【作者】 姜萌；

【导师】 曲元明；

【作者基本信息】 山东大学 ， 外科学， 2012， 博士

【摘要】 第一部分MVD、TSP-1、TGF-β1、PPAR-γ、MMP-9、VEGF及CD36在胶质瘤中表达情况的研究研究背景胶质瘤是发生于神经外胚层的肿瘤,故亦称神经外胚层肿瘤或神经上皮肿瘤,是最常见的成人脑肿瘤,约占所有颅内肿瘤的45%。据文献报道,中国脑胶质瘤年发病率为3-6人/10万人,年死亡人数达3万人。高分级的胶质瘤患者五年生存率低于3%,中位生存时间小于一年。目前其治疗主要依赖手术,辅以放化疗等,虽然外科手术和其他辅助治疗措施进展很快,但仍很难完全根治。其预后主要取决于肿瘤发展速度及部位。近来,胶质瘤化疗发展迅速,其中以替莫唑胺为代表的化疗药物已投入临床使用。目前认为,实体肿瘤的生长是血管依赖性的,肿瘤血管生成增加意味着肿瘤细胞的快速增殖,局部侵袭甚至转移。肿瘤血管生成是受多种促血管生成因子及抗血管生成因子调节的。通过研究各种促血管生成因子及抗血管生成因子在胶质瘤中的表达与各项临床、病理指标的联系,可以为研究胶质瘤提供了一个新的思路,为今后的靶向治疗奠定理论基础。血小板反应蛋白1/凝血酶敏感蛋白1(thrombospondin-1, TSP-1)是最早被发现的自然存在的血管生成抑制因子。TSP-1在各生理病理过程中起着调节血管内皮细胞增生、迁移、调亡等作用,主要作用表现为调节血管生成。其抑制血管生成的主要机制在与其受体CD36结合,从而影响内皮细胞的增殖、粘附及运动性,从而影响血管生成。其他可能抑制血管生成的机制包括促进TGF-β1由前体向其活性形式的转化、抑制VEGF及MMP-9的活性等。同时有证据表明,TSP-1亦可直接引起肿瘤细胞的调亡。TSP-1对肿瘤的影响随肿瘤细胞类型的不同而存在差异。目前国内尚无对TSP-1在胶质瘤中表达及与其他各种血管生成调节因子联系的研究。转化生长因子-β1(transforming growth factor-β1,TGF-β1)是属于转化生长因子超家族蛋白。转化生长因子-β1与细胞表面的转化生长因子-β受体结合而激活其受体。其作用主要为抑制免疫监视作用,调节细胞增殖、分化和细胞外基质产生,促进肿瘤血管形成。转化生长因子-β受体是丝氨酸/苏氨酸激酶受体。其信号传递可以通过SMAD信号通路和/或DAXX信号通路,从而可以引起包括内皮细胞在内的细胞调亡。但同时TGF-β1亦可引起VEGF及纤溶酶原激活物抑制剂生成增多,促进血管再塑,从而加强血管生成。其对肿瘤生长的影响最终取决于何种机制占主导地位。过氧化物酶体增殖因子活化受体(peroxisome Proliferator-activated receptor gama, PPAR-γ)属于Ⅱ型核受体超家族成员,是一类细胞核转录的调节因子,包括α,β,γ三种亚型。PPAR的生物学功能复杂,可参与调节脂质代谢与糖代谢、脂肪细胞分化和能量平衡、炎症反应、动脉粥样硬化及肿瘤细胞的分化与凋亡等生理和病理过程。其中PPAR-γ研究最多。越来越多的证据表明,PPAR-γ激动剂具有显著的抗肿瘤作用。其作用机制涉及抑制细胞增殖、诱导细胞凋亡及分化、抑制血管生成和降低肿瘤侵袭能力等各个方面。目前在胶质瘤中关于PPAR-γ的研究主要侧重于其抑制细胞增殖的作用,而对PPAR-γ抑制血管生成的机制研究较少。金属基质蛋白酶9(matrix metalloproteinases9, MMP-9)是一种锌依赖性的水解细胞外基质的蛋白裂解酶,包括基质中以及整合于质膜中的各种胶原酶和弹性蛋白酶等。其在伤口愈合、血管生成及肿瘤重塑、侵袭、转移方面均发挥重要作用。MMP-9可以使内皮细胞穿过以Ⅳ型胶原为主的基底膜,与肿瘤间质血管生成关系密切,被认为是血管生成激活的开关之一。血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种重要的血管生长因子。它主要作用于血管内皮细胞,使其分裂和产生趋化作用,从而促进内皮细胞的增殖、迁移,并增加微血管的通透性,引起血浆蛋白渗漏到细胞外基质,为成纤维细胞和血管内皮细胞的迁入提供条件基质。同时VEGF能诱导内皮细胞表达尿激酶型纤溶酶原激活因子(uPA)、组织型纤溶酶原激活因子(uPAR)及尿激酶型纤溶酶原激活因子受体(uPAR)等,从而促使渗透到血管外区域的血浆蛋白形成血浆蛋白凝块,作为血管新生的支持物。目的本实验通过免疫组化、RT-PCR及Western Blotting方法,研究TSP-1、TGF-β1、PPAR-γ、MMP-9、VEGF在胶质瘤中的表达情况,比较其在各病理分级与各项临床指标之间分布与表达差异,进一步探讨其潜在的临床治疗价值。材料和方法：1、材料随机选取2009年1月～2011年4月期间山东省立医院神经外科胶质瘤手术标本62例,其中男性34例,女性28例,年龄5-71岁,平均年龄(52.3±1.3)岁；所有病例诊断均有术后的免疫组化病理证实。2、方法采用免疫组织化学SP法检测MVD、TSP-1、TGF-β1、PPAR-γ MMP-9、VEGF及CD36在胶质瘤石蜡标本中的蛋白表达,采用荧光实时定量PCR检测TSP-1、TGF-β1、PPAR-γ、MMP-9、VEGF及CD36mRNA水平表达,采用Western Blotting方法检测其在组织标本中蛋白表达。结果：1、胶质瘤中血管生成程度与肿瘤性质相关,恶性程度越高则血管生成程度越高。2、TSP-1在脑胶质瘤表达与病人性别、年龄无关,与胶质瘤恶性程度负相关。TSP-1表达与MVD负相关。3、PPARγ在脑胶质瘤表达与病人性别、年龄无关,与胶质瘤恶性程度负相大。PPARγ表达与MVD负相关。4、TGF-β1在脑胶质瘤表达与病人性别、年龄无关,与胶质瘤恶性程度正相关。TGF-β1表达与MVD正相关。5、MMP-9在脑胶质瘤表达与病人性别、年龄无关,与胶质瘤恶性程度正相关。MMP-9表达与MVD正相关。6、VEGF在脑胶质瘤表达与病人性别、年龄无关,与胶质瘤恶性程度正相关。VEGF表达与MVD正相关。7、TSP-1表达与TGF-β1、MMP-9、VEGF表达均呈负相关,TGF-β1与MMP-9、VEGF表达正相关,PPAR-γ表达与CD36呈正相关,MMP-9与VEGF表达正相关。