小鼠室管膜下区神经干细胞增殖及分化研究

发布时间：2018-04-22 09:39

  本文选题：室管膜下区 + 神经干细胞　； 参考：《中国修复重建外科杂志》2015年06期

【摘要】：目的探讨新生小鼠室管膜下区(subventricular zone,SVZ)神经干细胞(neural stem cells,NSCs)体外培养方法,为治疗神经系统疾病寻找合适的种子细胞。方法取SPF级新生ICR小鼠SVZ组织,采用机械法和酶消化法分离培养NSCs并传代,倒置显微镜下观察细胞形态。取第3代细胞行抗SOX-2和抗巢蛋白(Nestin)免疫荧光染色鉴定,Brd U标记法及MTT法检测比较培养3、7 d细胞增殖能力。以未添加b FGF和EGF的无血清培养基诱导第3代NSCs分化,抗β-微管蛋白Ⅲ(Tuj-1)、GFAP免疫荧光染色检测比较诱导3、7 d NSCs向神经元及星形胶质细胞分化的能力。结果成功分离培养新生小鼠SVZ组织中细胞,倒置显微镜下见原代培养3 d即有神经球形成,至7 d时见大量悬浮生长神经球,且体积较前增大,部分神经球出现融合及贴壁分化现象。细胞呈典型NSCs形态。免疫荧光染色鉴定获得细胞为NSCs。细胞增殖能力检测显示,体外培养3 d的NSCs中Brd U阳性细胞数为(75.817±2.961)个、吸光度(A)值为0.478±0.025,均显著高于培养7 d的(56.600±4.881)个、0.366±0.032(t=3.366,P=0.028;t=2.752,P=0.011)。经诱导培养后,NSCs能分化为神经元、星形胶质细胞;细胞分化能力检测显示,诱导分化3 d时Tuj-1、GFAP阳性细胞百分比分别为23.1%±3.7%、23.7%±3.8%,显著低于诱导分化7 d(40.1%±3.6%、37.1%±4.5%)(t=3.285,P=0.030;t=3.930,P=0.017)。结论体外培养的新生小鼠SVZ处NSCs时具有自我增殖和多分化潜能,且体外培养时间不同,细胞增殖、分化能力亦不同。
[Abstract]:Objective to explore the culture method of neural stem cells (NSCs) from subventricular subventricular zone (SVZ) of newborn mice in vitro, and to find suitable seed cells for the treatment of nervous system diseases. Methods the SVZ tissues of newborn ICR mice of SPF grade were isolated and cultured by mechanical method and enzyme digestion method. The morphology of the cells was observed under inverted microscope. The proliferative ability of the third passage cells was assayed by Brd-U labeling method and MTT method by immunofluorescence staining of anti-nestin and anti-nestin. The third generation of NSCs differentiation was induced by serum-free medium without b FGF and EGF, and the ability of inducing NSCs to differentiate into neurons and astrocytes was compared by immunofluorescence staining with anti 尾 tubulin 鈪，

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