本文選題：膠質(zhì)瘤 + 外泌體　； 參考：《河北醫(yī)科大學(xué)》2017年博士論文

【摘要】：腦膠質(zhì)瘤在中樞神經(jīng)系統(tǒng)中高發(fā),且預(yù)后很差。即使給予術(shù)后放療、化療或聯(lián)合治療,腦膠質(zhì)瘤患者的復(fù)發(fā)率仍然很高,治療效果始終不能令人滿意。隨著基因水平研究的不斷深入,有關(guān)膠質(zhì)瘤的基因分子機(jī)制得到深入的探索。O6-甲基鳥嘌呤-DNA甲基轉(zhuǎn)移酶(O6-methylguanine-DNA methyhransferase,MGMT)啟動(dòng)子甲基化、1p/19q雜合性缺失、異檸檬酸脫氫酶1(Isocitrate dehydrogenase 1,IDH1)基因突變、ATRX表達(dá)缺失等均已經(jīng)成為膠質(zhì)瘤病理學(xué)診斷的重要分子生物學(xué)標(biāo)記,也豐富了人們對(duì)于腫瘤基因變異的認(rèn)識(shí)。Stephen Paget于1889年提出腫瘤生長的“種子-土壤”學(xué)說,奠定了腫瘤微環(huán)境的概念。外泌體是腫瘤微環(huán)境中的重要成分。它通過釋放自身攜帶的細(xì)胞因子參與了腫瘤的發(fā)生、增殖、侵襲和化療抵抗等生物學(xué)過程。而腫瘤細(xì)胞來源的外泌體(Tumor cells derived exosome,Texo)由于它的結(jié)構(gòu)特征和運(yùn)輸功能已經(jīng)成為近年來腫瘤領(lǐng)域的研究熱點(diǎn)。2013年,美國、德國3位科學(xué)家憑借他們所發(fā)現(xiàn)的細(xì)胞囊泡運(yùn)輸?shù)恼{(diào)節(jié)機(jī)制,榮獲2013年諾貝爾生理學(xué)或醫(yī)學(xué)獎(jiǎng)。外泌體(exosome)作為人體內(nèi)一類重要運(yùn)輸囊泡,也開始受到越來越多的關(guān)注。科學(xué)家們已發(fā)現(xiàn),外泌體會(huì)參與到腫瘤細(xì)胞的生長、增殖、侵襲、凋亡、血管生成、炎癥反應(yīng)、免疫應(yīng)答等重要的生物過程,細(xì)胞會(huì)通過分泌外泌體,將一些信號(hào)分子分泌到較遠(yuǎn)的組織或細(xì)胞中,以起到調(diào)控作用。外泌體作為藥物傳遞系統(tǒng)(Drug delivery system,DDS)為高效藥物投遞提供天然的內(nèi)源性納米級(jí)載體,其靶向作用的潛力也逐漸被發(fā)現(xiàn)。miRNA是一類內(nèi)源性的具有調(diào)控功能的非編碼RNA,可以與靶mRNA的3'非翻譯區(qū)(Untranslated Regions,UTR)結(jié)合從而在轉(zhuǎn)錄后水平調(diào)節(jié)靶基因的表達(dá)。有研究顯示,miR-221在直腸癌、胰腺癌、乳腺癌和膀胱癌中是一種促癌基因。利用高通量篩選測(cè)序平臺(tái)篩選腦膠質(zhì)瘤組織miRNA差異表達(dá)情況,篩選出了膠質(zhì)瘤組織內(nèi)miR-221高表達(dá),特別是膠質(zhì)母細(xì)胞瘤組織�？紤]到膠質(zhì)瘤細(xì)胞外泌體內(nèi)miR-221的表達(dá)情況以及調(diào)節(jié)機(jī)制尚未進(jìn)行過研究,故在本實(shí)驗(yàn)中我們選取膠質(zhì)瘤細(xì)胞來源的外泌體miR-221作為研究對(duì)象,進(jìn)行體內(nèi)外細(xì)胞和外泌體miR-221表達(dá)的檢測(cè),并通過CCK-8實(shí)驗(yàn),流式細(xì)胞技術(shù)以及Transwell實(shí)驗(yàn)探討其對(duì)于膠質(zhì)瘤的作用。然后進(jìn)一步尋找miRNA的靶基因。DNM3(Dynamin 3)是發(fā)動(dòng)蛋白超家族(包括DNM1、DNM2、DNM3)中的一員,參與許多膜轉(zhuǎn)運(yùn)功能,例如胞質(zhì)分裂、吞噬作用、轉(zhuǎn)運(yùn)囊泡出芽和細(xì)胞流動(dòng)等。最近的研究表明,DNM3的高表達(dá)可抑制細(xì)胞增殖及誘導(dǎo)凋亡�；趯�(duì)目前已有文獻(xiàn)的充分分析,我們推測(cè)miRNA-DNM3途徑有可能成為抑制神經(jīng)膠質(zhì)瘤生長及放化療增敏的治療靶點(diǎn)。由于DNM3出現(xiàn)在Targetscan的預(yù)測(cè)結(jié)果中,我們進(jìn)一步研究其與miR-221的關(guān)系。實(shí)驗(yàn)中我們確定了miR-221的靶基因DNM3以及miR-221的轉(zhuǎn)錄調(diào)控因子RELA,并通過雙熒光素酶報(bào)告基因?qū)嶒?yàn)得以證實(shí),并分析它們表達(dá)量的相關(guān)性,最后通過病毒轉(zhuǎn)染實(shí)驗(yàn)證實(shí)miR-221對(duì)膠質(zhì)瘤的作用是通過靶向調(diào)控DNM3引起的,并受上游RELA的轉(zhuǎn)錄調(diào)控。第一部分miR-221在人腦膠質(zhì)瘤組織和GBM源性外泌體內(nèi)的表達(dá)及其與膠質(zhì)瘤WHO病理分級(jí)的關(guān)系目的:研究miR-221在人腦膠質(zhì)瘤組織和GBM源性外泌體內(nèi)的表達(dá),并分析miR-221的表達(dá)與膠質(zhì)瘤WHO病理分級(jí)的關(guān)系。方法:1 48例膠質(zhì)瘤患者及11例非腫瘤患者組織標(biāo)本均于2015年采集自河北醫(yī)科大學(xué)第二醫(yī)院神經(jīng)外科。包括23名女性,36名男性,年齡從30至68歲。標(biāo)本采集后迅速放至液氮中冷凍,然后-80℃低溫保存。診斷結(jié)果均由2位病理醫(yī)師根據(jù)WHO分級(jí)指導(dǎo)得出。2抽取上述患者的靜脈血,室溫靜置后獲得血清或者采集后立即離心獲得血漿上清液,提取外泌體。