【作者】 宋平；

【导师】 葛春林；

【作者基本信息】 中国医科大学 ， 普通外科学， 2007， 硕士

【摘要】 前言胰腺癌为临床较常见的恶性肿瘤，早期难以发现，晚期常有周围组织、血管神经的浸润和淋巴结转移。在肿瘤细胞的恶性增殖和转移过程中，iNOS被认为是最重要的因子之一。现已有研究表明，iNOS具有通过多种途径促进肿瘤生长、发展和转移的作用。NO作为肿瘤细胞信号转导途径中重要的信使分子，调节与细胞增殖相关基因的表达，增加肿瘤血供的血管生成，促进肿瘤生长、侵袭和转移。VEGF是参与血管生成过程的血管生成因子中，唯一能特异性地作用于血管内皮细胞的有丝分裂原，在肿瘤血管生成的各个环节中起着重要作用。VEGF对内皮细胞移行、增殖和管腔形成的作用部分是通过其诱导的信号转导分子NO来完成。研究发现VEGF与NO之间存在密切的关系。肿瘤的NO可上调VEGFmRNA水平，VEGF水平与肿瘤血管化程度正相关。因此，NO及其合酶可能是VEGF调节血管生成的下游通道和介质。近期有研究发现肿瘤相关巨噬细胞(tumour-associated macrophages，TAMs)也与淋巴结转移有密切关系。许多肿瘤组织特别是上皮组织包括肺、乳腺、胃的实体瘤内有大量的TAMs存在。TAMs主要来源于外周血单核细胞，也有一部分是固定的组织巨噬细胞。浸润的部位主要位于肿瘤的基质部和血管内皮。巨噬细胞有抗肿瘤作用，可肿瘤组织尽管有大量TAMs的浸润，却能继续生长，说明TAMs可能具有促进肿瘤生长的作用。TAMs对肿瘤组织的作用机制目前尚不完全清楚。免疫组织化学染色及微血管计数发现某些肿瘤的TAMs与血管形成有关。有研究表明食道癌中TAMs的数量和iNOS的表达有关，同时证实iNOS并和瘤周的淋巴管密度及淋巴结转移有关。但在胰腺癌中TAMs和iNOS与淋巴结转移的关系尚无报告。本实验用免疫组化方法对比进行了研究。材料与方法收集1999～2005年中国医科大学附属一院手术切除胰腺癌标本35例，其中男24例，女11例，年龄34～76岁，平均54.2岁。患者术前均未经任何治疗。所有标本均经病理证实。高分化腺癌19例，中分化腺癌9例，低分化腺癌7例。临床TNM分期：Ⅰ期8例，Ⅱ期12例，Ⅲ期10例，Ⅳ期5例。有淋巴结转移15例，无淋巴结转移20例。收集了因胆管下段癌行胰十二指肠切除术而切除的正常胰腺组织6例，其中男4例，女2例，年龄39～75岁，平均58.4岁。以上标本均经过脱水，福尔马林固定，石蜡包埋，连续切片(厚度4μm)。抗原修复后，常规SP法正常胰腺和胰腺癌组织免疫组化染色。鼠抗人CD68单克隆抗体标记巨噬细胞，鼠抗人iNOS单克隆抗体标记iNOS。设阳性及阴性对照，并进行常规HE染色。以光镜下随机3个癌组织高倍视野(400倍)内TAMs细胞数的均值为该病例TAMs值。iNOS的表达以其评分值表示：以癌组织染色强度评分(无，0分；弱，1分；中，2分；强，3分)和阳性细胞率评分(小于5％，0分；5～10％，1分；10～20％，2分；20～50％，3分；大于50％，4分)之和为该病例评分值，将评分值小于或等于2分定为阴性病例，大于2分定为阳性病例。用SPSS 12.0统计软件进行统计学分析，计量资料用((?)±s)表示。两组之间比较采用t(t’)检验，三组或三组以上两两比较采用方差分析(Newman-keuls法，q检验)。采用Pearson相关系数检验VEGF评分与TAMs计数之间的关系，P＜0.05有统计学意义。结果1．免疫组织化学染色结果正常组中巨噬细胞分布明显稀少，胰腺癌组中CD68表达呈黄色或棕黄色颗粒状，主要位于巨噬细胞的胞膜和胞浆。在癌组织及癌周组织广泛分布。iNOS的表达也呈黄色或棕黄色颗粒，主要位于癌细胞的胞浆中，在癌细胞中表达比较明显，在癌周围及正常组织中较少表达。2．iNOS的表达与各项临床指标的关系iNOS在胰腺癌组中的表达阳性率为54％(19／35)评分值3.17±1.22，明显高于正常组(P＜0.01)。在胰腺癌各病理分级组和临床分期组之间的差别无统计学意义(P＞0.05)。淋巴结转移组的iNOS表达高于淋巴结未转移组，差别有统计学意义(P＜0.05)。3．TAMs计数与各项临床指标的关系胰腺癌中TAMs计数均值为112.27±24.57。在胰腺癌各病理分级组和临床分期组之间的差别均有统计学意义(P＜0.05)。淋巴结转移组中TAMs计数高于淋巴结未转移组，其差别有统计学意义(P＜0.05)。4．胰腺癌中iNOS的表达与TAMs计数之间的关系在胰腺癌中iNOS的表达和巨噬细胞计数呈直线正相关关系(γ=0.551，P＜0.05)。即随肿瘤组织内巨噬细胞计数的增加，iNOS的表达也随之增加。讨论胰腺癌是较为常见的恶性肿瘤。而血管新生是恶性肿瘤生长和转移的前提。iNOS是作用较强的促血管生长因子之一。本研究显示iNOS主要表达于胰腺癌组织中，癌周和正常组织较少表达。说明胰腺癌细胞含有大量iNOS以促进肿瘤血管形成。各临床分期组和病理分级组中iNOS的表达无明显差异，而转移组iNOS表达较未转移组明显增高，说明iNOS的表达与淋巴结转移有关，提示iNOS可能通过某种机制促进淋巴管生长，导致淋巴结转移的发生。目前发现在许多肿瘤组织内有大量的巨噬细胞浸润。本研究显示胰腺癌组织中有大量肿瘤相关巨噬细胞(TAMs)浸润，主要位于胰腺癌的间质中。各临床分期组和病理分级组中TAMs的表达均有明显差异，而且转移组TAMs计数均值也明显高于未转移组，说明TAMs与胰腺癌的分化转移有密切关系。TAMs也可能分泌促进肿瘤生长和转移的因子，参与肿瘤的生长和转移。TAMs计数越高，可能胰腺癌分化越低，越易发生转移。本研究还发现胰腺癌中iNOS的表达与TAMs计数呈密切正相关关系，提示iNOS的分泌与TAMs的浸润有着密切的关系，且均与胰腺癌的转移有关。而且有研究证实iNOS是TAMs浸润的诱导趋化因子。可能是癌细胞产生iNOS促进血管新生，诱导TAMs浸润，TAMs也产生iNOS促进肿瘤生长。但在胰腺癌中TAMs是否产生iNOS尚需进一步研究。本研究还发现TAMs的表达与胰腺癌的分期分级有关，iNOS的表达与胰腺癌的分期分级无关，说明iNOS和TAMs的表达是不完全相同的，可能还分别与其它因子的作用有关，而TAMs在胰腺癌的分化和转移可能会更为重要。在今后探索胰腺癌的治疗时抑制TAMs的浸润可能是一个有意义的方向。结论iNOS和TAMs的表达均与胰腺癌的淋巴结转移有密切关系，而且二者之间也存在密切联系。iNOS的表达与胰腺癌的分期分级无关，而TAMs的表达与胰腺癌的分期分级有关，说明TAMs在胰腺癌的分化和转移可能更为重要。更多还原

