本文關(guān)鍵詞： 神經(jīng)干細(xì)胞 白細(xì)胞介素-2 增殖　出處：《浙江大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：目的提取胚胎大鼠(E14)大鼠腦室下區(qū)、海馬等部位的神經(jīng)干細(xì)胞體外條件下進(jìn)行原代培養(yǎng)和傳代培養(yǎng),并研究白細(xì)胞介素-2(interleukin-2, IL-2)對(duì)神經(jīng)干細(xì)胞增殖的影響。 方法提取胚胎大鼠(E14)大鼠腦室下區(qū)、海馬等部位的神經(jīng)干細(xì)胞,體外條件下在完全培養(yǎng)基中進(jìn)行原代培養(yǎng)和傳代培養(yǎng),培養(yǎng)后行nestin染色,檢測(cè)其是否為神經(jīng)干細(xì)胞。將細(xì)胞分為四組,實(shí)驗(yàn)組內(nèi)分別添加一定量的IL-2使其濃度分別為50ng/ml、100ng/ml和150ng/ml;對(duì)照組不添加IL-2。相同條件下培養(yǎng)3天后,加入BrdU,終濃度10μmol/L;繼續(xù)培養(yǎng)1天后行免疫細(xì)胞化學(xué)染色；顯微鏡下觀察,并計(jì)數(shù)陽(yáng)性細(xì)胞和陰性細(xì)胞數(shù),計(jì)算各組神經(jīng)干細(xì)胞的增殖率。 結(jié)果原代培養(yǎng)的神經(jīng)干細(xì)胞數(shù)目較少,培養(yǎng)第一天就可見大量細(xì)胞貼壁,少數(shù)細(xì)胞伸出突起,從培養(yǎng)的第4-5天開始,貼壁的細(xì)胞破裂、死亡,而未貼壁的細(xì)胞則懸浮于培養(yǎng)基中,細(xì)胞數(shù)目增多,形成小的神經(jīng)球(Neurosphere),神經(jīng)球逐漸增大。傳代細(xì)胞呈球形懸浮于培養(yǎng)瓶中,細(xì)胞形態(tài)規(guī)則,未見明顯突起生長(zhǎng),傳代7天時(shí)神經(jīng)球已生長(zhǎng)成為數(shù)百個(gè)細(xì)胞的大克隆。神經(jīng)球nestin免疫細(xì)胞化學(xué)染色基本全部呈陽(yáng)性,證實(shí)其為神經(jīng)干細(xì)胞。加入IL-2后神經(jīng)干細(xì)胞的增殖率顯著增加,其中IL-2濃度為150ng/ml時(shí),神經(jīng)干細(xì)胞的增殖率最大,明顯高于對(duì)照組；IL-2濃度為50ng/ml時(shí)神經(jīng)干細(xì)胞的增殖率較IL-2濃度為150ng/ml時(shí)低,但仍顯著高于對(duì)照組；IL-2濃度為100ng/ml時(shí)神經(jīng)干細(xì)胞的增殖率介于前兩組之間,且顯著高于對(duì)照組。 結(jié)論一定濃度的IL-2可以促進(jìn)神經(jīng)干細(xì)胞的增殖。
[Abstract]:Objective to extract neural stem cells (NSCs) from the subventricular and hippocampal regions of embryonic rat (E14) in vitro and to study the effects of interleukin-2 (IL-2) on the proliferation of neural stem cells (NSCs). Methods the neural stem cells were extracted from the subventricular area and hippocampus of the embryonic rat (E14). The neural stem cells were cultured and subcultured in a complete culture medium in vitro. Nestin staining was performed after culture. The cells were divided into four groups. In the experimental group, a certain amount of IL-2 was added to the cells at the concentrations of 50 ng / ml 100 ng / ml and 150 ng / ml, respectively, while the control group was not supplemented with IL-2.After the same conditions, the cells were cultured for 3 days. BrdU was added at the final concentration of 10 渭 mol / L; immunocytochemical staining was performed after 1 day of culture; the number of positive and negative cells was observed under microscope, and the proliferation rate of neural stem cells in each group was calculated. Results the number of primary cultured neural stem cells was relatively small. On the first day of culture, a large number of cells adhered to the wall and a few cells protruded. From the 4-5 days after culture, the adherent cells broke down and died. However, the unattached cells were suspended in the culture medium, the number of cells increased, and the small neurosphere was formed, and the neurosphere gradually increased. The passage cells were spherical suspension in the culture flask, the morphology of the cells was regular, and there was no obvious protruding growth. After 7 days of passage, the nerve ball had grown into a large clone of hundreds of cells. The immunocytochemical staining of nestin was positive, which confirmed that it was a neural stem cell. The proliferation rate of neural stem cells increased significantly after the addition of IL-2. When the concentration of IL-2 was 150ng / ml, the proliferation rate of neural stem cells was the highest, which was significantly higher than that of the control group when the concentration of IL-2 was 50ng / ml, and the proliferation rate of neural stem cells was lower than that of IL-2 (150ng / ml). But the proliferation rate of neural stem cells was significantly higher than that of the control group when the concentration of IL-2 was 100 ng / ml, and the proliferation rate of neural stem cells was significantly higher than that of the control group. Conclusion certain concentration of IL-2 can promote the proliferation of neural stem cells.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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本文編號(hào)：1501972