组蛋白去乙酰化酶-3与胶质瘤血管生成拟态的相关性

发布时间：2018-01-05 05:21

本文关键词：组蛋白去乙酰化酶-3与胶质瘤血管生成拟态的相关性　出处：《南方医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：脑胶质瘤是颅内最常见的原发性肿瘤,约占颅内所有肿瘤的40-60%。其具有发病率高、预后差和复发率高等特点。目前,临床上主要的治疗手段为手术切除病灶联合术后放化疗,然而患者仍然难以获得较长的生存期。由于肿瘤的发生和生长依赖充足的血液供应,因此,血管靶向治疗被认为是最有效的治疗方法之一,甚至科研机构以此作为药物研发和临床试验的基本原则。但近年来的研究发现,抗血管治疗使肿瘤细胞长时间处于缺氧与缺血环境中,不仅不能有效控制肿瘤甚至可能增加肿瘤的恶性程度,加速肿瘤转移。这些研究提示我们,恶性肿瘤的发生发展还潜藏着其他机制。1999年,Maniotis等发现并提出了血管生成拟态现象(vasculogenic mimicry,VM)。血管生成拟态是一种与经典的内皮细胞依赖的肿瘤血管构成不同的、不需要依赖内皮的、全新的肿瘤微循环模式,是肿瘤细胞为了满足自身生长对血供的需求,通过变形和细胞外基质重塑而形成的一种与血管类似的、具有供血功能的管道结构。目前,已在黑色素瘤、肝癌、侵袭性卵巢癌、胃肠道间质瘤、乳腺癌、胶质瘤、结直肠癌、前列腺癌、喉头、鳞状细胞癌等多种肿瘤组织中发现血管生成拟态的存在。我们实验室研究发现血管生成拟态现象与患者临床的生存预后呈负相关。然而,有关VM形成机制仍然没有十分明确,但自从该概念被提出后,Maniotis等众多研究者相继在不同的肿瘤中发现并证实了血管生成拟态相关信号转导机制的分子,例如：血管内皮细胞钙粘着蛋白(vascularendothelial cadherin, VE-cadherin)、Eph受体酪氨酸激酶A2(Erythropoietin-producing hepatocellular carcinoma-A2, EphA2)、基质金属蛋白酶(Matrix metalloproteinase, MMPs)、磷脂酰肌醇-3-激酶(Phosphoinositide 3-Kinase, PI3K)、层黏连蛋白(Laminin-5 γ2)、血管内皮因子(Vascular endothelial growth factor, VEGF)、缺氧诱导因子(Hypoxia-inducible factor, HIF)和组织因子途径抑制因子-2(tissue factor pathway inhibitor-2, TFPI-2)等。上述分子逐渐被深入研究并形成经典的血管生成拟态信号传导通路模型。其中Seftor, R.E等在2001年证明MMP-2和MT1-MMP (MMP-14)的表达能裂解laminin5 γ2链,从而产生分裂片段γ2'和γ2x,促进VM的生成。Hess, A .R等在2003年证明PI3K能调节MT1-MMP,从而影响VM。因此,PI3K、MMPs和LAMC2是VM信号转导通路上的重要分子。组蛋白去乙酰化酶(Histone deacetylase, HDAC)是一类对染色体结构修饰和基因表达调控发挥着重要作用的酶,其催化组蛋白的去乙酰化作用,与基因转录抑制密切相关,牵涉到促基因沉默的诸多过程,是抗肿瘤药物设计中的热门靶标。HDACs乙酰化不同种类的细胞核转录因子和蛋白等,抑制多种抑癌蛋白的表达且与多种癌基因密切关联,导致细胞过度增殖和肿瘤发生,有大量文献证实其与血管生成也密切相关；Liby等发现在胶质细胞瘤中HDAC3随着肿瘤恶性程度增高而表达增高,HDAC3可能是胶质瘤细胞增殖所必须的,在胶质瘤细胞的恶性转化和生长过程发挥了重要的作用；有文献研究证明组蛋白去乙酰化酶的抑制剂TSA、SAHA等药物可以通过PI3K等通路抑制肿瘤的周期复制,而目前认为PI3K通路是MMP促成VM的最后通路。另有文献指出HDAC3是HIF-1a的特定反应物；而HIF-1a等是引起VM的最主要因素之一。