NSCs缺氧模型的建立以及锂对缺氧NSCs的修复作用

发布时间：2018-07-22 12:25

【摘要】：目的 体外建立新生鼠NSCs缺氧模型，探讨锂对缺氧NSCs的修复作用，为临床应用锂治疗HIE提供实验依据。 方法 1.鼠NSCs分离培养及鉴定：生后24小时内SD大鼠脱颈处死，75%乙醇消毒，分离新生SD大鼠海马组织，投入冰浴的盛有D-hank's液的平皿中，去除脑膜和血管，用眼科剪将组织尽可能剪碎，0.25%胰蛋白酶水浴箱消化，玻璃管吹打成细胞悬液，200目筛网过滤，离心，获得单细胞悬液，用无血清培养技术培养神经干细胞。免疫组织化学技术检测其巢蛋白（Nestin）的表达；并用-溴脱氧尿嘧啶核苷（BrdU）掺入试验，免疫荧光双标技术观测神经干细胞的增殖状况；诱导神经干细胞分化，分别用神经元特异性稀醇化酶（NSE）抗体和胶质纤维酸性蛋白（GFAP）抗体进行免疫组织化学技术鉴定分化细胞。 2.体外NSCs缺氧模型的建立：即无糖培养基+低氧环境。无糖培养基采用与基础培养基成分相似的无糖DMEM培养基，低氧环境采用体积分数为5%CO2和95%N2的混合气体。细胞形态学观察、台盼蓝染色及cck-8检测法作为判断缺氧模型成功与否的标准。 3.锂对缺氧NSCs的修复作用：缺氧后的NSCs中加入不同浓度的氯化锂共同培养，通过细胞形态学观察和cck-8检测法探讨氯化锂对缺氧NSCs的修复作用。 结果 1.鼠NSCs分离培养及鉴定：原代培养第1天可见分离的细胞呈圆形，遮光性强。原代培养3d，可见近球形的细胞团，结构松散，大小不均，5-7d可见细胞连接紧密，呈球形增大。传代后神经球生长较快，球体较均匀，背景干净。通过免疫细胞化学检测Nestin阳性表达证实为NSCs，BrdU阳性表达证实NSCs处于增殖状态。血清培养基诱导NSCs3d后，可见NSE染色阳性的神经元，胞体呈三角形，有1-2个突起。GFAP染色阳性的星形胶质细胞，，胞体呈星形，突起粗短、较多分支。 2.NSCs缺氧模型的建立：在DMEM无糖培养基和95%N2和5%CO2混合气体中培养1h后，缺氧组与正常组比较，细胞形态学改变：缺氧组NSCs数量减少，形态不规则，甚至出现破裂，形成絮状物或碎片；台盼蓝计数：缺氧组死亡细胞数量明显增多（P0.05）；cck-8检测法：缺氧组NSCs活性明显降低（P0.05），且与缺氧时间呈正相关。 3．锂对缺氧后NSCs的修复作用：缺氧NSCs加入不同浓度的氯化锂后，同一时相点下锂干预组与缺氧组相比，细胞形态学观察：锂干预组NSCs数量多，折光性强；cck-8检测法：锂干预组NSCs活性明显高于缺氧组，3mM锂干预组NSCs活性高于1mM锂干预组（P0.05）；免疫细胞化学检测：锂干预组NSCs增殖明显高于缺氧组。 结论 1.生后1d正常SD鼠海马在体外可以培养出NSCs。 2.在无糖培养基和95%N2+5%CO2条件下缺氧1h可以建立NSCs缺氧模型。 3.氯化锂增加缺氧后NSCs细胞活力。 4.氯化锂促进缺氧后NSCs的增殖。 5.氯化锂对缺氧后NSCs的修复作用在一定范围内呈剂量依赖性。 6.氯化锂有希望成为临床治疗HIE的新药。
[Abstract]:Objective to establish anoxic model of neonatal rat NSCs in vitro and to explore the effect of lithium on the repair of hypoxic NSCs in order to provide experimental evidence for the clinical application of lithium in the treatment of HIE. Method 1. Isolation, culture and identification of NSCs: within 24 hours after birth, SD rats were killed with 75% ethanol to sterilize the hippocampus of newborn SD rats and put into a plate containing D-hankosine solution in ice bath to remove meninges and blood vessels. The tissue was shredded in 0.25% trypsin water bath with ophthalmic scissors, and the glass tube was blown into the suspension of the cells to filter and centrifuge. The single cell suspension was obtained, and the neural stem cells were cultured by serum-free culture. The expression of nestin was detected by immunohistochemistry, and the proliferation of neural stem cells was observed by using BrdU incorporation assay, and the differentiation of neural stem cells was induced. Neuron-specific dilute alcoholase (NSE) antibody and glial fibrillary acidic protein (GFAP) antibody were used to identify differentiated cells by immunohistochemistry. 2. Establishment of hypoxia model of NSCs in vitro: hypoxia-free medium. The sugar-free DMEM medium, similar to the basic medium, was used in the sugar-free medium, and the mixture of 5 CO2 and 95N2 was used in the hypoxic environment. Cell morphology, trypan blue staining and cck-8 were the criteria for judging the success of hypoxia model. The effect of lithium on the repair of hypoxic NSCs: different concentrations of lithium chloride were added to NSCs after hypoxia and the effects of lithium chloride on the repair of hypoxic NSCs were investigated by cell morphology observation and cck-8 detection. Result 1. Isolation and identification of rat NSCs: on the first day of primary culture, the isolated cells were round and strong shading. After 3 days of primary culture, the cells were found to be nearly globular, with loose structure and close connection between 5 and 7 days. The cells were spherical and enlarged. After passage, the nerve ball grows faster, the sphere is more uniform and the background is clean. The positive expression of nestin was confirmed by immunocytochemistry to confirm the proliferation of NSCs. After NSCs were induced by serum medium for 3 days, NSE positive neurons were found, with a triangular cell body, 1 or 2 GFAP positive astrocytes. 2. Establishment of anoxic model of NSCs: after cultured in DMEM sugar-free medium and mixed gas of 95N2 and 5CO 2 for 1 h, the morphological changes of NSCs in hypoxia group were compared with those in normal group: the number of NSCs in hypoxia group was decreased and the morphology was irregular. The count of Trypan blue: the number of dead cells in hypoxia group increased significantly (P0.05). The activity of cck-8 was significantly decreased in hypoxia group (P0.05), and positively correlated with hypoxia time. 3. The effect of lithium on the repair of hypoxic cck-8: after adding different concentrations of lithium chloride, lithium intervention group was compared with hypoxia group at the same time. Cell morphology observation: the number of NSCs in lithium intervention group was more than that in 1 mm lithium intervention group (P0.05). The activity of NSCs in lithium intervention group was significantly higher than that in hypoxia group (P0.05), and the activity of NSCs in lithium intervention group was significantly higher than that in anoxic group (P 0.05). Immunocytochemistry: the proliferation of NSCs in lithium intervention group was significantly higher than that in hypoxia group. Conclusion 1. NSCs. 2. 2 could be cultured in the hippocampus of normal SD rats 1 d after birth. Hypoxia model of NSCs could be established by hypoxia for 1 hour in glucose free medium and 95% N 22 + 5 CO 2. 3. Lithium chloride increased the viability of NSCs cells after hypoxia. 4. Lithium chloride promotes proliferation of NSCs after hypoxia. The effect of lithium chloride on the repair of NSCs after hypoxia was in a dose-dependent manner. 6. 6. Lithium chloride is a promising new drug for the treatment of HIE.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R722.1

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