论文部分内容阅读
目的:探讨成体嗅上皮神经干细胞(olfactory epithelium neural stem cells)的培养方法及其体外分化的多潜能性。方法:使用中性蛋白酶和Ⅰ型胶原酶分阶段酶解消化嗅上皮,富集嗅上皮神经干细胞,扩增细胞,用加入生长因子bFGF和EGF的无血清培养液促使其形成神经球。血清诱导神经球分化并用免疫细胞化学染色方法鉴定分化细胞。结果:原代培养的嗅上皮神经干细胞呈集落样增殖,集落中的细胞表达在体分子标记GBC2和细胞角蛋白5。传代细胞用含生长因子的无血清培养液培养可增殖形成神经球。用含血清的培养液可诱导神经球分化;从神经球迁移出的分化细胞具有很高的异质性,其中包含有TUJ1阳性的神经元、P75NGFR阳性的嗅鞘细胞、GFAP阳性的星形胶质细胞和NG2阳性的寡突胶前体细胞,同时也存在有nestin阳性的神经干细胞。结论:成体嗅上皮神经干细胞可体外培养增殖形成神经球,神经球细胞具有向神经元、星形胶质细胞和寡突胶前体细胞分化的潜能。
OBJECTIVE: To investigate the culture method of adult olfactory epithelium neural stem cells and its multipotency in vitro. Methods: Neutral protease and type Ⅰ collagenase were used to digest the epithelium of the olfactory epithelium in stages. The neural stem cells were enriched and the cells were expanded. The neurospheres were formed by using the serum - free medium containing bFGF and EGF. Serum induced neurosphere differentiation and differentiated cells were identified by immunocytochemical staining. RESULTS: Primary culture of olfactory epithelium-derived neural stem cells showed colony-like proliferation, and colonies expressed in vivo markers GBC2 and cytokeratin-5. Passage cells cultured with serum-free medium containing growth factors proliferate to form neurospheres. Neurosphere differentiation was induced by serum-containing medium; differentiated cells migrated from neurospheres were highly heterogeneous and included TUJ1-positive neurons, P75NGFR-positive olfactory ensheathing cells, GFAP-positive astrocytes Cells and NG2-positive oligodendrocyte precursor cells, but also there are nestin-positive neural stem cells. Conclusion: Adult olfactory epithelial neural stem cells can proliferate and form neurospheres in vitro. Neurosphere cells have the potential to differentiate into neurons, astrocytes and oligodendrocyte precursor cells.