【作者】 赵锐；

【导师】 徐克森；

【作者基本信息】 山东大学 ， 外科学， 2010， 博士

【摘要】 研究背景和意义恶性肿瘤的侵袭和转移是一个多步骤、多阶段的复杂过程。癌变的细胞和组织会发生一系列的变化,包括细胞转化、肿瘤生长、血管及淋巴管的诱导生成及癌细胞扩散到循环系统；然后在靶器官中粘附、侵袭和增殖。目前已经有数种细胞因子被证实参与肿瘤的转移；其中,血管内皮生长因子(vascular endothelial growth factor, VEGF)不仅参与肿瘤血管的生成,还参与伤口愈合及其他重要的病理过程。VEGF通过两种机制诱导肿瘤血管的生成,一是主要通过其同源性受体VEGFR2,二是通过非VEGFR途径,其中包括整合素的上调和(或)激活。整合素是一种异二聚体跨膜蛋白,通过VEGF及其受体的协调,能够诱导细胞粘附于细胞外基质(extracellular matrix,ECM)。整合素αvβ6在正常的上皮细胞几乎不表达,而在生长中的胚胎组织及许多上皮性恶性肿瘤中高表达。前期研究中,我们已经证实了整合素αvβ6可用来作为胃癌预后的指标之一。另外αvβ6在结肠癌中的表达可促进基质金属蛋白-9(matrix metalloprotein-9,MMP-9)分泌的增加。在胃癌组织中,MMP-9可降解细胞外基质,从而促进肿瘤细胞的播散和转移。由此可见,αvβ6通过MMP-9降解ECM屏障和介导肿瘤细胞的侵袭性。有研究证实整合素与VEGF在细胞的粘附、侵袭和转移过程中具有协同作用。整合素α1β1和α2β1可促进VEGF介导的信号转导和内皮细胞的侵袭作用。VEGF与整合素共同参与多发性骨髓瘤细胞的侵袭。在结肠癌细胞中,α9β1阻断抗体可以特异性的抑制VEGF-A诱导的血管生成。然而,整合素αvβ6在VEGF介导的肿瘤细胞侵袭转移过程中的作用尚未明确。VEGF-C是VEGF家族的新成员,是一种酪氨酸激酶受体,其主要表达在内皮组织的淋巴系统内。在一些恶性肿瘤,如：乳腺癌、胃癌以及肺癌中呈高表达,并且有研究证实VEGF-C的表达与肿瘤的侵袭以及淋巴转移密切相关。但是很少研究VEGF-C在胆管癌中的表达以及与胆管癌侵袭转移之间的关系。本次研究的目的是研究整合素αvβ6的表达和VEGF的关系,以及它们在体外对胃癌细胞侵袭性的影响；并且研究VEGF-C在胆管癌侵袭转移过程中所起的作用。第一部分VEGF及整合素αvβ6在胃癌中的表达目的研究VEGF以及整合素αvβ6在胃癌组织及细胞中的表达情况方法收集手术切除的胃癌组织标本,冰冻储存于-80℃低温冰箱中。应用免疫组化的方法检测VEGF以及整合素αvβ6在胃癌组织的表达情况。体外培养胃癌AGS细胞,采用流式细胞技术、免疫荧光以及免疫沉淀检测胃癌细胞中整合素的表达情况。结果30例胃癌标本的免疫组织化学分析显示,其中26例VEGF染色阳性,13例整合素αvβ6染色阳性。其中VEGF以及整合素αvβ6均表达阳性的有10例标本。流式细胞仪检测胃癌AGS细胞中整合素αvβ6呈现高表达,而且阳性率大于90%；细胞免疫荧光结果显示AGS细胞膜中整合素的表达,而应用免疫沉淀技术检查蛋白水平整合素αvβ6的表达情况。结论VEGF以及整合素αvβ6在胃癌组织中均有表达；整合素αvβ6在胃癌细胞系AGS细胞中呈现高表达。第二部分VEGF介导的胃癌细胞侵袭作用与整合素αvβ6相关目的探讨整合素αvβ6在VEGF促进胃癌细胞侵袭、转移能力的作用,寻求控制胃癌转移的靶点。方法体外培养AGS细胞,外源性VEGF(30ng／ml)刺激24h,应用流式细胞仪、Real-time PCR、Western blot以及免疫沉淀检测整合素αvβ6、P-ERK和MMP-9在mRNA以及蛋白水平的表达变化。再应用体外培养的AGS细胞,分别用ERK通路阻断剂U0126以及鼠抗人整合素αvβ6单克隆抗体10D5预处理后,外源性VEGF(30ng/ml)刺激24h,收集细胞并检测整合素αvβ6、P-ERK和MMP-9在mRNA以及蛋白水平的表达变化。针对人整合素基因序列设计其特异性siRNA序列。用荧光标记的阴性对照siRNA筛选转染条件,按照预实验筛选的转染条件将β6 siRNA转染到无血清培养液处理过的AGS细胞中,48h后收集细胞进行抑制效应分析,荧光定量PCR检测β6 mRNA的表达,以无血清培养基及对照siRNA为对照。AGS细胞转染β6 siRNA成功后,采用外源性VEGF刺激,收集细胞检测整合素β6、P-ERK和MMP-9在mRNA以及蛋白水平的表达。趋化小室试验检测外源性VEGF介导的AGS细胞侵袭能力。β6 siRNA转染的AGS细胞,以及分别用ERK通路阻断剂U0126以及鼠抗人整合素αvβ6单克隆抗体10D5预处理的AGS细胞,加入包被好的小室上层,在有无外源性VEGF介导的情况下,观察转移到小室下层的细胞个数,从而反应其不同条件下的侵袭能力。结果应用免疫沉淀、流式细胞仪以及细胞免疫荧光,可以发现体外培养的AGS细胞内有整合素αvβ6的表达,其主要表达在细胞膜,而且表达的阳性率在90%以上。AGS细胞在外源性VEGF(30ng/ml)刺激24h后,整合素αvβ6、P-ERK和MMP-9在mRNA以及蛋白水平的表达明显增加,与对照组相比具有统计学意义。而应用ERK通路阻断剂U0126以及鼠抗人整合素αvβ6单克隆抗体10D5预处理后,外源性VEGF(30ng/ml)刺激24h,整合素αvβ6、P-ERK和MMP-9在mRNA以及蛋白水平的表达显著被抑制,与对照组相比具有统计学意义。荧光显微镜观察见筛选后的转染条件下荧光标记siRNA的转染效率较高,超过90%。在β6 siRNA转染的无血清培养液处理过的AGS细胞中,在有无外源性VEGF作用的条件下,整合素αvβ6、P-ERK和MMP-9在mRNA以及蛋白水平的表达得到相同的结论。趋化小室实验检测到在外源性VEGF介导的情况下,转移至小室下层的AGS细胞数明显增加。而应用ERK通路阻断剂U0126、鼠抗人整合素αvβ6单克隆抗体10D5以及β6 siRNA转染预处理后,转移至小室下层的AGS细胞数显著减少,与对照组相比具有统计学意义。结论胃癌AGS细胞内有整合素αvβ6的表达；外源性VEGF (30ng/ml)作用于AGS细胞后,可以刺激整合素αvβ6、P-ERK和MMP-9在mRNA以及蛋白水平的表达显著增加以及提高AGS细胞的侵袭能力,而且这种作用可以被ERK通路阻断剂U0126、鼠抗人整合素αvβ6单克隆抗体10D5以及β6 siRNA转染预处理显著抑制,与对照组相比具有统计学意义。这说明整合素αvβ6参与了VEGF介导的胃癌侵袭转移作用,并且ERK通路在此过程中起着一定的作用。第三部分VEGF-C在胆管癌中的表达及侵袭转移中的作用研究目的研究VEGF-C在胆管癌癌组织中的表达情况及促进胆管癌癌细胞侵袭、转移能力的作用。方法收集手术切除的胆管癌组织标本,冰冻储存于-80℃低温冰箱中。应用免疫组化的方法检测VEGF-C在胆管癌组织的表达情况,回顾性分析这些病人的临床病例资料,并与免疫组化结果相结合,分析VEGF-C的表达与患者临床资料之间的关系；体外培养胆管癌FRH-0201细胞,并且在有无外源性VEGF-C作用的情况下,采用增殖实验以及趋化小室实验检测胆管癌细胞FRH-0201的侵袭转移能力。结果VEGF-C在胆管癌组织中呈高表达,表达率约为75.4%；其表达与病人的年龄、性别以及肿瘤大小、肿瘤的临床分期无明显关系,而与肿瘤的淋巴结转移明显相关。胆管癌细胞在外源性VEGF-C的作用的情况下,胆管癌细胞的增殖以及侵袭能力明显被增强,与对照组相比具有统计学意义。结论VEGF-C在胆管癌组织中呈高表达并且在其侵袭转移过程中起着一定的作用。更多还原

