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目的 探索提高神经干细胞单克隆形成率的方法并证实所分离培养的神经干细胞仍具有多分化潜能。 方法 对神经干细胞单克隆方法进行改良 ,即在单克隆培养液中加入 1 2原代克隆培养液。将所得到的单细胞克隆球消化、分离、增殖成大量的神经干细胞球 ,用含血清的DMEM分化培养液促其分化。 14d后 ,分别用神经元和胶质细胞的特异性标记物MAP 2标记神经元、GFAP标记星形胶质细胞和CNP标记少突胶质细胞。 结果平均每块含 1 2原代克隆培养液的 96孔培养板中有 2～ 3只孔可形成克隆球 ,而含纯新鲜培养液的培养板中仅 0 5～ 1 0只孔可形成克隆球。这些单细胞克隆球增殖后得到的大量亚细胞系克隆球分化后分别呈MAP 2、GFAP和CNP免疫荧光阳性。 结论 单细胞克隆实验中加入 1 2原代克隆培养液可提高神经干细胞的单克隆形成率 ,单克隆球增殖后得到的大量亚细胞系克隆球亦具有多分化潜能。
Objective To explore a method to improve the rate of monoclonal cell formation of neural stem cells and to confirm that the neural stem cells cultured in vitro still have the potential of differentiation. Methods Monoclonal methods of neural stem cells were modified, that is, 12 primary culture broths were added to the monoclonal culture medium. The resulting single cell clone ball digestion, separation, proliferation into a large number of neural stem cells, with serum-containing DMEM differentiation medium to promote differentiation. After 14 days, MAP 2-labeled neurons, GFAP-labeled astrocytes and CNP-labeled oligodendrocytes were respectively labeled with specific markers of neurons and glial cells. Results On average, each clone contained 1-2 wells in a 96-well culture plate containing 12 primary culture broths, while only 0-50 cells in the culture plate containing pure fresh culture medium formed colonies ball. A large number of subcellular clonal spheres obtained after proliferation of these single-cell clone spheres were positive for immunofluorescence of MAP 2, GFAP and CNP, respectively. Conclusions Monoclonal growth rate of neural stem cells can be increased by adding 1 2 primary cloning medium to single cell cloning experiments. A large number of subcellular clonal spheroids obtained by monoclonal spheres also have multipotency.