發(fā)布時間：2018-01-05 05:21

  本文關(guān)鍵詞：組蛋白去乙酰化酶-3與膠質(zhì)瘤血管生成擬態(tài)的相關(guān)性　出處：《南方醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 膠質(zhì)瘤U87細(xì)胞 血管生成擬態(tài) 組蛋白去乙�；�3 (HDAC3) 基質(zhì)金屬蛋白酶(MMPs) 磷脂酰肌醇-3-激酶(PI3K) 層黏連蛋白(Laminin-5γ2)

【摘要】：腦膠質(zhì)瘤是顱內(nèi)最常見的原發(fā)性腫瘤,約占顱內(nèi)所有腫瘤的40-60%。其具有發(fā)病率高、預(yù)后差和復(fù)發(fā)率高等特點。目前,臨床上主要的治療手段為手術(shù)切除病灶聯(lián)合術(shù)后放化療,然而患者仍然難以獲得較長的生存期。由于腫瘤的發(fā)生和生長依賴充足的血液供應(yīng),因此,血管靶向治療被認(rèn)為是最有效的治療方法之一,甚至科研機構(gòu)以此作為藥物研發(fā)和臨床試驗的基本原則。但近年來的研究發(fā)現(xiàn),抗血管治療使腫瘤細(xì)胞長時間處于缺氧與缺血環(huán)境中,不僅不能有效控制腫瘤甚至可能增加腫瘤的惡性程度,加速腫瘤轉(zhuǎn)移。這些研究提示我們,惡性腫瘤的發(fā)生發(fā)展還潛藏著其他機制。1999年,Maniotis等發(fā)現(xiàn)并提出了血管生成擬態(tài)現(xiàn)象(vasculogenic mimicry,VM)。血管生成擬態(tài)是一種與經(jīng)典的內(nèi)皮細(xì)胞依賴的腫瘤血管構(gòu)成不同的、不需要依賴內(nèi)皮的、全新的腫瘤微循環(huán)模式,是腫瘤細(xì)胞為了滿足自身生長對血供的需求,通過變形和細(xì)胞外基質(zhì)重塑而形成的一種與血管類似的、具有供血功能的管道結(jié)構(gòu)。目前,已在黑色素瘤、肝癌、侵襲性卵巢癌、胃腸道間質(zhì)瘤、乳腺癌、膠質(zhì)瘤、結(jié)直腸癌、前列腺癌、喉頭、鱗狀細(xì)胞癌等多種腫瘤組織中發(fā)現(xiàn)血管生成擬態(tài)的存在。我們實驗室研究發(fā)現(xiàn)血管生成擬態(tài)現(xiàn)象與患者臨床的生存預(yù)后呈負(fù)相關(guān)。然而,有關(guān)VM形成機制仍然沒有十分明確,但自從該概念被提出后,Maniotis等眾多研究者相繼在不同的腫瘤中發(fā)現(xiàn)并證實了血管生成擬態(tài)相關(guān)信號轉(zhuǎn)導(dǎo)機制的分子,例如：血管內(nèi)皮細(xì)胞鈣粘著蛋白(vascularendothelial cadherin, VE-cadherin)、Eph受體酪氨酸激酶A2(Erythropoietin-producing hepatocellular carcinoma-A2, EphA2)、基質(zhì)金屬蛋白酶(Matrix metalloproteinase, MMPs)、磷脂酰肌醇-3-激酶(Phosphoinositide 3-Kinase, PI3K)、層黏連蛋白(Laminin-5 γ2)、血管內(nèi)皮因子(Vascular endothelial growth factor, VEGF)、缺氧誘導(dǎo)因子(Hypoxia-inducible factor, HIF)和組織因子途徑抑制因子-2(tissue factor pathway inhibitor-2, TFPI-2)等。上述分子逐漸被深入研究并形成經(jīng)典的血管生成擬態(tài)信號傳導(dǎo)通路模型。其中Seftor, R.E等在2001年證明MMP-2和MT1-MMP (MMP-14)的表達能裂解laminin5 γ2鏈,從而產(chǎn)生分裂片段γ2'和γ2x,促進VM的生成。Hess, A .R等在2003年證明PI3K能調(diào)節(jié)MT1-MMP,從而影響VM。因此,PI3K、MMPs和LAMC2是VM信號轉(zhuǎn)導(dǎo)通路上的重要分子。組蛋白去乙�；�(Histone deacetylase, HDAC)是一類對染色體結(jié)構(gòu)修飾和基因表達調(diào)控發(fā)揮著重要作用的酶,其催化組蛋白的去乙酰化作用,與基因轉(zhuǎn)錄抑制密切相關(guān),牽涉到促基因沉默的諸多過程,是抗腫瘤藥物設(shè)計中的熱門靶標(biāo)。HDACs乙酰化不同種類的細(xì)胞核轉(zhuǎn)錄因子和蛋白等,抑制多種抑癌蛋白的表達且與多種癌基因密切關(guān)聯(lián),導(dǎo)致細(xì)胞過度增殖和腫瘤發(fā)生,有大量文獻證實其與血管生成也密切相關(guān)；Liby等發(fā)現(xiàn)在膠質(zhì)細(xì)胞瘤中HDAC3隨著腫瘤惡性程度增高而表達增高,HDAC3可能是膠質(zhì)瘤細(xì)胞增殖所必須的,在膠質(zhì)瘤細(xì)胞的惡性轉(zhuǎn)化和生長過程發(fā)揮了重要的作用；有文獻研究證明組蛋白去乙酰化酶的抑制劑TSA、SAHA等藥物可以通過PI3K等通路抑制腫瘤的周期復(fù)制,而目前認(rèn)為PI3K通路是MMP促成VM的最后通路。另有文獻指出HDAC3是HIF-1a的特定反應(yīng)物；而HIF-1a等是引起VM的最主要因素之一。由此可見,HDAC與血管生成擬態(tài)形成的信號通路中多種分子之間存在一定的交叉(crosstalk),然而目前尚無關(guān)于其與血管生成擬態(tài)之間關(guān)系的報道。若能進一步明確HDAC3是否與腦膠質(zhì)瘤血管生成擬態(tài)形成相關(guān),進一步確定HDAC3與血管生成擬態(tài)信號通路之間的分子關(guān)聯(lián),既有利于進一步推動血管生成擬態(tài)形成的機制研究進展,又有望獲從中得新的治療靶點和方法,對于臨床治療腦膠質(zhì)瘤患者具有非常重要的意義。第一章臨床數(shù)據(jù)中腦膠質(zhì)瘤VM與HDAC3表達的相關(guān)性目的分別探討腦膠質(zhì)瘤VM和HDAC3表達與臨床數(shù)據(jù)的相關(guān)性,并進一步分析血管生成擬態(tài)與HDAC3表達之間的關(guān)聯(lián)性。方法收集南方醫(yī)科大學(xué)珠江醫(yī)院2010-2013年之間102例腦膠質(zhì)瘤患者的病歷資料,并獲取相應(yīng)病理組織。按照標(biāo)準(zhǔn)流程對病理組織進行再次免疫組織化學(xué)染色。