【作者】 洪景芳；

【导师】 丁学华；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 背景垂体腺瘤是一种严重危害人类身心健康的脑良性肿瘤,部份垂体腺瘤具有向周围结构尤其是海绵窦侵袭的特征。目前侵袭性垂体腺瘤向海绵窦侵犯的机制尚不清楚,其生物学特征和组织学特征相背离的现象令人困惑,难以找到合适的理论和作用机制来解释。海绵窦特殊的微环境可能是肿瘤侵袭的重要因素。细胞表面受体DDR1具有接受肿瘤微环境信号,启动磷酸化通路并上调MMP-2/9表达、促进肿瘤细胞侵袭的能力,可能在垂体腺瘤细胞和肿瘤的微环境的相互作用中起到重要作用。本课题利用基因工程技术,采取真核表达系统纯化出DDR1的可溶性特异竞争阻断蛋白NDDR1。课题以临床磁共振影像学为侵袭性判断标准,在大量组织标本免疫组化结果的基础上,以NDDR1和nilotinib对原代培养的垂体腺瘤细胞DDR1进行干预研究,旨在明确垂体腺瘤中DDR1对MMP-2/9和侵袭能力的调节作用,探讨DDR1在垂体腺瘤海绵窦侵袭发病过程中的作用和相关机制。第一部分:DDR1和MMP-2/9在人垂体腺瘤组织中的表达和相关性研究目的:明确DDR1、MMP-2/9在各类型垂体腺瘤组织中的表达及其同肿瘤生物学行为的关系,探讨DDR1和MMP-2/9的相关性。方法:肿瘤组织标本按MRI表现分为侵袭组和非侵袭组,按临床和免疫组化分为功能性腺瘤组和无功能腺瘤组。共聚焦显微镜下观察DDR1的细胞定位;免疫组化观察DDR1和MMP-2/9蛋白的表达情况;Western blot对DDR1和MMP-2/9的表达进行半定量分析;同时采用即时荧光定量PCR检测各组的DDR1、MMP-2/9mRNA表达水平。结果:1.DDR1表达于细胞膜;2.侵袭组中DDR1蛋白和mRNA水平显著高于非侵袭组(P＜0.01),功能性腺瘤组显著高于无功能腺瘤组(P＜0.01);3.侵袭组MMP-2/9蛋白和mRNA水平显著高于非侵袭组(P＜0.01),功能性腺瘤组和无功能腺瘤组无显著差异(P＞0.05);4.DDR1和MMP-2之间有正相关性(r=0.857,P＜0.01),DDR1和MMP-9之间有正相关性(r=0.813,P＜0.01)。结论:DDR1和垂体腺瘤的侵袭性有关;DDR1可能通过上调MMP-2/9的表达,从而增强垂体腺瘤的侵袭性。第二部分:重组DDR1竞争阻断蛋白(NDDR1)的制备和活性鉴定目的:制备DDR1的可溶性特异竞争阻断蛋白(NDDR1);对重组NDDR1进行活性鉴定,确定其阻断垂体腺瘤原代培养细胞DDR1的量效关系。方法:扩增DDN1胞外区,构建真核表达质粒pcDNA3.1-NDDR1,瞬时转染293细胞,纯化获得NDDR1蛋白;ELISA竞争抑制实验验证NDDR1的生物学活性,确定阻断垂体腺瘤原代培养细胞DDR1的最佳剂量。结果:1.获得哺乳动物系统表达和纯化的NDDR1蛋白3mg/L;2.竞争抑制实验示NDDR1具有可竞争阻断细胞表面天然DDR1受体的能力;3.2μg/L的NDDR1即可阻断原代培养的垂体腺瘤DDR1。结论:可溶性竞争阻断蛋白NDDR1可特异性阻断原代培养的垂体腺瘤DDR1受体。第三部份原代培养细胞中胶原Ⅰ-DDR1-MMP-2/9环路的激活目的:确定垂体腺瘤原代培养细胞中诱发DDR1信号通路的胶原类型,研究启动DDR1信号通路后Tyr磷酸化水平的变化,探讨细胞水平DDR1调节MMP-2/9表达的作用,体外论证垂体腺瘤海绵窦侵袭中胶原Ⅰ-DDR1-MMP-2/9环路的激活过程。方法:原代培养垂体腺瘤细胞中,给予外源性胶原Ⅰ、胶原Ⅱ、胶原Ⅲ、胶原Ⅳ刺激,细胞上清液行ELISA法以确定诱发DDR1信号通路的胶原类型;免疫沉淀法测定NDDR1和nilotinib干预前后Tyr磷酸化水平;明胶酶谱法和ELISA法测量刺激和阻断DDR1信号通路后MMP-2/9活性和表达水平的变化情况;建立Transwell侵袭模型小室,镜下计数观察NDDR1和nilotinib干预前后细胞侵袭能力的变化。结果:成功对9例垂体腺瘤进行原代培养。1.胶原Ⅰ是激发垂体腺瘤DDR1信号通路的主要胶原类型;2.胶原Ⅰ刺激后Tyr磷酸化水平明显升高(P＜0.05),NDDR1和nilotinib可抑制自磷酸化;3.胶原Ⅰ刺激后显著提高MMP-2/9的表达(P＜0.05),NDDR1和nilotinib可抑制该效应;4.NDDR1和nilotinib干预后细胞侵袭能力下降(P＜0.05)。结论:胶原Ⅰ通过诱发DDN1的磷酸化上调MMP-2/9的表达,从而增强垂体腺瘤的侵袭能力,阻断DDR1可有效降低肿瘤的侵袭性;体外证实了胶原Ⅰ-DDR1-MMP-2/9环路的激活可能是垂体腺瘤海绵窦侵袭的重要环节和发生机制。两侧海绵窦壁高表达胶原蛋白1可能是垂体腺瘤海绵窦壁侵袭的病理基础之一。更多还原

【Abstract】 BackgroundPituitary adenomas is a benign brain tumor which usually lead to severely health problems.Some of them could invade surrounding structures,especially cavernous sinus. The molecular mechanisms that dictate this local invasive behavior of Pas remain poorly understood by now.Few theory and mechanism can explain the discrepancy between biology characteristics and histologic characteristics in invasive pituitary adenomas. Special microenvironment in cavernous sinus is a possibly important factor that contribute to invasion.DDR1,a cell surface receptor,can receive signal from tumor microenvironment,induce autophosphorylation and upregrate expression of MMP-2/9, promote the invasiveness of pituitary adenomas.Thereby,DDR1 may play an impotant role between pituitary adenomas cells and tumor microenvironment.In the present investigation we expressed and purified soluble protein NDDR1,which have a capability to block DDR1 with competition