来源于人胚纹状体区神经干/祖细胞的培养、冻存及鉴定  

The cultivation、 cryopreservation and identification of human embryonic striatum tissue neural stem/progenitor cells

作　　者：高文勇[1] 姜晓荣[1] 肖娟[1] 郗海涛[1,2] 陈琳[1,3] 黄红云[1,4,2] 

机构地区：[1]北京市虹天济神经科学研究院 [2]首都医科大学附属北京康复医院 [3]清华大学玉泉医院,北京100144 [4]武警总医院神经修复研究所

出　　处：《解剖科学进展》2015年第6期642-644,共3页Progress of Anatomical Sciences

基　　金：国家自然科学基金(81171143);国家高技术研究发展计划(863计划;2012AA020905)

摘　　要：目的分离人胚胎纹状体组织的神经干/祖细胞,运用细胞培养上清液作为冻存液和贴壁法培养扩增、合理的冻存密度及有效的复苏时间,在体外建立稳定培养、保存体系,给中枢神经系统疾病的治疗提供技术支持。方法将来源于胚胎的纹状体神经干/祖细胞,利用无血清培养基进行体外培养扩增。采用细胞培养上清液作为冻存液,冻存密度为5×10~6/ml^2~5×10~7/ml,分别复苏冻存1、3、6个月的细胞各3支,并用台盼蓝染色计数,计算细胞存活率。将此细胞传代培养p3、p9、p15、p21,用Nestin免疫组化进行化学染色鉴定。结果应用贴壁培养法细胞能够稳定传代p21以上,冻存1、3、6个月后,细胞存活率可达87%左右,仍然保持生长分化能力,Nestin表达阳性率为85%以上。结论本方法培养人胚胎纹状体神经干/祖细胞能传代扩增、低温保存,为中枢神经系统疾病细胞治疗研究提供技术依据。Objective To establish the culture method of neural stem/progenitor cells derived from human embryonic striatum tissue to afford the technical support for the treatment of central nervous system diseases. Methods The neural stem/progenitor cells derived from human embryonic striatum were cultured using non-serum medium and cell 6 7culture supernatant as cell freezing medium, the cell cryopreservation density is 5×10 / ml ～ 5×10 /ml.The recovery cells were frozen for 1, 3, 6 months respectively, the cell survival rate was calculated with trypan blue staining. The expression of Nestin was determined by immunohistochemistry in different generations of p3, p9, p15, p21 cells. Results The cells of p21 generation were cultured successfully by application of adherent culture method, the cell survival rate reached 87% 1, 3, 6 months after cryopreserving, still with growth ability. The positive rate of Nestin expression was more than 85% in neural stem/progenitor cells. Conclusion The present method could culture the human embryonic striatal cells into neural stem/progenitor cells to provide the technical basis for the treatment of the central nervous system diseases.

关 键 词：神经干/祖细胞 细胞培养 免疫荧光细胞 超低温储存 

分 类 号：Q813.11[生物学—生物工程]