由此可见,HDAC与血管生成拟态形成的信号通路中多种分子之间存在一定的交叉(crosstalk),然而目前尚无关于其与血管生成拟态之间关系的报道。若能进一步明确HDAC3是否与脑胶质瘤血管生成拟态形成相关,进一步确定HDAC3与血管生成拟态信号通路之间的分子关联,既有利于进一步推动血管生成拟态形成的机制研究进展,又有望获从中得新的治疗靶点和方法,对于临床治疗脑胶质瘤患者具有非常重要的意义。第一章临床数据中脑胶质瘤VM与HDAC3表达的相关性目的分别探讨脑胶质瘤VM和HDAC3表达与临床数据的相关性,并进一步分析血管生成拟态与HDAC3表达之间的关联性。方法收集南方医科大学珠江医院2010-2013年之间102例脑胶质瘤患者的病历资料,并获取相应病理组织。按照标准流程对病理组织进行再次免疫组织化学染色。染色结果将由两位资深病理学专家再次诊断。所有的资料对诊断专家均不公开,诊断标准为2007年世界卫生组织关于中枢神经系统肿瘤分类的方法。102例病历资料入选标准为：1、所有患者均为首次发现罹患胶质瘤且术前未进行任何治疗；2、术中冰冻和术后病理结果一致且均为胶质瘤；3、患者病理信息完整无误。整理患者的临床数据包括年龄、性别、肿瘤大小、KPS评分和肿瘤WHO分级等。对患者的病理组织进行薄层(5μm)多张切片,每例分成两组,一组用于HDAC3免疫组织化学染色,分析HDAC3的表达水平；另一组进行CD34-PAS双重免疫组织化学染色,以鉴定标本中VM。石蜡块剩余部分进行荧光定量即时聚合酶链锁(qPCR)反应分析。染色结果收集完毕后,首先按照VM阳性和阴性结果与病例数据逐一进行统计学分析,然后对HDAC3表达情况与病例资料进行统计学分析,最后分析VM与HDAC3之间的关联性。P0.05在统计学上被认定为差异具有统计学意义。结果1、染色结果显示102例病例中,发现26例对应的病理组织切片中有血管生成拟态结构,占所有病例的25.49%；2、临床数据与血管生成拟态经卡方检验分析显示,在血管生成拟态阳性和阴性两组对比中,肿瘤分级有显著差异(χ2=2.902,P=0.048),而其他临床数据没有明显差异。在高级别脑胶质瘤中血管生成拟态结构出现的频率较高(III级和IV级分别为39.39%和40.43%,而I级和II级分别为0.00%和15.00%)；3、临床数据与HDAC3表达经卡方检验分析显示,在HDAC3表达强阳性、阳性和弱阳性三组比较中,肿瘤分级亦有显著性差(χ2=33.390,P0.001),而其他临床数据没有明显差异。HDAC3高表达(强阳性和阳性)比例在高级别胶质瘤中高于低级别胶质瘤中(III级和Ⅳ级分别为87.88%和73.91%,而I级和II级分别为20%和27.50%)；4、在血管生成拟态阳性和阴性两组对比中,HDAC3表达强弱各组有明显差异(χ2=6.203,P0.043)；5、qPCR结果显示,血管生成拟态阳性组中HDAC3的基因表达水平明显高于阴性组,有明显差异(P0.001)；6、显微镜下血管生成拟态结构计数结果显示,血管生成拟态结构中位数在高表达HDAC3的病理组织中比例更高。结论结果提示血管生成拟态和HDAC3表达与肿瘤分级(WHO)呈正相关,而与患者的一般临床病理特征,如性别、年龄、KPS评分、肿瘤大小等无关；血管生成拟态与HDAC3的表达呈正相关。第二章HIDAC3对胶质瘤U87MG细胞VM形成及其相关分子表达的影响目的建立脑胶质瘤细胞株U87MG的三维培养模型(血管生成拟态细胞模型),观察HDAC3对其血管生成拟态体外形成及其相关分子的影响,体内验证HDAC3与VM的相关性。方法1、使用不同剂量的HDAC3抑制剂和干扰RNA质粒(siRNAs)分别处理正常U87MG细胞后,建立血管生成拟态模型,对比各处理组体外血管生成拟态与正常组的差异。2、使用不同剂量的HDAC3抑制剂和HDAC3 siRNAs分别处理正常U87MG细胞,利用MTT和Transwell实验方法检测HDAC3是否影响胶质瘤瘤细胞的增值侵袭性。3、使用不同剂量的HDAC3抑制剂和HDAC3 siRNAs处理U87MG细胞后,建立血管生成拟态模型,验证LAMC2和MMP-14确实参与血管生成拟态过程。