体外培养及诱导胚兔视网膜及脑神经干细胞分化的研究

Invitro differentiation of embryonic neural stem cells from rabbit retina and brain

目的比较体外不同培养条件对视网膜及脑神经干细胞（NSCs）视紫红质的纯化能力及不同诱导条件对NSCs的诱导分化能力。方法取胚兔视网膜及大脑皮质制备单细胞悬液，分别在5种不同培养基中进行体外培养并纯化。选取无血清条件下传3代的NSCs，分别在2种培养基中诱导8～10d。采用免疫荧光法及流式细胞仪检测所获得细胞的神经干细胞和视网膜神经上皮细胞抗原的表达。结果免疫荧光法显示无血清培养条件下2种组织来源的NSCs均部分表达巢蛋白。流式细胞术显示2种诱导方式均部分细胞表达巢蛋白，较诱导前明显降低，而视紫红质及Thyl．1的表达均较诱导前明显增高。经5％胎牛血清（FBS）诱导的细胞表达视紫红质较联合应用全反式视黄酸（ATRA）时高，差异均有统计学意义（X2=30．59，6．76；P〈0．01），但Thyl．1表达低于后者，差异也有统计学意义（X2=6．19，22．92；P=0．01）。结论无血清培养基添加N2、EGF、bFGF、LIF4种因子可以获得最佳纯化效果。2种诱导培养基都能够诱导NSCs的分化，视网膜NSCs分化为视网膜神经上皮细胞的能力高于大脑皮质NSCs。

Objective To isolate neural stem cells （NSCs） from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and induced-retinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens. Results Immunofluorescence showed that NSCs from retina and brain, cultured in serum-free media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thyl. 1 positive cells were increased after induction. Compared with the combined induction of all-trans retinoid acid （ATRA） and serum, 5 ~ FBS （fetal bovine serum） led to higher expression of Rhodopsin （P〈0.01）, but lower expression of Thyl. 1 （P=0.01）. Conclusion Serum-free media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate. Retina NSCs have higher potentials to differentiate into retinal neuro-epithelial cells than brain NSCs.