新生大鼠脑源性神经干细胞培养方法的优化

目的寻求较理想的新生大鼠脑源性神经干细胞(neuralstemcells,NSCs)分离培养方法。方法用不同的接种密度和多种配方的培养基:细胞种植密度采用1×104、1×105、1×106和1×107个/ml4个浓度;培养基除基础成分DMEM/F12外,按照是否添加2%B27、碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)、表皮生长因子(epidermalgrowthfactor,EGF)以及开始培养时胎牛血清浓度不同而分别记为第1～8组。分离培养新生大鼠脑源性NSCs并诱导其分化,应用相差显微镜和免疫细胞化学法对其进行观察和比较。结果分离培养出的细胞聚集成神经球,可在体外大量增殖和长期存活,可诱导分化为神经元和神经胶质,具有神经干细胞特性。开始培养时培养液中加入5%胎牛血清有利于NSCs存活,当血清浓度采用10%时,克隆球迅速分化,干细胞收获量极少;干细胞增生速度在种植密度采用1×106个/ml时优于采用其它种植密度时,适当提高其种植密度可加快增生速度;培养基中加入2%B27、bFGF、EGF是必需的,2%B27、20ng/ml的bFGF和EGF同时加入时NSCs生长最好,bFGF和EGF对加快NSCs的增生具有协同作用。结论成功建立了较理想的新生大鼠脑源性NSCs分离培养方法,为进一步研究和应用奠定了基础。

THE OPTIMIZATION OF THE METHOD OF CULTURING NEURAL STEM CELLS IN NEONATAL RAT BRAIN

OBJECTIVE: To establish a better method of isolating and culturing of neural stem cells (NSCs) in neonatal rat brain. METHODS: Tissue of brain was isolated from neonatal rats. Different medium and culture concentration were used to culture NSCs of neonatal rat. The culture concentration used were 1 x 10(4), 1 x 10(5), 1 x 10(6), and 1 x 10(7)/ml respectively. Ingredient of medium was classified into group 1 to 8 respectively according to whether to add 2% B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as well as the difference in culture concentration. The cells were induced to differentiate as to be confirmed as NSCs, and then were checked by phase contrast microscopy and identified by immunocytochemistry. RESULTS: The cells isolated and cultured gathered into neurospheres. The cells were capable of proliferating and maintaining long-term survival in vitro. The cells could be differentiated into neurons and glia. It was to the benefit of the survival of NSCs to add 5% fetal bovine serum(FBS) into the medium at the beginning of the culturing. When 10% FBS was added into the medium, the neurospheres differentiated quickly. When concentration 1 x 10(6)/ml was used, the growth rate of the cells was the highest of all the concentrations. Reasonably higher cell concentration promoted the proliferation of NSCs. It was necessary to add 2% B27, EGF, and bFGF into the medium. The cells had the best growth when 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF were added into the culture medium. EGF and bFGF had cooperative effect. CONCLUSION: A better method of isolating and culturing of NSCs in neonatal rat brain is established and the foundation for future research is laid.