3 SHG-44、U87MG、HEB和U251的培養(yǎng),及細(xì)胞外泌體的提取。4定量反轉(zhuǎn)錄聚合酶鏈反應(yīng)(qRT-PCR)方法測(cè)定神經(jīng)膠質(zhì)瘤組織或外泌體內(nèi)miR-221含量。5應(yīng)用t檢驗(yàn)分析神經(jīng)膠質(zhì)瘤中miR-221含量與膠質(zhì)瘤級(jí)別或不同細(xì)胞系之間的關(guān)系。結(jié)果:1 miRNA-221在人腦膠質(zhì)瘤組織中表達(dá)上調(diào):與對(duì)照組織比較,膠質(zhì)瘤組織中miRNA-221均呈高表達(dá)(P0.01),其中高度惡性膠質(zhì)瘤組織中miRNA-221表達(dá)量均明顯高于低度惡性者(P0.01)。在WHO III和WHO IV膠質(zhì)瘤中表達(dá)量較WHO II或非腫瘤組織明顯增高(P0.01)。miR-221的表達(dá)與膠質(zhì)瘤的病理分級(jí)(WHO分級(jí))明顯相關(guān)。2比較在無細(xì)胞的血清和血漿外泌體內(nèi)miR-221的差異表達(dá),可見膠質(zhì)瘤患者與非腫瘤患者miR-221表達(dá)量具有顯著性差異(P0.01)。3血漿中的檢測(cè)結(jié)果與組織檢測(cè)結(jié)果相似:膠質(zhì)瘤患者(n=48)與非腫瘤患者(n=11)相比,血漿外泌體miR-221表達(dá)量具有顯著性差異(P0.01)。膠質(zhì)瘤患者血漿外泌體miR-221表達(dá)量隨膠質(zhì)瘤病理級(jí)別升高表達(dá)量顯著升高。4 miR-221在四種膠質(zhì)瘤細(xì)胞系中均高表達(dá),且miR-221在U87MG細(xì)胞來源的外泌體中表達(dá)最高,U251、SHG-44、HEB細(xì)胞外泌體內(nèi)表達(dá)依次降低。結(jié)論:1 miR-221在人腦膠質(zhì)瘤組織中高表達(dá),在血漿來源或膠質(zhì)瘤細(xì)胞系來源的外泌體內(nèi)也存在高表達(dá),且其表達(dá)量隨著膠質(zhì)瘤WHO病理分級(jí)呈遞增趨勢(shì)。高級(jí)別(III和IV級(jí))膠質(zhì)瘤miR-221的表達(dá)量明顯高于低級(jí)別(II級(jí))膠質(zhì)瘤,差別具有統(tǒng)計(jì)學(xué)意義。2 miR-221的表達(dá)量與膠質(zhì)瘤WHO病理分級(jí)呈顯著的正相關(guān),預(yù)示外泌體miR-221可能用于膠質(zhì)瘤相關(guān)的臨床檢測(cè)。第二部分U87MG細(xì)胞源性的外泌體miR-221對(duì)SHG-44細(xì)胞生物學(xué)活性的影響目的:研究U87MG細(xì)胞源性外泌體miR-221對(duì)SHG-44細(xì)胞的增殖、侵襲、化療抵抗等方面的影響。方法:1培養(yǎng)U87MG和SHG-44細(xì)胞系,提取U87MG細(xì)胞系外泌體。2向SHG-44細(xì)胞系轉(zhuǎn)染anti-miR-221(miR-221 ASO)以降低細(xì)胞內(nèi)miR-221的表達(dá),轉(zhuǎn)染anti-miR-NC(Ctrl ASO)作對(duì)照。實(shí)驗(yàn)組SHG-44細(xì)胞培養(yǎng)基中同時(shí)加入外泌體。3進(jìn)行CCK-8實(shí)驗(yàn)研究降低miR-221表達(dá)后膠質(zhì)瘤細(xì)胞增殖能力的變化。4進(jìn)行流式細(xì)胞實(shí)驗(yàn)研究降低miR-221表達(dá)對(duì)膠質(zhì)瘤細(xì)胞凋亡能力和替莫唑胺耐藥的變化。5進(jìn)行Transwell實(shí)驗(yàn)和劃痕實(shí)驗(yàn)研究降低miR-221表達(dá)后膠質(zhì)瘤細(xì)胞侵襲能力的變化。結(jié)果:1 qRT-PCR結(jié)果顯示,轉(zhuǎn)染anti-miR-221后,SHG-44細(xì)胞內(nèi)miR-221的表達(dá)量顯著降低(P0.01)。2 CCK-8實(shí)驗(yàn)結(jié)果顯示,anti-miR-NC+Exo組的增殖活力最高,anti-miR-221組的增殖活力最低,即敲低miR-221的表達(dá)后,膠質(zhì)瘤細(xì)胞SHG-44的增殖能力顯著降低(P0.01)。3流式細(xì)胞實(shí)驗(yàn)結(jié)果顯示,敲低miR-221表達(dá)后,SHG-44細(xì)胞凋亡能力無明顯改變,但是對(duì)替莫唑胺的敏感性顯著增高(P0.01)。4 Transwell實(shí)驗(yàn)和劃痕實(shí)驗(yàn)結(jié)果顯示,敲低miR-221表達(dá)后,SHG-44細(xì)胞侵襲能力顯著降低(P0.01)。結(jié)論:敲低miR-221表達(dá),可以使SHG-44細(xì)胞增殖及侵襲能力顯著降低,凋亡能力雖無改變,但是對(duì)替莫唑胺的敏感性增高。miR-221可以影響膠質(zhì)瘤細(xì)胞的增殖、侵襲及對(duì)替莫唑胺的化療耐藥。第三部分miR-221對(duì)下游靶基因DNM3的調(diào)控以及RELA對(duì)miR-221轉(zhuǎn)錄能力的影響目的:尋找miR-221的下游作用靶點(diǎn)以及miR-221的轉(zhuǎn)錄調(diào)控因子,深入了解miR-221對(duì)膠質(zhì)瘤生物學(xué)影響的作用機(jī)制。方法:1使用Targetscan數(shù)據(jù)庫預(yù)測(cè)miR-221的下游作用靶點(diǎn),并搜集相關(guān)文獻(xiàn)篩選有意義結(jié)果。2構(gòu)建miR-221過表達(dá)慢病毒質(zhì)粒,建立miR-221過表達(dá)穩(wěn)定SHG-44細(xì)胞系,進(jìn)行穩(wěn)定細(xì)胞系的篩選。3雙熒光素酶報(bào)告基因檢測(cè)用于驗(yàn)證miR-221與DNM3的作用關(guān)系。