本文選題：膠質(zhì)瘤干細(xì)胞 + PAX3　； 參考：《蘇州大學(xué)》2016年博士論文

【摘要】：第一部分 轉(zhuǎn)錄因子PAX3在膠質(zhì)瘤干細(xì)胞中的表達(dá)及生物學(xué)作用背景與目的轉(zhuǎn)錄因子PAX3作為配對(duì)盒轉(zhuǎn)錄子家族一員,其不僅在神經(jīng)系統(tǒng)胚胎發(fā)育過(guò)程中發(fā)揮重要作用,而且在神經(jīng)膠質(zhì)瘤的發(fā)生發(fā)展過(guò)程中起著重要作用。我們前期工作中發(fā)現(xiàn)PAX3在人腦神經(jīng)膠質(zhì)瘤組織中呈高表達(dá),且與神經(jīng)膠質(zhì)瘤惡性程度密切相關(guān)。膠質(zhì)瘤干細(xì)胞作為種子細(xì)胞是膠質(zhì)瘤發(fā)生、發(fā)展、治療失敗和復(fù)發(fā)的根源。本部分旨在研究轉(zhuǎn)錄因子PAX3在人腦膠質(zhì)瘤干細(xì)胞中表達(dá)及對(duì)人腦膠質(zhì)瘤干細(xì)胞凋亡、增殖、侵襲及分化等生物學(xué)行為的影響,進(jìn)一步探討其在人腦膠質(zhì)瘤發(fā)生發(fā)展過(guò)程中所起的作用。方法 1、對(duì)人腦膠質(zhì)瘤干細(xì)胞系GSC11進(jìn)行細(xì)胞培養(yǎng)及干細(xì)胞鑒定。通過(guò)單克隆實(shí)驗(yàn)驗(yàn)證膠質(zhì)瘤干細(xì)胞的克隆形成能力；2、采用實(shí)時(shí)熒光定量PCR (RT-PCR)與免疫印跡技術(shù)(Western Blot)檢測(cè)人腦膠質(zhì)瘤干細(xì)胞系GSC11、人腦膠質(zhì)瘤細(xì)胞系U87及人腦神經(jīng)膠質(zhì)細(xì)胞系1800中轉(zhuǎn)錄因子PAX3的表達(dá)；3、通過(guò)轉(zhuǎn)染PAX3真核過(guò)表達(dá)質(zhì)粒及小分子干擾RNA技術(shù)(siRNA)分別對(duì)GSC11細(xì)胞中轉(zhuǎn)錄因子PAX3進(jìn)行過(guò)表達(dá)和干擾表達(dá),采用RT-PCR和Western Blot檢測(cè)過(guò)表達(dá)和干擾效果。采用Western Blot檢測(cè)干擾PAX3表達(dá)后對(duì)膠質(zhì)瘤干細(xì)胞標(biāo)志物CD133及Nestin表達(dá)的影響。流式細(xì)胞儀檢測(cè)GSC11細(xì)胞凋亡率；CCK-8法檢測(cè)GSC11細(xì)胞增殖能力；微孔濾膜培養(yǎng)小室及雙室聯(lián)合培養(yǎng)系統(tǒng)(Transwell小室實(shí)驗(yàn))檢測(cè)GSC11細(xì)胞侵襲能力；建立膠質(zhì)瘤干細(xì)胞誘導(dǎo)分化模型,采用細(xì)胞免疫熒光法檢測(cè)對(duì)GSC11細(xì)胞分化的影響。結(jié)果1、細(xì)胞免疫熒光檢測(cè)顯示GSC11細(xì)胞中CD133和Nestin表達(dá)陽(yáng)性,單克隆實(shí)驗(yàn)證實(shí)細(xì)胞具有良好的克隆形成能力,懸浮生長(zhǎng)的膠質(zhì)瘤干細(xì)胞聚集成球；2、RT-PCR與Western Blot發(fā)現(xiàn)GSC11細(xì)胞與U87細(xì)胞中PAX3的mRNA和蛋白表達(dá)水平均顯著高于1800細(xì)胞。3、轉(zhuǎn)染或者干擾后GSC11細(xì)胞中的PAX3的mRNA及蛋白表達(dá)水平較對(duì)照組明顯增高或者降低。4、干擾PAX3表達(dá)后GSC11細(xì)胞中CD133及Nestin表達(dá)水平明顯下降。5、過(guò)表達(dá)PAX3后明顯抑制GSC11細(xì)胞凋亡,細(xì)胞增殖能力明顯增強(qiáng),且細(xì)胞侵襲能力顯著提高；而抑制PAX3表達(dá)則明顯促進(jìn)GSC11細(xì)胞凋亡,細(xì)胞增殖能力明顯下降,且細(xì)胞侵襲能力顯著降低；6、細(xì)胞免疫熒光法發(fā)現(xiàn)過(guò)表達(dá)PAX3的GSC11細(xì)胞分化明顯減弱,GFAP陽(yáng)性細(xì)胞率顯著降低；而抑制PAX3表達(dá)的GSC11細(xì)胞分化明顯增強(qiáng),GFAP陽(yáng)性細(xì)胞率顯著升高。