本文選題：小鼠胚胎干細(xì)胞 + 神經(jīng)分化�。� 參考：《浙江大學(xué)》2011年碩士論文

【摘要】：目的：探索異戊烯基黃酮異補(bǔ)骨脂甲素(Isobavachin, IBA)是否具有促小鼠胚胎干(embryonic stem, ES)細(xì)胞定向分化為神經(jīng)細(xì)胞的作用,并探索其相應(yīng)的分化機(jī)制。 方法：采用懸滴培養(yǎng)4-/4+法模擬體內(nèi)胚胎發(fā)育狀態(tài),形成擬胚體,繼而進(jìn)行貼壁培養(yǎng)。IBA以終濃度為10-7mol/L誘導(dǎo)小鼠ES細(xì)胞定向分化為神經(jīng)細(xì)胞,10-7mol/L全反式維甲酸(Retinoic acid, RA)為陽性對照,0.1%二甲基亞砜(DMSO)為溶劑對照。在分化培養(yǎng)終點(diǎn),利用光鏡和免疫熒光成像法鑒定神經(jīng)元形成(軸突長度是胞體長度的3倍及以上)。收集ES細(xì)胞、擬胚體(embryoid bodies, EBs)、鋪展培養(yǎng)d 8+0、d 8+5、d 8+10各時(shí)期細(xì)胞樣本,用Western-blot以及RT-PCR法,評價(jià)神經(jīng)細(xì)胞微管蛋白(β-tubulinⅢ)和神經(jīng)膠質(zhì)纖維酸性蛋白(GFAP),作為ES細(xì)胞定向分化為神經(jīng)細(xì)胞的指標(biāo)；觀察巢蛋白基因(nestin),神經(jīng)纖維蛋白(NEFM),干細(xì)胞未分化狀態(tài)基因(OCT3/4)等在各個神經(jīng)分化時(shí)期表達(dá)特征。同時(shí)利用、Vestern-blot (?)去檢測MAPK通路中p38, ERK, JNK磷酸化等相關(guān)分子事件的變化,以探索神經(jīng)分化的可能機(jī)制。添加IBA時(shí)同步添加異戊稀基酶Ⅰ型抑制劑GGTI-298,通過條件摸索確定終濃度為10-6mol/L,觀察其是否對IBA的促神經(jīng)分化作用有影響,驗(yàn)證蛋白異戊稀化是否與IBA促小鼠ES細(xì)胞定向分化為神經(jīng)細(xì)胞呈相關(guān)性。 結(jié)果：IBA誘導(dǎo)ES細(xì)胞分化為神經(jīng)細(xì)胞過程中,光鏡下多數(shù)呈典型神經(jīng)元表型,即軸突長度是胞體長度的3倍及以上。免疫熒光成像結(jié)果表明,EBs期呈nestin,OCT3/4陽性表達(dá)；分化后期大多數(shù)呈神經(jīng)元表型,并分別表達(dá)β-tubulinⅢ, ChAT,GABA等陽染,少量未呈神經(jīng)元表型者GFAP陽染。Western blot (?)去觀察在分化過程中,β-tubulinⅢ, GFAP表達(dá)均隨分化時(shí)程延長而呈上調(diào)趨勢,尤以β-tubulinⅢ陽性表達(dá)為甚。同步添加異戊稀基酶抑制劑GGTI-298能顯著減少IBA促ES細(xì)胞分化為神經(jīng)細(xì)胞,而未明顯影響RA促神經(jīng)細(xì)胞分化,提示兩種化合物誘導(dǎo)神經(jīng)分化作用機(jī)制不盡相同。MAPK通路中p38和JNK的磷酸化表達(dá)下調(diào),ERK磷酸化表達(dá)上調(diào),提示該通路不同蛋白的磷酸化與否與IBA誘導(dǎo)ES細(xì)胞分化為神經(jīng)細(xì)胞呈相關(guān)性。 結(jié)論：IBA具有促進(jìn)小鼠ES細(xì)胞定向分化為神經(jīng)細(xì)胞的作用,主要為神經(jīng)元,并有一定比例的星形膠質(zhì)細(xì)胞,異戊稀基是IBA促神經(jīng)分化的功能基團(tuán)。IBA通過蛋白異戊烯化,下調(diào)MAPK通路中p38,JNK磷酸化,上調(diào)ERK磷酸化促進(jìn)小鼠ES細(xì)胞定向分化為神經(jīng)細(xì)胞。 目的：探索異補(bǔ)骨脂甲素(Isobavachin, IBA)促小鼠ES細(xì)胞神經(jīng)元分化過程中是否伴隨可興奮細(xì)胞運(yùn)動記憶相關(guān)蛋白Junctophilin 3,4(JP-3和JP-4)的表達(dá)。 方法：采用懸滴培養(yǎng)4-/4+法模擬體內(nèi)胚胎發(fā)育狀態(tài),形成擬胚體(embryoid bodies, EBs),繼而進(jìn)行貼壁培養(yǎng)。