胚胎中脑神经干细胞的培养及分化条件探讨

Investigation on cultural conditions of neural stem cells from embryonic midbrain

摘要:

目的：探讨神经干细胞（neural stem cells，NSCs）的培养方法以及各种条件对NSCs生长状况的影响。方法：取SD大鼠胚胎中脑，在含细胞因子碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）和内皮细胞生长因子（epidermal growth factor，EGF）的无血清培养基中培养扩增，得到的神经球用神经巢蛋白nestin抗体鉴定和5-溴脱氧尿嘧啶核苷（5-bromo-2-de－oxy uridine，Brdu）掺入检测增殖能力。血清诱导分化后用免疫荧光做星形胶质细胞酸性蛋白（glial fibrillary acidic protein，GFAP）和神经元特异性Ⅲ型微管蛋白（neuron-specific class Ⅲ β-tubulin，β-Ⅲ-tublin）的双标检测。研究进一步观察了细胞因子 bFGF和 EGF单用及合用对克隆形成数量的影响及不同接种密度和白血病抑制因子（leukemia inhibitory factor，LIF）对细胞生长的影响。结果：神经球 nestin免疫荧光表达显示强阳性；Brdu掺入实验显示神经球细胞具有很好的增殖能力；分化后免疫双标结果得到了GFAP（68.32±5.70）%和 β-Ⅲ-tublin（21.65±3.40）%的阳性表达；当２种因子同时存在时细胞克隆数量优于单因子作用组（P﹤0.05，与EGF组比较；P＜0.01，与bFGF组比较）；与对照组比较LIF作用组细胞数量明显增多（F=2 630.114，P=0.000）；细胞接种密度对神经球的数量有明显影响（F=155.1００，P=0.001），在（0.5～16.0）×105个/ml密度范围内，神经球数量随着接种密度增加而增加。结论：bFGF和 EGF ２种细胞因子对NSCs的生长是必须的；长期传代过程中LIF 能促进NSCs的生长；NSCs生长有密度依赖性，但接种密度应该控制在适宜范围内。

Abstract:

Objective：To investigate the cultural condition of neural stem cells（NSCs） from embryonic midbrain. Methods：Midrains of embryonic SD rice（12.5 d） were used. Cells were maintained and expanded in the medium with serum-free supplement containing basic fibroblast growth factor（bFGF） and epidermal growth factor（EGF）. The cultured neurospheres were identified by neuroepithelial stem cell protein（nestin）. 5-bromodeoxyuridine（Brdu） was added into the medium to see the proliferative ability. Differentiation of cultured NSC was checked by immunocytochemistry to see the expression of glial fibrillary acidic protein（GFAP），and β-Ⅲ tublin. Clone forming of different cell densities was checked. The effects of separated and combined use of FGF and EGF on cell growth were investigated. Effect of different inoculation density（0.5-16.0）×105/ml and leukemia inhibitory factor（LIF） on cell growth was also taken into consideration. Results：The cultured neurospheres positively expressed nestin. Brdu tests showed that most NSC in neuro－spheres had proliferative capacity. After differentiation，NSCs differentiated into cells expressing GFAP（68.32±5.70）% or β-Ⅲ tublin（21.65±3.40）%. Cell clone numbers were higher in EGF and bFGF combined use group than in separated use group（P＜0.05，compared with those of EGF group；P＜0.01，compared with those of bFGF group）. Number of cells was significantly increased in LIF group than in control group（F=2 630.114，P=0.000）. The results of different inoculation density showed that cell density had signifi－cant impact on the number of neural spheres（F=155.1００，P=0.001）. The number of neural spheres increased within the inoculation density from 0.5×105/ml to 16.0×105/ml. Conclusion：Cytokines bFGF and EGF is necessary for the growth of NSCs. LIF promotes the growth of NSCs during long-term culture. In a suitable range，NSCs clone forming shows density-dependent.