染色結(jié)果將由兩位資深病理學(xué)專家再次診斷。所有的資料對診斷專家均不公開,診斷標(biāo)準(zhǔn)為2007年世界衛(wèi)生組織關(guān)于中樞神經(jīng)系統(tǒng)腫瘤分類的方法。102例病歷資料入選標(biāo)準(zhǔn)為：1、所有患者均為首次發(fā)現(xiàn)罹患膠質(zhì)瘤且術(shù)前未進行任何治療；2、術(shù)中冰凍和術(shù)后病理結(jié)果一致且均為膠質(zhì)瘤；3、患者病理信息完整無誤。整理患者的臨床數(shù)據(jù)包括年齡、性別、腫瘤大小、KPS評分和腫瘤WHO分級等。對患者的病理組織進行薄層(5μm)多張切片,每例分成兩組,一組用于HDAC3免疫組織化學(xué)染色,分析HDAC3的表達水平；另一組進行CD34-PAS雙重免疫組織化學(xué)染色,以鑒定標(biāo)本中VM。石蠟塊剩余部分進行熒光定量即時聚合酶鏈鎖(qPCR)反應(yīng)分析。染色結(jié)果收集完畢后,首先按照VM陽性和陰性結(jié)果與病例數(shù)據(jù)逐一進行統(tǒng)計學(xué)分析,然后對HDAC3表達情況與病例資料進行統(tǒng)計學(xué)分析,最后分析VM與HDAC3之間的關(guān)聯(lián)性。P0.05在統(tǒng)計學(xué)上被認(rèn)定為差異具有統(tǒng)計學(xué)意義。結(jié)果1、染色結(jié)果顯示102例病例中,發(fā)現(xiàn)26例對應(yīng)的病理組織切片中有血管生成擬態(tài)結(jié)構(gòu),占所有病例的25.49%；2、臨床數(shù)據(jù)與血管生成擬態(tài)經(jīng)卡方檢驗分析顯示,在血管生成擬態(tài)陽性和陰性兩組對比中,腫瘤分級有顯著差異(χ2=2.902,P=0.048),而其他臨床數(shù)據(jù)沒有明顯差異。在高級別腦膠質(zhì)瘤中血管生成擬態(tài)結(jié)構(gòu)出現(xiàn)的頻率較高(III級和IV級分別為39.39%和40.43%,而I級和II級分別為0.00%和15.00%)；3、臨床數(shù)據(jù)與HDAC3表達經(jīng)卡方檢驗分析顯示,在HDAC3表達強陽性、陽性和弱陽性三組比較中,腫瘤分級亦有顯著性差(χ2=33.390,P0.001),而其他臨床數(shù)據(jù)沒有明顯差異。HDAC3高表達(強陽性和陽性)比例在高級別膠質(zhì)瘤中高于低級別膠質(zhì)瘤中(III級和Ⅳ級分別為87.88%和73.91%,而I級和II級分別為20%和27.50%)；4、在血管生成擬態(tài)陽性和陰性兩組對比中,HDAC3表達強弱各組有明顯差異(χ2=6.203,P0.043)；5、qPCR結(jié)果顯示,血管生成擬態(tài)陽性組中HDAC3的基因表達水平明顯高于陰性組,有明顯差異(P0.001)；6、顯微鏡下血管生成擬態(tài)結(jié)構(gòu)計數(shù)結(jié)果顯示,血管生成擬態(tài)結(jié)構(gòu)中位數(shù)在高表達HDAC3的病理組織中比例更高。結(jié)論結(jié)果提示血管生成擬態(tài)和HDAC3表達與腫瘤分級(WHO)呈正相關(guān),而與患者的一般臨床病理特征,如性別、年齡、KPS評分、腫瘤大小等無關(guān)；血管生成擬態(tài)與HDAC3的表達呈正相關(guān)。第二章HIDAC3對膠質(zhì)瘤U87MG細(xì)胞VM形成及其相關(guān)分子表達的影響目的建立腦膠質(zhì)瘤細(xì)胞株U87MG的三維培養(yǎng)模型(血管生成擬態(tài)細(xì)胞模型),觀察HDAC3對其血管生成擬態(tài)體外形成及其相關(guān)分子的影響,體內(nèi)驗證HDAC3與VM的相關(guān)性。方法1、使用不同劑量的HDAC3抑制劑和干擾RNA質(zhì)粒(siRNAs)分別處理正常U87MG細(xì)胞后,建立血管生成擬態(tài)模型,對比各處理組體外血管生成擬態(tài)與正常組的差異。2、使用不同劑量的HDAC3抑制劑和HDAC3 siRNAs分別處理正常U87MG細(xì)胞,利用MTT和Transwell實驗方法檢測HDAC3是否影響膠質(zhì)瘤瘤細(xì)胞的增值侵襲性。3、使用不同劑量的HDAC3抑制劑和HDAC3 siRNAs處理U87MG細(xì)胞后,建立血管生成擬態(tài)模型,驗證LAMC2和MMP-14確實參與血管生成擬態(tài)過程。4、使用不同劑量的HDAC3抑制劑和HDAC3 siRNAs分別處理正常U87MG細(xì)胞,然后利用qPCR和Western blot檢測血管生成擬態(tài)相關(guān)分子的表達水平,分析HDAC3與血管生成擬態(tài)的關(guān)系。5、建立裸鼠荷瘤模型,將裸鼠分為U87MG和穩(wěn)定低表達HDAC3的U87MG組進行喂養(yǎng),然后利用免疫組化、PAS-CD34雙重染色、Western blot等方法驗證HDAC3在體內(nèi)參與血管生成擬態(tài)的過程。結(jié)果1、U87MG三維培養(yǎng)模型建立成功；2、除siRNA對照組外,處理組細(xì)胞形成的管道結(jié)構(gòu)與正常組的對比,網(wǎng)狀管道結(jié)構(gòu)的交點數(shù)和管道長度均下降；3、處理組細(xì)胞均未能形成管道結(jié)構(gòu)；4、qPCR檢測VM相關(guān)分子：使用HDAC3干擾RNA處理的細(xì)胞組與對照及正常組對比,HDAC3 mRNA、MMP-2 mRNA. MMP-14 mRNA、LAMC2 mRNA表達水平均有明顯下調(diào)。Western blot技術(shù)檢測VM相關(guān)分子：(1)應(yīng)用HDAC3抑制劑SAHA處理的細(xì)胞組與未加入抑制劑的對照組相比,HDAC3蛋白水平、MMP-2蛋白水平、MMP-14蛋白水平、LAMC2蛋白水平均有明顯下調(diào)。(2)使用HDAC3干擾RNA處理后的細(xì)胞組與對照及正常組對比,HDAC3蛋白水平、MMP-2蛋白水平、MMP-14蛋白水平、LAMC2蛋白水平表達水平均有明顯下調(diào)。4、Western blot和qPCR檢測結(jié)果顯示應(yīng)用HDAC3抑制劑和siRNAs處理細(xì)胞后,LAMC2和MMP2/14的蛋白和mRNA表達量均明顯下降。