结论：1、胶质瘤组织中TSP-1、PPAR-γ mRNA及蛋白表达随胶质瘤恶性程度增加均显著下降,且二者均与MVD负相关,提示TSP-1与PPAR-γ为胶质瘤血管生成过程中的负性调节因子,其负性血管生成作用可能导致其抑制胶质瘤恶性进展。TSP-1抑制血管生成作用除与CD36结合直接作用于血管内皮细胞外,可能还包括其抑制MMP-9、VEGF的表达。PPAR-γ抑制血管生成作用可能包括上调CD36表达,从而重建TSP-1对血管内皮细胞的抑制作用。2、胶质瘤组织中TGF-β1、MMP-9、VEGF mRNA及蛋白表达随胶质瘤恶性程度增加均显著增高,且三者均与MVD正相关,提示TGF-β1、MMP-9、 VEGF为胶质瘤血管生成过程中的正性调节因子,其正性血管生成作用可能导致其促进胶质瘤恶性进展。第二部分：联合应用TSP-1与PPARγ激动剂对HUVEC及U251细胞影响的研究研究背景：靶向血管治疗肿瘤具有不易产生耐药性、毒副作用少、可长期用药、可用于多种肿瘤、可与多种传统化疗药物共用等优点。尽管在细胞及动物实验中表现优异,一些靶向性抗血管生成药物单药在临床应用过程中并未能取得预期的疗效。主要原因在于对抗血管治疗而言,肿瘤微环境十分复杂,其间各种细胞可能分泌多种促血管生成因子和血管生成抑制因子,从而影响抗血管药物的疗效。TSP-1是最早被发现的血管抑制因子,其作用主要通过与与其受体CD36结合,抑制血管内皮细胞黏附,增殖,从而抑制血管生成的形成。在促进血管内皮细胞凋亡的体外实验中,CD36介导的TSP-1能够抑制内皮细胞迁移和管腔形成,且这一作用可被CD36类似物GST融合蛋白及CD36的抗体所抑制。CD36转染入人脐静脉内皮细胞(HUVECs,内皮细胞)后,可使其对TSP-1抑制迁移作用更敏感。但CD36的表达在体内是是不均质的。它是在大血管上较少表达,在某些动脉上不表达,而且即便是在微小血管上其表达也差异较大。而TSP-1对血管内皮细胞的抑制作用主要取决其中CD36阳性的血管内皮细胞的比例。PPARΓ激动剂已被研究证实与受体结合后,可提高CD36受体在血管内皮细胞的表达。但目前PPARγ上调CD36表达的机制还不甚确定。由第一部分研究可知,TSP-1与PPARΓ均与胶质瘤恶性程度及MVD成负相关,说明TSP-1与PPARΓ在胶质瘤中的作用为抑制血管生成。且此前研究亦表明CD36在胶质瘤细胞及血管内皮细胞中均有表达。本研究部分主要着重于体外细胞上验证TSP-1与PPARΓ激动剂是否在抑制血管生成及相关因子分泌上有协同作用。人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)是从人脐静脉血中提取的细胞,因其人体血管内皮细胞的特性,故在实验室中,多被用来进行关于血管生成、血管内皮细胞方向的研究。U251细胞属于人类星形胶质细胞瘤细胞系,它具有星形胶质细胞特性,同时有较强的增殖能力及侵袭能力,常在实验中模拟人恶性星形细胞瘤。本部分研究即选用两种细胞,观察PPARγ激动剂及TSP-1联合作用对血管内皮细胞的凋亡及对肿瘤细胞分泌的各种血管生成调节因子的影响。目的：通过对PPARγ激动剂及TSP-1作用过的HUVEC及U251的检测,观察其对血管内皮细胞的凋亡影响及对肿瘤细胞分泌的各种血管生成调节因子的影响,以验证第一部分研究的结论,并为下一步的动物实验打下基础。方法：联合应用不同浓度的PPARγ激动剂罗格列酮及TSP-1作用于HUVEC及U251,使用RT-PCR与Western Blotting检测HUVEC细胞CD36表达情况,MTT法及流式细胞检测HUVEC凋亡情况；使用RT-PCR与Western Blotting检测U251中TGF β1、VEGF、MMP-9的表达情况。结果：在HUVEC细胞中,RT-PCR与Western Blotting显示CD36蛋白的表达随罗格列酮浓度的增加而增高。MTT实验表明,罗格列酮与TSP-1联合应用可有效的抑制HUVEC细胞的生长,其作用随药物浓度提高和作用时间延长而增强,呈现一定的剂量、时间依赖性。当此诱导凋亡的作用可被CD36抗体FA6-152所拮抗。流式细胞检测结果表明,罗格列酮与TSP-1作用于HUVEC细胞后,可明显诱导细胞凋亡,且随作用浓度和作用时间的延长,凋亡率显著增加。在U251细胞中,RT-PCR与Western Blotting显示TSP-1可降低VEGF的表达。结论：本实验结果表明：PPARγ激动剂可提高HUVEC细胞CD36的表达,与TSP-1协同应用可提高其诱导细胞凋亡的能力；而TSP-1可引起U251细胞表达VEGF的下降。以上提示PPARγ激动剂与TSP-1协同作用后有较强的抗血管生成能力。更多还原

【Abstract】 Parts1expression of MVD、TSP-1、TGF-β1、PPAR-γ、MMP-9、VEGFand CD36in gliomasBackgrounds:Gliomas is derived from neuroectodermal cells, thus it is also called Neuroectodermal neoplasms or Neuroepithelial neoplasms. It is the most common intracranial tumor, accounting for about forty-five percent of all intracranial tumors in adult. It is reported that in China the incidence of disease is about3-6persons/100,000persons, and almost30,000patients died of gliomas each year. The survival rate for5years of patients with gliomas of high grade is less than3%, and the median live time is less than one year. The current treatment