提高胚胎大鼠神经干细胞数量的实验方法

Experimental method for enhancing the number of neural stem cells of fetal rats

目的:通过培养了解神经干细胞的生物学特性,着重从胎龄选择、优化分离和换液方法上提高神经干细胞的增殖能力和生长数量。方法:实验于2004-04/2005-09在锦州医学院中心实验室完成。①雌性SD大鼠35只,每日做阴道细胞涂片,排卵期雌雄合笼,以出现精栓计为受孕第0.5天。分别于孕10.5,11.5,12.5,13.5,14.5,16.5,18.5d取出大鼠胚胎体,每时间点各5只。②通过采用机械分离和酶消化分离相结合的方法分离培养不同胎龄大鼠脑神经干细胞。③换液首次采用分瓶法以后采用低速离心半量换液。④采用免疫细胞化学技术分别检测神经干细胞特征性标志巢蛋白表达和用血清诱导分化为大量神经元微管组合蛋白2和神经胶质纤维酸性蛋白表达。结果:①胎龄为12.5d的胎鼠提取的神经干细胞集落最多,其次为13.5d。②在上述条件下培养及传代的细胞不断分裂增殖,形成悬浮生长的呈巢蛋白阳性的神经球。③用血清诱导分化为大量表达微管组合蛋白2阳性的神经元和胶质纤维酸性蛋白阳性的神经胶质细胞。结论:考虑到生长因子在孕13～15d能够很好地发挥作用,故选择胎龄为13.5d胎鼠作为实验材料较为合适。通过采用机械分离和消化分离相结合的方法分离和原液首次采用分瓶法显著提高了神经干细胞的培养数量,培养出的细胞具有自我更新能力和增殖能力,可分化为神经元、神经胶质细胞及少突胶质细胞。

AIM： To investigate the biological characters of neural stem cells through culture, and enhance the proliferation ability and quantity of neural stem cell mainly from the choice of fetal age, optimization and isolation and method of medium replacing. METHODS： This experiment was conducted at the Central Laboratory of Jinzhou Medical College from April 2004 to September 2005. ①Totally 35 female SD rats were chosen and vagina film preparation was performed every day. Female and male rats at the ovulation period were put in the same cage. Seen vaginal plug was set as 0.5 day after coitus. Embryos of the rats were taken out on days 10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and 18.5, 5 rats at each time point. ② Brain neural stem cells of rats with different gestational ages were isolated and cultured with combination method of mechanical separation and enzyme digestion separation . ③ The method of separating flask was adopted in the first replace medium , later the medium was replaced haft with low- speed centrifugalization. ④ Immucytachemical method was used to detect the expression of neural stem cell specific marker nestin and the expression of serum-induced neuron tubulin 2 and glial fibrillary acidic protein. RESULTS： ①The neural stem cells amount of rat with 12.5 days of gestational age was the largest , and that of 13.5 days was the second. ② Cells under above culture conditions continuously proliferated and formed neurospheres which were Nestin positive. ③Serum-induced differentiated cells took on the shapes of neuron and astrocyte cell and were neuron tubulin 2 and glial fibrillary acidic protein positive respectively. CONCLUSION： Because growth factor at pregnancy of 13 to 15 days can well exert its role, so it is suitable to choose fetal rats with 13.5 days of gestational age as experimental materials. The aforementioned tips greatly enhance the number of neural stem cells which have the characteristics of self-renewal and proliferated ability and could differentiate into neurons, astrocytes and oligodendracytes.