚赖氨酸盖玻片的培养皿,诱导分化。③以免疫荧光细胞化学技术,用神经干细胞的特异性单克隆抗体(巢蛋白)和增殖细胞单克隆抗体(5-溴脱氧尿苷嘧啶)鉴定克隆细胞,以鉴定神经干细胞的自我更新增殖能力。④以免疫荧光细胞化学技术,用神经细胞的特异性抗体(神经元特异性烯醇化酶、胶质纤维酸性蛋白、半乳糖脑苷脂)鉴定分化细胞,以鉴定神经干细胞的多向分化潜能。结果:①从胎鼠大脑皮质及皮质下分离的组织,经原代和传代培养均可形成细胞克隆,并具有增殖的能力,表达神经上皮干细胞蛋白抗原和增殖细胞抗原。②在血清诱导下,分化后的细胞表达神经元、星形胶质细胞和少突胶质细胞3种神经细胞的特异性抗原。结论:运用上述方法分离培养的细胞具有自我更新和增殖能力,并具有多向分化潜能和具有神经干细胞的特异性抗原。
AIM： To isolate and culture the brain-derived neural stem cells （NSCs） from rat＇s embryos and identify the ability of proliferation and differentiation of NSCs.
METHODS： The experiment was completed at the Laboratory of Professor Zhou Jiang-ning, School of Life Science, University of Science and Technology of Chiria between June and December 2005. ①Cerebral cortex and tissues under cortex were dissected mechanically from embryos Wistar rats with 14-day old. The dissociated single cells were resuspended in DMEM/F12 （1：1） conditional culture media containing recombinating human basic fibroblast growth factor （rhbFGF）, recombinating human epidermal growth factor （rhEGF） and B27 supplements. Cell clones were acquired by using serum-free primary and passage culture. Limiting dilution was used to obtain subcell line clone from the same cells. ②The growth factors and B27 were removed from the DMEM/F12 （1：1） media, and the differentiation of NSCs were induced by plating the neurospheres on the polylysine-precoated coverslips in DMEM/F12 （1：1） media containing 0.1 volume of fetal bovine serum. ③ The clone cells were identified with nestin and 5-bromodeoxyuridine （5-BrdU）, specific monoclonal antibody, by immunofluorescence staining, so the ability of self-renewal and proliferation of NSCs was confirmed. ④ The differentiated cells were identified with mature neural cells specific monoclonal .or polyclonal antibody （neuron specific enolase, glial fibrillary acidic protein, galactocerebroside） by immunofluorescence staining, so the potential ability of multipotent differentiation of NSCs was confirmed.
RESULTS： ① Cell clones could be obtained from the cerebral cortex and tissues under cortex of rat＇s embryos after primary and passage culture, and the clones pessesed the ability of proliferation, could be identified by their expression of nestin and BrdU specific antigen. ②The differentiated cells could express 3 specific antigens of mature neural cells （neurons, astrocytes and oligodendrocytes） under the induction of serum . CONCLUSION： The isolated cells from the cerebral cortex and tissues under cortex in rat＇s embryos posses the potential ability of self-renewal, proliferation and rnultipotent differentiation, which are brain-derived neural stern cells.
出处
《中国临床康复》
CSCD
北大核心
2006年第11期36-38,i0001,共4页
Chinese Journal of Clinical Rehabilitation
基金
安徽省自然科学研究项目基金(050430904)
安徽省教育厅自然科学研究项目基金(2006KJ382B)
北京中医药大学吕亚芳博士研究生科研资助基金~~