膠質(zhì)瘤干細(xì)胞分化過(guò)程中轉(zhuǎn)錄因子PAX3表達(dá)與GFAP陽(yáng)性細(xì)胞數(shù)呈負(fù)相關(guān)。結(jié)論轉(zhuǎn)錄因子PAX3在膠質(zhì)瘤干細(xì)胞中呈高表達(dá)。轉(zhuǎn)錄因子PAX3表達(dá)與膠質(zhì)瘤干細(xì)胞生物學(xué)行為密切相關(guān)。抑制PAX3的表達(dá)可促進(jìn)膠質(zhì)瘤干細(xì)胞凋亡與分化,抑制膠質(zhì)瘤干細(xì)胞增殖與侵襲能力。轉(zhuǎn)錄因子PAX3有望成為膠質(zhì)瘤干細(xì)胞治療新的靶分子。第二部分轉(zhuǎn)錄因子PAX3對(duì)膠質(zhì)瘤干細(xì)胞中膠質(zhì)纖維酸性蛋白基因的轉(zhuǎn)錄調(diào)控機(jī)制背景與目的膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)是星形膠質(zhì)細(xì)胞中主要的中間纖維素蛋白,參與構(gòu)成細(xì)胞骨架,在中樞神經(jīng)系統(tǒng)多能干細(xì)胞向星形細(xì)胞分化的過(guò)程中起著關(guān)鍵作用,GFAP基因表達(dá)功能狀態(tài)對(duì)于膠質(zhì)瘤細(xì)胞的生物學(xué)功能也有重要影響。本文第一部分研究發(fā)現(xiàn)在膠質(zhì)瘤干細(xì)胞向星型膠質(zhì)細(xì)胞分化過(guò)程中轉(zhuǎn)錄因子PAX3與GFAP陽(yáng)性細(xì)胞率呈負(fù)相關(guān)。本部分旨在進(jìn)一步研究轉(zhuǎn)錄因子PAX3對(duì)膠質(zhì)瘤干細(xì)胞中GFAP基因的轉(zhuǎn)錄調(diào)控機(jī)制。方法1、采用RT-PCR與Western Blot檢測(cè)人腦膠質(zhì)瘤干細(xì)胞系GSC 11、人腦膠質(zhì)瘤細(xì)胞系U87及人腦神經(jīng)膠質(zhì)細(xì)胞系1800中GFAP的表達(dá)；2、RT-PCR與Western Blot檢測(cè)轉(zhuǎn)染過(guò)表達(dá)PAX3及siRNA干擾降低PAX3表達(dá)后GSC11細(xì)胞中GFAP表達(dá),并分析PAX3與GFAP表達(dá)相關(guān)性；3、通過(guò)染色質(zhì)免疫共沉淀法(ChIP Assay)檢測(cè)轉(zhuǎn)錄因子PAX3在GSC11細(xì)胞中與GFAP基因啟動(dòng)子相應(yīng)區(qū)域結(jié)合情況；4、予以構(gòu)建正常及突變的人GFAP啟動(dòng)子報(bào)告基因質(zhì)粒,通過(guò)熒光素酶報(bào)告分析系統(tǒng)(Luciferase Assay)檢測(cè)轉(zhuǎn)錄因子PAX3對(duì)GFAP基因啟動(dòng)子相應(yīng)結(jié)合位點(diǎn)的轉(zhuǎn)錄調(diào)控活性；5、依據(jù)測(cè)得的GFAP基因啟動(dòng)子PAX3轉(zhuǎn)錄結(jié)合位點(diǎn)來(lái)設(shè)計(jì)探針序列,采用電泳遷移率試驗(yàn)(EMSA)進(jìn)一步驗(yàn)證轉(zhuǎn)錄因子PAX3與GFAP基因啟動(dòng)子相應(yīng)位點(diǎn)結(jié)合情況。結(jié)果1、RT-PCR與Western Blot發(fā)現(xiàn)GSC11細(xì)胞與U87細(xì)胞中GFAP的mRNA和蛋白表達(dá)水平明顯低于1800細(xì)胞；2、轉(zhuǎn)染過(guò)表達(dá)PAX3后GSC11細(xì)胞中GFAP的mRNA和蛋白表達(dá)水平明顯下降,而siRNA干擾降低PAX3表達(dá)后GSC11細(xì)胞中GFAP的mRNA和蛋白表達(dá)水平明顯增加。膠質(zhì)瘤干細(xì)胞中轉(zhuǎn)錄因子PAX3表達(dá)與GFAP表達(dá)呈負(fù)相關(guān)；3、ChIP實(shí)驗(yàn)結(jié)果證實(shí)轉(zhuǎn)錄因子PAX3在GSC11細(xì)胞中能與GFAP基因啟動(dòng)子相應(yīng)區(qū)域結(jié)合；4、在成功構(gòu)建正常與突變的GFAP啟動(dòng)子報(bào)告基因質(zhì)粒后,通過(guò)Luciferase Assay檢測(cè)發(fā)現(xiàn)過(guò)表達(dá)PAX3后正常GFAP啟動(dòng)子報(bào)告基因熒光素酶活性較對(duì)照組明顯提高。