【Abstract】 Pancreatic cancer is a clinically common malignant tumor which is difficult to be detected at early stage and often encroach upon neighbour cells, blood vessels and nerves, even metastasize to lymphatics at advanced stage. During the proliferation and metastasis of malignant cells, iNOS is regarded as one of the most important factors. Not only does it directly contribute to the increase of vasopermeability and the proliferation of vascular endothelial cells, it is also an indispensable pro-vasculogenesis factor. Considerable amount of investigations showed that iNOS is expressed by almost all malignant tumours and is rarely seen in surrounding normal tissues. High iNOS expression is significant correlated to the infiltrative growth of tumors, plasma membrane involvement, lymphatic and liver metastasis, high recurrence rate and bad prognosis.Recent studies, however, suggested that macrophages infiltrated into the tumors (also known as tumor-associated macrophages, TAMs) are also closely related to lymphatic metastasis. In many tumors, especially solid tumors of epithelial tissue, i e. lung, mammary and stomach, there exist a great number of TAMs which substantially come from peripheral blood monouclear cells and partly formed by fixed tissue macrophage and which mainly infiltrate the ground substance of tumors and endothelium of vessels. Macrophages have antitumors ruction. Even given the fact that tumors are invaded by a great number of macrophages, they continue to growth. Therefore, TAMs may help to promote the development and advancement of tumors. The exact effects of TAMs on tumors are not very clear. Immunohistochemical staining and microvessel counts show that TAMs may be associated with the formation of tumor blood vessels. Other investigations show that the number of TAMs in oral squamous carcinoma is correlated to iNOS expression and TAMs themselves express iNOS. Schoppman et al. also gave evidence to the expression of iNOS in TAMs in carcinoma of cervix and the relation between TAMs and the density of lymphoducts around tumor and lymphatic metastasis. Thus iNOS produced by TAMs may play an important role in the generation of lymphoducts surrounding tumor. However, to the best of our knowledge, there’s no report on the relationship between iNOS and TAMs and lymphatic metastasis in pancreatic cancer. Therefore, in this report, we investigate it with immunohistologic methods.Methods and materialsA total of 35 patients who underwent surgical resection for pancreatic cancer in the First Affiliated Hospital of China Medical University between 1999 and 2005 were examined. Among them(age from 34 to 76, average 54.2), 24 were male and 11 were female. None of them underwent any therapy before operation. All surgical specimens were confirmed by pathology review with 19 cases of high-differentiated adenocarcinoma, 9 cases of middle-differentiated adenocarcinoma and 7 cases of lowdifferentiated adenocarcinoma. According to the clinical TNM staging, 8 were ofⅠstage, 12 ofⅡstage, 10 ofⅢstage and 5 ofⅣstage. 