【作者】 陈钢；

【导师】 王洲；

【作者基本信息】 山东大学 ， 外科学， 2010， 博士

【摘要】 研究背景肿瘤的转移是恶性肿瘤最重要的生物学特性之一,是一个多步骤、多因素参与的极其复杂的过程,但其具体机制尚未明确。研究证实,肿瘤的转移不是随意的,而是具有选择性的,即器官转移特异性。1889年Peget提出的“种子—土壤”学说(the "seed and soil" hypothesis)认为,肿瘤的转移是特定的肿瘤细胞(种子)在适宜其生长的器官环境(土壤)中生长发展的结果。1928年Ewing的“解剖或机械”理论(the anatomical or mechanical theory)以器官的血液、淋巴引流方向解释转移的发生。而后来在肿瘤特异性转移的研究中出现了新的概念：“归巢”理论(the homing theory)；该理论认为,肿瘤细胞转移到特定器官是由于不同器官通过趋化作用捕获或吸引特定类型肿瘤细胞而使肿瘤细胞“归巢”的结果。近年来,化学趋化因子受体—配体信号轴的研究发现为“归巢”理论提供了有利的证据。趋化因子是细胞因子超家族中一类具有化学趋化作用的分泌型小分子蛋白,由70～125个氨基酸组成,分子量8-15kD不等。迄今为止至少已发现50余种趋化因子及20余种受体。根据其N末端4个保守半胱氨酸(Cys)残基的前两个半胱氨酸残基的位置,可将趋化因子分为4个亚家族——CXC、CC、C、CX3C亚家族。趋化因子受体是一类介导趋化因子行使功能的GTP蛋白耦联的跨膜受体,通常表达于免疫细胞、内皮细胞等细胞膜上；7个跨膜区将分子分成细胞外自由的N端、3个细胞外环和C端几个部分。趋化因子与细胞表面的特异性受体结合后可参与多种生理和病理过程,如细胞生长、发育、分化、凋亡、组织损伤、肿瘤的生长和转移等。2001年Muller A首次提出了肿瘤细胞可以利用趋化因子及其受体的特异性结合实现器官特异性转移,研究发现乳腺癌细胞高表达趋化因子受体CXCR4和CCR7,而CXCR4的配体CXCL12多表达于乳腺癌的特异转移部位肺、肝脏、骨髓等,而CCR7的配体CCL21则多表达于淋巴结组织,从而导致了乳腺癌细胞的定向转移。越来越多的研究表明,趋化因子CXCL12／CXCR4信号轴在肿瘤的器官特异性转移中与趋化因子受体CCR7及其配体在肿瘤淋巴结转移中均发挥着愈加重要的作用。肺癌是当今全世界最严重威胁人民健康的恶性疾病之一,随着我国工业化的进展,肺癌的发病率呈上升趋势,现已位居恶性肿瘤发病率和死亡率之首,其中80%以上是非小细胞肺癌(non-small cell lung cancer, NSCLC)。肺癌早期可发生淋巴结转移,晚期可发生远处器官的特异性转移,常见的转移部位有脑、骨、肝和肾上腺等,其中脑转移最为常见。尽管目前在肺癌早期诊断及综合治疗方面取得了一定的进步,肺癌的转移仍是临床治疗所面对的最大难题,直接影响着患者的生存预后。所以研究肺癌器官特异性转移的分子生物学机制,包括淋巴结转移及远处转移,对指导肺癌患者选择治疗方案和判定预后具有重要的临床价值。目前,国内对趋化因子与肺癌器官特异性转移机制的研究仍较为少见,尤其是与肺癌脑转移的研究未见相关文献报道。本课题通过应用RT-PCR、免疫组化及Western blot三种方法联合检测趋化因子受体CCR7与VEGF-C在NSCLC患者肺癌组织及转移淋巴结中的表达情况,意在探索肺癌淋巴结转移的可能机制。另外,本课题在国内首次检测趋化因子受体CXCR4在NSCLC并脑转移患者的脑转移灶中的表达情况,以期初步探索肺癌脑特异性转移的可能机制。第一部分趋化因子受体CCR7及VEGF-C表达对非小细胞肺癌淋巴结转移潜能的影响目的：肺癌淋巴结转移的发生与肺癌细胞的趋化迁移及肿瘤淋巴管生成关系密切。本文旨在于探讨趋化因子受体CCR7及VEGF-C的表达对非小细胞肺癌淋巴结转移潜能的影响。方法：临床收集2008年1月至2008年8月间在山东大学附属省立医院胸外科施行肺癌完全切除加系统淋巴结清扫的65例NSCLC患者的标本,包括肺癌组织、淋巴结组织及正常肺组织。应用RT-PCR方法检测标本中CCR7mRNA及VEGF-C mRNA的表达,并进行基因测序验证；应用免疫组化与Western blot方法检测CCR7及VEGF-C蛋白的表达情况,单克隆抗体D2-40标记淋巴管内皮细胞以计数淋巴管密度(LVD)。建立数据库,应用x2检验比较CCR7及VEGF-C表达情况的差异,u检验比较LVD的差异情况,并应用Logistic多因素回归分析判定影响非小细胞肺癌淋巴结转移潜能的独立相关危险因素。结果：在65例患者的肺癌组织中,RT-PCR方法检测有47例(72.3%)CCR7 mRNA及39例(60.0%)VEGF-C mRNA表达阳性,免疫组化方法检测有43例(66.2%)CCR7蛋白及36例(55.4%)VEGF-C蛋白表达阳性,Western blot方法检测有44例(67.7%)CCR7蛋白及37例(56.9%)VEGF-C蛋白表达阳性。CCR7及VEGF-C在肺癌组织中的表达均无明显差异(P>0.05),且均与淋巴结转移状态明显相关(P<0.05),而CCR7与VEGF-C的共同表达较单一表达与淋巴结转移状态有更为显著的相关性(P<0.05)。肺癌组织中LVD较正常肺组织中明显增高(P<0.001),且与CCR7、VEGF-C的表达及肺癌淋巴结转移状态显著相关(P<0.05)。35例有转移的淋巴结中CCR7、VEGF-CmRNA的表达率为85.7%、74.3%,分别高于无转移的淋巴结(20.0%、13.3%),并与肺癌组织具有高度同源性。20例正常肺组织中均未见CCR7及VEGF-C蛋白的明显表达。Logistic多因素回归分析结果显示：肺癌组织中CCR7阳性表达(OR=10.275,P=0.005)及VEGF-C阳性表达(OR=5.550,P=0.021)是影响NSCLC患者淋巴结转移潜能的独立危险因素。结论：CCR7及VEGF-C在NSCLC患者的肺癌组织及淋巴结转移灶中均表达明显增高,并与肺癌淋巴结转移状态密切相关；CCR7与VEGF-C的共同表达可能促进了NSCLC淋巴结转移,亦可作为预测NSCLC患者淋巴结转移潜能的调控基因。第二部分趋化因子受体CXCR4过度表达与非小细胞肺癌脑转移相关性的临床研究目的：肺癌可发生远处器官特异性转移,其中以脑转移最为常见,但其具体机制仍未明确。本文旨在于探讨趋化因子受体CXCR4的表达情况与非小细胞肺癌脑转移的相关性。方法：选取1998年6月至2008年9月期间在山东大学附属省立医院行肺癌及脑转移瘤切除的32例单发脑转移(M1组)的NSCLC患者的肺癌组织及脑转移灶石蜡标本；并选取近十年期间条件最大限度一致配对的32例无远处转移(M0组)的NSCLC患者的肺癌组织及另30例原发性脑肿瘤组织作为对照。应用免疫组化方法(SP法)检测各标本中CXCR4蛋白的表达情况,Mcnemar配对x2检验或Fisher精确概率法比较CXCR4蛋白表达的差异。随访患者,Kaplan-Meier法计算生存率,绘制生存曲线,Log-rank检验比较生存差别,Cox多因素回归分析判定单发脑转移NSCLC患者预后的独立危险因素。结果：在32例单发脑转移(M1组)的NSCLC患者中,有29例(90.6%)肺癌组织中CXCR4蛋白阳性表达,而全部32例(100%)脑转移灶中CXCR4蛋白均阳性表达,二者之间无显著差异(P=0.238)。在32例无远处转移(M0组)的NSCLC患者的肺癌组织中CXCR4蛋白阳性表达率为68.8%(22／32),明显低于M1组NSCLC患者肺癌组织(P=0.000)。30例原发性脑肿瘤中有19例(63.3%)CXCR4蛋白阳性表达,明显低于脑转移灶中的表达(P=0.000)。32例单发脑转移的NSCLC患者术后1年、3年、5年累积生存率分别为62.5%、25%、15.6%；而32例无远处转移的NSCLC患者术后1年、3年、5年累积生存率分别为84.4%、53.1%、40.6%；Log-rank检验显示两者有显著差异(P=0.002)。同时相脑转移的18例NSCLC患者脑部手术后1年、3年及5年生存率分别为72.2%、27.7%、16.7%；异时相脑转移的14例NSCLC患者脑部手术后1年、3年及5年生存率分别为50%、21.4%、14.3%；Log-rank检验显示两组患者无显著差异(P=0.39)。Cox多因素回归分析显示结果显示,性别、年龄、组织学类型、肿瘤分化程度、T分期、N分期、转移方式及CXCR4在肺癌组织中的表达均与单发脑转移的NSCLC患者预后无明显相关性(P>0.05)。结论：趋化因子受体CXCR4在NSCLC患者脑转移灶及肺癌组织中均存在过度表达,提示了CXCL12／CXCR4信号轴可能在NSCLC特异性脑转移中起到一定的趋化作用。更多还原

