【作者】 杨广运；

【导师】 徐克森； 牛军；

【作者基本信息】 山东大学 ， 外科学， 2008， 博士

【摘要】 研究背景和意义结肠癌为目前常见的影响人类健康的恶性肿瘤之一。癌细胞侵袭浸润性生长和远隔器官转移是恶性肿瘤区别于良性肿瘤的重要生物学特征,也是影响结肠癌预后的关键因素,如何抑制癌肿侵袭浸润性生长以及如何控制结肠癌肝脏转移是目前癌症研究中最具有挑战性的关键课题。大量研究资料证明,结肠癌的发生发展过程与细胞表面的生物学特性密切相关,是受细胞粘附分子和基质降解酶调控的,但是这些蛋白分子是如何在结肠癌肝脏转移过程中发挥作用,如何调控结肠癌侵袭性生长,我们却知之甚少。在癌细胞表面表达的粘附分子家族中,整合素家族是最具有特征性的,特别是整合素αvβ6在结肠癌细胞的表达模式与结肠癌的发生发展密切相关。整合素αvβ6在健康成人上皮细胞不表达或极少表达,但是在肿瘤发生发展过程中却诱导高表达。文献报道:在口腔白斑病整合素αvβ6诱导表达而且是恶性变为鳞状细胞癌的必须的前提条件;在口腔鳞状细胞癌和结肠癌也观察到整合素αvβ6的新生表达。前期研究证明:无论在体内还是在体外,整合素αvβ6促进了结肠癌细胞的增殖和生长。此外,αvβ6可以诱导基质金属蛋白酶-9(MMP-9)的分泌,通过提高对细胞周围基质的降解,增强癌细胞的浸润迁徙能力。不难想象,癌细胞能克服和逃避正常细胞拥挤和密度积压障碍,而无限制的侵袭性生长和远处转移,可能是通过癌细胞的诱导表达αvβ6而介导MMP-9分泌实现的。本实验的研究目的是探讨αvβ6在调控结肠癌肝脏转移和侵袭性生长方面的作用机制,为进行有关结肠癌的进一步研究提供全新的思路和理论基础,有理由相信特异性针对整合素αvβ6的靶向治疗策略,对进展期结肠癌病人的治疗将起到重要的作用。第一部分整合素αvβ6调控结肠癌细胞肝脏转移和肝脏定植潜能的实验研究目的结肠癌肝脏转移是结肠癌特征性的恶性行为之一,整合素αvβ6与结肠癌的发生发展密切相关,然而,整合素αvβ6在结肠癌细胞表面的表达程度与结肠癌肝脏转移之间的关系我们知之甚少。本研究的目的是探讨整合素αvβ6与结肠癌细胞肝脏转移和肝脏定植潜能的关系。材料与方法1.免疫组织化学染色是本实验检测整合素αvβ6在结肠癌组织和肝脏转移癌组织表达水平的重要方法,为了验证免疫组化结果的特异性,首先选择不同的结肠癌细胞系(WiDr细胞、SW480 wild细胞、SW480β6转染细胞)采用免疫沉淀分析检测单克隆抗体2G2(美国Biogen公司)的特异性。2.随机选择63例的结肠癌肝脏转移病人(1995年1月至2005年1月经过手术治疗)的肝脏转移癌标本,用抗αvβ6的单克隆抗体2G2作为一抗,采用Elivision法进行免疫组织化学染色。然后从2001年1月至2002年12月经手术治疗的结肠癌病人中,随机选取结肠癌组织标本358例,构建成2张200点的组织芯片(在陕西超英生物公司制作),采用ABC法进行αvβ6免疫组化染色,根据免疫组化结果(αvβ6阳性或者αvβ6阴性),将手术后病人分为两组,进行3年手术后肝脏转移随访。3.为了探讨在进展期结肠癌中整合素αvβ6免疫反应性与癌细胞在肝脏定植和肝脏转移的关系,我们选用了肝脏定植实验(肝脏组织内直接注射结肠癌WiDr转染细胞)和肝脏转移成瘤实验(经脾脏注射结肠癌HT29转染细胞)。4.为了探讨整合素αvβ6调节结肠癌肝脏转移的机制,分别用明胶酶谱法和Biotrak MMP-9活性分析系统检测人工培养的结肠癌细胞(WiDr和HT29转染细胞)的基质金属蛋白酶-9(MMP-9)的分泌水平;同时采用Transwell小室侵袭实验检测不同结肠癌细胞(HT29和WiDr转染细胞)的侵袭转移能力。结果1.免疫沉淀分析显示:不同的结肠癌细胞(WiDr细胞、SW480 wild细胞,SW480β6转染细胞)表面的整合素αvβ6受体特异性的被单克隆抗体2G2(美国Biogen公司)识别。2.免疫组化结果显示:63例肝脏转移癌组织的αvβ6阳性表达率是71.4%(45/63),明显高于结肠癌组织(358例)的αvβ6阳性表达率(34.1%,122/358)(p＜0.01)。3年随访的结果显示αvβ6阳性结肠癌患者的肝脏转移率(17%,21/122)明显高于αvβ6阴性结肠癌患者的肝脏转移率(3%,7/236(p＜0.01)。3.肝脏定植试验(肝脏组织内直接注射结肠癌WiDr转染细胞)显示:55%(11 of 20)裸鼠注射结肠癌WiDr细胞(表达αvβ6)6周后肝脏有癌瘤形成。肝脏转移成瘤试验(经脾脏注射结肠癌HT29转染细胞)显示:90%(18 of 20)裸鼠注射结肠癌HT29转染细胞(表达αvβ6)4周后肝脏都观察到转移癌形成。而采用上述两种实验方法,经肝脏注入反义β6转染的WiDr A/Sense细胞,或者经脾脏注入反义β6转染HT29 A/Sense细胞,肝脏都没有肿瘤形成。动物实验进一步证明αvβ6有助于结肠癌细胞在肝脏新环境的定植存活能力,有助于提高结肠癌细胞的肝脏转移能力。4.明胶酶谱法和Biotrak MMP-9活性分析仪检测显示,WiDr细胞和HT29细胞在人工培养状态下随着αvβ6免疫反应性的下调,MMP-9分泌水平和活性水平明降低(p＜0.01)。体外Transwell小室侵袭实验也表明:表达αvβ6的WiDr细胞和HT29细胞在纤维连接蛋白基质上的趋化迁移能力较反义β6转染的WiDr A/Sense和HT29 A/Sense细胞明显增强(p＜0.01)。结论1.抗αvβ6单克隆抗体2G2特异性的识别结肠癌细胞表面的整合素αvβ6受体。2.结肠癌肝脏转移组织比结肠癌组织具有较高的αvβ6阳性表达率,整合素αvβ6阳性表达的结肠癌患者比αvβ6阴性患者具有较高的肝脏转移率。说明结肠癌细胞的αvβ6的免疫反应性与结肠癌肝脏转移密切相关。3.结肠癌肝脏转移的转归取决于结肠癌细胞表面的生物学特性及肝脏宿主器官微环境。整合素αvβ6有助于结肠癌细胞在肝脏的定植存活,有助于增强癌细胞的肝脏转移能力。4.整合素αvβ6能促进MMP-9分泌,加快细胞外基质降解;整合素αvβ6能提高结肠癌细胞在纤维连接蛋白基质上的趋化迁移能力;二者构成了结肠癌肝脏转移的先决条件。根据本实验研究的结论,特异性的针对抑制癌细胞αvβ6表达的策略可以降低结肠癌细胞的肝脏定植和肝脏转移能力,为最终控制结肠癌肝脏转移提供了全新的理论依据。第二部分整合素αvβ6调控结肠癌细胞侵袭性生长机制的实验研究目的结肠癌细胞的侵袭性生长是区别于结肠良性肿瘤的重要特征,也是影响预后的关键因素。本实验的目的是研究结肠癌在细胞高密度和细胞挤压状态对结肠癌细胞表面整合素αvβ6表达的影响效果,探讨αvβ6对结肠癌细胞分泌基质金属蛋白酶-9(MMP-9)的影响,以及αvβ6对癌细胞侵袭性生长的影响,进一步揭示结肠癌细胞永生化侵袭性生长的分子机制。材料与方法1.用流式细胞仪检测结肠癌细胞系WiDr和SW480细胞株以及正常人角质细胞系HaCaT细胞株,分别在高、低细胞密度培养条件下各细胞表面的αvβ6表达水平。2.将100例结肠癌标本的肿瘤边缘部分(癌细胞密集相对高细胞密度)及肿瘤中心部分(癌细胞稀疏相对低细胞密度)制作成200点的组织芯片(陕西超英生物),用免疫组织化学染色方法检测分析整合素αvβ6在结肠癌不同部位的分布差异和表达强度区别。3.用Biotrak MMP-9分析系统和明胶酶谱法分别测定不同的结肠癌WiDr细胞株和SW480细胞株,以及正常人角质细胞系HaCaT细胞株,在高、低细胞密度培养条件下,MMP-9的活性和分泌水平。结果1.