P1位點(diǎn)突變的GFAP啟動(dòng)子報(bào)告基因熒光素酶活性明顯下降,而P2位點(diǎn)突變的GFAP啟動(dòng)子報(bào)告基因熒光素酶活性無(wú)明顯下降。轉(zhuǎn)錄因子PAX3對(duì)GFAP基因啟動(dòng)子具有轉(zhuǎn)錄調(diào)控作用,且P1為其轉(zhuǎn)錄調(diào)控位點(diǎn)；5、EMSA實(shí)驗(yàn)進(jìn)一步證實(shí)了轉(zhuǎn)錄因子PAX3能與GFAP基因啟動(dòng)子結(jié)合位點(diǎn)P1特異性結(jié)合。結(jié)論膠質(zhì)瘤干細(xì)胞中轉(zhuǎn)錄因子PAX3表達(dá)與GFAP表達(dá)呈負(fù)相關(guān)。轉(zhuǎn)錄因子PAX3通過(guò)與膠質(zhì)瘤干細(xì)胞中的GFAP基因啟動(dòng)子區(qū)域特異性結(jié)合,對(duì)GFAP基因表達(dá)起轉(zhuǎn)錄調(diào)控作用。
[Abstract]:The expression of the first partial transcription factor PAX3 in human glioma stem cells and its biological function background and the target transcription factor PAX3 play an important role in the development of glioma .
2 . The expression of transcription factor PAX3 in human glioma stem cell line GSC11 , human glioma cell line U87 and human brain glioma cell line 1800 was detected by real - time fluorescence quantitative PCR ( RT - PCR ) and Western Blot .
3 . After transfection of PAX3 eukaryotic overexpression plasmid and small interfering RNA technology ( siRNA ) , the expression and interference of PAX3 in GSC11 cells were detected by RT - PCR and Western Blot . Western Blot was used to detect the effect of PAX3 expression on the expression of CD133 and Nestin in glioma stem cells .
CCK - 8 method was used to detect the proliferation of GSC11 cells .
The cell invasion ability of GSC11 cells was detected by a micro - filtration membrane culture chamber and a dual - chamber combined culture system ( Transwell chamber experiment ) .
The differentiation model of glioma stem cells was established . The effect of cell immunofluorescence was used to detect the differentiation of GSC11 cells . Results 1 . The expression of CD133 and Nestin in GSC11 cells was positive by immunofluorescence assay .
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本文編號(hào)：1734961