【摘要】：目的探討過表達Mash-1基因?qū)π∈笈咛ジ杉毎?embryonic stem cells,ESC)向神經(jīng)細胞分化的影響。方法采用病毒感染技術(shù)將MSCV-Mash-1基因、MSCV空載體感染至小鼠ESC(CE3細胞)(MSCV-Mash-1-CE3組、MSCV-CE3組),RT-PCR鑒定細胞Mash-1基因的表達情況;然后采用懸滴培養(yǎng)法使其形成擬胚體,再經(jīng)神經(jīng)培養(yǎng)液貼壁誘導分化。培養(yǎng)7、21 d于倒置相差顯微鏡下觀察細胞形態(tài)改變;免疫熒光染色檢測細胞貼壁后其神經(jīng)干細胞和神經(jīng)元標記蛋白巢蛋白(nestin)和β-微管蛋白Ⅲ(β-tubulinⅢ)的陽性率;實時熒光定量PCR檢測誘導培養(yǎng)0、1、7、14、21 d時,細胞甲胎蛋白(α-fetal protein,AFP)(內(nèi)胚層標記基因)、Brachyury(中胚層標記基因)、FGF-5(外胚層標記基因)、Oct3/4(ESC標記基因)、nestin(神經(jīng)干細胞標記基因)和β-tubulinⅢ(神經(jīng)元標記基因)表達情況。以正常CE3細胞作為對照(CE3組)。結(jié)果與MSCV-CE3組和CE3組比較,MSCVMash-1-CE3組細胞Mash-1基因表達明顯升高。經(jīng)神經(jīng)培養(yǎng)液誘導培養(yǎng)7、21 d后,MSCV-Mash-1-CE3組可見許多細胞折光性增強,長出細長軸突,出現(xiàn)單極、雙極或多極形態(tài),呈神經(jīng)干細胞和神經(jīng)元細胞形態(tài);MSCV-CE3組和CE3組僅能觀察到少數(shù)細胞長出細長軸突。免疫熒光染色檢測示MSCV-Mash-1-CE3組誘導分化7 d nestin陽性率和21 dβ-tubulinⅢ陽性率顯著高于MSCV-CE3組及CE3組(P0.05)。實時熒光定量PCR檢測示,與CE3組及MSCV-CE3組比較,培養(yǎng)1 d后MSCV-Mash-1-CE3組Brachyury表達明顯降低(P0.05),FGF-5和nestin表達增高(P0.05);7 d后β-tubulinⅢ表達亦顯著增高(P0.05);CE3組及MSCV-CE3組以上指標比較,差異均無統(tǒng)計學意義(P0.05)。3組間各時間點AFP和Oct3/4表達比較,差異均無統(tǒng)計學意義(P0.05)。結(jié)論過表達Mash-1基因能促進小鼠ESC向神經(jīng)細胞方向分化。
[Abstract]:Objective to investigate the effect of overexpression of Mash-1 gene on the differentiation of mouse embryonic stem cells (embryonic stem cells,ESC) into neural cells. Methods MSCV-Mash-1 gene and MSCV empty vector were infected into ESC (CE3 cells) of mice (MSCV-Mash-1-CE3 group, MSCV-CE3 group) by virus infection technique. RT-PCR was used to identify the expression of Mash-1 gene. Then suspension culture method was used to induce the formation of embryoid body, and then differentiation was induced by adherent neural media. The morphological changes of the cells were observed under inverted phase-contrast microscope for 21 days, and the positive rates of nestin (nestin) and 尾 -tubulin 鈪，

本文編號：2420970