【Abstract】 BackgroundTumor metastasis is one of the most important biological behaviors of the tumor, which involves a series of complex processes with many factors. But the mechanism of metastasis is still unclear. Tumor metastasis results from a non-random process, but a selective process——Organ specificity. In 1889 Paget developed the theory of "seed and soil", and He hypothesized that certain tumor cells (seeds) colonize selectively distant organs (soil) with a favorable environment facilitating survival of tumor cells. Then in 1928, Ewing explained metastases by the "anatomical or mechanical" theories such as tumor cell trapping or lodgement of tumor emboli into organ vascular bed. However, in the study of Tumor metastasis a new theory——the "homing" theory appeared. The theory holds that different organs have special abilities to arrest or attract through chemotactic factors specific types of cancer cells.In recent years, the research of chemokine receptor-ligand signaling axis provided compelling support for the chemoattraction part of the homing theory. Chemokines are a large family of small secreted proteins associated with the leukocyte trafficking during host defense and pathological immune responses. Chemokines are generally 8-15 kDa in size and contain 70～125 amino acids. The human chemokine system currently includes more than 50 different chemokines and 20 different chemokine receptors. They are classified into which are grouped into four families (C, CC, CXC, and CX3C) based on the spacing of key cysteine residues near the N terminus of these proteins. Chemokines act through both specific and shared receptors that all belong to the superfamily of G protein-coupled receptors (GPCRs) with seven-transmembrane domains. Chemokines and the specific receptors on cell surface may participate in a variety of physiological and pathological processes, such as cell growth, development, differentiation, apoptosis, tissue injury, tumor growth and metastasis. In 2001, Muller A and the colleagues first demonstrated a potential mechanism for site-specific metastasis which is related to expression of chemokines and their receptors. They reported that the chemokine receptors CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-la and CCL21/6Ckine exhibited peak levels of expression in organs representing the first destinations of breast cancer metastasis. Their findings indicated that chemokines and their receptors have a critical role in determining the metastatic destination of tumour cells.Nowadays, lung cancer is one of the most serious malignant diseases threaten to people’s health in the world. In China, lung cancer occurs at very high frequencies and has been the leading cause of tumor-specific death, and more than 80% are non-small cell lung cancer (NSCLC). Clinically, it appears that lymph node metastasis of lung cancer may occur early, and latter lung cancer can metastasize to distant specific target organs, such