4應(yīng)用qRT-PCR及蛋白質(zhì)印跡(Western blot)方法檢測(cè)miR-221與DNM3在SHG-44細(xì)胞中的表達(dá)量,并分析表達(dá)量的相關(guān)性。5通過CCK-8實(shí)驗(yàn)、流式細(xì)胞術(shù)、Transwell遷移和侵襲實(shí)驗(yàn)等方法,研究上調(diào)DNM3對(duì)膠質(zhì)瘤SHG-44細(xì)胞增殖、侵襲和凋亡等生物學(xué)行為的影響。6通過生物信息學(xué)方法,預(yù)測(cè)miR-221啟動(dòng)子區(qū)域的轉(zhuǎn)錄因子結(jié)合位點(diǎn);利用熒光素酶報(bào)告實(shí)驗(yàn),在SHG-44細(xì)胞中驗(yàn)證此轉(zhuǎn)錄因子與miR-221啟動(dòng)子的結(jié)合情況;通過上調(diào)或下調(diào)這一轉(zhuǎn)錄因子,采用實(shí)時(shí)定量PCR方法檢測(cè)miR-221的表達(dá)變化。結(jié)果:1根據(jù)Targetscan的預(yù)測(cè)結(jié)果,并結(jié)合相關(guān)文獻(xiàn)資料,發(fā)現(xiàn)DNM3上存在兩個(gè)與miR-221的作用靶點(diǎn)。2成功構(gòu)建了miR-221過表達(dá)慢病毒載體和穩(wěn)轉(zhuǎn)膠質(zhì)瘤細(xì)胞系,miR-221的表達(dá)水平顯著升高(P0.01),是對(duì)照組的45.5倍。3雙熒光素酶報(bào)告檢測(cè)結(jié)果表明,相對(duì)于miR-NC,miR-221可以顯著抑制熒光素酶的活性(P0.01),而在突變靶位點(diǎn)后此現(xiàn)象消失(P0.01)。4 qRT-PCR檢測(cè)膠質(zhì)瘤組織中的DNM3表達(dá)量:DNM3表達(dá)量隨膠質(zhì)瘤病理級(jí)別升高表達(dá)量反而降低。Spearman秩相關(guān)分析表明,膠質(zhì)瘤細(xì)胞DNM3與miR-221的表達(dá)量呈負(fù)相關(guān)(Spearman r=-0.908,P0.01)。5轉(zhuǎn)染miR-221過表達(dá)載體后,在高表達(dá)miR-221的細(xì)胞中,DNM3低表達(dá);CCK-8實(shí)驗(yàn)提示,增加DNM3的表達(dá)后,細(xì)胞增殖能力顯著降低。流式細(xì)胞技術(shù)檢測(cè)發(fā)現(xiàn),加入替莫唑胺后,DNM3促進(jìn)了膠質(zhì)瘤細(xì)胞的早期凋亡(P0.01)。Transwell實(shí)驗(yàn)和劃痕實(shí)驗(yàn)結(jié)果表明,轉(zhuǎn)染DNM3能夠抑制膠質(zhì)瘤細(xì)胞的遷移和侵襲能力。6通過生物信息學(xué)方法,預(yù)測(cè)到miR-221編碼基因的啟動(dòng)子區(qū)域存在RELA的結(jié)合位點(diǎn);雙熒光素酶報(bào)告基因檢測(cè)證實(shí)RELA能夠與miR-221基因的啟動(dòng)子區(qū)域相結(jié)合;實(shí)時(shí)定量PCR結(jié)果表明,轉(zhuǎn)染RELA后,能誘導(dǎo)miR-221的過表達(dá)。結(jié)論:DNM3的mRNA中存在miR-221在膠質(zhì)瘤中的靶向作用位點(diǎn),轉(zhuǎn)錄因子RELA調(diào)節(jié)miR-221基因的轉(zhuǎn)錄。第四部分miR-221促進(jìn)SHG-44人腦膠質(zhì)瘤細(xì)胞系生長的體內(nèi)研究目的:動(dòng)物體內(nèi)證實(shí)miR-221對(duì)膠質(zhì)瘤生物學(xué)活性影響。方法:1穩(wěn)定轉(zhuǎn)染miR-221的SHG-44膠質(zhì)瘤細(xì)胞系,進(jìn)行穩(wěn)定細(xì)胞系的篩選,對(duì)照組轉(zhuǎn)染miR-NC。2應(yīng)用反轉(zhuǎn)錄定量聚合酶鏈反應(yīng)(qRT-PCR)方法檢測(cè)miR-221在SHG-44細(xì)胞中的表達(dá)量。3裸小鼠皮下人腦膠質(zhì)瘤模型的建立。4腫瘤生長情況觀察:每隔4天用游標(biāo)卡尺測(cè)量一次腫瘤的長徑(a)及寬徑(b),計(jì)算腫瘤體積。結(jié)果:1成功構(gòu)建miR-221過表達(dá)的SHG-44膠質(zhì)瘤細(xì)胞系,qRT-PCR檢測(cè)穩(wěn)定細(xì)胞系內(nèi)miR-221的相對(duì)表達(dá)量為47±1.45,明顯高于Vector轉(zhuǎn)染細(xì)胞系(P0.01)。2 miR-221過表達(dá)腦膠質(zhì)瘤模型證實(shí)腫瘤生長速度明顯增快。帶瘤生存20天后取下腫瘤組織,發(fā)現(xiàn)miR-221過表達(dá)組腫瘤重量為對(duì)照組腫瘤重量的2倍余(P0.05)。結(jié)論:miR-221過表達(dá)促進(jìn)膠質(zhì)瘤細(xì)胞體內(nèi)生長,miR-221是一種促癌因子。
