本文選題：中腦/皮質(zhì) + 神經(jīng)干細(xì)胞　； 參考：《武漢大學(xué)》2005年碩士論文

【摘要】：目的 在己成功分離培養(yǎng)皮質(zhì)神經(jīng)干細(xì)胞的基礎(chǔ)上探索小鼠胚胎中腦區(qū)神經(jīng)干細(xì)胞體外培養(yǎng)的方法,并觀察比較二者在生長(zhǎng)和分化性狀方面的差異,以及它們?cè)贗L-1β、AA作用下向TH陽(yáng)性神經(jīng)元的誘導(dǎo)分化,為神經(jīng)干細(xì)胞的基礎(chǔ)理論和帕金森病細(xì)胞移植治療的應(yīng)用研究提供一定的實(shí)驗(yàn)資料。 方法 1.無(wú)菌條件下分離E12-E13天小鼠胚腦腹側(cè)中腦曲,胰酶消化和機(jī)械吹打制成單細(xì)胞懸液,在bFGF和B27存在的無(wú)血清培養(yǎng)基中培養(yǎng)擴(kuò)增,機(jī)械分離法傳代,免疫細(xì)胞化學(xué)染色方法鑒定神經(jīng)干細(xì)胞及其子代細(xì)胞的分化方向。 2.無(wú)菌條件下同時(shí)分離培養(yǎng)胚鼠大腦皮質(zhì)與腹側(cè)中腦神經(jīng)干細(xì)胞,倒置顯微鏡觀察比較二者生長(zhǎng)狀況;含10%血清的DMEM/F12培養(yǎng)基接種分化,觀察比較它們分化子代細(xì)胞中TH陽(yáng)性神經(jīng)元的情況。 3.體外培養(yǎng)中腦神經(jīng)干細(xì)胞和皮質(zhì)神經(jīng)干細(xì)胞,分別傳代各分為四組,在有血清條件下分別予以AA、IL-1β及AA+IL-1β誘導(dǎo)分化,并設(shè)空白對(duì)照,DAPI細(xì)胞核標(biāo)記,抗酪氨酸羥化酶(TH)免疫細(xì)胞化學(xué)鑒定分化結(jié)果并細(xì)胞計(jì)數(shù)陽(yáng)性細(xì)胞比例。 結(jié)果 1.從E12-E13鼠胚腦腹側(cè)中腦曲分離的小部分細(xì)胞在B27和bFGF存在的無(wú)血清培養(yǎng)基中可以在體外培養(yǎng)和傳代,這些細(xì)胞表達(dá)神經(jīng)干細(xì)胞特異性抗原nestin,在撤除B27和bFGF的有血清培養(yǎng)基中能夠分化為神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞。 2.小鼠胎腦大腦皮質(zhì)部位所含神經(jīng)干細(xì)胞明顯多于中腦,體外相同條件下皮質(zhì)神經(jīng)干細(xì)胞也更宜成球;有血清條件下分化,中腦神經(jīng)干細(xì)胞可少量分化出TH陽(yáng)性神經(jīng)元;皮質(zhì)神經(jīng)干細(xì)胞則未見(jiàn)分化出TH陽(yáng)性神經(jīng)元。 3.在誘導(dǎo)分化實(shí)驗(yàn)中,AA、IL-1β及AA+IL-1β作用下中腦神經(jīng)干細(xì)胞分化出TH陽(yáng)性神經(jīng)元比例分別約為10%、9%和11%,明顯高于對(duì)照組(約1%,P0.01);三實(shí)驗(yàn)組間相對(duì)比較無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);加AA誘導(dǎo)組中分化出的TH陽(yáng)性神經(jīng)元細(xì)胞形態(tài)更成熟。皮質(zhì)神經(jīng)干細(xì)胞各實(shí)驗(yàn)組均未見(jiàn)分化出TH陽(yáng)性神經(jīng)元。
[Abstract]:Objective to explore the method of in vitro culture of neural stem cells from mouse embryonic mesencephalic region on the basis of successful isolation and culture of cortical neural stem cells, and to observe and compare the differences of growth and differentiation between them. And their induction and differentiation into th positive neurons under the action of IL-1 尾 -AA provide some experimental data for the basic theory of neural stem cells and the application of transplantation therapy for Parkinson's disease. Method 1. Single cell suspension was isolated from the ventral midbrain of E12-E13 day mouse embryo under aseptic conditions, digested by trypsin and mechanically blown. The suspension was cultured and amplified in serum-free medium of bFGF and B27, and was subcultured by mechanical separation. Immunocytochemical staining was used to identify the differentiation of neural stem cells and their progenies. 2. Embryonic rat cerebral cortex and ventral mesencephalic neural stem cells were isolated and cultured simultaneously under aseptic condition. The growth status of the two cells was observed and compared by inverted microscope. DMEM/F12 medium containing 10% serum was inoculated and differentiated. The th positive neurons in the differentiated progenies were observed and compared. 3. Neural stem cells (NSCs) and cortical neural stem cells (NSCs) were cultured in vitro and were subcultured into four groups respectively. Under the condition of serum, they were induced to differentiate by AA-IL-1 尾 and AA IL-1 尾, respectively. Antityrosine hydroxylase (THH) immunocytochemistry was used to identify the differentiation and the percentage of positive cells counted. Result 1. A small number of cells isolated from the ventral midbrain of E12-E13 mouse embryos could be cultured and subcultured in serum-free medium containing B27 and bFGF in vitro. These cells express the specific antigen of neural stem cells, Neisin, and can differentiate into neurons and glial cells in serum-containing medium that removes B27 and bFGF. 2. The neural stem cells contained more neural stem cells in the cortex of the fetal brain than in the mesencephalon, and in the same conditions in vitro, the neural stem cells in the cortex were more suitable for forming spheres, and in the presence of serum, the neural stem cells in the mesencephalon could differentiate into a small amount of th positive neurons. No th positive neurons were found in cortical neural stem cells. 3. The percentage of th positive neurons induced by IL-1 尾 and AA IL-1 尾 were 10 9% and 11%, respectively, which were significantly higher than those in the control group (P 0.01); there was no significant difference among the three experimental groups (P 0.05); The differentiated th-positive neurons were more mature in morphology. No th-positive neurons were found in all experimental groups of cortical neural stem cells.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R329

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本文編號(hào)：1907618