as brain, bone, liver and adrenal glands. Among them, brain metestasis occurs most frequently. Metastatic spread constitutes the primary source of morbidity and mortality, and the prognosis of patients with metastasizing lung cancer leaves much to be desired. Therefore, a thorough understanding of the organ-specific metastatic process, including lymph node metastasis and distant metastasis, is likely to be crucial to developing effective new therapies for lung cancer.While nowadays the reports on the role of the chemokine-receptor axis in organ-specific metastasis, especially brain-specific metastasis, of lung cancer were very rare. Then this study was to investigate the relationship between CCR7 expression and lymph node metastasis of NSCLC by RT-PCR, immunohistochemistry, and western blot assays and explore the mechanism of lymph node metastasis of lung cancer. And then this study was the first time to investigate the correlation between CXCR4 expression and brain-specific metastasis of NSCLC and explore the mechanism of brain metastatic specificity of lung cancer initially.Part I Effect of CCR7 and VEGF-C on lymph node metastatic potentiality of non-small cell lung cancerObjective:The incidence of lymph node metastasis of lung cancer was shown to be positive correlated with tumor chemotactic migration and lymphangiogenesis. This study was to investigate the relationship between lymph node metastatic potentiality of non-small cell lung cancer(NSCLC) and expression of CCR7 and VEGF-C.Methods:The samples of cancer tissues, lymph nodes and normal lung tissues from 65 patients with NSCLC, who underwent complete resection in Provincial Hospital Affiliated to Shandong University from January 2008 to August 2008, were collected. And all samples were examined by RT-PCR, immunohistochemistry and western blot assays to detect CCR7 and VEGF-C expression. Monoclonal antibody D2-40 was used to assessment of lymphatic vessel density (LVD). The SSPS 11.5 software package was used for statistical analysis. According to the clinicopathologic factors, the difference of CCR7 and VEGF-C expression was compared byχ2 test, and the difference of LVD was compared by u-test. Logistic regression analysis was performed to determine the independent risk factors of influencing lymph node metastatic potentiality of NSCLC.Results:CCR7 mRNA and VEGF-C mRNA expression was observed in 47(72.3%) and 39 (60.0%) lung cancer tissues by RT-PCR assay; CCR7 protein and VEGF-C protein expression was observed in 43 (66.2%) and 36 (55.4%) lung cancer tissues by immunohistochemistry; and CCR7 protein and VEGF-C protein expression was also observed in 44 (67.7%) and 37 (56.9%) lung cancer tissues by western blot, respectively. No significant difference was found between CCR7 and VEGF-C expression(P>0.05), and each was positive related to lymph node metastasis of NSCLC (P<0.05), furthermore co-expression of CCR7 and VEGF-C expression was more significantly related to lymph node metastasis of NSCLC (P<0.05). In lung cancer tissues, LVD was higher than that in normal lung tissues (P<0.001); and LVD in lung cancer was positively related to CCR7 expression, VEGF-C expression and lymph node metastasis. The expression rate of CCR7 mRNA and VEGF-C mRNA in metastatic lymph nodes were 85.7% and 74.3%, which were higher than that in non-metastatic