5、荷瘤模型建立成功,離體腫瘤無論在體積還是重量上,穩(wěn)定低表達HDAC3的U87MG組均低于正常U87MG組；免疫組化鑒定VM結(jié)果顯示正常U87MG組VM陽性率高于穩(wěn)定低表達HDAC3的U87MG組；Western blot 和qPCR檢測VM相關(guān)分子顯示,穩(wěn)定低表達HDAC3的U87MG組的VM相關(guān)分子表達量明顯低于正常U87MG組。結(jié)論1、HDAC3在細(xì)胞水平、分子水平、體內(nèi)均與VM相關(guān)。第三章HDAC3參與腦膠質(zhì)瘤VM的信號轉(zhuǎn)導(dǎo)機制目的初步研究HDAC3參與惡性膠質(zhì)瘤血管生成擬態(tài)的信號轉(zhuǎn)導(dǎo)機制,證明HDAC3通過調(diào)控信號通路影響血管生成擬態(tài)的形成。PI3K/ERK-MMPs-LAMC2方法1、使用分別處理正常U87MG細(xì)胞后,用HDAC3 siRNAs方法檢測western blot以及血管生成擬態(tài)相關(guān)分子MMP-14、HDAC3、ERK、ρ-ERK、Akt、ρ-AktLAMC2的表達,分析PI3K和ERK是否參與HDAC3調(diào)節(jié)VM的過程；2、使用PI3k抑制劑LY294002以及ERK抑制劑U0126分別或聯(lián)合處理正常U87MG細(xì)胞,然后用方法檢測western blot以及血管生成擬HDAC3、ERK、ρ-ERK、Akt、ρ-Akt態(tài)相關(guān)分子的表達,分析PI3K和ERK如何參與此過程。MMP-14、LAMC2結(jié)果1、經(jīng)過siRNAs處理的U87MG細(xì)胞組,血管生成擬態(tài)相關(guān)分子MMP-14和LAMC2蛋白表達量明顯下降,同時可見p-ERK和p-Akt蛋白表達量明顯下降,但ERK和Akt未見明顯下降；2、使用U0126抑制劑處理的細(xì)胞組,p-ERK表達水平明顯下降,血管生成擬態(tài)相關(guān)分子MMP-14和LAMC2蛋白表達水平均有一定程度下降,其他未見變化；使用LY294002抑制劑處理的細(xì)胞組,p-Akt蛋白表達水平明顯下降,血管生成擬態(tài)相關(guān)分子MMP-14和LAMC2表達水平均有一定程度下降,其他未見變化；使用U0126和LY294002抑制劑聯(lián)合處理的細(xì)胞組,p-ERK和p-Akt蛋白表達水平均明顯下降,血管生成擬態(tài)相關(guān)分子MMP-14和LAMC2表達水平均有明顯下降,其他未見變化；結(jié)論ERK和P13K信號通路作為并列的兩條獨立通路參與血管生成擬態(tài)的形成。
[Abstract]:Glioma is the most common primary brain tumor, intracranial tumor all 40-60%. which has high incidence rate, high recurrence rate and poor prognosis. Currently, chemotherapy and clinical treatment were the main surgical excision combined with postoperative patients, however, is still difficult to obtain longer survival due. The tumor occurrence and growth depends on sufficient blood supply, therefore, vascular targeting therapy is considered to be one of the most effective method of treatment, or scientific research institutions as a basic principle of the drug development and clinical trials. But recent studies have found that anti angiogenesis therapy of tumor cells in long time hypoxia and ischemia in malignant not only can not effectively control tumor may even increase tumor, accelerate tumor metastasis. These studies suggest that the development of malignant tumors also suggests other mechanisms in.1999, Maniotis Found the vasculogenic mimicry (vasculogenic mimicry, VM). Vasculogenic mimicry is a kind of dependence with the classical endothelial cells of tumor vessels constitute different, does not need to rely on the endothelium, a new mode of tumor microcirculation, tumor cells in order to meet the needs of their own growth of blood supply, which is formed by deformation and the remodeling of the extracellular matrix with a similar structure with blood vessels, blood supply pipeline function. At present, has been in melanoma, liver cancer, invasive ovarian cancer, gastrointestinal stromal tumor, breast cancer, glioma, colorectal cancer, prostate cancer, larynx, vasculogenic mimicry exists that squamous cell cancer and other tumor tissues. We found that survival was negatively correlated with clinical