[Abstract]:The brain glioma is high in the central nervous system and has poor prognosis. The recurrence rate of the patients with glioma is still high and the therapeutic effect is still not satisfactory even after the postoperative radiotherapy, chemotherapy or combined therapy. The molecular mechanism of glioma has been deeply explored with the molecular mechanism of the gene level..O6- methyl guanine is deeply explored. Methotrexate -DNA methyltransferase (O6-methylguanine-DNA methyhransferase, MGMT) promoter methylation, 1p/19q heterozygosity deletion, ISO citrate dehydrogenase 1 (Isocitrate dehydrogenase 1, IDH1) gene mutation and ATRX expression loss have become important molecular biomarkers for pathological diagnosis of glioma, and also enrich people's tumor base. .Stephen Paget proposed the "seed soil" theory of tumor growth in 1889, which established the concept of tumor microenvironment. Exocrine is an important component of tumor microenvironment. It participates in the biological processes of tumor occurrence, proliferation, invasion and chemotherapeutic resistance by releasing its own cytokine, and tumor cells. Tumor cells derived exosome (Texo), because of its structural characteristics and transport function, has become a research hotspot in the field of cancer in recent years.2013. In the United States, 3 scientists in Germany were awarded the 2013 Nobel prize for physiology or medicine for their discovery of cellular vesicle transport. The exocrine (exosome) was awarded. As an important type of transport vesicles in the human body, more and more attention has been paid. Scientists have found that exocrine experience is involved in the biological processes such as growth, proliferation, invasion, apoptosis, angiogenesis, inflammatory response, immune response and other important biological processes, and the cells secrete some signal molecules into more distant tissues. Drug delivery system (DDS) provides natural endogenous nanoscale carriers for high efficiency drug delivery. The potential of its targeting is also gradually found to be a kind of endogenous non coded RNA with regulatory function, which can be used with the 3'non translation region of the target mRNA (Untrans). Lated Regions, UTR) combined to regulate the expression of target genes at post transcriptional levels. Studies have shown that miR-221 is a oncogene in rectal cancer, pancreatic cancer, breast and bladder cancer. High throughput screening sequencing platform is used to screen the differential expression of miRNA in brain glioma tissue, and the high expression of miR-221 in glioma tissues is screened, especially in glioma tissues. It is a glioblastoma tissue. Considering the expression of miR-221 in the extracellular secretory of glioma cells and the regulation mechanism has not been studied, in this experiment, we selected the external secretory miR-221 derived from glioma cells as the research object to detect the expression of miR-221 in the cells and exosecrete in vitro and in vivo, and through