本文選題：膠質(zhì)母細(xì)胞瘤　切入點(diǎn)：EGFR　出處：《天津醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目的:1.構(gòu)建mi R-21抑制劑的慢病毒系統(tǒng)以及PPARA和VHL的熒光素酶報(bào)告質(zhì)粒,驗(yàn)證PPARA和VHL為mi R-21的靶基因并闡述mi R-21通過PPARA和VHL對(duì)EGFR信號(hào)通路調(diào)控的詳細(xì)分子機(jī)制;在體內(nèi)實(shí)驗(yàn)和體外實(shí)驗(yàn)中聯(lián)合mi R-21抑制劑和EGFR單克隆抗體--尼妥珠單克隆抗體治療膠質(zhì)瘤,為優(yōu)化膠質(zhì)瘤的基因治療提供新的思路。2.利用生物信息學(xué)方法找出可能受lnc RNA HOTAIR調(diào)控的信號(hào)通路,通過小分子抑制劑阻斷以及表達(dá)載體構(gòu)建等方法闡明LSD1復(fù)合物和PRC2復(fù)合物在lnc RNA HOTAIR對(duì)膠質(zhì)瘤細(xì)胞周期進(jìn)展調(diào)控中的分子機(jī)制。方法:1.用表達(dá)mi R-21抑制劑的慢病毒感染膠質(zhì)瘤細(xì)胞系U87、LN229和U251,Western blot方法檢測EGFR、p-EGFR、AKT、p-AKT的表達(dá)水平變化;細(xì)胞克隆形成實(shí)驗(yàn)、細(xì)胞流式分析以及transwell實(shí)驗(yàn)檢測mi R-21對(duì)膠質(zhì)瘤細(xì)胞增殖、細(xì)胞周期進(jìn)展、細(xì)胞侵襲以及細(xì)胞凋亡的影響。2.通過靶點(diǎn)預(yù)測找出mi R-21可能的靶點(diǎn);熒光素酶報(bào)告實(shí)驗(yàn)驗(yàn)證PPARA和VHL為mi R-21的靶基因;免疫熒光和蛋白質(zhì)免疫共沉淀揭示β-catenin和PPARα之間的調(diào)控網(wǎng)絡(luò)。3.構(gòu)建膠質(zhì)瘤顱內(nèi)動(dòng)物模型,研究mi R-21抑制劑和尼妥珠單抗聯(lián)合應(yīng)用的體內(nèi)抑瘤效果。4.生物信息學(xué)分析HOTAIR可能調(diào)控的下游信號(hào)傳導(dǎo)通路;利用含lnc RNA HOTAIR干擾序列的慢病毒、EZH2小分子抑制劑DZNep、LSD1小分子抑制劑2-PCPA分別處理膠質(zhì)瘤細(xì)胞U87和LN229,分析它們對(duì)膠質(zhì)瘤細(xì)胞周期的影響。5.在敲低HOTAIR的U87和LN229細(xì)胞系中分別過表達(dá)HOTAIR的3’及5’結(jié)構(gòu)域,驗(yàn)證si RNA HOTAIR引起的細(xì)胞周期阻滯能否部分恢復(fù)。6.構(gòu)建顱內(nèi)原位膠質(zhì)瘤動(dòng)物模型,檢測HTOAIR siRNA的體內(nèi)抑瘤效果。結(jié)果:1.感染mi R-21抑制劑病毒的膠質(zhì)瘤細(xì)胞中p-EGFR和p-AKT表達(dá)明顯下調(diào),細(xì)胞的增殖受到抑制,細(xì)胞周期被阻滯在G1期,細(xì)胞凋亡明顯增加(p0.01)。2.Western blot實(shí)驗(yàn)和熒光素酶報(bào)告實(shí)驗(yàn)證實(shí)VHL及PPARA為mi R-21的直接作用靶點(diǎn)。3.感染mi R-21抑制劑或轉(zhuǎn)染VHL表達(dá)質(zhì)粒均能降低經(jīng)典型Wnt/β-catenin信號(hào)通路的活性。Western blot和免疫熒光結(jié)果提示mi R-21通過靶定PPARα進(jìn)而上調(diào)轉(zhuǎn)錄因子AP-1的轉(zhuǎn)錄活性調(diào)節(jié)EGFR/AKT信號(hào)通路。4.蛋白質(zhì)免疫共沉淀結(jié)果證明β-catenin和AP-1能形成復(fù)合體,且復(fù)合體的形成受mi R-21的調(diào)控。5.mi R-21抑制劑聯(lián)合尼妥珠單抗治療膠質(zhì)瘤效果優(yōu)于單藥治療。6.生物信息學(xué)結(jié)果顯示HOTAIR調(diào)節(jié)的基因主要參與細(xì)胞周期的調(diào)控。7.EZH2抑制劑DZNep能模擬si RNA HOTAIR的功能,將膠質(zhì)瘤細(xì)胞周期阻滯在G1期。8.HOTAIR敲低的細(xì)胞系中過表達(dá)其5’結(jié)構(gòu)域能部分的回復(fù)受si RNA HOTAIR引起的細(xì)胞周期抑制作用。9.膠質(zhì)瘤顱內(nèi)模型證實(shí)siRNA HOTAIR能抑制膠質(zhì)瘤的生長。結(jié)論:1.mi R-21通過靶定VHL/β-catenin和PPARα/AP-1調(diào)節(jié)EGFR/AKT信號(hào)傳導(dǎo)通路。2.EGFR和mi R-21之間存在反饋調(diào)節(jié)環(huán)路。3.體內(nèi)外研究顯示聯(lián)合尼妥珠單克隆抗體和mi R-21抑制劑治療膠質(zhì)瘤優(yōu)于單藥治療。4.Lnc RNA HOTAIR調(diào)控的基因主要參與細(xì)胞周期過程;HOTAIR主要通過其5’結(jié)構(gòu)域和PRC2復(fù)合物的結(jié)合調(diào)節(jié)膠質(zhì)瘤細(xì)胞周期進(jìn)展。5.敲低HOTAIR的表達(dá)能在體內(nèi)抑制膠質(zhì)瘤的惡性增殖。
[Abstract]:Objective: to construct mi 1. R-21 inhibitors and PPARA lentiviral system and VHL luciferase reporter plasmid, verification of PPARA and VHL as the target gene of MI R-21 and MI R-21 by PPARA and VHL on the regulation of EGFR signaling pathway with molecular mechanism; in vivo and in vitro experiments in MI R-21 inhibitors and EGFR monoclonal antibody nimotuzumab treatment of glioma, and provide new ideas using.2. biological information by LNC signaling pathway may find RNA HOTAIR control method for gene therapy of glioma by optimization of small molecule inhibitors block and expression vector construction method of LSD1 complex and PRC2 complex in LNC RNA HOTAIR on the molecular mechanism of progress glioma cell cycle regulation. Methods: 1. expression by Mi inhibitors of R-21 lentivirus infected glioma cell