vasculogenic mimicry. However, the formation mechanism of VM is still not very clear, but since the concept was put After Maniotis, many researchers have been found in different tumors and confirmed the molecular, vasculogenic mimicry related signal transduction mechanisms such as vascular endothelial cadherin (vascularendothelial, cadherin, VE-cadherin), Eph receptor tyrosine kinase A2 (Erythropoietin-producing hepatocellular carcinoma-A2, EphA2), matrix metalloproteinases (Matrix metalloproteinase, MMPs), phosphatidylinositol -3- kinase (Phosphoinositide, 3-Kinase, PI3K), laminin (Laminin-5 gamma 2), vascular endothelial growth factor (Vascular endothelial, growth factor, VEGF), hypoxia inducible factor (Hypoxia-inducible, factor, HIF) and tissue factor pathway inhibitor -2 (tissue factor pathway inhibitor-2, TFPI-2). The molecular has been thoroughly studied and the formation of vascular mimicry signal pathway. The classic model of Seftor, R. E in 2001 that MMP-2 and MT1-MMP (MMP-14) expression can split laminin5 gamma 2 chain, resulting in 2'and 2x gamma gamma fission fragment, promote the formation of.Hess VM, A.R in 2003 that PI3K MT1-MMP can be adjusted, thus affecting the VM. therefore, PI3K, MMPs and LAMC2 is an important molecule of VM signal transduction pathway the histone deacetylase (Histone deacetylase HDAC) is a kind of chromosome structure modification and regulation of gene expression plays an important role in the enzyme, the catalytic acetylation of histone, closely related gene transcription inhibition involves many processes, promote gene silencing, anti-tumor drug design in the hot target.HDACs acetylation of different nuclear transcription factors and protein expression, inhibit a variety of tumor suppressor proteins and many oncogenes are closely related, leading to excessive cell proliferation and tumorigenesis, have confirmed that the It is closely related with angiogenesis; Liby found HDAC3 in human glioma with malignant and higher expression of HDAC3 may be essential for glioma cell proliferation and play an important role in glioma cell malignant transformation and growth process; literature research proved that histone deacetylase inhibitors TSA, SAHA and other drugs can inhibit tumor by PI3K pathway and replication cycle, now that the PI3K pathway is the final pathway of MMP contributed to VM. Otherliterature indicated that HDAC3 is a specific reaction of HIF-1a; and HIF-1a is one of the main reasons for VM. Thus, there is a certain cross between a variety of molecular signaling pathway HDAC with the formation of vasculogenic mimicry (crosstalk), but there is no about its relationship with vasculogenic mimicry reported. If we can further clarify whether HDAC3 and brain glue Stromal tumor vasculogenic mimicry formation, further to determine the molecular association between vasculogenic