摘要
目的:探讨获取和培养人胚胎神经干细胞的方法。方法:实验于2003-09/2004-12在重庆医科大学基础研究所完成。人胚胎来源为7～12周人工药物流产胎儿,由重庆医科大学附属第二医院及重庆市妇幼保健院提供,征得产妇本人同意。①从人胚胎大脑皮质中分离、培养、纯化原代神经干细胞,经体外反复传代培养以获得能在体外长期生存的神经干细胞。②免疫细胞化学鉴定神经干细胞特异性表达抗原。③观察不同种植细胞密度对神经干细胞增殖的影响,分析其生长特点并绘制生长曲线。④采用悬浮细胞培养法扩增细胞,冷冻复苏后观察细胞的生物学特性。结果:①人胚胎神经干细胞体外生长特点:从7～12周流产胎儿大脑皮质中获得了能在体外长期生存的神经干细胞。经体外传至15代,神经干细胞细胞数量扩增至200倍。②神经干细胞接种密度对增生的影响:当种植细胞的密度为1×105L-1,1×107L-1时,体外培养30d后仍呈单细胞贴壁状态。以1×109L-1细胞密度接种时,12h后即见多个单细胞聚成的小球,悬浮生长。③神经干细胞自我更新能力的检测结果:多数单个细胞在培养后24～48h分裂为2～4个细胞的小克隆,7～10d均发展为上百个细胞的较大的克隆。④nestin抗原的表达:培养的神经干细胞神经球nestin免疫细胞染色均呈阳性。⑤神经干细胞多向分化能力检测:NSE和GFAP免疫细胞化学染色显示,被测细胞胞浆有棕黄色颗粒沉着,呈阳性表达。⑥神经干细胞复苏后的存活率:冻存1,4,8,12周的神经干细胞存活率基本相似[(80.2±1.8)%,(80.1±1.2)%,(79.9±0.8)%,(80.1±2.1)%,P>0.05]。结论:成功地从人胚胎大脑皮质中分离得到了神经干细胞。经体外培养证明:人胚胎神经干细胞具有自我更新增殖能力和向神经元样细胞、星形胶质样细胞分化的潜能。
AIM： To investigate the method of obtaining and culturing human embryonic neural stem cells （NSC）. METHODS： The experiment was conducted at Institute of Basic Medical Sciences, Chongqing University of Medical Sciences from September 2003 to December 2004. Abortus aged 7-12 week were collected from drug induced abortion in the Second Affiliated Hospital of Chongqing University of Medical Sciences and Chongqing Health Center for Woman and Child. The sample were consented to use in scientific research by donator. ① Primary NSC were isolated, cultured and purified from embryo brain cortex, and the cells were passaged repeatedly in vitro in order to culture the NSC for long period. ② The technique of immunochemistry was employed to detect special expressive antigen of NSC. ③The effect of different number of planted NSC on proliferation of NSC was observed. The feature of its growth was analyzed and growth curve was drawn. ④Cell proliferation was conducted with suspended cell culture method; Biological characteristics of NSC was observed after recovering from cryopreservation. RESULTS： ①Growth characteristics of human embryonic NSC in vitro： NSC isolated from the 7-12 weeks embryonic cerebral cortex could survive in vitro for long period. The total number of NSC increased 200 folds after 15 passages. ②Effect of NSC concentration on proliferation： The NSC planted at the concentration of 1 ×10^5 L^-1 or 1 ×10^7 L^-1 in vitro could maintain single layer even culture for 30 days, when NSC were planted at the concentration of 1×10^9 L^-1, some small spherical colonies associated with several cells were observed suspending in the media after 12 hours culture. ③ The capability of self-renewal of NSC： The single cell proliferated into small spherical clone consisting of 2-4 cells after 24-48 hours culture, and the small clone developed into large spherical clone consisting of hundred of cells. ④ Expression of nestin antigen： The spherical clone nestin immunologic cell staining showed positi
出处
《中国临床康复》
CSCD
北大核心
2006年第45期21-23,共3页
Chinese Journal of Clinical Rehabilitation