lines U87, LN229 and U251, Western blot EGFR detection method , p-EGFR, AKT, p-AKT expression level changes; cell clone formation assay, flow cytometry analysis and Transwell assay of MI R-21 on glioma cell proliferation, cell cycle progression, invasion and apoptosis effect of.2. cells to identify targets of MI R-21 may be through the target prediction; luciferase reporter experiments of PPARA and VHL the target gene mi R-21; reveal the regulation between.3. and PPAR network construction of alpha beta -catenin glioma animal model of intracranial immunofluorescence and immunoprecipitation of MI protein, and R-21 inhibitor nimotuzumab combined in vivo antitumor effect of.4. bioinformatics analysis of the downstream HOTAIR signaling pathway may regulate the LNC containing RNA; HOTAIR interference sequence with lentivirus, EZH2 small molecule inhibitor DZNep, small molecule inhibitors of LSD1 were treated with 2-PCPA U87 and LN229 glioma cells, analysis of glioma fine Effect of.5. cell cycle in U87 and LN229 cell lines at low HOTAIR were over expression of HOTAIR 3 'and 5' domain, can verify the Si RNA cell cycle arrest caused by HOTAIR partially restored.6. construction in situ intracranial glioma animal model, detection of HTOAIR siRNA in vivo antitumor effect. Results: the down-regulation of p-EGFR and p-AKT expression in glioma cells infected with MI virus R-21 1. inhibitor, cell proliferation was inhibited, cell cycle arrest in G1 phase, cell apoptosis increased significantly (P0.01).2.Western blot experiment and luciferase reporter assay demonstrated that VHL PPARA and MI R-21 for the direct targets of.3. infection or transfection of VHL R-21 inhibitor mi the expression plasmid could reduce the transcriptional activity of.Western regulating activity of blot and immunofluorescence results of classical Wnt/ beta -catenin signaling pathway mi R-21 by targeting PPAR alpha and upregulation of the transcription factor AP-1 Festival The results prove that -catenin and AP-1 can form complex beta EGFR/AKT signaling pathway.4. protein co immunoprecipitation, and complex formation is better than the control effect of.5.mi R-21 inhibitor combined with nimotuzumab treatment of glioma by Mi R-21 monotherapy.6. bioinformatics results showed that regulation of.7.EZH2 inhibitor DZNep HOTAIR regulating genes mainly involved in cell cycle can simulation of Si RNA HOTAIR, the glioma cell cycle arrest in G1 phase of.8.HOTAIR knockdown cell lines overexpressing the 5 'domain can reply by cell cycle Si RNA HOTAIR induced inhibition of.9. glioma model of intracranial confirmed that siRNA HOTAIR can inhibit the growth of glioma. Conclusion: 1.mi by R-21 target feedback loop.3. in vitro and in vivo studies have shown that combination existed between VHL/ and PPAR alpha /AP-1 beta -catenin regulating EGFR/AKT signal transduction pathways of.2.EGFR and MI R-21 With MI monoclonal antibody and R-21 inhibitor for the treatment of glioma is superior to monotherapy.4.Lnc RNA HOTAIR regulated genes mainly involved in cell cycle process; combined with the expression of HOTAIR mainly through the 5 'domain and PRC2 complexes regulate glioma cell cycle progression.5. knockdown of HOTAIR in vivo inhibition of glioma malignant proliferation.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.4