mimicry and HDAC3 signaling pathway, is conducive to further promote the progress of research on the mechanism of vasculogenic mimicry formation, and is expected to obtain new therapeutic targets and methods from it, has very important significance for the clinical treatment of patients with glioma. Chapter related clinical data expression of glioma VM and HDAC3 respectively to investigate the correlation between brain glioma VM and HDAC3 expression and clinical data, and further analysis between the expression of vasculogenic mimicry and HDAC3 correlation. Methods 102 cases of brain glioma patients 2010-2013 years in Zhujiang Hospital of Southern Medical University, and obtain the corresponding pathology organization. According to the standard process of pathological re immunohistochemical staining. The staining results will be composed of two senior experts of Pathology Again. All the data on the diagnosis expert diagnosis are not open, diagnostic criteria for method of WHO in 2007 on central nervous system tumor classification.102 cases were selected for: 1, all patients were first diagnosed with glioma before operation and without any treatment; 2, intraoperative and postoperative pathological results and all for glioma; 3 patients with pathological information is complete and accurate. Consolidation of patient clinical data including age, gender, tumor size, KPS score and WHO tumor grading. Thin layer of pathological tissue of the patient (5 m) more slices, each cases were divided into two groups, one group used HDAC3 immunohistochemistry chemical staining, analysis the expression level of HDAC3; another group of CD34-PAS double immunohistochemistry, fluorescence quantitative real-time polymerase chain to identify VM. in the samples of paraffin block remainder (qPCR) staining reaction analysis. The collection is completed, according to the positive and negative results of VM and clinical data were statistically analyzed one by one, and then statistical analysis of HDAC3 expression and clinical data, finally.P0.05 analysis of the correlation between VM and HDAC3 was identified as a statistically significant difference. The results of 1 staining showed in 102 cases. 26 cases were found the corresponding pathological sections have the structure of vascular mimicry in all cases, accounting for 25.49%; 2, the clinical data and vasculogenic mimicry by chi square test analysis showed that in vasculogenic mimicry in two groups with positive and negative contrast, tumor grade had significant difference (2=2.902, P=0.048) and other clinical data. There is no significant difference in high-grade gliomas angiogenesis in the high frequency structure of mimicry (III level and IV level were 39.39% and 40.43%, while I and II were 0% and 1 5%); 3, after the chi square test showed that the expression of HDAC3 and clinical data, the