结肠癌细胞WiDr(表达αvβ6)出现细胞密度依赖的αvβ6表达增加,而结肠癌细胞SW480(缺乏αvβ6表达)和正常人角质细胞系HaCaT细胞(良性上皮细胞),在高、低细胞密度培养条件下αvβ6表达没有改变。2.组织芯片免疫组化结果,结肠癌αvβ6阳性表达率为38%(38/100);在肿瘤边缘部分(癌细胞密集、相对高细胞密度区域),αvβ6阳性高表达率是73.7%(28/38),且αvβ6密布于癌肿生长侵袭的边缘;而癌细胞相对稀疏的肿瘤中央区域组织,以αvβ6低表达为主,高表达率只有28.9%(11/38)。3.Biotrak MMP-9活性检测分析显示,表达αvβ6的WiDr细胞和SW480 p6转染细胞的MMP-9分泌水平,在高密度培养条件下比低密度培养明显增加(p＜0.01),而缺乏αvβ6表达的SW480 wild细胞,没有细胞密度依赖的增加。明胶酶谱分析也显示SW480β6转染细胞的MMP-9分泌水平,在高密度培养条件下比低密度培养明显增加,而SW480 wild(缺乏αvβ6表达)和HaCaT细胞(良性上皮细胞)的MMP-9分泌水平没有增加。结论1.细胞高密度和细胞挤压状态诱导癌细胞αvβ6表达,是结肠癌侵袭性生长的启动子。2.在结肠癌细胞,整合素αvβ6的表达呈密度依赖性增加的机制,与正常人上皮细胞不同。在高细胞密度培养条件下,结肠癌细胞MMP-9分泌量的增加也是由整合素αvβ6调控的,特异性的封闭整合素αvβ6受体可以减少癌细胞的MMP-9分泌水平。结肠癌细胞MMP-9的分泌量呈密度依赖性增加的机制,对人正常细胞也是无效的。基于这一全新的理论,可以研发特异性的针对癌细胞高效的而对正常细胞无毒的抗癌药物,可以通过特异性的抑制αvβ6表达,达到控制结肠癌永生化侵袭性生长的目的。3.细胞高密度诱导结肠癌细胞αvβ6持续表达,促进MMP-9的分泌,加速细胞外基质的降解,是构成结肠癌细胞永生化侵袭性生长的分子生物学基础。更多还原

【Abstract】 Backgroud and significationColon cancer is one of the commonest malignant tumors affecting men and women together in our society.Invasion growth and distant metastasis are two key factors distinguishing from the benign tumors,which affect the prognosis of patients with colon cancer.How to restrain tumors invasive growth and how to prevent colonic metastasis to liver in patients with colon carcinoma remains one of the most challenging issues in cancer research.Colon cancer progression is closely correlated with the biological feature of cell surface,is thought to be regulated,at least in part,by cell adhesion molecules and matrix-degrading enzymes.Relatively little is known about how these factors might act to enhance invasive growth of colon cancer and mediate the process of colonic metastasis to liver in patients with colon carcinoma.Of all the families of cell adhesion molecules expressed on the surface of cancer cells,the family known as integrins has been best characterized.Integrinαvβ6 expression patterns appear to be directly implicated in the progression of colon cancer in particular.Theαvβ6 integrin is either not expressed or expressed at low levels in normal adult epithelia;however,it becomes highly expressed during tumourigenesis.For example,induction ofαvβ6 expression in oral leukoplakia appears to be a necessary prerequisite for progression to squamous cell cancer and de novo expression ofαvβ6 has been observed in oral squamous and colon cancers. Leading up to this series research,αvβ6 had been reported to enhance proliferation and growth of colon cancer cell in vitro and vivo.In addition,αvβ6 had also been shown to induce secretion of matrix metalloproteinase-9(MMP-9),a matrix-degrading enzymes,in these cell types and enhance the colon cancer cells migration by degeneration of surrounding matrix.This indicated that cancer cells might escape the growth constraints imposed on normal cells when crowded together was by autocrine up-regulation of theαvβ6-mediating MMP-9 secretion.In this study,the major aim of the study was to explore some of the possible mechanisms by which integrinαvβ6 mediates colonic metastasis to liver and invasive growth in colon cancer progression,which provides a new idea and basis for further study.Strategies targeting the integrinαvβ6 can play key roles in the management of patients with advanced colon cancer in the near future.PARTⅠIntegrinαvβ6 mediates the