无血清培养新生Wistar大鼠神经干细胞及免疫荧光鉴定

目的在无血清条件下,分离培养新生1d的Wistar大鼠神经干细胞,观察其生长及分化情况,并对其生物学特性进行鉴定。方法取新生1d的大鼠海马组织,机械分离法分离神经干细胞,加入含有表皮生长因子、碱性成纤维生长因子、B27的DMEM/F12无血清培养基中培养、增殖。倒置显微镜下观察神经干细胞的增殖分化情况,采用免疫荧光染色鉴定其生物特性。结果从新生大鼠海马组织分离培养的神经干细胞在无血清培养基中不断增殖,免疫荧光染色显示巢蛋白呈阳性表达。诱导分化后,免疫荧光染色可见高分子量神经丝蛋白、胶质纤维酸性蛋白和髓鞘碱性蛋白阳性表达细胞。结论无血清条件分离培养的神经干细胞具有自我更新和增殖能力,能分化为神经元、星形胶质细胞和少突胶质细胞。

Serum-free culture and immunofluorescent identification of neural stem cells in hippocampus of newborn Wistar rat

Objective To isolate and culture neural stem cells （NSCs） from hippoeampus of newborn Wistar rats to observe the growth and differentiation of NSCs and to identify thier biological properties. Methods NSCs were isolated from hippocampus of newborn Wistar rats and cultured in serum-free DMEM/F12 medium, supplemented with basic fibroblast growth factor （bFGF） , epidermal growth factor （EGF） and B27. The proliferation and differentiation of NSCs were observed using inverted microscope, and thier biological properties were identified using immunofluoreseenee staining. Results The proliferation of NSCs cultured in serum-free medium was found with positive expression of nestin by immunofluorescence staining. Immunofluorescence staining also showed the expressions of high molecular weight neurofilament protein （NF-H） , glial fibrillary acidic protein （GFAP） and myelin basic protein （MBP） in NSCs cultured in serum-free medium after differentiation induction. Conclusions NSCs cultured in seerum-free medium have self- renewal and proliferative ability, and could be induced and differentiated into neurons, astrocytes and oligodendrocytes.