神经干细胞的培养鉴定及分化

Cultivation, identification and differentiation of neural stem cells

背景:神经干细胞在神经损伤修复和退行性疾病中有广泛的应用前景,体外培养鉴定及促神经元诱导分化是后续研究的基础。目的:采用悬浮神经球培养法分离培养神经干细胞并对其鉴定,了解生物学特性。方法:采用悬浮神经球培养法从C57BL/6胎鼠大脑半球分离培养神经干细胞,观察形态特征及超微结构,CCK-8法绘制生长曲线,流式细胞仪检测细胞周期,免疫荧光法检测特异性标志蛋白Nestin的表达;用体积分数为1%和10%的血清诱导分化后,免疫荧光法检测GFAP、βⅢ-tubulin和MBP的表达。结果与结论:(1)体外培养得到悬浮生长的神经球,生长曲线和细胞周期表明细胞增殖力强;(2)透射电镜观察到神经干细胞核浆比高,呈未分化状态;(3)Nestin免疫荧光阳性;(4)不同体积分数的血清诱导后均可分化为星形胶质细胞、神经元和少突胶质细胞,体积分数为1%血清能诱导分化得到更多的神经元细胞;(5)结果表明,采用悬浮神经球培养法成功分离培养得到了神经干细胞,低体积分数血清有利于神经干细胞向神经元分化。

BACKGROUND： Neural stem cell transplantation is an emerging therapeutic option in the recovery of neural lesions and neurodegenerative diseases. Neural stem cell culture and differentiation lay a foundation for the further study. OBJECTIVE： To improve the techniques for the isolation, cultivation, differentiation and identification of neural stem cells, and to explore the biological characteristics of cells. METHODS： The neural stem cells from C57BL/6 fetal rats were isolated and cultured in vitro using neurophere culture method followed by morphological and ultrastucture examination. The growth curve and cell cycle of passage 3 cells were drawn and analyzed. Nestin expression was tested by immunofluorescence. Neural stem cells induced in 1% and 10% fetal bovine serum were identified using anti-GFAP, anti-βⅢ-tubulin and anti-MBP by immunofluorescence. RESULTS AND CONCLUSION： The neurospheres exhibited strong cell proliferation ability. Under transmission electron microscope, there was a high nuclear/cytoplasmic ratio in the neural stem cells, indicating a low differentiation degree. Immunofluorescence analysis revealed that neural stem cells were positive for Nestin. The induced cells were positive for GFAP, βⅢ-tubulin, and MBP, indicating these cells were induced to differentiate into astrocytes, neurons and oligodendrocytes, and there were more neurons in 1% fetal bovine serum than those in 10% fetal bovine serum. In conclusion, we could successfully isolate neural stem cells in C57BL/6 mice, and low concentration of fetal bovine serum contributes to more neurons differentiated from neural stem cells.