potential for colon cancer cells to colonize in and metastasize to the liverObjectiveColon cell carcinoma is characterized by the propensity for distant metastasis to liver.How to prevent colonic metastasis to liver in patients with colon carcinoma remains one of the most challenging issues in cancer research.Integrin alphavbeta6(αvβ6)is closely correlated with colon cancer progression.However, the link betweenαvβ6 expression in primary colon cancer and colonic metastasis to liver in patients with colon carcinoma remains unknown.Our experimental aims were to detect the effects of integrinαvβ6 on colonization in and colonic metastasis to liver in colon cancer progression.Materials and Methods1.Immunohistochemistry is the most important methodology to detect the effects of integrinαvβ6 expression on both colon cancer and liver metastatic carcinoma specimen in this experimental study.To demonstrate the specificity of the immunohistochemical results,the specificity of againstαvβ6 monoclonal antibody 2G2(obtained from Biogen Idec.)was examined with various colon cancer cells(WiDr cells,SW480 wild cells,SW480β6 transfectants)by immunoprecipitation assay.2.Integrinαvβ6-immunoreactivity(IR)in liver metastasis tissues(63 cases of hepatic metastatic specimens obtained randomly from Jan.1995 to Jan.2005) was performed with Elivision method.Then 358 cases of colon carcinoma specimens obtained randomly from Jan.2001 to Dec.2002 were constructed into two tissue microarrays(200 points / each,constructed by Xi’an Chaoying Biochip Company,Ltd.).Integrinαvβ6 immunoreactivity(IR)was examined according to the Avidin-Biotin Complex protocol.Two groups were divided according to the immunohistochemical results(αvβ6-IR positive orαvβ6-IR negative)to investigate postoperative liver metastases for 3 years.3.To confirm the effect of integrinαvβ6 on enhanced liver colonization and metastatic potential for colon cancer cells in progression,we performed in vivo studies using the liver colonization model(directly liver injection for WiDr cells), and the liver metastatic model(intrasplenic injection for HT29 cell lines).4.To examine the underlying mechanisms associated withαvβ6 regulating colonic metastasis to liver,matrix metalloproteinase-9(MMP-9)levels in the cultures of both HT29 and WiDr transfectants were detected by the Biotrak MMP-9 activity assay system and gelatin zymography assay.The migration capability on fibronectin(FN)for HT29 / WiDr transfectants was examined also by Transwell migration assay in vitro.Results1.The immunoprecipitation assay of various colon cancer cells(WiDr cells, SW480 wild cells,SW480β6 transfectants)for the monoclonal antibody 2G2 showed that integrin avβ6 was specifically recognized by the anti-β6 antibody 2G2.2.Integrinαvβ6 positive rate in liver metastatic tissues(71.4%,45/63)was higher than that in primary colon cancer(34.0%,122/358)(p＜0.01).The results of 3-year follow up for postoperative liver metastasis showed that liver metastasis rates(17%,21/122)for patients withαvβ6 positive was higher than those withαvβ6 negative(only 3%,7/236)(p＜0.01).3.Liver colonization assay(directly liver injection of WiDr transfectants)in nude mice showed that 55%(11 of 20)of the mice injected