for gliomas is removal of tumor surgically, assisted by radiotherapy and chemotherapy. Although there is rapid progress made in the surgery technique and adjuvant radiotherapy and chemotherapy, it is still difficult to bring gliomas under permanent control. The tumor location and progression speed is the main prognostic factors. Recently progress were made in adjuvant chemotherapy, chemotherapeutics such as Temozolomide were put into clinical use. However, chemotherapeutic drugs being used now are designed for killing the residual tumor cells so as to prevent a quick recurrence of gliomas. It is more effective in some patients with gliomas of specific hereditary features, such as positive for O6-methylguanine-DNA methyl-transferase (MGMT), or loss of heterozygosity (LOH). It now demonstrated that the growth of tumor entity is angiogenesis-dependent. Angiogenesis plays a crucial role in tumor proliferation, invasion and even metastasis. The angiogenesis process is regulated by pro-angiogenesis factors and anti-angiogenesis factors in the microenvironment. In the present study, we investigate the expression of pro-angiogenesis factors and anti-angiogenesis factors and its association with clinicopathologic factors in gliomas, so that we might find new targets for gliomas treatment.Thrombospondin-1(TSP-1) was the first identified natural occurring protein for angiogenesis inhibition. Subsequent studies indicated that TSP-1regulated proliferation, migration, and apoptosis in various physiological and pathological events. It mainly play an important role in the regulation of angiogenesis. The mechanisms may include binding with CD36, in turn affects the proliferation, adhesion and motility of endothelial cells, and regulates angiogenesis eventually. There were findings that indicate TSP-1can induce cell apoptosis directly. Thus, the effect of TSP-1on tumor growth may vary from different tumor types.Transforming growth factor-β1(TGF-β1) is a member of transforming growth factor superfamily. It can bind to the TGF-β receptors in cell membrane and activate the receptors. The signaling pathway includes the SMAD pathway and DAXX pathway; therefore induce cell apoptosis including endothelial cells. However, TGF-β1can also increase the production of VEGF and plasminogen activator inhibitor, which promote the remodeling of tumor vessels, resulting in angiogenesis. So the major effects of TGF-β1on tumors depend on which mechanism is dominant in the microenvironment.Peroxisome Proliferator-activated receptor gamma (PPAR-γ) is a member of type Ⅱ nuclear receptor superfamily, functioned as regulatory