15 cases had lymphatic metastasis and 20 had no lymphatic metastasis. Six 39 to 75 year-old (average age 58.4) cases of normal pancreatic tissue were collected as negative control with 4 male and 2 female.All specimens went through anhydration, formalin fixation, paraffin imbedding, serial cross-sectioning of 4μm. After antigen retrieval, sections of normal pancreatic tissue and pancreatic cancer were stained with regular SP immunohistochemical methods. Macrophages were labeled by mouse anti-human CD68 monoclone antibody while iNOS by mouse anti-human iNOS monoclonal antibody. Positive and negative controls and HE staining were included in each experiment. The TAMs value of a case was defined as the average number of TAMs of 3 random high power field (×400) in the same carcinoma tissue under light microscope, iNOS scoring was used for the valuation of iNOS expression as counted by the sum of staining intensity score, with 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining in the relevant cell types and positive cell score with 0 for positive staining cells＜5% of all cells, 1 for 5～10%, 2 for 10～20%, 3 for 20～50%,and 4 for＞50%. The cases with iNOS score＜2 was defined as iNOS positive cases, those with iNOS score≥2 as iNOS negative cases.Statistical analyses were carried out using SPSS 12.0 statistical packages. Counts data are shown as means±SD. Differences between 2 groups were determined by t (t’)-test, those between 3 or more groups by one-way ANOVA(Newman-keuls q-test). Correlation between iNOS score and TAMs counts was also analyzed by analysis of covariance (Pearson correlation). Statistical significance was accepted at the P＜0.05 level.Results1. Results of immunohistochemical staining: Macrophages are scarcely seen in control group, while cells staining for CD68 show yellow or brown granules located on cell membrane or in cytoplasm extensively and unevenly distributed in carcinoma and surrounding tissues. Cells staining for iNOS also show yellow or brown granules in cytoplasm which mainly distribute in carcinoma cells and less present in normal tissues and tissues surrounding carcinoma.2. Relationship between iNOS expression and clinical data: iNOS positive rate (54%, 19/35) and iNOS score (3.17±1.22) of pancreatic cancer group are obviously higher than control group (P＜0.01). But there are no significant differences between patients with high, middle and low-differentiated carcinoma (P＞0.05) and among patients with different clinical stages (P＞0.05). iNOS expression in the group with lymphatic metastasis is significantly higher than that without lymphatic metastasis (P＜0.05).3. Relationship between TAMs counts and clinical data: Mean counts of TAMs in pancreatic cancer is 112.27±24.57. TAMs counts of low- differentiated group is higher than middle-differentiated group, and the latter is higher than high-differentiated group (P＜0.05). TAMs counts of lymphatic metastasis group is also significantly higher than that without metastasis (P＜0.05).4. Correlation between iNOS expression and TAMs counts in pancreatic cancer: In pancreatic cancer, iNOS expression showed a straight-line correlation with TAMs counts(γ=0.551, P＜0.05), i e, with the increase of TAMs, iNOS expression increases in carcinoma tissue.DiscussionPancreatic cancer is a common malignant tumor