本文關(guān)鍵詞：缺氧對新生大鼠海馬神經(jīng)干細胞增殖分化的影響　出處：《中國醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 缺氧 新生大鼠 海馬 神經(jīng)干細胞 增殖 分化

【摘要】： 目的 探討單純性缺氧對新生大鼠海馬組織神經(jīng)干細胞(NSCs)增殖和分化的影響。 材料和方法 將36只新生1d Wistar大鼠隨機分為N組(正常對照組)、Q2組(缺氧2h組)、Q5組(缺氧5h組),每組12只。 1、原代培養(yǎng) 新生1天Wistar大鼠腦海馬組織細胞在含有堿性成纖維生長因子(bFGF)、表皮生長因子(EGF)和B27的無血清培養(yǎng)液中進行原代培養(yǎng)。 2、單克隆培養(yǎng) 當(dāng)原代培養(yǎng)克隆球直徑約為200μm時,利用有限稀釋法進行單細胞克隆培養(yǎng)。 3、傳代培養(yǎng) 單細胞克隆培養(yǎng)形成的克隆球直徑約200μm時進行傳代培養(yǎng)。 4、神經(jīng)干細胞鑒定 (1)巢蛋白(Nestin)和煙酸己可堿33342(Hoechst33342)免疫熒光染色 當(dāng)?shù)?代細胞大多數(shù)克隆球直徑約為200μm時,部分克隆球進行巢蛋白(Nestin)和煙酸己可堿33342(Hoechst33342)免疫熒光染色。 (2)誘導(dǎo)分化及染色 當(dāng)?shù)?代細胞大多數(shù)克隆球直徑約為200μm時,部分克隆球在含10%胎牛血清的DMEM/F12培養(yǎng)基中誘導(dǎo)分化3d。誘導(dǎo)分化3d的細胞分別行神經(jīng)元特異性烯醇化酶(NSE)、膠質(zhì)纖維酸性蛋白(GFAP)和煙酸己可堿33342(Hoechst33342)免疫熒光染色。 5、統(tǒng)計分析 記錄各組神經(jīng)干細胞克隆球生長至直徑達到200μm時所需時間;計數(shù)每100個有核細胞中NSE陽性的神經(jīng)元的個數(shù),計算每組神經(jīng)干細胞向神經(jīng)元分化的比率,進行統(tǒng)計學(xué)分析。 實驗結(jié)果 1、神經(jīng)干細胞的鑒定結(jié)果 單細胞克隆培養(yǎng)形成的克隆球表達Nestin,誘導(dǎo)分化3d后細胞表達NSE、GFAP。 2、缺氧對神經(jīng)干細胞增殖影響的結(jié)果 Q2組、N組、Q5組細胞原代培養(yǎng)克隆球直徑達為200μm所需時間分別為:7d、、10d、13d。 3、缺氧對神經(jīng)干細胞分化影響的結(jié)果 Q2組、N組、Q5組神經(jīng)干細胞分化為神經(jīng)元的比例分別為22.57±4.08%、16.14±6.20%、10.86±2.54%。各組之間均存在顯著差異,p＜0.05。 結(jié)論 短時間缺氧可促進在體神經(jīng)干細胞增殖及神經(jīng)干細胞向神經(jīng)元分化;長時間缺氧則抑制在體神經(jīng)干細胞增殖及神經(jīng)干細胞向神經(jīng)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 鹵4.08 and 16.14 鹵6.20% respectively. 10.86 鹵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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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本文編號：1360763