条件培养液对Wistar大鼠海马神经干细胞单细胞克隆增殖的影响

Effect of conditioned culture medium on the proliferation of neural stem cell single cell clone in hippocampus of Wistar rat

目的探讨神经干细胞(NSCs)条件培养液对其单细胞克隆增殖的影响。方法采用无血清培养法体外培养Wistar大鼠海马神经干细胞并进行传代培养,然后收集神经干细胞条件培养液。第四代细胞进行单细胞克隆,并将单细胞克隆细胞分为条件培养液组、无血清培养液对照组。通过MTT法比较两组细胞的增殖情况。单克隆培养细胞行巢蛋白(nestin)免疫细胞化学染色,诱导分化后细胞行神经元特异性烯醇化酶(NSE)、胶质原纤维酸性蛋白(GFAP)、半乳糖脑苷脂(Galc)免疫细胞化学染色。结果单克隆培养后克隆球表达nestin,诱导分化后细胞表达NSE、GFAP、Galc,条件培养液组细胞增殖速度高于对照组。结论条件培养液能提高Wistar大鼠海马神经干细胞单细胞克隆的增殖速度。

Objective To study the effect of conditioned culture medium （CM） on the proliferation of neural stern cells （NSCs） single cell clone in hippocampus of Wistar rat. Methods The serum-free medium was used to culture NSCs isolated from hippocampus of rats, the conditioned-medium was collected for further use. Cells clusters from passage 4 were performed for mono-cloning and divided into control （serum-free medium） and CM groups. The proliferation of NSCs was evaluated by MTF. Immunocytochemistry for nestin, neuron specific enolase （NSE）, glial fibrillary acidic protein （GFAP） and galactocerebroside （Galc） was used to identify monoclonal cells, neurons, astrocytes and oligodendrocytes respectively. Results The cultured monoclonal cells expressed nestin, and expressed NSE, GFAP and Galc one week after induced differentiation.The proliferation of NSCs was faster in CM group than in the control group. Conclusions CM can enhance the proliferation of the single cell clone of NSCs single cell clone in hippocampus of Wistar rat.