【作者】 章必成；

【导师】 陈正堂；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2008， 博士

【摘要】 目前研究认为,活化的巨噬细胞是可以执行不同功能的复杂群体。其中,替代性活化的巨噬细胞(aaMphi)参与了细胞生长、血管生成、免疫抑制、组织修复和间质形成等过程。肺腺癌间质中有大量肿瘤相关巨噬细胞(TAM)浸润,且与其进展和转移密切相关,而经淋巴道转移是肺腺癌播散的主要途径之一。但是,肺腺癌间质中TAM是否存在替代性活化表型、该活化表型在淋巴管生成中有否作用及其机制尚不明确。本课题拟首先寻找人肺腺癌间质TAM呈替代性活化的证据;进而通过建立小鼠Lewis肺癌(LLC)移植瘤模型等实验明确aaMphi在小鼠肺腺癌淋巴管生成中的作用;最后,探讨aaMphi促进小鼠肺腺癌淋巴管生成的作用机制。本课题旨在为阐明肺腺癌的转移机制提供新的证据,为拮抗肺腺癌淋巴管生成提供新靶点。目的鉴定肺腺癌TAM的活化表型,探讨aaMphi促进小鼠肺腺癌淋巴管生成的机制。方法1、免疫组织化学SP法检测人肺腺癌组织中CD68、血管内皮生长因子(VEGF)-C和血管内皮生长因子受体(VEGFR)-3表达,并计数CD68阳性TAM、VEGF-C阳性指数和VEGFR-3阳性微淋巴管密度(LMVD),分析其相关性及与临床病理特征之间的关系。2、以CD68阳性作为TAM之标记,以CD68和巨噬细胞甘露糖受体(MMR)双阳性作为aaMphi之标记,以CD68和诱导型一氧化氮合酶(iNOS)双阳性作为经典活化的巨噬细胞(caMphi)之标记,采用双标免疫荧光染色和激光共聚焦显微镜鉴定人肺腺癌TAM的活化表型。3、以IFN-γ+LPS或IL-4处理小鼠RAW264.7巨噬细胞,分别建立caMphi或aaMphi模型;观察CpG+IL-10RA能否逆转aaMphi表型。将aaMphi与LLC细胞混合注射至小鼠颈部背侧皮下,建立移植瘤模型;免疫组织化学SP法检测并计数移植瘤LYVE-1阳性LMVD,HE染色检测淋巴结和远处器官转移情况;另取小鼠随机分组计算生存率。4、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,置于自制鼠尾胶包被的培养瓶(板)中培养。免疫荧光化学法检测LEC表达VEGFR-3和LYVE-1,3H-TdR掺入法检测LEC增殖活力,淋巴管形成实验判定LEC能否形成淋巴管样结构。5、采用transwell系统共培养aaMphi和小鼠LEC,绘制LEC生长曲线,细胞迁移实验观察LEC迁移,基质胶实验观察LEC管样结构形成;实时定量RT-PCR分别检测共培养前后aaMphi表达VEGF-C和VEGF-D mRNA,以及LEC表达VEGFR-3和Prox1 mRNA。之后,选择浓度为100ng/ml的VEGF-C建立aaMphi转分化系统;分别于第0、7、14和28天以实时定量RT-PCR法检测aaMphi表达VEGFR-3、Prox1和Fizz1;连续28天观察管样结构形成情况。最后,采用transwell系统共培养aaMphi和小鼠LLC细胞,免疫细胞化学检测LLC细胞表达VEGF-C,3H-TdR掺入法和transwell侵袭模型分别观察aaMphi对LLC细胞增殖和侵袭的影响。结果1、人肺腺癌TAM计数(103.36±15.31)、VEGF-C阳性指数(20.60±11.96)均明显高于肺良性病变(32.15±5.48,3.82±3.21)(P<0.01);人肺腺癌VEGFR-3阳性率(54.7%)和肺良性病变VEGFR-3阳性率(50.0%)之间无统计学差异(P>0.05),但前者VEGFR-3阳性LMVD(11.56±10.73)明显高于后者(4.29±4.18)(P<0.01)。人肺腺癌TAM计数、VEGF-C阳性指数均与淋巴结转移、P-TNM分期密切相关,VEGFR-3阳性LMVD只与淋巴结转移密切相关。人肺腺癌TAM计数和VEGF-C阳性指数、VEGFR-3阳性LMVD之间均呈正相关(r=0.338,P<0.05;r=0.410,P<0.01);VEGF-C阳性指数和VEGFR-3阳性LMVD之间也呈正相关(r=0.653,P<0.01)。2、40例人肺腺癌组织中TAM均同时表达CD68和MMR,MMR阳性巨噬细胞占全部TAM的74.7~98.2%;其中,有3例肺腺癌TAM同时表达CD68和iNOS,但iNOS阳性巨噬细胞占全部TAM的比例仅为2.4~12.7%。在10例肺良性病变中,8例同时表达CD68和iNOS,2例仅表达CD68;未见有同时表达CD68和MMR的病例。3、成功地建立了小鼠caMphi和aaMphi模型,且CpG+IL-10RA能逆转aaMphi为caMphi。与空白对照组比较,aaMphi组和RAW264.7组移植瘤LMVD增高(14.80±3.64,10.50±3.17),淋巴结转移数较多(10.80±1.32,7.30±0.95)(P<0.01),双肺转移结节数增多(32.50±4.91,29.30±4.42)(P<0.01),荷瘤小鼠生存率降低(P<0.01);其中,aaMphi组LMVD和淋巴结转移数明显高于RAW264.7组(P<0.05),但双肺转移结节数和荷瘤小鼠生存率之间无统计学差异(P>0.05)。