發(fā)布時(shí)間：2018-04-22 09:39

  本文選題：室管膜下區(qū) + 神經(jīng)干細(xì)胞��； 參考：《中國(guó)修復(fù)重建外科雜志》2015年06期

【摘要】：目的探討新生小鼠室管膜下區(qū)(subventricular zone,SVZ)神經(jīng)干細(xì)胞(neural stem cells,NSCs)體外培養(yǎng)方法,為治療神經(jīng)系統(tǒng)疾病尋找合適的種子細(xì)胞。方法取SPF級(jí)新生ICR小鼠SVZ組織,采用機(jī)械法和酶消化法分離培養(yǎng)NSCs并傳代,倒置顯微鏡下觀察細(xì)胞形態(tài)。取第3代細(xì)胞行抗SOX-2和抗巢蛋白(Nestin)免疫熒光染色鑒定,Brd U標(biāo)記法及MTT法檢測(cè)比較培養(yǎng)3、7 d細(xì)胞增殖能力。以未添加b FGF和EGF的無(wú)血清培養(yǎng)基誘導(dǎo)第3代NSCs分化,抗β-微管蛋白Ⅲ(Tuj-1)、GFAP免疫熒光染色檢測(cè)比較誘導(dǎo)3、7 d NSCs向神經(jīng)元及星形膠質(zhì)細(xì)胞分化的能力。結(jié)果成功分離培養(yǎng)新生小鼠SVZ組織中細(xì)胞,倒置顯微鏡下見(jiàn)原代培養(yǎng)3 d即有神經(jīng)球形成,至7 d時(shí)見(jiàn)大量懸浮生長(zhǎng)神經(jīng)球,且體積較前增大,部分神經(jīng)球出現(xiàn)融合及貼壁分化現(xiàn)象。細(xì)胞呈典型NSCs形態(tài)。免疫熒光染色鑒定獲得細(xì)胞為NSCs。細(xì)胞增殖能力檢測(cè)顯示,體外培養(yǎng)3 d的NSCs中Brd U陽(yáng)性細(xì)胞數(shù)為(75.817±2.961)個(gè)、吸光度(A)值為0.478±0.025,均顯著高于培養(yǎng)7 d的(56.600±4.881)個(gè)、0.366±0.032(t=3.366,P=0.028;t=2.752,P=0.011)。經(jīng)誘導(dǎo)培養(yǎng)后,NSCs能分化為神經(jīng)元、星形膠質(zhì)細(xì)胞;細(xì)胞分化能力檢測(cè)顯示,誘導(dǎo)分化3 d時(shí)Tuj-1、GFAP陽(yáng)性細(xì)胞百分比分別為23.1%±3.7%、23.7%±3.8%,顯著低于誘導(dǎo)分化7 d(40.1%±3.6%、37.1%±4.5%)(t=3.285,P=0.030;t=3.930,P=0.017)。結(jié)論體外培養(yǎng)的新生小鼠SVZ處NSCs時(shí)具有自我增殖和多分化潛能,且體外培養(yǎng)時(shí)間不同,細(xì)胞增殖、分化能力亦不同。
[Abstract]:Objective to explore the culture method of neural stem cells (NSCs) from subventricular subventricular zone (SVZ) of newborn mice in vitro, and to find suitable seed cells for the treatment of nervous system diseases. Methods the SVZ tissues of newborn ICR mice of SPF grade were isolated and cultured by mechanical method and enzyme digestion method. The morphology of the cells was observed under inverted microscope. The proliferative ability of the third passage cells was assayed by Brd-U labeling method and MTT method by immunofluorescence staining of anti-nestin and anti-nestin. The third generation of NSCs differentiation was induced by serum-free medium without b FGF and EGF, and the ability of inducing NSCs to differentiate into neurons and astrocytes was compared by immunofluorescence staining with anti 尾 tubulin 鈪，

本文編號(hào)：1786671