摘要
目的:探索胚胎大鼠神经干细胞的分离培养、鉴定及标记的方法。方法:分离胚胎大鼠海马组织,用无血清培养技术培养神经干细胞,免疫组织化学荧光技术检测其巢蛋白(Nestin)的表达;5-溴脱氧尿嘧啶(BrDU)掺入试验,并用免疫荧光双标技术观测神经干细胞的增殖状况。采用荧光染料Hoechest33342标记神经干细胞并诱导其分化,用神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)免疫组织化学荧光染色鉴定分化细胞。结果:①获得大量未分化呈巢状悬浮生长的神经干细胞团,并能分化为神经元和神经胶质细胞,且经传代培养8代后仍具干细胞特性;②荧光染料的标记率可达97%,细胞传8代后荧光亮度无明显衰减,并且分化后的细胞核中仍有荧光表达。结论:成功培养出胚胎大鼠神经干细胞,培养出的细胞具有增殖、自我更新及核多潜能分化能力,可分化为神经元及神经胶质细胞;荧光染料的标记可获得较高的标记率,可作为神经干细胞移植实验研究的供体细胞。
Objective：To explore the methods of culture,identification and label of embryonic rat neural stem cells. Method：The cells isolated from fetal rat hippocampus were identified with nestin immunocytochemical fluorescent staining. The cellular multiplication was observed by immunocytochemical fluorescence co-label after accession of BrDU. The neural stem cells （NSCs） were marked with fluorescent dye, bisbenzimide （Hoechest33342） and induced to differentiate. The differentiated cells were detected with Neuron Specific Enolase（NSE） and Glial Fibrillary Acidic Protein（GFAP）immunocytochemical fluorescent staining respectively. Result： Nest-like clusters of neural stem cells were obtained in suspension and the cells could be differentiated into neurons and astrocytes which maintaining the main characteristics of NSCs after 8 passages of culture. The label efficiency of cells with Hoechest33342 was 97% and no attenuation of fluorescent brightness was observed after 8 passages of culture. The cellular fluorescence was observed in the NSCs and the differentiated cells. Conclusion： The cells from embryonic rat hippocampus possessed the abilities of division, multiplication and self-renew, which were believed to be the main characteristics of NSCs of the central nervous system. The cells could be efficiently labeled with fluorescent dye and could be used as donor cells in experimental research on NSCs transplantation.
出处
《临床耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2007年第4期172-175,共4页
Journal of Clinical Otorhinolaryngology Head And Neck Surgery
基金
国家自然科学基金资助项目(No:30672308)
第四届教育部项目"高校青年教师奖"
