【作者】 赵伟鹏；

【导师】 陈正堂；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2009， 博士

【摘要】 研究背景淋巴管生成不仅参与了胚胎淋巴管发育、组织损伤修复、慢性炎症等生理和病理过程，而且在肿瘤淋巴道转移也具有十分重要的作用。大量临床病理研究证实：非小细胞肺癌(NSCLC)、乳腺癌、结肠癌、恶性黑色素瘤、头颈部肿瘤和前列腺癌等实体肿瘤高表达VEGF-C和VEGF-D，并与淋巴管密度(lymphatic microvesse l density,LVD)、淋巴管侵袭(lymphatic vessel invasion, LVI)，以及淋巴结转移密切相关。肺腺癌极易发生区域性淋巴结和远处器官转移，难以手术切除，对放、化疗敏感性较低，预后很差，目前认为淋巴管生成可能是肺腺癌淋巴结转移的早期核心机制。近年研究证实，肿瘤诱导的淋巴管生成促进了癌细胞的转移性扩散，导致肿瘤患者预后差、生存率低。血管内皮生长因子(VEGF)-C、VEGF-D、成纤维细胞生长因子(FGF)、血小板衍生的生长因子(PDGF)等是目前公认的淋巴管生成因子，它们通过结合淋巴管内皮细胞(lymphatic endothelial cell , LEC)上的受体，继而促进LEC增殖、迁移和管状结构形成，从而诱导淋巴管生成。目前被广泛认可的LEC特异性标记物包括血管内皮生长因子受体-3 (VEGFR-3)、Prox-1、淋巴管内皮透明质酸受体-1 (LYVE-1)和Podoplanin等，LEC特异性标记物的发现促进了LEC生物学功能研究的发展。随着LEC的成功分离和多种用于研究淋巴管生成的动物模型的建立，淋巴管生成迅速成为肿瘤领域的研究热点。我们研究小组此前的研究也表明NSCLC能够通过表达VEGF-C诱导瘤周淋巴管生成；瘤周淋巴管生成对NSCLC的发生、发展起重要作用；瘤内的淋巴管呈塌陷状态，并不具有介导淋巴道转移的能力。临床资料显示，虽然肺腺癌极易发生淋巴道转移，但是临床患者淋巴结转移存在很大的差异性；很多情况下原发肿瘤的大小与肿瘤是否发生远处转移不平行。通过对肿瘤细胞在淋巴管生成中发挥诱导作用的复习，我们设想除VEGF-C、VEGF-D外是否存在其他的淋巴管生成促进因子？另一方面，是否存在直接抑制淋巴管生成的因子？本课题旨在筛选与肺腺癌淋巴管生成相关的基因，为控制肺腺癌淋巴管转移寻找新的作用靶点。目的利用全基因组芯片筛选技术，采用Human Genome U133 Plus 2.0 Array差异筛选肺腺癌淋巴管生成相关的基因。通过GenBank数据库生物信息学分析，结合已有文献报道，确定被选的基因，初步验证其在肿瘤淋巴管生成中的作用。方法1.免疫组织化学法检测淋巴管内皮标记物Podoplanin在34例肺腺癌组织和5例肺良性病变组织中的表达，计算LVD，按LVD评分对肺腺癌淋巴管高、低转移进行分组，分析LVD与肿瘤分期、分化程度及淋巴结转移等的关系。2.采用全基因组芯片筛选的方法，构建肺腺癌高淋巴管生成和低淋巴管生成差异表达的基因文库(cDNA library)，采用Human Genome U133 Plus 2.0 Array筛选肺腺癌淋巴管生成相关的基因，GenBank数据库生物信息学分析，结合文献报道选取上调和下调基因，采用real time PCR进一步验证差异表达情况。3.以NSCLC为研究对象，利用免疫组织化学方法检测NSCLC组织中上调基因（以IGFBP7为对象）、下调基因（以CDH1为对象）的表达情况，分析IGFBP7、CDH1表达与患者年龄、性别、肿瘤分期、分化程度及淋巴结转移的关系。4. RT-PCR检测人肺癌细胞株和小鼠肺癌细胞株(lewis lung cancer, LLC）的IGFBP7的表达。5.构建IGFBP7干扰载体pGenesil-si IGFBP7，通过siRNA干扰IGFBP7表达。6.应用不完全弗氏佐剂(IFA)诱导小鼠腹腔淋巴管瘤形成，消化法分离获得LEC，以鼠尾胶为粘贴剂原代培养。淋巴管形成实验判定LEC能否形成管样结构。7.建立LLC与LEC共培养体系，用CCK-8细胞活性实验和Transwell小室细胞迁移实验，分析敲减IGFBP7前后LLC细胞对LEC生长和迁移的影响。8.利用C57BL/6小鼠建立敲减IGFBP7的LLC和未敲减IGFBP7的LLC皮下移植瘤模型，利用免疫荧光和共聚焦显微镜观察肿瘤新生淋巴管情况，分析敲减IGFBP7对移植瘤生长、淋巴管生成、淋巴结转移和肺转移的影响。结果1. Podoplanin阳性淋巴管LVD在5例肺炎性假瘤为10.8±4.3，在34例肺腺癌瘤内为11.2±5.0，肺腺癌瘤周为23.5±8.4，瘤周LVD明显高于前二者(P <0.05)。肿瘤分期N1-2与N0瘤周的LVD分别是24.3±8.7、18.4±6.3，前者明显高于后者(P <0.01)；Ⅰ-Ⅱ期与Ⅲ-Ⅳ瘤周的LVD分别是18.4±5.6、24.1±8.2，前者明显低于后者(P <0.01)。2.按LVD评分对6例肺腺癌新鲜手术标本进行了分组，具体为LVDhigh组和LVDlow组。利用全基因表达谱芯片技术，对两组进行差异性表达基因的筛选。以差异2倍为标准，得到上调基因即促进淋巴管生成的基因94个，下调基因即抑制淋巴管生成的基因81个。我们分别选取IGFBP7、CDH1进行了real time PCR的验证，与表达谱芯片的筛选结果基本一致。3.免疫组化结果显示：97例NSCLC患者中IGFBP7的表达阳性54例，瘤周LVD为23.1±8.5；而IGFBP7的表达阴性的43例，瘤周LVD为16.9±6.0，IGFBP7的表达阳性的瘤周LVD明显高于阴性的瘤周LVD (P <0.05)。淋巴结转移组IGFBP7阳性率(45.2%, 28/62)显著高于无淋巴结转移组(25.7%, 9/35) (P <0.05)。Spearman相关性分析表明，IGFBP7阳性率与LVD呈正相关(r =0.423，P <0.01)。97例NSCLC中CDH1的表达阳性48例，瘤周LVD为21.2±10.4；而CDH1的表达阴性的49例，LVD为17.2±7.9，CDH1的表达阳性的瘤周LVD与阴性的瘤周LVD无显著性差异(P >0.05)。4. RT-PCR检测IGFBP7在人肺癌细胞株和小鼠肺癌细胞株的高表达，在NIH3T3和L929未见表达。5.成功构建了pGenesil-siIGFBP7干扰载体；载体转染LLC细胞可下调IGFBP7表达。6. IFA能诱导小鼠腹腔淋巴管瘤形成，所获LEC活细胞数大于96%，高表达VEGFR-3。在自制鼠尾胶包被的培养瓶(板)中，LEC生长状况良好，在鼠尾胶凝胶中，LEC可形成淋巴管样结构。