本文選題：sonic + hedgehog�。� 參考：《南方醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 神經(jīng)干細(xì)胞(neural stem cells,NSCs)指能自我增殖和自我更新,并具有向神經(jīng)元和膠質(zhì)細(xì)胞分化潛能的細(xì)胞。在神經(jīng)系統(tǒng)發(fā)育的過程中,神經(jīng)干細(xì)胞大部分分化成膠質(zhì)細(xì)胞而只有少部分分化為神經(jīng)元。大量關(guān)于神經(jīng)干細(xì)胞體外分化實(shí)驗(yàn)或體內(nèi)移植后檢測(cè)的結(jié)果也顯示,在沒有特殊干預(yù)的情況下,大部分神經(jīng)干細(xì)胞都將分化為星形膠質(zhì)細(xì)胞,而分化成神經(jīng)元或少突膠質(zhì)細(xì)胞的比率很小。然而,移植神經(jīng)干細(xì)胞治療中樞神經(jīng)損傷的主要目的卻是希望它能在分化后替代死亡或退化的神經(jīng)元或少突膠質(zhì)細(xì)胞,所以如何促進(jìn)神經(jīng)干細(xì)胞向神經(jīng)元或少突膠質(zhì)細(xì)胞分化已經(jīng)成為目前大家重點(diǎn)關(guān)注的問題。 Sonic Hedgehog(SHH)是胚胎發(fā)育過程中由脊索產(chǎn)生的一種重要的發(fā)育調(diào)控因子,根據(jù)胚胎發(fā)育時(shí)期SHH的作用機(jī)制及目前的研究進(jìn)展,我們?cè)O(shè)想SHH有可能會(huì)對(duì)神經(jīng)干細(xì)胞的增殖與分化發(fā)生一定的作用,但是,迄今為止尚未見相關(guān)的報(bào)道。為此,本課題擬通過體外實(shí)驗(yàn),探討SHH對(duì)神經(jīng)干細(xì)胞的增殖與分化的影響,為神經(jīng)干細(xì)胞定向誘導(dǎo)分化研究及體內(nèi)移植實(shí)驗(yàn)提供基礎(chǔ)。 本課題分為兩部分: 第一部分 目的:從大鼠胚胎端腦分離培養(yǎng)神經(jīng)干細(xì)胞,并對(duì)所獲得的神經(jīng)干細(xì)胞進(jìn)行特異性蛋白表達(dá)以及增殖與分化能力鑒定。 方法:從大鼠胚胎(E9.5d)端腦分離培養(yǎng)神經(jīng)干細(xì)胞,用含堿性成纖維細(xì)胞生長(zhǎng)因子和表皮生長(zhǎng)因子的無血清培養(yǎng)液進(jìn)行培養(yǎng)。第2代細(xì)胞采取熒光免疫細(xì)胞化學(xué)檢測(cè)神經(jīng)干細(xì)胞特異性標(biāo)志物nestin的表達(dá);在培養(yǎng)液中添加BrdU 3h后檢測(cè)BrdU在神經(jīng)干細(xì)胞中的表達(dá);在含血清而無生長(zhǎng)因子的分化條件培養(yǎng)液中培養(yǎng)7d后經(jīng)βⅢ-tubulin、GFAP、Rip和nestin熒光免疫細(xì)胞化學(xué)檢測(cè)細(xì)胞分化情況。 結(jié)果:體外培養(yǎng)的神經(jīng)干細(xì)胞能快速增殖并形成神經(jīng)球,其中的細(xì)胞均表達(dá)神經(jīng)干細(xì)胞特異性標(biāo)志物nestin。所獲得的細(xì)胞能將BrdU結(jié)合到細(xì)胞核中;部分細(xì)胞能分化為神經(jīng)元、星型膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞。 結(jié)論:我們獲得的細(xì)胞在特異性標(biāo)志物、增殖與分化能力等方面均符合神經(jīng)干細(xì)胞的特性。 第二部分 目的:探討sonic hedgehog(SHH)對(duì)神經(jīng)干細(xì)胞增殖與分化的影響。 方法:在分別將SHH、維甲酸(retinoic acid,RA)或PBS添加入培養(yǎng)液的情況下,對(duì)第2代神經(jīng)干細(xì)胞的增殖能力(BrdU陽性細(xì)胞核的百分率)、分化能力(βⅢ-tubulin、GFAP、Rip和nestin陽性細(xì)胞百分率)進(jìn)行檢測(cè),用SPSS13.0做One-way ANOVA分析,組間多重比較方差齊性時(shí)用LSD方法,方差不齊時(shí)使用Dunnett T3方法。 結(jié)果:SHH組的BrdU、βⅢ-tubulin和Rip陽性率均顯著高于RA組和PBS組,nestin陽性率顯著低于RA組和PBS組,GFAP陽性率各組間無顯著差異。 結(jié)論:SHH可以提高神經(jīng)干細(xì)胞的增殖能力,并可促進(jìn)其向神經(jīng)元和少突膠質(zhì)細(xì)胞分化。
[Abstract]:Neural stem cells (NSCs) are cells that can proliferate and renew themselves and have the potential to differentiate into neurons and glial cells. During the development of the nervous system, most neural stem cells differentiate into glial cells and only a few differentiate into neurons. Numerous studies of neural stem cell differentiation in vitro or after in vivo transplantation also show that most neural stem cells will differentiate into astrocytes without special intervention. The rate of differentiation into neurons or oligodendrocytes is small. However, the main purpose of transplantation of neural stem cells in the treatment of central nerve injury is to hope that it can replace dead or degenerated neurons or oligodendrocytes after differentiation. Therefore, how to promote the differentiation of neural stem cells into neurons or oligodendrocytes has become the focus of attention. Sonic Hedgehog is an important developmental regulator produced by the notochord during embryonic development. According to the mechanism of SHH during embryonic development and its current research progress, Hedgehog is one of the most important developmental regulators in embryonic development. We hypothesized that SHH might play a role in the proliferation and differentiation of neural stem cells. Therefore, this study aims to explore the effect of SHH on the proliferation and differentiation of neural stem cells in vitro, and to provide a basis for the study of neural stem cell differentiation in vitro and in vivo transplantation. This topic is divided into two parts: Part one Aim: to isolate and culture neural stem cells from rat embryonic terminal brain and to identify the specific protein expression, proliferation and differentiation ability of the obtained neural stem cells. Methods: neural stem cells (NSCs) were isolated and cultured from the rat embryonic stem cells (E9.5 d) and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The expression of nestin, a specific marker of neural stem cells, was detected by fluorescence immunocytochemistry in the second generation of cells, and the expression of BrdU in neural stem cells was detected after adding BrdU for 3 h. The differentiation of the cells was detected by fluorescence immunocytochemistry of 尾 -tubulin Gapp Rip and nestin after being cultured in the medium containing serum but without growth factor for 7 days. Results: neural stem cells cultured in vitro could proliferate rapidly and form neurospheres, and all of the cells expressed the specific marker of neural stem cells, Neisin. The obtained cells can bind BrdU to the nucleus, and some of the cells can differentiate into neurons, astrocytes and oligodendrocytes. Conclusion: the obtained cells accord with the characteristics of neural stem cells in specific markers, proliferation and differentiation. Part two Objective: to investigate the effect of sonic hedgehog on the proliferation and differentiation of neural stem cells. Methods: under the condition of adding SHH, retinoic acid PBS or PBS into the culture medium, the percentage of BrdU positive nuclei and the differentiation ability (尾 鈪，

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