and vasculogenesis are prerequisite for the growth and metastasis of it. iNOS is one of the strongest and most specific pro-vasculogenesis factors. Our results show that iNOS is mainly expressed in pancreatic cancer and less in surrounding and normal tissues which may suggest carcinoma cells contain a great amount of iNOS which can promote the tumor to generate new vessels. There’s no significant difference in iNOS expression among different clinical stages and pathological degrees, but it is obviously higher in metastasis group than in non-metastasis group which suggest the association between iNOS expression and lymphatic metastasis in pancreatic cancer and that iNOS may be able to promote the growth of lymph vessel which in turn result in metastasis.Modern researches found that there are a great many macrophages in many kinds of tumors which is also the case in pancreatic cancer in our research. We found that the main part of macrophages infiltration in pancreatic cancer is within the stroma. The mean values of TAMs counts in pancreatic cancer are significantly different among the three different differentiated groups, and is the highest in low-differentiated group and the lowest in high-differentiated group. TAMs counts of metastasis group is also obviously higher than non-metastasis group. Thus TAMs may be closely related to the differentiation and metastasis of pancreatic cancer by secreting some factors or other mechanisms. The higher TAMs counts is, the poorer is pancreatic tumor differentiation, the easier is metastasis, and the worse is the prognosis.Our study also show that there is a straight-line relationship between iNOS expression and TAMs counts in pancreatic cancer, which suggest the close relationship between iNOS secretion and TAMs infiltration and the association between the former two and the metastasis of pancreatic tumor. Some research show that iNOS is the chemotatic factor to TAMs infiltration. Therefore, cancer cells may produce iNOS and induce vasculogenesis and TAMs infiltration, and TAMs may also produce iNOS to promote tumor growth, which become a infernal circle. But if TAMs in pancreatic cancer could produce iNOS remains to be elucidated. Our study also shows that TAMs counts is related to the stages and grades of pancreatic cancer, but iNOS expression is not. So, iNOS expression is not the same as TAMs expression, and they may separately related to other different factors. TAMs may be more important in the differentiation and metastasis of pancreatic cancer. It may be a promising way to prevent TAMs infiltration in the treatment of pancreatic cancer.ConclusionNot only expression of iNOS but also TAMs counts may relate to the lymphatic metastasis of pancreatic cancer. Moreover, the positive relationship exist between scores of iNOS expression and TAMs counts. TAMs counts are related to the clinical stages and pathological degrees of pancreatic cancer, but iNOS expression is not, which show that TAMs may be more important in the differentiation and metastasis of pancreatic cancer.更多还原