expression of HDAC3 in strong positive, positive and weakly positive in three groups, there was also a significant difference in tumor grade (2=33.390, P0.001), and other clinical data have no significant difference of.HDAC3 expression (Qiang Yang and positive) in the proportion of high grade gliomas are higher than the low grade gliomas (grade III and grade were 87.88% and 73.91%, while I and II were 20% and 27.50%); 4 in vasculogenic mimicry in two groups with positive and negative contrast, HDAC3 expression in each group were significantly different (2=6.203, P0.043); 5, the results of qPCR showed that HDAC3 positive group of vasculogenic mimicry in gene expression level was significantly higher than that of negative group, there was significant difference (P0.001); 6, under the microscope structure of vasculogenic mimicry counting results showed that vasculogenic mimicry in the pathological structure of median HDAC3 expression ratio Were higher. Conclusion the results suggest that the vasculogenic mimicry and HDAC3 expression and tumor grade (WHO) was positively correlated with the general clinical and pathological features of patients, such as gender, age, KPS score, tumor size; vasculogenic mimicry and HDAC3 expression were positively related. The second chapter HIDAC3 influence the formation and expression of related molecules objective to establish a three-dimensional culture of glioma cell line U87MG model of U87MG glioma cells (VM cells vasculogenic mimicry model), the effect of HDAC3 on the formation of vasculogenic mimicry in vitro and related molecules, HDAC3 and VM in vivo correlation test. The method of 1 HDAC3 inhibitors and RNA interference plasmid using different doses (siRNAs) were treated in normal U87MG cells, the establishment of vasculogenic mimicry model, comparing the differences between each treatment group in vitro vasculogenic mimicry and normal groups.2 and HDAC3 inhibitors with different dosages HDAC3 and siRNAs were treated with normal U87MG cells, whether using MTT and Transwell assay HDAC3 glioma cell proliferation invasion of.3, using different doses of HDAC3 inhibitors and HDAC3 siRNAs in U87MG cells after treatment, the establishment of vasculogenic mimicry model, the LAMC2 and MMP-14 did participate in vasculogenic mimicry process.4, HDAC3 inhibitor HDAC3 and siRNAs were treated with different dosages of normal U87MG cells, then the expression level of qPCR and Western blot detection of vasculogenic mimicry related molecules, analysis of HDAC3 and angiogenesis in quasi.5 state, the establishment of nude mice model of the nude mice were divided into U87MG and stable low expression of U87MG group HDAC3 for feeding, and then use the immunohistochemistry, PAS-CD34 double staining, Western blot verification method for HDAC3 in vivo in vasculogenic mimicry. Results 1 U87MG, three-dimensional