inhibition effection,by eukaryotic expression system and genetic engineering.We used MRI finding as criterion to assess the clinical invasiveness of the pituitary adenomas,Based on Immunohistochemistry founding of large pituitary adenomas sample,NDDR1 and nitotinbib was used to inhibit DDR1 in pituitary adenornas primary cultured cells.Our study aimed to investigate the effects of DDR1 on the regulation of expression of MMP-2 and MMP-9 in pituitary adenomas,and to explore its potential role and mechanism of action of carvernous sinus invasion by pituitary adenomas.Part one:Expression of DDR1 and MMP-2/9 in human pituitary adenomas and their significaneesObjective:To determine the expression of DDR1 and MMP-2/9 in pituitary adenomas and their relationships with biological behaviour of pituitary adenomas;to explore the significances between DDR1 and MMP-2/9.Methods:All adenomas samples were divided into invasion group and non-invasion group according to MRI finding or divided into functional adenomas group and non-functional adenomas group on the basis of clinic manifestation and results of Immunohistochemistry.Cellular localization of DDR1 was detected by confocal microscopy observaion,Immunohistochemistry was used to confirm the expression of DDR1 protein and MMP-2/9 protein.Semiquantitative analysis of DDR1 protein and MMP-2/9 protein was evaluated by Western blot;the expressions of DDR1 mRNA and MMP-2/9 mRNA were also analysised by the method of reverse transcriptase poly merase chin reaction(RT-PCR).Results:1.DDR1 immunoreactivity was located in cellular membrane of pituitary adenomas cells.2.The expression of DDR1 protein and mRNA in invasive pituitary adenomas was significantly higher than in noninvasive pituitary adenomas(P＜0.01);the expression of DDR1 in functional pituitary adenomas was significantly higher than in non-functional pituitary adenomas(P＜0.01);3.There was no significant difference of MMP-2/9 protein and mRNA between functional and non-functional pituitary adenomas(P＞0.05).In invasive pituitary adenomas,there were more expression of MMP-2/9 protein and mRNA than in corresponding noninvasive adenomas(P＜0.01).4.A positive correlation existed between expression of DDR1 and MMP-2(r=0.857,P＜0.01) in functional pituitary adenomas,either between DDR1 and MMP-9(r=0.813,P＜0.01).Conclusions:DDR1 may play an important role in the invasiveness of pituitary adenomas. DDR1 could increase the invasiveness of pituitary adenomas by up-regulating expression of MMP-2/9.Part two:Express and purify of competition inhibition protein(NDDR1) and identify the biologic activity of NDDR1Objective:To express and purify soluble protein(NDDR1) which have a capability to block DDR1 with competition inhibition effection,to identify the biologic activity of NDDR1 and determine the dose-effect relationship between NDDR1 and pituitary adenomas primary cultured cells.Motheds:Extracellular domain of DDR1 was amplify by PCR and an eukaryotic expression plasmid pcDNA3.1-NDDR1 was