【作者】 肖科；

【导师】 汤恢焕；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 背景：胆管癌(cholangiocarcinoma)是较为少见的消化道恶性肿瘤,约占人类全部消化道恶性肿瘤病例总数的2～3%左右。最早由Durand Fardel于1840年报道,其来源于胆管上皮细胞,在肝脏及胆道系统的恶性肿瘤中较为常见,约占肝胆系统肿瘤的10%～15%左右。在世界范围内其发病率约为1.2/10万,但近十年来胆管癌的发病率有逐年升高的趋势,在我国其发病率以每年递增5%的速度上升,是消化道肿瘤中上升速度最快的肿瘤。尽管近年来外科手术、化疗、放疗、免疫治疗等在胆管癌的治疗上取得了长足的进步,但由于胆管癌特殊的病理特征和病灶特殊的解剖位置等诸多原因,导致胆管癌患者缺乏早期特异性临床表现及有效的早期诊断手段,当患者出现症状就诊时多数已处于中晚期,难以获得根治性切除,这使得手术后患者3年生存率仅为35%至50%,而5年生存率仅有不到10%,在我国仅有5%左右。因其缺乏有效的综合治疗手段,故诊治水平至今未取得突破性的进展,严重危害了我国人民的健康。因此,研究胆管癌发生、发展、侵袭转移的具体机制,了解胆管癌进展过程中可能的分子步骤,寻求胆管癌恶性转化的靶点及胆管癌的靶向治疗已成为当前研究的热点。血管内皮生长因子(vascular endothelial growth factor, VEGF)参与血管淋巴管生成的各个环节,是目前公认最有效也是最重要的一种促血管生长的特异性因子。在众多肿瘤的发生、发展过程中,VEGF都扮演极为重要的角色,而且是肿瘤血管形成最为成重要的始动因子之一。MMP-2基因是基质金属蛋白酶(matrix metalloproteinase, MMPs)家族中最主要也是分布最广的成员,其主要作用是降解细胞外基质,破坏肿瘤细胞侵袭过程中的组织学屏障,进而导致癌细胞突破细胞外基质及基底膜屏障,向临近纤维结缔组织浸润并发生远处转移。在肿瘤细胞的侵袭转移上发挥重要功能。RNAi是一种典型的转录后基因调控方法,因其作用机制为细胞内通过mRNA特异基因序列降解而使目的基因沉默(gene silencing),故具有极高的靶向性和有效性。目前RNAi技术己经被广泛应用于基因表达转录后调控、基因功能鉴定等热门研究领域,并在许多疾病的基因治疗上显示了巨大的应用前景。目的：第一部分,通过分别检测’VEGF-A、VEGF-C和MMP-2在胆管癌组织标本中的表达情况,分析VEGF-A、VEGF-C和MMP-2表达与胆管癌临床病理因素、临床预后的相关性。进一步检测胆管癌组织标本中微血管密度(microvascular density, MVD)和微淋巴管密度(micro lymphatic vessel density, MLVD),以此来说明VEGF-A、 VEGF-C和MMP-2蛋白表达在胆管癌的发生、发展和侵袭转移过程中所起到的作用。第二部分,通过RNAi技术构建特异性VEGF-siRNA表达载体抑制胆管癌细胞中VEGF基因的表达,并从中筛选出干扰效率最高的小RNA干扰表达质粒。第三部分,通过VEGF-siRNA表达载体下调QBC939胆管癌细胞中VEGF-A、VEGF-C和MMP-2基因的表达,从细胞水平探讨三者在胆管癌发生、发展及侵袭转移的作用机制。方法：1.采用免疫组织化学法检测47例胆管癌组织、40例邻近非癌胆管组织及15例正常胆管组织中VEGF-A、VEGF-C和MMP-2蛋白的表达情况,分析胆管癌组织中VEGF-A、VEGF-C和MMP-2的表达与胆管癌临床病理因素及患者预后之间的关系。2.用CD105和D2-40分别对47例胆管癌组织、40例邻近非癌胆管组织及15例正常胆管组织中的血管内皮细胞和淋巴管内皮细胞进行标记,并采用免疫组织化学法检测其中CD105和D2-40蛋白的表达情况。显微镜下分别对47例胆管癌组织、40例邻近非癌胆管组织及15例正常胆管组织的MVD和MLVD进行计数,以此分析MVD和MLVD与VEGF-A、VEGF-C及MMP-2间的相互关系,进一步说明VEGF-A、VEGF-C和MMP-2蛋白表达在胆管癌的发生、发展和侵袭转移过程中所起到的作用。3.构建特异性VEGF-siRNA表达载体,使用脂质体载体将其分别转染至胆管癌细胞系QBC939、HCCC-9810和RBE中,筛选出针对VEGF基因抑制效率最高的VEGF-siRNA序列。4.通过RT-PCR和Western blotting法分别检测VEGF-siRNA转染后胆管癌QBC939细胞中VEGF-A、VEGF-C及MMP-2mRNA和蛋白的表达水平。5.分别采用Transwell侵袭实验、MMT法和流式细胞分析技术(flow cytometry, FCM)检测VEGF-siRNA转染后胆管癌QBC939细胞侵袭转移和增殖的能力及肿瘤细胞发生凋亡的情况。结果：1.与邻近非癌胆管组织和正常胆管组织相比,VEGF-C及MMP-2蛋白在胆管癌组织中阳性表达率明显增多(P<0.01)。VEGF-A、VEGF-C蛋白高表达与胆管癌浸润深度、TNM分期、淋巴结转移密切相关。而MMP-2蛋白高表达胆管癌浸润深度、分化程度、TNM分期、淋巴结转移密切相关。对胆管癌组织中VEGF-A、VEGF-C和MMP-2的进行两两比较,其蛋白表达均为正相关(P值均<0.05)。Cox多因素分析结果发VEGF-A、VEGF-C和MMP-2的蛋白表达水平均是影响胆管癌患者预后的独立因素。2.胆管癌组织中MVD显著高于邻近非癌胆管组织及正常胆管组织(P<0.01),而胆管癌组织和邻近非癌胆管组织中MLVD均显著高于正常胆管组织(P<0.01)。MVD和MLVD计数水平的高低与胆管癌浸润深度、分化程度、TNM分期、淋巴结转移密切相关。VEGF-A和MMP-2的表达与MVD水平呈正相关,三者可作为反映胆管癌微血管生成状况的指标；VEGF-C和MMP-2的表达与MLVD水平呈正相关,三者可作为反映胆管癌微淋巴管生成状况的指标。3.成功构建了VEGF-siRNA表达载体并能高效的转染进胆管癌细胞系QBC93、HCCC-9810和RBE内。与空白对照组和阴性对照组相比,VEGF-siRNA表达载体能有效地抑制VEGF mRNA及蛋白在胆管癌细胞系QBC939、HCCC-9810和RBE中的表达。其中抑制效率最高的VEGF-siRNA-1干扰片段对VEGF基因mRNA和蛋白表达的抑制率分别达到86.37%和79.72%。4.当胆管癌QBC939细胞系中的VEGF基因被沉默后,其VEGF-A. VEGF-C及MMP-2mRNA和蛋白的表达水平较之空白对照组和阴性对照组也明显下调(P<0.01),并可诱导肿瘤细胞产生凋亡,使肿瘤细胞的增殖、侵袭和迁移的能力明显减弱。结论：1. VEGF-A、VEGF-C及MMP-2在胆管癌组织中呈高表达,并在胆管癌的发生、发展及浸润转移过程中可能起协同作用。2. VEGF-A、VEGF-C及MMP-2的高表达,与胆管癌浸润深度、分化程度、TNM分期、淋巴结转移等生物学行为关系密切。3. VEGF-A、VEGF-C及MMP-2参与调控胆管癌中微血管密度和微淋巴管密度的水平,并与之水平呈正相关,提示其在胆管癌新生血管和淋巴管形成过程中发挥重要作用。4. VEGF-siRNA表达载体能特异性抑制胆管癌细胞系QBC939、 HCCC-9810和RBE中VEGF基因,并下调其mRNA和蛋白的表达水平。5.当胆管癌QBC939细胞系中的VEGF基因被沉默后,其VEGF-A、 VEGF-C及MMP-2mRNA和蛋白的表达水平也同时下调。6. VEGF-siRNA表达载体可抑制胆管癌QBC939细胞的增殖,诱导其细胞的凋亡,并降低其细胞迁移和侵袭的能力。提示通过VEGF-siRNA治疗胆管癌在理论上是可行的。7. VEGF-A、VEGF-C及MMP-2蛋白和mRNA的表达水平,与胆管癌发生、发展、预后密切相关,联合测定这三者和CD105、D2-40的表达水平可能成为评价预后,判断复发的重要而有效的指标。更多还原