culture model A successful; 2, in addition to the siRNA group, compared with normal group treated with pipeline structure cell formation, node number and length of pipe network pipeline structure were decreased; 3, treatment group cells failed to form a pipeline structure; 4, qPCR detection of VM related molecules: HDAC3 cells treated with RNA interference group with the control and normal group, HDAC3 mRNA, MMP-2 mRNA., MMP-14 mRNA, LAMC2 mRNA expression levels were significantly reduced VM.Western blot detection technology related molecules: (1) cell group using HDAC3 inhibitor SAHA treatment and not adding inhibitors compared to the control group, levels of HDAC3 protein and MMP-2 protein level, the protein level of MMP-14, LAMC2 protein levels were significantly reduced. (2) and the control cell group and normal group comparison using HDAC3 interference after RNA treatment, the protein level of HDAC3, MMP-2 protein, MMP-14 protein, LAMC2 protein expression level Obviously down regulated.4, Western blot and qPCR showed that the application of HDAC3 inhibitors and siRNAs cells treated with LAMC2 and MMP2/14 mRNA and protein expression decreased obviously.5 tumor model was established successfully in vitro, both in tumor volume and weight, low and stable expression of U87MG in group HDAC3 were lower than the normal U87MG group; immunohistochemical identification of VM showed that the positive rate of U87MG was higher than that of normal group VM stable low expression of U87MG group HDAC3; Western blot and qPCR VM molecular detection showed that the stable low expression of U87MG group of VM related molecules expression of HDAC3 was significantly lower than that in normal U87MG group. Conclusion: 1, HDAC3 at the cellular level, molecular level in vivo. Was associated with VM. The mechanism of signal transduction to the signal transduction mechanism of the third chapter HDAC3 in glioma VM preliminary study of HDAC3 in malignant glioma vasculogenic mimicry, proved that the HDAC3 signal pass through the regulation The way of vasculogenic mimicry formation.PI3K/ERK-MMPs-LAMC2 method 1, respectively, using normal U87MG cells after treatment with HDAC3, Western blot and siRNAs for the detection of vasculogenic mimicry related molecules MMP-14, HDAC3, ERK, P -ERK, Akt, the expression of P -AktLAMC2, PI3K and ERK analysis process is involved in HDAC3 regulation of VM; 2, PI3k LY294002 and ERK inhibitor U0126 inhibitor respectively or combined treatment of normal U87MG cells, and then use the western method for the detection of blot and angiogenesis of quasi HDAC3, ERK, Akt, P -ERK, P -Akt expression state of related molecules, how to analyze PI3K and ERK participate in the process of.MMP-14, LAMC2 1, by

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.7

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