IBA以終濃度為10-7mol/L誘導(dǎo)小鼠ES細(xì)胞定向分化為神經(jīng)細(xì)胞,10-7mol/L全反式維甲酸(Retinoic acid, RA)為陽性對照,0.1%二甲基亞砜(DMSO)為溶劑對照。在分化培養(yǎng)終點(diǎn),利用光鏡和免疫熒光成像法鑒定神經(jīng)元,JP-3和JP-4的表達(dá)。收集ES、EBs、鋪展培養(yǎng)d 8+0、d 8+5、d 8+10各時(shí)期細(xì)胞樣本,用Western-blot法,評價(jià)神經(jīng)細(xì)胞微管蛋白(β-tubulinⅢ)和運(yùn)動記憶相關(guān)蛋白(JP-3和JP-4)的表達(dá)與疊加特征,作為ES細(xì)胞定向分化為可興奮性神經(jīng)細(xì)胞的指標(biāo)。觀察巢蛋白基因(nestin),干細(xì)胞多潛能基因(OCT3/4)等隨著神經(jīng)分化的變化特征。 結(jié)果：IBA誘導(dǎo)ES細(xì)胞分化為神經(jīng)細(xì)胞過程中,免疫熒光成像表明,高度表達(dá)nestin的EBs同時(shí)高度表達(dá)JP-3和JP-4;隨著神經(jīng)分化時(shí)程,JP-3和JP-4逐步減少,分化末期d 8+10,DMSO組幾乎無JP-3和JP-4表達(dá),RA和IBA組尚有少量蛋白表達(dá)。JP-3和JP-4多數(shù)表達(dá)于神經(jīng)元胞體中,鮮有出現(xiàn)在軸突及突觸中。 結(jié)論：IBA促小鼠ES細(xì)胞分化的神經(jīng)元具有表達(dá)運(yùn)動記憶相關(guān)蛋白的功能。JP-3和JP-4在神經(jīng)分化早期高度表達(dá),成熟神經(jīng)元表達(dá)量相對減少,且分布存在一定的區(qū)域性。
[Abstract]:Objective : To explore the effect of isobavachin ( IBA ) on the orientation and differentiation of embryonic stem ( ES ) cells into nerve cells and to explore its corresponding differentiation mechanism .

Methods : The embryonic development of mouse ES cells was simulated by suspension culture 4 - / 4 + method . The cells were cultured with IBA at a final concentration of 10 - 7 mol / L .
We observed the expression of nestin , NEFM and OCT3 / 4 in different stages of neurodifferentiation . In order to explore the possible mechanism of neurodifferentiation , we determined whether the final concentration was 10 - 6 mol / L by adding IBA to determine whether the final concentration was 10 - 6 mol / L .

Results : IBA induced ES cell differentiation into nerve cells , most of them were typical neuronal phenotype , that is , the axon length was 3 times or more of the cell length . Immunofluorescence imaging showed that EBs had nestin and OCT3 / 4 positive expression .
In the later stage of differentiation , most of the neurons were in neuronal phenotype , and the positive staining of 尾 - ATPase 鈪，

本文編號：1763916