4、使用不同剂量的HDAC3抑制剂和HDAC3 siRNAs分别处理正常U87MG细胞,然后利用qPCR和Western blot检测血管生成拟态相关分子的表达水平,分析HDAC3与血管生成拟态的关系。5、建立裸鼠荷瘤模型,将裸鼠分为U87MG和稳定低表达HDAC3的U87MG组进行喂养,然后利用免疫组化、PAS-CD34双重染色、Western blot等方法验证HDAC3在体内参与血管生成拟态的过程。结果1、U87MG三维培养模型建立成功；2、除siRNA对照组外,处理组细胞形成的管道结构与正常组的对比,网状管道结构的交点数和管道长度均下降；3、处理组细胞均未能形成管道结构；4、qPCR检测VM相关分子：使用HDAC3干扰RNA处理的细胞组与对照及正常组对比,HDAC3 mRNA、MMP-2 mRNA. MMP-14 mRNA、LAMC2 mRNA表达水平均有明显下调。Western blot技术检测VM相关分子：(1)应用HDAC3抑制剂SAHA处理的细胞组与未加入抑制剂的对照组相比,HDAC3蛋白水平、MMP-2蛋白水平、MMP-14蛋白水平、LAMC2蛋白水平均有明显下调。(2)使用HDAC3干扰RNA处理后的细胞组与对照及正常组对比,HDAC3蛋白水平、MMP-2蛋白水平、MMP-14蛋白水平、LAMC2蛋白水平表达水平均有明显下调。4、Western blot和qPCR检测结果显示应用HDAC3抑制剂和siRNAs处理细胞后,LAMC2和MMP2/14的蛋白和mRNA表达量均明显下降。5、荷瘤模型建立成功,离体肿瘤无论在体积还是重量上,稳定低表达HDAC3的U87MG组均低于正常U87MG组；免疫组化鉴定VM结果显示正常U87MG组VM阳性率高于稳定低表达HDAC3的U87MG组；Western blot 和qPCR检测VM相关分子显示,稳定低表达HDAC3的U87MG组的VM相关分子表达量明显低于正常U87MG组。结论1、HDAC3在细胞水平、分子水平、体内均与VM相关。第三章HDAC3参与脑胶质瘤VM的信号转导机制目的初步研究HDAC3参与恶性胶质瘤血管生成拟态的信号转导机制,证明HDAC3通过调控信号通路影响血管生成拟态的形成。PI3K/ERK-MMPs-LAMC2方法1、使用分别处理正常U87MG细胞后,用HDAC3 siRNAs方法检测western blot以及血管生成拟态相关分子MMP-14、HDAC3、ERK、ρ-ERK、Akt、ρ-AktLAMC2的表达,分析PI3K和ERK是否参与HDAC3调节VM的过程；2、使用PI3k抑制剂LY294002以及ERK抑制剂U0126分别或联合处理正常U87MG细胞,然后用方法检测western blot以及血管生成拟HDAC3、ERK、ρ-ERK、Akt、ρ-Akt态相关分子的表达,分析PI3K和ERK如何参与此过程。MMP-14、LAMC2结果1、经过siRNAs处理的U87MG细胞组,血管生成拟态相关分子MMP-14和LAMC2蛋白表达量明显下降,同时可见p-ERK和p-Akt蛋白表达量明显下降,但ERK和Akt未见明显下降；2、使用U0126抑制剂处理的细胞组,p-ERK表达水平明显下降,血管生成拟态相关分子MMP-14和LAMC2蛋白表达水平均有一定程度下降,其他未见变化；使用LY294002抑制剂处理的细胞组,p-Akt蛋白表达水平明显下降,血管生成拟态相关分子MMP-14和LAMC2表达水平均有一定程度下降,其他未见变化；使用U0126和LY294002抑制剂联合处理的细胞组,p-ERK和p-Akt蛋白表达水平均明显下降,血管生成拟态相关分子MMP-14和LAMC2表达水平均有明显下降,其他未见变化；结论ERK和P13K信号通路作为并列的两条独立通路参与血管生成拟态的形成。
[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

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

【共引文献】