the CCK-8 experiment, flow formula. Cell technology and Transwell experiments explore its role in glioma. Then further search for the target gene.DNM3 (Dynamin 3) of miRNA is a member of the superfamily of the promoter protein (including DNM1, DNM2, DNM3), and participates in many membrane transport functions, such as cytokinesis, phagocytosis, transport vesicle buds and cell flow. Recent studies have shown that The high expression of DNM3 inhibits cell proliferation and induces apoptosis. Based on a full analysis of current literature, we speculate that the miRNA-DNM3 pathway may be a therapeutic target for inhibiting the growth of glioma and chemosensitization. As DNM3 appears in the prediction of Targetscan, we further study the relationship with miR-221. We identified the target gene DNM3 of miR-221 and the transcriptional regulator RELA of miR-221, and confirmed by the double luciferase reporter gene experiment, and analyzed the correlation of their expression. Finally, the effect of miR-221 on the glioma was confirmed by the targeting regulation of DNM3 by the virus transfection experiment, and was regulated by the upstream RELA. Control. Part 1 expression of miR-221 in human glioma tissue and GBM derived exocrine and its relationship with pathological grade of glioma WHO: To study the expression of miR-221 in human glioma tissue and GBM derived exocrine, and to analyze the relationship between the expression of miR-221 and the pathological grade of glioma WHO. Methods: 148 patients with glioma and 11 cases of glioma. The tissue specimens of non tumor patients were collected from the Department of Neurosurgery at the second hospital of Hebei Medical University in 2015, including 23 women, 36 men, aged from 30 to 68. The specimens were quickly frozen in liquid nitrogen after collection, and then stored at -80 C. The diagnosis results were guided by 2 pathologists according to the guidance of.2 to extract the above patients. Blood serum, after room temperature statics, obtained serum or immediately after collection, centrifugation to obtain plasma supernatant, extract exocrine.3 SHG-44, U87MG, HEB and U251, and extract.4 quantitative reverse transcriptional polymerase chain reaction (qRT-PCR) method for the determination of glioma tissue or exocrine miR-221 content.5 application of t test and analysis of T analysis of neuroglia The relationship between the miR-221 content of the tumor and the grade of glioma or different cell lines. Results: 1 miRNA-221 was up-regulated in the human glioma tissue. Compared with the control tissue, the expression of miRNA-221 in the glioma tissues was highly expressed (P0.01), and