摘要
目的:在已成功分离培养皮质神经干细胞的基础上,体外分离培养鼠胚中脑区的神经干细胞,并进行鉴定,为中脑神经干细胞的基础与应用研究建立细胞培养平台。方法:实验于2005-11/2006-07在武汉工业学院完成。①选取孕12～13d昆明小鼠1只,处死后无菌条件下分离胚胎腹侧中脑,胰酶消化和机械吹打制成单细胞悬液,在碱性成纤维生长因子和B27存在的无血清培养基中培养扩增,机械分离法传代,观察细胞生长状况。②将传至第2代的神经球以20～30个/cm2接种于置有预先经左旋多聚赖氨酸包被盖玻片的24孔培养板中,加入含体积分数为0.1胎牛血清的DMEM/F12(不加任何生长因子)诱导分化。③采用免疫细胞化学染色方法鉴定神经干细胞及其传代细胞的分化方向。结果:①孕12～13d的鼠胚中脑细胞体外培养36h后,可见2～5个细胞的团块;48h后有明显球状团块产生,胞间界限不很清楚,出现神经细胞球;培养6d后传代,少部分单细胞于传代2d后出现分裂相,随后渐形成细胞团及神经球,生长性状基本同原代。②将培养的神经细胞球转入有血清培养时,约1周球体可完全分化为神经细胞和胶质细胞。免疫荧光染色显示神经球细胞表达巢蛋白,且神经元特异性烯醇化酶染色和胶质纤维酸性蛋白染色均呈阳性。结论:孕12～13d鼠胚胎腹侧中脑区存在神经干细胞,这些细胞在B27和碱性成纤维生长因子存在的无血清培养基中能够成功的在体外培养和传代。
AIM： To isolate and culture in vitro neural stem cells from the mesencephalon of embryonic mice on the basis of successful cultivation of neural stem cells from the cortex, and then determine them and establish a cell culture platform for the research on basis and application of neural stem cells from mesencephalon. METHODS： The experiment was performed at the Wuhan Polytechnic University from November 2005 to July 2006. ① Embryonic 12-13 days mesencephalon of a Kunming mouse was dissociated sterilely and single cell suspensions were achieved by trypsin digestion and mechanical dissociation. The cells were amplified in serum-free medium supplemented with basic fibroblast growth factor （bFGF） and B27, and then passaged by mechanical methods. Cell growth was observed. ②Neurospheres of the second generation at the density of 20-30/cm^2 were inoculated in 24-well culture plate with cover glass coated with poly L lysine, and then induced with DMEM/F12 （no growth factor） containing fetal bovine serum （FBS） of 0.1 volume fraction. ③lmmunocytochemical technique was used to identify the neural stem cells and their progeny. RESULTS： ①2-5 blocks of cells appeared 36 hours after in vitro culture in embryonic 12-13 days mesencephalon of a mouse, obviously globular masses appeared, but the margin was not clear and neurospheres were found 48 hours later. Passaging was conducted 6 days after culture. Metaphases appeared in a few cells, cell mass and neurospheres formed and growth traits were mostly the same as primary generation 2 days after passage. ②Neurosphere at about 1 week could completely differentiate into nerve cells and gliacytes when neurospheres were cultivated in serum medium. Immunofluorescent staining showed that neurospheres expressed nidogen. Cells were positive for neuron-specific enolase staining and glial flbrillary acidic protein staining. CONCLUSION： The neural stem cells present in the mesencephalon of embryonic 12-13 days mice, can be cultured and passaged in vitro in serum-free medium containing B27 and bFGF.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第24期4666-4669,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