【Abstract】 Background and significationTumor metastasis is a complex and multi-stage process. A number of cellular and tissue changes occur as a cancer spreads, including cellular transformation and tumor growth, angiogenesis and lymphangiogenesis, and release of cancer cells into the circulation; then attachment, invasion, and proliferation within the new target organ. Several cytokines are now known to be involved in the process of tumor metastasis. Among these is vascular endothelial growth factor (VEGF), a factor that is associated not only with the angiogenesis of cancers but also with those involved in wound healing and other important pathologies. There are two mechanisms involved in VEGF-mediated angiogenesis; one is through its cognate receptor VEGFR2, the other is via up-regulation of the expression and/or activation of integrins.Integrins are heterodimeric transmembrane proteins that can induce cell adhesion to the extracellular matrix (ECM), coordinated by VEGF and its receptors. The avP6 integrin is restricted to epithelial cells and is highly expressed in developing fetal tissues as well as in a number of epithelial carcinomas and cancers. In our previous studies, we demonstrated that integrin avP6 can be used as a prognostic indicator of gastric carcinoma. In addition,αvβ6 expression in colon cancer cells leads to the increase in secretion of matrix metalloprotein-9 (MMP-9). In gastric cancer, degradation of ECM barriers by MMP-9 is important in facilitating tumor cellular dissemination and metastasis. Thus, integrinαvβ6 appears to have roles in promoting ECM degradation and in mediating tumor cell invasiveness via MMP-9.There are several confirmed collaborative relationships between integrins and VEGF in mediation of cell adhesion, migration, and proliferation. Theα1β1 andα2β2 integrins support VEGF-stimulated signal transduction and endothelial cell migration. Migration of multiple myeloma cells is induced by VEGF and associated withβ1 integrin. In colon cancer cells,α9β1-blocking antibody has been demonstrated to specifically inhibit the angiogenesis induced by VEGF-A. However, a role for integrinαvβ6 in VEGF-induced tumor progression has not yet been established.VEGF-C is a new member of the VEGF family, a tyrosine kinase receptor that is predominantly expressed in the endothelium of lymphatic vessels. The expression of VEGF-C is high in several types of human malignant tumors, including breast, lung, and gastric carcinomas and some investigators have shown that VEGF-C expression was closely associated with tumor invasion and lymph node metastasis. However, there have been very few reports on the correlation between VEGF-C gene expression and invasive phenotype in cholangiocarcinoma.The goal of the present study was therefore to investigate the relationship between VEGF and expression of integrinαvβ6 and their effects on invasiveness of gastric carcinoma cells in vitro. And the effect of VEGF-C in invasiveness and metastasis of cholangiocarcinoma.PARTⅠExpression of VEGF and integrinαvβ6 in gastric carcinoma tissues and cells Objective To study the expression of VEGF and integrinαvβ6 in gastric carcinoma tissues