發(fā)布時(shí)間：2018-06-18 20:50

  本文選題：神經(jīng)干細(xì)胞 + 飼養(yǎng)層　； 參考：《廣西醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:探討替代細(xì)胞因子培養(yǎng)神經(jīng)干細(xì)胞的適合技術(shù)和條件,建立神經(jīng)干細(xì)胞體外培養(yǎng)體系,降低實(shí)驗(yàn)成本,為建立細(xì)胞資源庫打下基礎(chǔ)。 方法: 1.分離海馬制成單細(xì)胞懸液。用含2%B27的DMEM/F12無血清培養(yǎng)液(bFGF+EGF各20ng/ml)培養(yǎng)細(xì)胞。采用Nestin的免疫細(xì)胞化學(xué)方法鑒定原代和傳代細(xì)胞中的NSCs。免疫細(xì)胞化學(xué)方法檢測(cè)誘導(dǎo)分化細(xì)胞的β-Tublin、GFAP。采用Brdu標(biāo)記原代神經(jīng)干細(xì)胞,傳代后檢測(cè)Brdu。采用Brdu標(biāo)記神經(jīng)干細(xì)胞,并進(jìn)行同質(zhì)小鼠大腦海馬和側(cè)腦室內(nèi)移植,檢測(cè)細(xì)胞存活情況。 2.選擇胚胎成纖維細(xì)胞、Sertoli細(xì)胞、骨髓基質(zhì)細(xì)胞,建立細(xì)胞培養(yǎng)體系,使用10μg/ml的絲裂霉素C制備飼養(yǎng)層。 3.實(shí)驗(yàn)分為五組,倒置顯微鏡下觀察計(jì)數(shù)細(xì)胞,并繪制生長曲線。 結(jié)果: 1.添加細(xì)胞因子培養(yǎng)可以形成大量的懸浮方式生長的細(xì)胞球。細(xì)胞球經(jīng)過多次傳代可以形成次代細(xì)胞球。原代、次代細(xì)胞經(jīng)免疫細(xì)胞化學(xué)染色檢測(cè)Nestin,結(jié)果為陽性。免疫細(xì)胞化學(xué)染色分別檢測(cè)分化細(xì)胞的β-Tublin、GFAP,結(jié)果為陽性。采用Brdu標(biāo)記神經(jīng)干細(xì)胞,免疫細(xì)胞化學(xué)檢測(cè)Brdu,原代、次代NSC均為陽性。體內(nèi)移植7d后在海馬和側(cè)腦室可檢測(cè)到Brdu標(biāo)記陽性的細(xì)胞。 2.分離培養(yǎng)小鼠胚胎成纖維細(xì)胞、Sertoli細(xì)胞及骨髓基質(zhì)細(xì)胞,并用絲裂霉素C處理,成功制備飼養(yǎng)層。 3.三種飼養(yǎng)層都能使NSC增殖,在胚胎成纖維細(xì)胞、骨髓基質(zhì)細(xì)胞飼養(yǎng)層上的生長狀況優(yōu)于Sertoli細(xì)胞飼養(yǎng)層。細(xì)胞因子組培養(yǎng)效果優(yōu)于飼養(yǎng)層組,對(duì)照組細(xì)胞分化明顯。 結(jié)論: 1.新生昆明小鼠大腦海馬能分離出NSC。 2.NSC大腦內(nèi)移植能繼續(xù)存活。 3.NSC細(xì)胞在三種飼養(yǎng)層上均能穩(wěn)定的增殖并保持未分化狀態(tài),在胚胎成纖維細(xì)胞、骨髓基質(zhì)細(xì)胞飼養(yǎng)層上的生長狀況優(yōu)于Sertoli細(xì)胞飼養(yǎng)層。 4.細(xì)胞因子組培養(yǎng)NSC效果優(yōu)于飼養(yǎng)層組,但細(xì)胞因子的價(jià)格昂貴,大規(guī)模培養(yǎng)NSC成本過高。飼養(yǎng)層組可較好地模擬體內(nèi)細(xì)胞生長微環(huán)境,且培養(yǎng)費(fèi)用低廉、增殖穩(wěn)定等方面較細(xì)胞因子組有優(yōu)勢(shì),適宜在普通實(shí)驗(yàn)室推廣。
[Abstract]:Objective: to explore the suitable techniques and conditions for alternative cytokine culture of neural stem cells, to establish the culture system of neural stem cells in vitro, to reduce the experimental cost, and to lay a foundation for the establishment of cell resource bank. Methods: 1. The hippocampus was isolated and made into single cell suspension. The cells were cultured in the serum-free medium of DMEM / F _ (12) containing 27% bFGF EGF (20 ng / ml). Nestin immunocytochemistry was used to identify NSCsin primary and passage cells. Immunocytochemical method was used to detect 尾 -Tublinus GFAPs. Primary neural stem cells (NSCs) were labeled with Brdu. Neural stem cells (NSCs) were labeled with Brdu and transplanted into hippocampus and lateral ventricle of homogenous mice. Sertoli cells and bone marrow stromal cells were selected from embryonic fibroblasts to establish a cell culture system. The feeder layer was prepared with mitomycin C of 10 渭 g/ml. The experiment was divided into five groups. The count cells were observed under inverted microscope and the growth curve was drawn. Results: 1. The addition of cytokines can form a large number of suspension growth cells. The cell ball can form the secondary cell ball after multiple passages. Nestin was detected by immunocytochemical staining in primary and secondary cells. The 尾-Tublinus GFAPs of differentiated cells were detected by immunocytochemical staining, and the results were positive. Brdu labeled neural stem cells were used to detect Brdu. primary and secondary NSC were all positive by immunocytochemistry. Brdu labeled positive cells were detected in hippocampus and lateral ventricle 7 days after transplantation in vivo. 2. 2. Sertoli cells and bone marrow stromal cells were isolated from mouse embryonic fibroblasts and treated with mitomycin C. All the three feeding layers could make NSC proliferate. The growth of NSC in the feeder layer of embryonic fibroblasts and bone marrow stromal cells was better than that of Sertoli cell feeder layer. The culture effect of cytokine group was better than that of feeder layer group, and the cell differentiation of control group was obvious. Conclusion: 1. NSC2. 2. NSC can survive. 3. NSC cells can proliferate stably and remain undifferentiated in the three feeder layers, and NSC cells can be transplanted into embryonic fibroblasts. The growth status of bone marrow stromal cell feeder layer was better than that of Sertoli cell feeder layer. 4. The effect of cultured NSC in cytokine group was better than that in feeder layer group, but the price of cytokines was high, and the cost of large scale culture of NSC was too high. The feeding layer group can simulate the microenvironment of cell growth in vivo, and the culture cost is low, the proliferation is stable and so on, which is suitable to be popularized in the common laboratory.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

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