缺氧对新生大鼠海马神经干细胞增殖分化的影响

发布时间：2017-12-31 17:33

本文关键词：缺氧对新生大鼠海马神经干细胞增殖分化的影响　出处：《中国医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的探讨单纯性缺氧对新生大鼠海马组织神经干细胞(NSCs)增殖和分化的影响。材料和方法将36只新生1d Wistar大鼠随机分为N组(正常对照组)、Q2组(缺氧2h组)、Q5组(缺氧5h组),每组12只。 1、原代培养新生1天Wistar大鼠脑海马组织细胞在含有碱性成纤维生长因子(bFGF)、表皮生长因子(EGF)和B27的无血清培养液中进行原代培养。 2、单克隆培养当原代培养克隆球直径约为200μm时,利用有限稀释法进行单细胞克隆培养。 3、传代培养单细胞克隆培养形成的克隆球直径约200μm时进行传代培养。 4、神经干细胞鉴定 (1)巢蛋白(Nestin)和烟酸己可碱33342(Hoechst33342)免疫荧光染色当第3代细胞大多数克隆球直径约为200μm时,部分克隆球进行巢蛋白(Nestin)和烟酸己可碱33342(Hoechst33342)免疫荧光染色。 (2)诱导分化及染色当第3代细胞大多数克隆球直径约为200μm时,部分克隆球在含10%胎牛血清的DMEM/F12培养基中诱导分化3d。诱导分化3d的细胞分别行神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和烟酸己可碱33342(Hoechst33342)免疫荧光染色。 5、统计分析记录各组神经干细胞克隆球生长至直径达到200μm时所需时间;计数每100个有核细胞中NSE阳性的神经元的个数,计算每组神经干细胞向神经元分化的比率,进行统计学分析。实验结果 1、神经干细胞的鉴定结果单细胞克隆培养形成的克隆球表达Nestin,诱导分化3d后细胞表达NSE、GFAP。 2、缺氧对神经干细胞增殖影响的结果 Q2组、N组、Q5组细胞原代培养克隆球直径达为200μm所需时间分别为:7d、、10d、13d。 3、缺氧对神经干细胞分化影响的结果 Q2组、N组、Q5组神经干细胞分化为神经元的比例分别为22.57±4.08%、16.14±6.20%、10.86±2.54%。各组之间均存在显著差异,p＜0.05。结论短时间缺氧可促进在体神经干细胞增殖及神经干细胞向神经元分化;长时间缺氧则抑制在体神经干细胞增殖及神经干细胞向神经元分化。
[Abstract]:Purpose To investigate the effect of hypoxia on the proliferation and differentiation of neural stem cells (NSCs) in hippocampal tissue of neonatal rats. Materials and methods Thirty-six neonatal Wistar rats on day 1 were randomly divided into N group (n = 12) (n = 12) (n = 12) in the control group (n = 12). 1. Primary culture The hippocampal tissue cells of neonatal Wistar rats were cultured in serum-free medium containing basic fibroblast growth factor (bFGFF), epidermal growth factor (EGF) and B27. 2, Monoclonal culture When the diameter of the primary culture was about 200 渭 m, the single cell clone culture was carried out by finite dilution method. 3. Subculture Single cell clone culture was carried out when the diameter of clone sphere was about 200 渭 m. 4. Identification of neural stem cells (1) Nestin) and hexadine nicotinate 33342 Hoechst33342) immunofluorescence staining When the diameter of most clones in the third passage was about 200 渭 m. Some clones were stained with Nestin (Nestin) and H333342 (Hoechst33342) immunofluorescence staining. Differentiation and staining When the diameter of most clones in the third passage was about 200 渭 m. Some cloned spheres were induced to differentiate in DMEM/F12 medium containing 10% fetal bovine serum for 3 days. The cells which were induced to differentiate for 3 days were treated with neuron-specific enolase (NSE). Glial fibrillary acidic protein (GFAP) and nicotinic acid alkaloid 33342Hoechst 33342) immunofluorescence staining. 5. Statistical analysis The time required for the growth of neural stem cell clone spheres to reach 200 渭 m in diameter was recorded. The number of NSE positive neurons per 100 nucleated cells was counted and the ratio of neural stem cells to neuron differentiation was calculated and analyzed statistically. Experimental results 1. Identification of neural stem cells The clone spheres formed by single cell clone culture expressed Nestin, and after 3 days of differentiation, the cells expressed NSEFGFAPs. Effect of hypoxia on proliferation of neural stem cells The time required for the primary culture of the cloned spheres to reach 200 渭 m in Q _ 2 / N group was 10 d / 10 d ~ (13) d, respectively. Effect of hypoxia on differentiation of neural stem cells The percentage of neural stem cells differentiated into neurons in Q2 group was 22.57 卤4.08 and 16.14 卤6.20% respectively. 10.86 卤2.54. There was a significant difference between the two groups (P < 0.05). Conclusion Hypoxia for a short time can promote the proliferation of neural stem cells and the differentiation of neural stem cells into neurons in vivo, while hypoxia for a long time can inhibit the proliferation of neural stem cells and the differentiation of neural stem cells into neurons in vivo.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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