constructed.Then the plasmid was transiently transfected into 293 cells,purified the expressed protein NDDR1.Competitive blinding inhibition assay by ELISA was used to demonstrate the biologic activity of NDDR1 and determine the optimum dose blocking DDR1 activity of pituitary adenomas primary cultured cells.Results:1.3mg/L protein of NDDR1 expressed and purified by mammalian express system was obtained.2.Competitive blinding inhibition assay demonstrated that expressed NDDR1 could block the blinding of DDR1 and natural DDR1 receptors on primary culture cells.3.2μg/L of NDDR1 could block the blinding of DDR1 and natural DDR1 receptors on primary cultured cells.Conclusions:Soluble protein NDDR1 could block the blinding of DDR1 and natural DDR1 receptors on primary cultured cells with a competitive inhibition effection.Part three:Activation of circuit loop of collagenⅠ-DDR1-MMP-2/9 in pituitary adenomas primary cultured cellsObjective:To determine the collagen subtypes that induces the signal pathway of DDR1 in pituitary adenomas primary cultured cells.To investigate autophosphorylation of DDR1 after DDR1 signal was started.To investigate regulation on MMP-2 and MMP-9 of DDR1. To demonstrate the activation of circuit loop of collagenⅠ-DDR1-MMP-2/9 in cavernous sinus invasion by pituirary adenomas.Motheds:The MMP-2/9 level of the supernatants of primary cultured human pituitary adenoma cells was detectd by ELISA after stimulated by different collage subtypes. Autophosphorylation of DDR1 was evaluated by immuno-precipitation after blocked by NDDR1 and nilotinib.Gelatine zymography was used to detected MMP-2/9 activity and protein expression after DDR1 signal pathway was stimulated or blocked.An invasion model was established by Transwell chambers and cell invasion was observed with microscope after intervention by NDDR1 and nilotinib.Results:Nine cases of functional pituitary adenomas primary cultured cells were obtaind successully.1.CollagenⅠwas the main collagen subtypes that induces the signal pathway of primary cultures cell of pituitary adenomas.2.Autophosphorylation of DDR1 was significant higher after stimulated by collagenⅠ(P＜0.05),NDDR1 and nilotinib could inhibit autophosphorylation of DDR1.3.CollagenⅠstimulation increased the activity and protein expression of MMP-2/9(P＜0.05) and this effection could be blocked by NDDR1 and nilotinb.4.Invasiveness of primary cultured cells decreased after intervention by NDDR1 and nilotinib.Conclusions:CollagenⅠcould increase the invasiveness of pituitary adenomas by inducing autophosphorylation of DDR1 and up-regulating expression of MMP-2/9. blocking DDR1 could decrease the invasiveness of adenomas efficiently.Our results demonstrated that activation of circuit loop of collagenⅠ-DDR1-MMP-2/9 maybe an important factor and mechanism of action in cavernous sinus invasion by pituitary adenomas.Prominent deposition of collagenⅠin cavernous sinus could be one of pathology foundation in cavernous sinus invasion by pituirary adenomas.更多还原