the expression of miRNA-221 in the high malignant glioma tissues was significantly higher than that of the low-grade malignant (P0.01). In WHO The expression of III and WHO IV glioma was significantly higher than that of WHO II or non tumor tissue (P0.01), and the expression of.MiR-221 was significantly correlated with the pathological grade of glioma (WHO grading). The difference of.2 in the expression of miR-221 in the non cellular and plasma exocrine bodies showed significant difference between the miR-221 expression of glioma and non tumor patients (P0). .01) the detection results in.3 plasma were similar to that of tissue detection. Compared with non tumor patients (n=11), the miR-221 expression of plasma exocrine miR-221 was significantly different (P0.01). The expression of miR-221 expression in plasma exocrine miR-221 in glioma patients increased with the pathological grade of glioma significantly increased.4 miR-221 in four gliomas The expression of miR-221 in the Exocyst of U87MG cells was the highest, and the expression of U251, SHG-44 and HEB cells decreased in turn. Conclusion: 1 miR-221 is highly expressed in human glioma tissue, and high expression in the external secretory of plasma source or glioma cell line, and its expression is along with glioma WHO disease. The expression of miR-221 in high grade (III and IV) glioma was significantly higher than that of low grade (II grade) glioma, and the difference was statistically significant positive correlation between the expression of.2 miR-221 and the pathological grade of glioma WHO, indicating that the exocrine miR-221 may be used in the clinical detection of glioma. Second part U87MG cells The effect of source miR-221 on biological activity of SHG-44 cells: To study the effects of U87MG cell derived exocrine miR-221 on proliferation, invasion and chemotherapy resistance of SHG-44 cells. Methods: 1 culture U87MG and SHG-44 cell lines, and the extraction of U87MG cell line exocrine.2 to SHG-44 cell lines for anti-miR-221 (miR-221) Expression of miR-221 in low cell and transfection of anti-miR-NC (Ctrl ASO) as control. In the experimental group SHG-44 cell culture medium also added exosecreting.3 to carry out CCK-8 experimental study to reduce the proliferation ability of glioma cells after miR-221 expression and.4 performed by flow cytometry to reduce the apoptosis ability of miR-221 expression to glioma cells and temozolomide Drug resistance changes.5 conducted Transwell experiment and scratch test to reduce the invasion ability of glioma cells after miR-221 expression. Results: 1 qRT-PCR results showed that after transfection of anti-miR-221, the