NSCs缺氧模型的建立以及鋰對缺氧NSCs的修復(fù)作用
發(fā)布時間：2018-07-22 12:25
【摘要】：目的 體外建立新生鼠NSCs缺氧模型，探討鋰對缺氧NSCs的修復(fù)作用，為臨床應(yīng)用鋰治療HIE提供實驗依據(jù)。 方法 1.鼠NSCs分離培養(yǎng)及鑒定：生后24小時內(nèi)SD大鼠脫頸處死，75%乙醇消毒，分離新生SD大鼠海馬組織，投入冰浴的盛有D-hank's液的平皿中，去除腦膜和血管，用眼科剪將組織盡可能剪碎，0.25%胰蛋白酶水浴箱消化，玻璃管吹打成細胞懸液，200目篩網(wǎng)過濾，離心，獲得單細胞懸液，用無血清培養(yǎng)技術(shù)培養(yǎng)神經(jīng)干細胞。免疫組織化學(xué)技術(shù)檢測其巢蛋白（Nestin）的表達；并用-溴脫氧尿嘧啶核苷（BrdU）摻入試驗，免疫熒光雙標(biāo)技術(shù)觀測神經(jīng)干細胞的增殖狀況；誘導(dǎo)神經(jīng)干細胞分化，分別用神經(jīng)元特異性稀醇化酶（NSE）抗體和膠質(zhì)纖維酸性蛋白（GFAP）抗體進行免疫組織化學(xué)技術(shù)鑒定分化細胞。 2.體外NSCs缺氧模型的建立：即無糖培養(yǎng)基+低氧環(huán)境。無糖培養(yǎng)基采用與基礎(chǔ)培養(yǎng)基成分相似的無糖DMEM培養(yǎng)基，低氧環(huán)境采用體積分數(shù)為5%CO2和95%N2的混合氣體。細胞形態(tài)學(xué)觀察、臺盼藍染色及cck-8檢測法作為判斷缺氧模型成功與否的標(biāo)準(zhǔn)。 3.鋰對缺氧NSCs的修復(fù)作用：缺氧后的NSCs中加入不同濃度的氯化鋰共同培養(yǎng)，通過細胞形態(tài)學(xué)觀察和cck-8檢測法探討氯化鋰對缺氧NSCs的修復(fù)作用。 結(jié)果 1.鼠NSCs分離培養(yǎng)及鑒定：原代培養(yǎng)第1天可見分離的細胞呈圓形，遮光性強。原代培養(yǎng)3d，可見近球形的細胞團，結(jié)構(gòu)松散，大小不均，5-7d可見細胞連接緊密，呈球形增大。傳代后神經(jīng)球生長較快，球體較均勻，背景干凈。通過免疫細胞化學(xué)檢測Nestin陽性表達證實為NSCs，BrdU陽性表達證實NSCs處于增殖狀態(tài)。血清培養(yǎng)基誘導(dǎo)NSCs3d后，可見NSE染色陽性的神經(jīng)元，胞體呈三角形，有1-2個突起。GFAP染色陽性的星形膠質(zhì)細胞，，胞體呈星形，突起粗短、較多分支。 2.NSCs缺氧模型的建立：在DMEM無糖培養(yǎng)基和95%N2和5%CO2混合氣體中培養(yǎng)1h后，缺氧組與正常組比較，細胞形態(tài)學(xué)改變：缺氧組NSCs數(shù)量減少，形態(tài)不規(guī)則，甚至出現(xiàn)破裂，形成絮狀物或碎片；臺盼藍計數(shù)：缺氧組死亡細胞數(shù)量明顯增多（P0.05）；cck-8檢測法：缺氧組NSCs活性明顯降低（P0.05），且與缺氧時間呈正相關(guān)。 3．鋰對缺氧后NSCs的修復(fù)作用：缺氧NSCs加入不同濃度的氯化鋰后，同一時相點下鋰干預(yù)組與缺氧組相比，細胞形態(tài)學(xué)觀察：鋰干預(yù)組NSCs數(shù)量多，折光性強；cck-8檢測法：鋰干預(yù)組NSCs活性明顯高于缺氧組，3mM鋰干預(yù)組NSCs活性高于1mM鋰干預(yù)組（P0.05）；免疫細胞化學(xué)檢測：鋰干預(yù)組NSCs增殖明顯高于缺氧組。 結(jié)論 1.生后1d正常SD鼠海馬在體外可以培養(yǎng)出NSCs。 2.在無糖培養(yǎng)基和95%N2+5%CO2條件下缺氧1h可以建立NSCs缺氧模型。 3.氯化鋰增加缺氧后NSCs細胞活力。 4.氯化鋰促進缺氧后NSCs的增殖。 5.氯化鋰對缺氧后NSCs的修復(fù)作用在一定范圍內(nèi)呈劑量依賴性。 6.氯化鋰有希望成為臨床治療HIE的新藥。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R722.1
本文編號：2137460
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R722.1
【共引文獻】
相關(guān)期刊論文 前3條
1 趙舒武;高英茂;汪濤;王曉玲;魏斌;;葡萄糖對神經(jīng)干細胞缺氧性損傷保護作用的實驗研究[J];中國組織化學(xué)與細胞化學(xué)雜志;2010年02期
2 龔敏;李樹清;;體外培養(yǎng)細胞缺氧模型及特點[J];臨床合理用藥雜志;2011年01期
3 楊友,陳惠金,錢龍華;血糖水平對缺氧缺血新生大鼠體重和腦重的影響觀察[J];中國實驗動物學(xué)報;2004年01期
相關(guān)博士學(xué)位論文 前1條
1 趙舒武;缺氧對神經(jīng)干細胞的損傷及EPO、葡萄糖對其保護作用的實驗研究[D];山東大學(xué);2006年
相關(guān)碩士學(xué)位論文 前1條
1 于佳瀾;苦碟子對缺氧損傷血管內(nèi)皮細胞的保護作用及PKCδ/MARCKS通路的調(diào)節(jié)[D];北京中醫(yī)藥大學(xué);2013年
本文編號：2137460
本文鏈接：http://sikaile.net/yixuelunwen/eklw/2137460.html
最近更新
教材專著