7.经过对transwell小室的insert外表面的LEC进行HE染色发现，LLC组透膜细胞百分比明显高于LLCSiRNA-IGFBP7组(P <0.05)；LLCSiRNA-IGFBP7百分比明显高于空白对照组(P <0.05)。结果表明，IGFBP7敲减的LLC能够明显增强LEC的迁移能力，但其促进能力小于未敲减IGFBP7的LLC细胞。8. IGFBP7敲减实验表明：pGenesil-siIGFBP7组同侧颈上、对侧颈上及腋窝淋巴结转移率分别为100%、33.3%和66.7%；而未转染组分别为100%、83.3%和100%；空质粒转染组分别是100%、66.7和83.3%。pGenesil-siIGFBP7组腋窝淋巴结转移率明显低于未转染组和空质粒转染组(P <0.05)。pGenesil-siIGFBP7组移植瘤瘤周LVD(9.4±2.3)显著低于未转染组(16.7±3.5)和空质粒转染组(13.2±2.8)转染组(P <0.05)。免疫荧光和共聚焦显微镜显示，移植瘤组织中新生淋巴管多位于瘤周，结构不完整，成窦状。结论1、利用全基因组表达基因表达芯片对LVDhigh与LVDlow组进行了差异性的筛选，差异性筛选出IGFBP7等上调基因94个、CDH1等下调基因81个。2、人肺腺癌组织IGFBP7表达与其LVD呈显著正相关，且淋巴结转移组IGFBP7阳性率(45.2%, 28/62)显著高于无淋巴结转移组(25.7%, 9/35) (P <0.05)；IGFBP7能够促进LEC迁移、淋巴管生成和淋巴结转移。CDH1与肿瘤淋巴管转移无明显相关性。更多还原

【Abstract】 BackgroundLymphangiogenesis was not only involved in the lymphatic vessels of em bryonicdevelopment, tissue repair, chronic inflammation, but also in tumor lymph node metastasis.Metastatic tumor spread through lymphatic vessels occurs in lung carcinoma, with regionallymph-node metastasis often being the most important prognostic fac tor for carcinomapatients. Vascular endothelial growth factor (VEGF) -C, vascular endothelial growth factor(VEGF)-D, fibroblast growth factor (FGF) and platelet -derived growth factor (PDGF) arepotent secreted activators critical for tumor -induced lymphangiogenesis.High expression of VEGF-C and VEGF-D were confirmed in clinicopathological study,such non-small cell lung cancer (NSCLC), breast cancer, colon cancer, malignantmelanoma, head and neck cancer and prostate cancer, furthermore lymphatic microve sseldensity (LVD) and lymphatic vessel invasion (LVI) were closely related to lymph nodemetastasis. There were some specific molecular markers of lymphatic endothelial cells(LEC), such as (VEGFR-3), Prox-1, LYVE-1, Podoplanin and so on. With LEC isolationand establishment of lymphangiogenesis animal model, lymphangiogenesis study becomesa hot spot in tumor research. Our recent study showed that tumor cells overexpressingVEGF-C could induce lymphangiogenesis surrounding tumor cells and invasion oflymphatic vessels was a key step in the metastasis of primary tumors to draining lymphnodes.Although lymph node metastasis occurs frequently in lung adenocarcinoma, size ofprimary tumor and tumor occurrence of distant metastasis is not parallel in many cliniccases. We have reviewed mechanism of induction of lymphangiogenesis by tumor cells. Arethere other lymphangiogenesis promoting factors besides VEGF-C and VEGF-D? On theother hand, are there direct inhibitory lymphangiogenesis factors? Based on this hypothesis,we aimed at screening genes related with tumor lymphangiogenesiss. This research shows new approach for the lymphangiogenesis in lung adenocarcinoma and thus provides a newtarget for the inhibition of the metastasis of this kind of malignant car cinoma.ObjectiveTo