with WiDr mock transfectants expressingαvβ6,six weeks later,developed liver tumors,and experimental liver metastasis assay in nude mice(intrasplenic injection of HT29 transfectants)showed that 90%(18of 20)of the mice injected intrasplenically with HT29 mock transfectants,four weeks later,developed visible liver metastatic tumors.However,none of visible tumors in liver was observed in the mice injected with either WiDr antisenseβ6 or HT29 antisenseβ6 transfectants.These results demonstrated thatαvβ6 contributed to promote metastatic potential and survive in liver environment in colon cancer progression.4.Matrix metalloproteinase-9(MMP-9)levels in the cultures of both HT29 and WiDr cells were detected by the Biotrak MMP-9 activity assay system and gelatin zymography assay,showed that suppression ofαvβ6-IR inhibited MMP-9 activity and secretion(p＜0.01).Transwell migration assay in vitro also showed thatαvβ6 promoted migration on fibronectin(FN)for HT29 / WiDr mock compared with HT29 / WiDr antisenseβ6 transfects(p＜0.01),respectively.Conclusions1.Integrin avβ6 in colon cancer cell surface was specifically recognized by the anti- avβ6 monoclone antibody 2G2.2.There is relatively higherαvβ6 positive rate in liver metastatic tissues than in colon cancer tissues,and there is relatively higher liver metastasis rates for patients withαvβ6 positive compared with those withαvβ6 negative,which indicate that integrinαvβ6 is closely correlated with colonic metastasis to liver in progression.3.The progression of liver metastasis was dependent on the biological features of colon cancer cell surface and the microenvironment of targeting organ(liver). Integrinαvβ6 contributed to survive in liver environment and promote liver metastasis for colon cancer cells in progression.4.Integrinαvβ6 promoted secretion of MMP-9 resulting in extracellular matrix(ECM)degradation,and enhanced migration on fibronectin(FN),which are two prerequisite conditions for colon cancer cells of liver metastasis in progression.Based on this view,strategies by targeting to inhibit the avβ6-IR in colon cancer cells could,at least partly,prevent the potential for colon cancer cells of liver colonization and liver metastasis,which provides the novel theory and basis to control the process of colonic metastasis to liver in progression.PARTⅡIntegrinαvβ6 mediates tumor invasive growth in colon cancer progressionObjectiveInvasive growth is one of the most important features distinguishing from the benign tumors and the key factors affecting the prognosis of patients with colon cancer.Our experimental aims were to detect the effects of cell density on both integrinαvβ6 expression and matrix metalloproteinase-9(MMP-9)secretion in colon cancer cells,and the mechanism by which cancer cells sustain invasive growth via a self-perpetuating manner in colon cancer progression.Methods1.Flow cytometry was applied to analyze avβ6 expression in human colon cancer line,WiDr,SW480 cells and the normal human keratinocyte cell line, HaCaT cells,respectively,at low and high cell density culture.2.To detect the disparity of both distribution and intense for integrin avβ6 expression,an immunohistochemical study of integrin avβ6 was performed on tissue