本文關(guān)鍵詞：胚胎大鼠神經(jīng)干細(xì)胞體外培養(yǎng)、分化與鑒定的實(shí)驗(yàn)研究　出處：《廣西醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胚胎大鼠 神經(jīng)干細(xì)胞 細(xì)胞培養(yǎng) 誘導(dǎo)分化

【摘要】： 目的: (1)建立簡(jiǎn)便、易行的胚胎大鼠NSCs體外分離、培養(yǎng)和擴(kuò)增的方法。 (2)鑒定NSCs及誘導(dǎo)分化后的子代細(xì)胞,為NSCs的體內(nèi)移植研究奠定基礎(chǔ)。 方法: (1)從胚胎大鼠腦組織中分離NSCs,采用神經(jīng)干細(xì)胞克隆懸浮法,選用DMEM/F12培養(yǎng)液,添加無(wú)血清培養(yǎng)添加劑b27,細(xì)胞生長(zhǎng)因子(bFGF和/或EGF)進(jìn)行體外擴(kuò)增培養(yǎng),機(jī)械分離連續(xù)傳代培養(yǎng),倒置相差顯微鏡觀察細(xì)胞形態(tài),細(xì)胞生長(zhǎng)曲線觀察NSCs的增殖能力,并采用免疫熒光細(xì)胞化學(xué)技術(shù)檢測(cè)NSCs標(biāo)志物Nestin的表達(dá)。 (2)觀察不同濃度的bFGF、EGF對(duì)NSCs增殖的影響,選擇最適合NSCs生長(zhǎng)的細(xì)胞生長(zhǎng)因子濃度。 (3)以多聚賴氨酸包被蓋玻片置于24孔培養(yǎng)板中,將傳代培養(yǎng)的NSCs接種于蓋玻片上,滴加5%胎牛血清的DMEM/F12培養(yǎng)液,誘導(dǎo)分化培養(yǎng)7天后,用免疫熒光細(xì)胞化學(xué)技術(shù)和免疫細(xì)胞化學(xué)染色,觀察GFAP、β-tubulin-Ⅲ的表達(dá)。 結(jié)果: (1)從胚胎大鼠腦組織分離的細(xì)胞在含b27及細(xì)胞生長(zhǎng)因子bFGF、EGF的無(wú)血清DMEM/F12培養(yǎng)液中,能夠形成大量懸浮的NSCs克隆球,并可在體外大量擴(kuò)增和連續(xù)傳代培養(yǎng),克隆球經(jīng)Nestin檢測(cè)鑒定證實(shí)為NSCs。 (2)發(fā)現(xiàn)不同的細(xì)胞生長(zhǎng)因子濃度對(duì)NSCs的增殖有影響,隨著細(xì)胞生長(zhǎng)因子濃度的增加,NSCs的增殖速度亦隨之加快,濃度在20-30ng/ml時(shí)細(xì)胞增殖速度達(dá)到較高水平,因子濃度大于50ng/ml時(shí)細(xì)胞增殖速度反而下降。 (3)胎牛血清共培養(yǎng)7天后,分別做β-tubulin-Ⅲ,GFAP抗體檢測(cè),結(jié)果顯示分化后的細(xì)胞部分表達(dá)神經(jīng)元、星形膠質(zhì)細(xì)胞的特異性抗原。
[Abstract]:Objective: To establish a simple and easy method for isolation, culture and amplification of embryonic rat NSCs in vitro. 2) Identification of NSCs and induced differentiation of progeny cells, which laid a foundation for in vivo transplantation of NSCs. Methods: 1) NSCs were isolated from embryonic rat brain tissue. Neural stem cell clone suspension method was used, DMEM/F12 culture medium was used and serum-free culture additive b27 was added. Cell growth factor bFGF and / or EGF were cultured in vitro and cultured by mechanical isolation and subculture. The morphology of cells was observed by inverted phase contrast microscope and the proliferative ability of NSCs was observed by cell growth curve. The expression of NSCs marker Nestin was detected by immunofluorescence cytochemistry. (2) to observe the effect of different concentrations of bFGF EGF on the proliferation of NSCs, and to select the concentration of cell growth factor that is most suitable for the growth of NSCs. The covered glass was coated with poly-lysine and placed in a 24-well culture plate. The NSCs cultured by passage was inoculated on the cover glass, and the DMEM/F12 medium of 5% fetal bovine serum was added to the culture medium. The expression of GFAP, 尾 -tubulin- 鈪，

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