and cells.Methods Thirty gastric cancer tissues were collected from patients requiring clinically indicated surgical resection in QiLu Hospital of Shandong University (Jinan, China), with informed patient consent. Frozen gastric cancer tissue samples were stored at -80℃until processing. Immunohistochemical analysis was used to detect the expression of VEGF and integrinαvβ6 in gastric carcinoma tissues. The human gastric cancer cell line AGS was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin, 10μg/ml streptomycin, and 2mM L-glutamine. Immuno-precipitation, FAC scanning and immunofluorescence staining were used to confirme the presence ofαvβ6 in gastric cancer AGS cells.Results Immunohistochemical analysis of tumor samples from 30 patients revealed that 26/30 samples stained positively for VEGF and 13/30 samples stained positively for integrinαvβ6. In 10/30 patients tumors were positive for both VEGF and integrinαvβ6. High surface expression ofαvβ6 integrin was detected by flow cytometry analysis, and the positive rate was higher than 90%. Expression ofαvβ6 was detected with immunofluorescent staining at the cell membrane in AGS cells. AGS cells were examined by immunoprecipitation analysis using antibody of 10D5 for expression ofαvβ6 integrin at protein levels.Conclusions VEGF and integrinαvβ6 are all expressed in gastric carcinoma tissues. And the high surface expression ofαvβ6 integrin is detected in gastric cancer AGS cells. PARTⅡVEGF enhances gastric carcinoma invasiveness via integrinαvβ6Objective To investigate the effect of integrinαvβ6 on VEGF regulated invasiveness and metastasis in gastric carcinoma cells, to probe into the key point of regulating metastasis of gastric carcinoma.Methods The AGS cells were starved for 4h in medium without fetal bovine serum and then stimulated with VEGF (30ng/ml) for 24h at 37℃. The expression at both mRNA and protein levels was also observed for integrinαvβ6, P-ERK and MMP-9 by using the real-time PCR, western blot analysis and immunoprecipitation.The roles ofαvβ6, P-ERK and MMP-9 at both mRNA and protein levels were detected by using monoclonal antibody for the humanαvβ6 integrin (10D5) and an inhibitor of the ERK signaling pathway (U0126).Specific P6 siRNA was designed according to the cDNA sequence of integrin subunit beta6 and the BLAST search was used to avoid unintentional silencing and ensure the uniqueness of target. Cells were grown subconfluently onto 24-well plates and transfected with 1μg ofβ6 siRNA, with nonspecific siRNA as control. After 48h of transfection, cells were washed and analyzed further as specifically indicated. The VEGF-stimulated transfected AGS cells were detected to calculate the change of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels.Migration of AGS cells through Matrigel-coated filters was examined in 24-well transwell inserts. After trypsinization, 1×105 cells in 100μl were incubated with 10D5 or U0126 for 1h and then were placed in the upper wells. Alternatively,β6 siRNA-treated cells were directly added to the transwell inserts. VEGF was added to the lower wells in a final volume of 1 ml. After 4 hours of incubation, while non-migrating cells were scraped away gently and