Screen genes related lymphangiogenesis in lung adenocarcinoma by HumanGenome U133 Plus 2.0 Array, identify the selected genes for further experiment in vivo andin vitro based on bioinformatics analysis and fu nction reported in the literatures.Methods1. Podoplanin expression in 34 NSCLC tissues and 5 control benign pulmonary lesiontissues were detected by immunohistochemistry method. LVD score was determinedbetween LVDlow vs LVDhigh group, furthermore, the relationship LVD between tumorstaging, differentiation and pathologic N staging were analysesd.2. A high and low lymphangiogenesis in lung adenocarcinoma differentially expressedgenes cDNA library was constructed. Human Genome U133 Plus 2.0 Array was employedto screen differential expressed genes related to lymphangiogenesis in lung adenocarcinoma .Then differential expressed level was confirmed by real time PCR.3. IGFBP7 and CDH1 expression were determined with immunohistochemistry , aswell as IGFBP7 and CDH1 expression relat ionship was anlysesed with patient age, sex,tumor staging, differentiation degree and lymph node metastasis.4. RT-PCR detected expression of IGFBP7 in human lung cancer cell lines and mouselewis lung carcinoma cell (LLC).5. An interference plasmid, pGenesil-siIGFBP7 for a short hairpin siRNA directedIGFBP7 was constructed using the pGenesil -1 vector, and the effect of IGFBP7 knockdownthrough transfection the pGenesil-siIGFBP7 to LLC cells.6. Mice lymphangiomas in abdominal cavity were induced by intr aperitoneal injectionof incomplete Freund’s adjuvant (IFA). LEC were obtained from the inducedlymphangiomas after disruption and digestion, and then were cultured in the flask or platepreviously coated with self-made rat-tail collagen. The capability of LEC to form lymphaticvessel-like structures was assessed by the in vitro lymphatic vessel formation assay.7. Transwell experiment and CCK-8 assay were used to determine whether IGFBP7knockdown affects cell migration of LLC cells and cell growth, respectively.8. The tumor models of IGFBP7 knockdown were established via subcutaneous injection of LLC cells in the dorsal site of the right ear of C57BL/6 mice, andlymphangiogenesis, lymph node metastasis and lung metastasis in vivo was investigated.Immunofluorence assay and confocal microscopy was used to examine the neo-lymphaticvessels.Results1. Podoplanin positive LVD was significantly higher peritumoral LVD ( 23.5±8.4) thanthat in benign pulmonary lesion (10.8±4.3) (P <0.01), and significantly higher thanintratumoral LVD (11.2±5.0). There is a statistical significance between lymph nodemetastasis N1-2 (24.3±8.7) and N0 (18.4±6.3) (P <0.01), as well as tumor stages (P <0.01).2. By the LVD scoring, 94 promoting lymphangiogenesis genes and 81 inhibitinggenes were screening. We repeated real time PCR to conform IGFBP7 