目的 簡化神經(jīng)干細(xì)胞原代培養(yǎng)的取材及步驟,明確體外定向誘導(dǎo)分化條件,為進(jìn)一步神經(jīng)干細(xì)胞移植相關(guān)實(shí)驗(yàn)提供基礎(chǔ)條件。方法 新生24 h昆明種小鼠無菌條件下冰上取大腦組織,經(jīng)機(jī)械分離加胰酶消化吹打后,加入含有堿性成纖維細(xì)胞生長因子、表皮細(xì)胞生長因子和B27的DMEM/F12培養(yǎng)基中培養(yǎng);加入不同濃度胎牛血清誘導(dǎo)其分化,應(yīng)用免疫熒光技術(shù)行巢蛋白、膠質(zhì)纖維酸性蛋白、微管相關(guān)蛋白2染色,對培養(yǎng)細(xì)胞鑒定。結(jié)果 新生小鼠提取神經(jīng)干細(xì)胞可形成神經(jīng)球,并穩(wěn)定增殖傳代,經(jīng)誘導(dǎo)可定向分化為神經(jīng)元及星形膠質(zhì)細(xì)胞。結(jié)論 新生小鼠較胎鼠取材簡單,可培養(yǎng)高質(zhì)量神經(jīng)干細(xì)胞,血清濃度同向星型膠質(zhì)細(xì)胞方向的分化概率呈正相關(guān)。

Objective To simplify the original culture of neural stem cells in vitro and to determine the differentiation conditions. Provide basic conditions for the further study of neural stem cell transplantation. Methods The brain tissue of Kunming mice newborn for 24 h was isolated under sterile conditions on the ice, by mechanical separation and trypsin digestion and trituration, and added with alkaline fibroblast growth factor, epidermal growth factor, and B27 of DMEM/F12 culture medium; The brain tissue was also added with different concentration of fetal bovine serum to induce differentiation; The immune fluorescence technique for nestin, glial fibrillary acidic protein, and microtubule associated protein 2 staining of cell culture identification were also performed. Results The primary cultured cells of the neonatal mice could form the nerve bulb and proliferate and differentiate into neurons and astrocytes. Conclusion Compared with the fetal rats, the newborn mice are simple and neural stem cells with high quality could be cultured, and the serum concentration is positively correlated with the differentiation of the direction of the astrocytes.

經(jīng)鼻給予NGF 聯(lián)合NSC 移植治療運(yùn)動神經(jīng)元病的實(shí)驗(yàn)研究(13JCYBJC24100)