【Abstract】 BACKGROUND:Cholangiocarcinoma is an uncommonly malign-nant neoplasm in digestive tract, and accounts for about2%～3%of all digestive tract cancers. This cancer was described firstly by Durand Fardel in1840, which was originating from the biliary epithelium (cholangiocytes), and was most usually in the tumor of liver and biliary tract system, the incidence rate reached10%～15%in the liver and biliary cancer. The incidence rate of this disease was about1/100,000in the worldwide, but in the near decade this rate tended to increase. In our country, this rate of increase account to5%every year, and this increase rate of development became to the fastest in the all of digestive tract cancers. Therapeutic approach as well as surgical technology, radiothe-rapy, chemotherapy, and immunity treatment advanced more than imagine. Because of especially pathological features and exceptive anatomical position for this tumor, the patient with cholangiocarcinoma lack of specific clinical symptoms and diagnostic technology on the early stage. When many patients appear clinical symptoms, the ill was on the progression or advanced stage, due to they have no chance to achieve radical operation. So that the3-year survival rate only was35%to50%on the post surgery, and the5-year survival rate less than10%worldwide, as well as5%in our country. Due to the diagnostic level and therapeutic efficacy have no conspicuously improved, so these neoplasms extremely threaten the health of people. For those reasons, it’s vital significance to further investigation of mechanisms of cholangiocarcinoma carcinogen-nesis, progression, invasion and metastasis. Therefore, it is current research focus to understand cholangiocarcinoma pathogenesis in the molecular biology, and to seek the targets of malignant transformation of cholangiocarcinoma, as well as targeted therapy of this tumor.Vascular endothelial growth factor (VEGF) takes part in the most all of aspects of vessel and lymphatic formation. It was generally ackno-wledged a specificity factor, which was one of the most effectively and strongly to neo-angiogenesis of tumors. VEGF has been shown to make a most important contribution for the angiogenesis of a solid tumor and its metastasis, moreover to take an important initiation factor for angio-genesis.MMP-2gene was one of the most important and widely distributed members in the matrix metalloproteinase (MMPs) family. Its functions were to depredate extracellular matrix (ECM), to destroy the histological barriers in the process of invasion, and then to lead tumor cells breakthrough ECM and basilar membrane, infiltrate to closed fibrous connective tissue, take place in the metastasis. It plays a key role on the invasion and metastasis to tumor cells.RNA interference (RNAi) is the classic kind of post-transcriptional gene silencing method, which allows to specifically blocking the expres-sing of target genes at the mRNA level, so it have most of specificity and effectivity. RNAi is now being exploited as a powerful tool for reverse genetics, widely powerful tool for the analysis of gene function and shows great