cells on the lower surface were fixed and stained with Crystal Violet. The migrating cells were counted using a microscope.Results By using immunoprecipitation analysis, flow cytometry analysis and immunofluorescent staining, integrinαvβ6 was detected at the cell membrane in AGS cells and the positive rate was higher than 90%. AGS cells were treated with VEGF (30ng/ml) after separately pretreatment withβ6 siRNA, with an inhibitor of the ERK signaling pathway (U0126), or with a neutralizing antibody toβ6 (10D5). Expression of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels was measured by means of a real time PCR assay and immunoprecipitation analysis. A significant difference were observed (P<0.05) between VEGF-stimulated and un-stimulated cells. VEGF-induced expression of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels was significantly inhibited byβ6 siRNA, U0126, and 10D5 (P<0.05). Inhibition of the VEGF-induced migration of AGS. AGS cells were separately pretreated withβ6 siRNA, U0126, or 10D5 and were examined for their ability to migrate through a Matrigel filter in the absence/presence of VEGF (30ng/ml). Migration of AGS cells without VEGF or with VEGF stimulation was significantly inhibited byβ6 siRNA, U0126, and 10D5 (P<0.05).Conclusions Expression ofαvβ6 is detected at the cell membrane in AGS cells. The expression of integrinαvβ6, P-ERK and MMP-9 at both mRNA and protein levels and the invasiveness of AGS cells are stimulated by VEGF, but these effects are significantly inhibited by P6 siRNA, U0126, and 10D5 (P<0.05). These data suggest that VEGF is critical to the invasive process in human gastric cancer and that this occurs via up-regulation of integrin avP6 expression and activation of ERK.PARTⅢVEGF-C Expression and invasiveness in cholangiocarcinomaObjective To investigate the expression of VEGF-C in cholangiocarcinoma tissues and the VEGF-C induced invasiveness and metastasis in cholangiocarcinoma cells.Methods The cholangiocarcinoma tissues were collected from patients requiring clinically indicated surgical resection in QiLu Hospital of Shandong University (Jinan, China), with informed patient consent. Frozen cholangiocarcinoma tissue samples were stored at -80℃until processing. Immunohistochemical analysis was used to detect the expression of VEGF-C in cholangiocarcinoma tissues. And the relationships between the expression of VEGF-C and the clinical-pathological features of cholangiocarcinoma tissues were investigated. The human cholangiocarcinoma cell line FRH-0201 was maintained in RPMI 1640 medium. And the proliferation and invasiveness of FRH-0201 cells were detected by the MTT and Transwell experiment in the absence/presence of VEGF-C.Results The expression of VEGF-C was detected in 78.2% of cholangio-carcinoma tissues, and the expression was not associated with age, sex and the size of tumor, but associated with lymphatic invasion of the tumors. The proliferation and invasiveness of tumor cells were significantly promoted by VEGF-C (P<0.05).Conclusions Positive VEGF-C expression in cholangiocarcinoma is linked to significantly enhanced invasiveness, and its value as a prognostic marker is significant for early stage tumors.更多还原