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

1 馮世宇;朱偉杰;余新光;;lncRNA在膠質(zhì)瘤發(fā)生發(fā)展中的作用機(jī)制研究進(jìn)展[J];山東醫(yī)藥;2014年18期

2 吳紅;李國棟;梁尚棟;;神經(jīng)疾病關(guān)聯(lián)的長鏈非編碼RNA的生物標(biāo)記物作用[J];神經(jīng)解剖學(xué)雜志;2015年05期

3 莊彪;閔志均;王廷峰;張鵬;倪熊;瞿惠龍;丁育明;;miRNA-34a對(duì)人結(jié)腸癌細(xì)胞HCT116增殖及侵襲轉(zhuǎn)移的影響[J];實(shí)用癌癥雜志;2016年02期

相關(guān)博士學(xué)位論文 前8條

1 李金平;長鏈非編碼RNA在骨肉瘤患者中的差異表達(dá)研究[D];中南大學(xué);2013年

2 李耀華;重型腦外傷患者血液LncRNAs動(dòng)態(tài)變化及差異表達(dá)[D];天津醫(yī)科大學(xué);2014年

3 萬中元;長鏈非編碼RNA在退變?nèi)俗甸g盤髓核內(nèi)的異常表達(dá)[D];第四軍醫(yī)大學(xué);2014年

4 馮世宇;重編程相關(guān)IncRNA（lincRNA-ROR）在腦膠質(zhì)瘤中的表達(dá)及功能的研究[D];中國人民解放軍醫(yī)學(xué)院;2014年

5 秦曉勇;長鏈非編碼RNA-TSLC1-AS1在人腦膠質(zhì)瘤中的表達(dá)及功能研究[D];中國人民解放軍醫(yī)學(xué)院;2014年

6 胡國章;利用生物信息學(xué)方法篩選膠質(zhì)瘤的差異表達(dá)基因及其相關(guān)機(jī)制[D];吉林大學(xué);2015年

7 王蘇雅;BET表觀信號(hào)通路蛋白抑制劑JQ1對(duì)食管鱗癌細(xì)胞增殖抑制活性的研究[D];北京協(xié)和醫(yī)學(xué)院;2015年

8 鄭小國;水稻抗旱性馴化的表觀遺傳學(xué)研究[D];華中農(nóng)業(yè)大學(xué);2015年

相關(guān)碩士學(xué)位論文 前4條

1 宋皓軍;胃癌相關(guān)長鏈非編碼RNA的鑒定及其臨床意義的分析[D];寧波大學(xué);2013年

2 胡萍;Twist誘導(dǎo)乳腺上皮細(xì)胞MCF10A發(fā)生上皮間質(zhì)轉(zhuǎn)化的1ncRNAs表達(dá)譜研究[D];重慶醫(yī)科大學(xué);2014年

3 萬惷;小鼠成骨細(xì)胞分化中表達(dá)上調(diào)長鏈非編碼RNA的鑒定[D];中南大學(xué);2014年

4 宋尚卿;RCRF在腎癌中表達(dá)的臨床意義及對(duì)腎癌細(xì)胞生物學(xué)特性影響的研究[D];第二軍醫(yī)大學(xué);2014年

，

本文編號(hào)：1637446