factor in nuclear transcription, including α, β, γ sub-types. PPAR have complicated biological function, taking part in the process of lipid metabolism, glycometabolism, adipocyte differentiation, energy balance, inflammatory reactions, arthrosclerosis, tumor cell differentiation and necrosis. PPAR-γ is studied intensively. The main function of which is to promote the synthesis of lipid. Many evidence indicated that the PPAR-γ activator has a distinguished function against tumor. The mechanisms include inhibiting cell proliferation, inducing cell apoptosis and differentiation, inhibiting angiogenesis and reduce the invasive ability of tumor.Matrix metalloproteinases9(MMP-9) is a zinc-dependent proteolytic enzyme that can lyse the extracellular matrix, including collagenase and elastase. It plays an important role in wound healing, neovascularization, tumor remodeling, invasion and metastasis. MMP-9can facilitate the tumor cells to penetrate the basal membrane that is mainly formed by type Ⅳ collagen, thus promote the neovascularization in the stroma. Vascular endothelial growth factor (VEGF) is an important factor in regulating angiogenesis. VEGF promotes cell division and Chemotaxis, causing the proliferation and migration of endothelial cells. It also increase the permeability of microvessels, leading to leaking of plasma protein to extracellular stroma, facilitating the migration of fibroblasts and endothelial cells. Moreover, VEGF can induce the production of urokinase-type plasminogen activator, tissue plasminogen activator and urokinase-type plasminogen activator receptor, causing coagulation of plasma proteins in the stroma, thus provide braces for neovessels.Objectives:In the present study, we investigated the expression of TSP-1, TGF-β1, PPAR-γ, MMP-9, VEGF and CD36in gliomas. The expression difference was compared between groups to find the potential target for new therapy.Material and methods:Patients and tissues:Gliomas removed surgically in Shandong Provincial Hospital from Jan2009to Aug2011were selected randomly. There were34male, and28female. The median average age is52.3±1.3y, ranging from5y to71y. The pathological diagnosis was confirmed by immunochemical staining.Methods:The expression of TSP-1, TGF-β1, PPAR-γ, MMP-9, VEGF and CD36in gliomas was detected by immunochemical staining, Realtime-PCR and Western Blotting.Results:1.The expression of TSP-1and PPAR-γ was found higher in low-grade gliomas than in high-grade gliomas. Both the expression of TSP-1and PPAR-γ was found reversely correlated to MVD.