提高成年大鼠神经干细胞单克隆形成率的方法

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。

INCREASING THE SINGLE-CLONE FORMED RATE OF NEURAL STEM CELLS FROM ADULT RATS

Objective Modified the medium that can increase single|clone formed rate and confirmed the single clone spheres had the multipotential of differentation. Methods We modified the medium, that is, the medium contained half of primary culture medium and half of fresh culture medium. A great deal of neurospheres dervied from a single cell were plated averagely into 24 well plates and added into the DMEM differentiation medium (containing serum). After culturing for 14 days, cultures were stained with the neuronal| ang glial|specific markers (MAP|2 for neurons, GFAP for astrocytes and CNP for oligodendrocytes). Results Each 96 well plate containing half of primary culture medium generated two to three single clone spheres, in control plate containing only fresh medium generated half to one single clone sphere. After differentiation, these cell clones expressed MAP|2, GFAP and CNP positive respectively.Conclusion Using half of primary culture medium can increase single|clone formed rate and these cell clones had the multipotential of differentiation.;[