promise for therapeutic applications.OBJECTIVE:In part one, to investigate the expression of VEGF-A, VEGF-C and MMP-2in the samples of cholangiocarcinoma tissues and analyze the relationship between expression of VEGF-A, VEGF-C, MMP-2and clinicopathological features, prognosis of cholangiocar-cinoma. In order to further explain the functions of VEGF-A, VEGF-C and MMP-2protein in the process of carcinogenesis, invasion, metastasis of cholangiocarcinoma, we detect the microvascular density (MVD) and micro lymphatic vessel density (MLVD) in the samples of cholangiocar-cinoma tissues. In part two, we successfully design and synthesize siRNA sequences of targeting VEGF gene, and chose a most effective siRNA to the next experiments. In part three, we use VEGF-siRNA to down-regu-late the expression of VEGF-A, VEGF-C and MMP-2gene in QBC939cell line, in order to explore the molecular mechanism of these3genes in the angiogenesis, proliferation, invasion and metastasis of cholangiocar-cinoma.METHODS:1. The expression of VEGF-A, VEGF-C and MMP-2in47specimens of cholangiocarcinoma tissues,40adjacent non-cancer specimens and15normal biliary tissues was detected by immunohistochemistry. The relationship between expression of VEGF-A, VEGF-C, MMP-2and clinicopathological features, prognosis was statistically analyzed.2. We used CD105and D2-40to mark the vascular and lymphatic endothelial cells in47cholangiocarcinoma tissues specimens,40adjacent non-cancer specimens and15normal biliary tissues, respectively. And then to count the number of MVD and MLVD under microscope, in order to analyze the relationship between MVD, MLVD and VEGF-A, VEGF-C, MMP-2, so that further explain the function of VEGF-A, VEGF-C and MMP-2protein in the process of carcinogenesis, invasion, metastasis of cholangiocarcinoma.3. We designed and synthesized siRNA sequences of targeting VEGF gene, cholangiocarcinoma cell line QBC939, HCCC-9810and RBE were respectively transfected with VEGF-siRNA plasmids for48h by LipofectamineTM2000. After the transfection, we measured the inhibited efficiency in VEGF expression; choose the most efficient one and QBC939cell line for further study.4. After the transfection, the RT-PCR and Western blotting test were used to measure the expression levels of mRNA and protein of VEGF-A, VEGF-C and MMP2in the QBC939cell line, respectively.5. The cell invasion potential was preformed by Transwell invasion and migration assay, and MTT assay was employed to detect the proliferation of the cholangiocarcinoma cell. Flow cytometry was employed to evaluate cell apoptosis and death.RESULTS:1. The expression of VEGF-A, VEGF-C and MMP-2proteins in cholangiocarcinoma tissues was significantly higher than that in adjacent non-cancer tissues and normal biliary tissues (P<0.01). Overexpression of VEGF-A, VEGF-C was statistically significantly associated with the depth of invasion, TNM stage and lymphatic metastasis. Overexpression of MMP-2was statistically significantly associated with the depth of invasion, differentiation degree, TNM stage and lymphatic metastasis. There are positive correlation between the expressions of VEGF-A, VEGF-C and MMP-2on protein level (P<0.05). In Cox multivariate analysis, expression of VEGF-A, VEGF-C and MMP-2immunostaining