microarrays(TMAs)of 200 points(including 100 cases malignant colon specimens).Two cores were taken from each sample,one obtained from invasive tumor portions(tumor margin,especially cell crowding margin at high cell density);the other obtained from non-invasive portions(centre tumor foci at low cell density).3.The MMP-9 activity levels for various colon cancer lines,WiDr cells,SW480 cells,and the normal human keratinocyte cell line,HaCaT cells,respectively,at high- and low- cell density culture were analyzed using Biotrak MMP-9 activity assay system and gelatin zymography assay system.Results1.High cell density evidently enhances integrin avβ6 expression only for WiDr cells expressing avβ6 compared with low density,but no increase was observed for both SW480 cells(lack avβ6 expression)and HaCaT cells(benign epithelial cells).2.The avβ6 expression rate on immunoreactivity(IR)in overall was 38%(38 in 100 cases).High positive expression for avβ6 on immunoreactivity was observed in invasive tumor edged portions(relatively cell crowding margin and high cell density)in 73.7%(28/38)cases,particularly,preferential localization at the edges of infiltrating tumor edge.In non-invasive centre tumor portions (relatively cell loose and low cell density),low avβ6 positive expressive was frequently observed and high avβ6 expressive only seen in 28.9%(11/38)cases.3.Biotrak MMP-9 activity assay indicated that the amounts of MMP-9 secreted per cell were evidently higher for WiDr and SW480β6 cells at high cell density compared with at low cell density(p＜0.01),but no density-dependent increase observed for SW480 wild cells which lack avβ6(p＞0.05).Gelatin zymography assay also indicated that the levels of MMP-9 secreted for SW480β6 cells which expressing avβ6 was evidently higher at high density than at low density,however no density-dependent increase observed for both SW480 cells (lack avβ6 expression)and HaCaT cells(benign epithelial cells).Conclusions1.Cell crowding and dense induces integrin avβ6 expressed in colon cancer cells,which is the promoter of tumor invasive growth in colon cancer progression.2.The characterization of the role ofαvβ6 in colon cancer progression (integrinαvβ6 expressed in colon cancer cells was in a cell density-dependent way) was profoundly different from normal cells.The MMP-9 secretion increased for colon cancer cells in the high cell density culture was mediated inαvβ6 independent way.Blockage ofαvβ6 expressed in colon cancer cells can inhibit MMP-9 secretion.The mechanisms by which MMP-9 secretion increased for colon cancer cells in a cell density-dependent way was also invalid for normal cells.Based on this view,strategies by targeting to inhibit the avβ6 expressed in colon cancer cells,specifically,at least partly,prevent invasive growth for colon cancer in a self-perpetuating way,which provides the novel theory and basis to develop high-performance and asepsis anticancer medicine.3.High cell density induces integrin avβ6 expression,promotes MMP-9 secretion for colon cancer cells,increases the degradation of extracellular matrix (ECM),which constitutes the molecular biological basis for a self-perpetuating system of tumor invasive growth in colon cancer progression.更多还原