lymph nodes(20.0% and 13.3%). Obvious expression of CCR7 and VEGF-C protein was found in no normal lung tissuse. Logistic regression analysis revealed that CCR7 mRNA expression (OR=10.275, P=0.005) and VEGF-C mRNA expression in tumors(OR=5.550, P=0.021) were independent risk factors of lymph node metastasis of NSCLC.Conclusions:CCR7 and VEGF-C were highly expressed in lung cancer and the metastatic lymph nodes, with significant correlation with lymph node metastasis of NSCLC. The co-expression of CCR7 and VEGF-C might promote lymphatic metastasis of lung cancer and might be a clinical predictor of lymph node metastasis of patients with NSCLC.Part II High-level CXCR4 expression correlates with brain metastasis of non-small cell lung cancerObjective:Lung cancer might develop organ-specific metastasis, and brain metastasis occurs most frequently, while the mechanism is still unclear. This study was to investigate the correlation between CXCR4 expression and brain metastasis of non-small cell lung cancer. Methods:The metastatic brain tumors and lung cancer tissues from 32 patients with solitary brain metastasis of NSCLC (M1), who underwent combined surgical treatment from June 1998 to September 2008, and 32 paring patients without distant metastasis (MO) and 30 patients with primary brain tumor, were examined by immunohistochemistry to detect the expression of CXCR4 protein. The difference of CXCR4 expression was compared byχ2 test. Estimation of survival was calculated using the Kaplan-Meier method, and the statistical differences were analyzed using the Log-rank test. Cox regression analysis was performed to identify risk prognostic factors of patients with solitary brain metastasis of NSCLC.Results:CXCR4 protein expression was observed in 29 (90.6%) lung cancer with brain metastasis(M1) and in all metastatic brain tumors (100%), and no difference in them two(P=0.238). And CXCR4 protein expression was observed in 68.8%(22/32) paired lung cancer without brain metastasis(MO), which was significantly lower than that in M1 lung cancer(P=0.000). In 63.3%(19/30) primary brain tumors, CXCR4 protein expression was observed and was significantly lower than that in metastatic brain tumors(P=0.000). The 1-year,3-year and 5-year cumulative survival rates of 32 patients with solitary brain metastasis of NSCLC were 62.5%,25%, and 15.6%; and the survival rates of patients with M0 NSCLC were 84.4%,53.1%, and 40.6%; Log-rank test showed that the rates of the two groups was significantly different(P=0.002). The 1-year,3-year and 5-year cumulative survival rates of 18 patients with synchronous brain metastasis were 72.2%,27.7%, and 16.7%; the survival rates of 14 patients with metachronous brain metastasis were 50%,21.4%, and 14.3%; Log-rank test showed there was no significant difference between the two groups(P=0.39). Cox regression analysis showed that gender, age, tumor histologic type or differentiation, T status, N status, brain metastasis pattern, adjuvant therapy and CXCR4 expression in lung cancer did not significantly affect survival of M1 NSCLC patients(P>0.05).Conclusion:CXCR4 protein expressed highly in lung cancer tissues and metastatic brain tumors of patients with M1 NSCLC, which indicated that CXCL12/CXCR4 signaling axis might play a chemotactic role in brain specific metastasis of NSCLC.更多还原