expression of miR-221 in SHG-44 cells decreased significantly (P0.01).2 CCK-8 experimental results, anti-miR-NC+Exo group was the highest proliferation activity. Anti-miR-2 The proliferation activity of the 21 groups was the lowest, that is, after the expression of low miR-221, the proliferation ability of SHG-44 in glioma cells decreased significantly (P0.01).3 flow cytometry results showed that the apoptosis ability of SHG-44 cells was not significantly changed after the knockout of low miR-221 expression, but the sensitivity to temozolomide increased significantly (P0.01).4 Transwell experiment and scratch test results. The invasion ability of SHG-44 cells decreased significantly (P0.01) after the knockout of miR-221. Conclusion: the proliferation and invasion ability of SHG-44 cells decreased significantly and the apoptosis ability was not changed, but the sensitivity to temozolomide increased, but.MiR-221 could affect the proliferation of glioma cells, invasion and chemotherapy of temozolomide. Resistance. The regulation of the downstream target gene DNM3 by part third miR-221 and the effect of RELA on the transcriptional ability of miR-221: to find the downstream target of miR-221 and the transcriptional regulator of miR-221, and to understand the mechanism of the effect of miR-221 on the biological effects of glioma. Method: 1 to predict the downstream effect of miR-221 with Targetscan database. Target, and collect relevant literature screening meaningful results.2 construction of miR-221 overexpression lentivirus plasmid, miR-221 overexpression of stable SHG-44 cell line, stable cell line screening,.3 double luciferase reporter gene detection is used to verify the relationship between miR-221 and DNM3,.4 application qRT-PCR and Western blot (Western blot) method for detecting M The expression of iR-221 and DNM3 in SHG-44 cells and the correlation of expression.5 through CCK-8 experiments, flow cytometry, Transwell migration and invasion experiments, the effect of up regulation of DNM3 on the proliferation, invasion and apoptosis of glioma SHG-44 cells,.6 through bioinformatics methods, and prediction of miR-221 promoter region The transcriptional factor binding site; using luciferase reporter assay to verify the binding of the transcription factor to the miR-221 promoter in SHG-44 cells. By up or down this transcription factor, the real-time quantitative PCR method was used to detect the changes in the expression of miR-221. Results: 1 according to the prediction results of Targetscan and the related literature, It was found that there were two target sites on DNM3 with miR-221,.2 successfully constructed the miR-221 overexpressed lentivirus and glioma cell lines, and the expression level of miR-221 increased significantly (P0.01), and the results of the 45.5 times.3 double Luciferase Report of the control group showed that miR-221 could significantly inhibit the activity of luciferase (P0.0) compared with miR-NC (P0.0). 