(P<0.05).2. The expression of TGF-β1, MMP-9, VEGF was found lower in low-grade gliomas than in high-grade gliomas. Both the expression of TSP-1and PPAR-γ was found positively correlated to MVD.(P<0.05).Conclusions:The expression of TSP-1and PPARγ was significantly decreased in high-grade gliomas compared to that in low-grade gliomas. The mechanism may due to their anti-angiogenesis effect. The mechanism underlying the anti-angiogenesis effect of TSP-1may involve the inhibition of MMP-9and VEGF. PPARy can increase the expression of CD36, which may help partly explain anti-angiogenesis effect of PPARy.2. The expression of TGF-β1, MMP-9and VEGF significantly higher in high-grade gliomas compared to that in low-grade gliomas. The mechanism may due to their pro-angiogenic effect. Part2The effect of TSP-1combined with PPAR Y ligands on HUVEC and U251BackgroundAnti-angiogenic therapy have many advantage, including seldom drug resistance, low toxicity, long-term medication, suitable for multiple tumors, co-existing with other chemotherapy drugs. Although Anti-angiogenic drugs made good performance in cell experiment and in vitro, many of them failed to reach the original expectancy. The main reason for this is that the microenvironment is very complicated. There are many cells which can produce pro/anti-angiogenic factor to undermine the effect of drugs.TSP.-1is proved to be function mainly by its binding to CD36. TSP-1can inhibit the adhesion, proliferation, and migration of endothelial cells, which can be agonized by CD36antibody. Transfection of CD36into HUVEC can sensitize ECs to the inhibitory effect of TSP-1on migration.The expression of CD36is heterogonous. It is less expressed in large vessels and some arteries. The inhibitory effects of TSP-1mainly depend on the portion of CD36-positive cells. PPAR can up-regulate the expression of CD36. The mechanism is still unknown.In the lab, HUVEC is often used for the test of ECs. U251is often used for test of gliomas. These two cells were used in our study. Objective:to determine the synergetic effect of TSP-1and PPAR γ in U251and HUVEC.Methods:After applying PPAR Y together with TSP-1, we conduct to determine the expression of CD36in HUVEC and expression of TGF β1、VEGF、MMP-9In U251. MTT and flow cytometry were also used to detect the apoptosis in HUVEC.Results:The CD36expression is elevated by PPAR Y in a time-dependent and dose-dependent way. PPAR Y and TSP-1induce the apoptosis in HUVEC. TSP-1down-regulates the expression of VEGF.Conclusions:PPAR Y and TSP-1have a synergetic effect and can inhibit angiogenesis by inducing CD36expression and ECs apoptosis. TSP-1can also down-regulates the expression of VEGF in vitro.更多还原