is found to be independent prognostic factors.2. MVD in cholangiocarcinoma tissues was significantly higher than that in adjacent non-cancer tissues and normal biliary tissues (P<0.01); as well as MLVD in cholangiocarcinoma tissues and adjacent non-cancer tissues both significantly higher than that in normal biliary tissues (P<0.01). The number of MVD and MLVD was statistically significantly associated with the depth of invasion, differentiation degree, TNM stage and lymphatic metastasis. There are positive correlation between the expressions of VEGF-A, MMP-2and MVD; and these three parameters can reflect the status of neo-angiogenesis in cholangiocarcinoma. There are positive correlation between the expressions of VEGF-C, MMP-2and MLVD; and these three parameters can reflect the status of neo-lymphan-giogenesis in cholangiocarcinoma.3. We designed and synthesized siRNA sequences of targeting VEGF gene, cholangiocarcinoma cell line QBC939, HCCC-9810and RBE were successfully transfected with VEGF-siRNA plasmids, respecti-vely. Compared to negative control group and blank control group, the mRNA and protein level of VEGF in VEGF-siRNA-1group were reduced by86.37%and79.72%, respectively.4. After the transfection of VEGF-siRNA-1, we can observe both the mRNA and protein of VEGF-A, VEGF-C and MMP2were signify-cantly down-regulated in the QBC939cell line, whereas the invasion, migration and proliferation of tumor cells were decreased greatly. The rate of apoptosis of tumor cells was increased obviously in the VEGF-siRNA group compared with the blank control and negative control group (P<0.01).CONCLUSION1. VEGF-A, VEGF-C and MMP-2are overexpression in the cholangiocarcinoma tissues, and maybe have synergetic effect in the process of carcinogenesis, invasion, and metastasis of cholangiocar-cinoma.2. Overexpression of VEGF-A, VEGF-C and MMP-2have significantly associated with the depth of invasion, differentiation degree, TNM stage and lymphatic metastasis in the cholangiocarcinoma.3. VEGF-A, VEGF-C and MMP-2involve within the regulation of MVD and MLVD level in the cholangiocarcinoma, and they have significant positive correlation. Moreover, they make an important contribution to the angiogenesis and lymphangiogenesis in the cholangiocarcinoma.4. VEGF-siRNA can efficiently and specifically inhibit expression of VEGF gene on mRNA and protein level in cholangiocarcinoma cell line QBC939, HCCC-9810and RBE, respectively.5. When VEGF gene was silenced, we can observe both the mRNA and protein of VEGF-A, VEGF-C and MMP2were significantly down-regulated in the QBC939cell line.6. Blocking the expression of VEGF gene via VEGF-siRNA can effectively inhibit the invasion, migration, proliferation, and induce apoptosis in the QBC939cell line. These findings suggest that the RNAi approach targeting VEGF maybe an effective therapeutic strategy for cholangiocarcinoma and provide a new idea for the treatment.7. The expressions of VEGF-A, VEGF-C and MMP-2on mRNA and protein level have significantly associated with carcinogenesis, progression, and prognosis of cholangiocarcinoma. In addition, detection and administration the serum level associated with VEGF-A, VEGF-C, MMP2, MVD and MLVD maybe a useful and significant prognostic indicator.更多还原