LaZ转基因小鼠腹侧中脑神经干细胞的扩增与诱导分化

目的构建LaZ转基因小鼠腹侧中脑神经干细胞的扩增与诱导分化技术平台,为进行体内移植携带报告基因的神经干细胞研究奠定基础.方法利用无血清培养基,分离并体外培养LaZ转基因胚胎小鼠腹侧中脑神经干细胞,连续稳定传代20代后,以免疫荧光、免疫细胞化学法及组织化学方法检测神经球Nestin和分化后细胞中神经纤维（NF）、胶质原纤维酸性蛋白（GFAP）、Galc和半乳糖苷酶（X-Gal）的表达.结果从LaZ转基因小鼠腹侧中脑分离的细胞群具有连续形成神经球的能力,其Nestin表达阳性.分化后的细胞可表达神经元、星形胶质细胞和少突胶质细胞的特异性抗原.所有细胞半乳糖苷酶组织化学均为阳性.结论LaZ转基因小鼠腹侧中脑神经干细胞可稳定扩增,具有多向分化潜能,分化后细胞携带LaZ报告基因,可用于进一步的移植实验研究.

The proliferation and differentiation of stem cells from the mesencephalon of embryonic LacZ trans - genetic mice

Objective To establish a technical platform of the cell culture for neural stem cells from the mesencephalon of embryonic LacZ trans-genetic mice,in which the stem cells can self-renew and proliferate.?Methods The suspension of mesencephalic cells was prepared from the brain of embryonic LacZ trans-genetic mice.The isolated cells were grown(primary culture) and passaged(for 20 generations) in serum-free culture medium.The cultured neural stem cells were identified after primary culture and stable passage by using immunocytochemistry.The stem cellc were induced to differentiate by adding fetal bovine serum(FBS) in the culture medium.The morphological changes in the cloned cells were observed continually for 5 d,and the differentiated cells were identified by immunocytochemistry for NF,glial fibrillary acidic protein(GFAP) and Galc.?Results The stem cells isolated from the mesencephalon of LacZ trans-genetic mouse embryo,which were positive for the expression of nestin,could be passaged for at least 20 generations.In the culture medium containing serum,the stem cells were induced to differentiate,and the differentiated cells showed positivity to GFAP,NF and Galc respectively.All of the cultured cells were positive for nestin and galactosidase(X-Gal).?Conclusion The stem cells from mesencephalon of LacZ trans-genetic mouse embryo can be successfully isolated and cultured in vitro.The stem cells can be passaged in series,and possess the potential of multi-directional differentiation and express the specific antigens of neuron,astrocyte and oligodendrocyte.The present study may serve as a basic step stone in the experimental study on neural stem cell transplantation.