and CDH1 genelevel.3. In 97 cases of NSCLC patients, positive expression of IGFBP7 was 54 cases,peritumoral LVD (23.1±8.5); negative 43 cases, peritumoral LVD (16.9±6.0). IGFBP7expression of tumor-positive LVD was significantly higher than negative tumor LVD ( P<0.05). Lymph node-positive group IGFBP7 positive rate (45.2%, 28/62) was significantlyhigher than node-negative group (25.7%, 9/35). But there was no significant associati onbetween CDH1 expression and lymph node metastasis, tumor stages, sex, age, histologicalclassification and differentiation stages ( P >0.05) was observed.4. RT-PCR detection of human lung cancer cell lines and mouse lewis lung carcinomacell over-expressed IGFBP7 gene, not detection in NIH3T3 and L929 cell lines.5. The vector pGenesil-siIGFBP7 targeting IGFBP7 was successfully constructed.6. Mice lymphangiomas in abdominal cavity were induced successfully by IFA, andmore than 96% living LEC were harve sted. The expression of VEGFR-3 was positive inLEC. In the gel formed by rat-tail collagen, LEC could form lymphatic vessel -likestructures.7. After the outer surface of the transwell insert of the LEC was found by HE staining,LLC group of cell membrane permeability was significantly higher than the percentage ofLLCSiRNA-IGFBP7 group (P <0.05); LLCSiRNA-IGFBP7 percentage was significantly higherthan the control group and the L929 group ( P <0.05). The results show that, IGFBP7knocked-down LLC can significantly enhance the capacity of LEC migration, but its capacity is less than the induction by not knock-down LLC. Immunofluorescence andconfocal microscopy showed that new -born lymphatic vessel is located peritumor and theirstructures were incomplete.8. At 45 days after tumor inoculation, the presence of supracervical node andcontralateral supracervical node metastases in control mice was 100% 33.3% and 66.7% inIGFBP7 knocked-down LLC inoculation, respectively, compared with nonfectcedgroup100% 83.3% and 100%; pGenesil-s1 group 100% 66.7 83.3%. In addition, themetastatic ratio of brachial nodes in IGFBP7 knocked-down tumors was significantly lowerthan that in control nonfiction and pGenesil -1 tumors. Immunochemical analysis forVEGFR3 expression LVD (9.4±2.3) was lower than that in control tumors was ( 16.7±3.5)and pGenesil-1(13.2±2.8) (P<0.05).Conclusions1. Human Genome Microarray was employed for comparison of expression profiles ofLVDhigh lung adenocarcinoma and LVDlow lung adenocarcinoma. IGFBP7 and CDH1 wereselected for further tumor lymphangiogesis study.2. IGFBP7 positive expression was correlated with tumor metastasis in lungadenocarcinoma, inducing LEC proliferation, migration and tube -like structure formation inLLC inoculated mice model, which indicating IGFBP7 should promote lymph nodemetastasis. CDH1 was not correlated with tumor lymphatic metastasis.更多还原