1), and the expression of DNM3 in glioma tissues was detected after the mutation target site (P0.01).4 qRT-PCR: the expression of DNM3 expression decreased with the histopathological level of glioma and decreased.Spearman rank correlation analysis, indicating that the expression of DNM3 in glioma cells was negatively correlated with the expression of miR-221 (Spearman r=-0.908, P0.01).5. After the carrier, the expression of DNM3 was low in the cells with high expression of miR-221. The CCK-8 experiment suggested that the proliferation of the cells decreased significantly after the increase of the expression of DNM3. Flow cytometry detected that DNM3 promoted the early apoptosis of glioma cells (P0.01).Transwell experiment and scratch test. The results showed that the transfection of DNM3 was inhibited. The migration and invasion ability of glioma cells.6 predicted the presence of RELA binding sites in the promoter region of the miR-221 encoding gene by bioinformatics method, and the double luciferase reporter gene detection confirmed that RELA could be combined with the promoter region of the miR-221 gene. The real-time quantitative PCR results showed that after transfection of RELA, miR-221 could be induced by the transfection of RELA. Expression. Conclusion: the targeting site of miR-221 in glioma is found in DNM3 mRNA. Transcription factor RELA regulates the transcription of miR-221 gene. Fourth part miR-221 promotes the growth of SHG-44 human glioma cell line in vivo. The objective of the study is to confirm the effect of miR-221 on the biological activity of glioma in vivo. Method: 1 stable transfection of miR-221 SHG- 44 glioma cell lines were screened for stable cell lines. The control group was transfected with miR-NC.2 using reverse transcriptional polymerase chain reaction (qRT-PCR) to detect the expression of miR-221 in SHG-44 cells. The growth of.4 tumor was observed in the subcutaneous human glioma model of.3 nude mice. The length of a tumor was measured every 4 days with a vernier caliper. A) and wide diameter (b). Tumor volume was calculated. Results: 1 the miR-221 overexpressing SHG-44 glioma cell line was successfully constructed, and the stable cells were detected by qRT-PCR.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

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