caMphi组和aaMphi+CpG+IL-10RA组移植瘤未见LYVE-1表达和淋巴结转移,双肺转移结节数较少(5.80±1.15,5.17±1.28) (P<0.01),荷瘤小鼠生存率提高(P<0.01)。4、IFA能诱导小鼠腹腔淋巴管瘤形成,所获LEC活细胞数大于98%,并高表达VEGFR-3和LYVE-1。在自制鼠尾胶包被的培养瓶(板)中,LEC生长状况良好,3H-TdR掺入率明显增加;在鼠尾胶凝胶中,LEC能形成淋巴管样结构。5、与aaMphi共培养后,小鼠LEC增殖、迁移和管样结构形成能力均明显高于空白对照组、caMphi组和RAW264.7细胞组。与共培养前比较,aaMphi表达VEGF-C明显增加(P<0.01),但表达VEGF-D无明显变化(P>0.05);LEC表达VEGFR-3和Prox1均明显增加(P<0.05~0.01)。建立转分化系统之后,小鼠aaMphi表达VEGFR-3和Prox1逐渐增加,而表达Fizz1逐渐下降,至第14天分别达到最高值和最低值;第28天和第14天的情况无明显差别。随着时间推移,aaMphi在基质胶中逐渐形成明显的管样结构。在4个共培养组中,aaMphi组小鼠LLC细胞表达VEGF-C最强,增殖速度最快,侵袭能力最强,RAW264.7组次之;而与空白对照组相比,caMphi组LLC细胞表达VEGF-C无明显变化,增殖和侵袭则受到抑制。结论1、TAM与人肺腺癌组织VEGF-C表达和淋巴管生成呈正相关,可能促进了肺腺癌淋巴结转移。2、人肺腺癌组织中TAM呈现出替代性活化的表型特征。3、aaMphi能促进小鼠LLC移植瘤淋巴管生成、淋巴结和肺转移,并降低荷瘤小鼠生存率。在形成移植瘤的过程中,未经处理的巨噬细胞可以朝着aaMphi的方向极化。CpG+IL-10RA能通过逆转aaMphi表型并最终拮抗小鼠肺腺癌淋巴管生成。4、以自制鼠尾胶为贴黏剂的体系是一种简便、廉价和稳定的小鼠LEC体外培养体系。5、aaMphi能够通过促进LEC增殖、迁移和管样结构形成,直接转分化为LEC和上调LLC细胞表达VEGF-C等三种方式促进小鼠肺腺癌淋巴管生成。更多还原

【Abstract】 Nowadays, activated macrophages are considered as complicated groups which can execute distinct functions. Among them, alternatively activated macrophages (aaMphi) participate in the process of cell growth, angiogenesis, immunosuppression, tissue repair and stroma formation. There is a great quantity of tumor-associated macrophages (TAM) infiltrating in the stroma of human lung adenocarcinoma, which are closely related to tumor development and metastasis. Lymphatic metastasis is one of the major routes of lung adenocarcinoma dissemination. However, the activated phenotypes of TAM and their roles in lymphangiogenesis remain unknown. This thesis will i) identify TAM in human lung adenocarcinoma to be aaMphi, ii) investigate the roles of aaMphi in lymphangiogenesis of lung adenocarcinoma based on the models of mouse Lewis lung carcinoma (LLC) transplantation tumors, iii) explore the possible mechanisms of the induction of lymphangiogenesis by aaMphi in lung adenocarcinoma. This research shows new approach for the lymphangiogenesis in lung adenocarcinoma and thus provides a new target for the inhibition of the metastasis of this kind of malignant carcinoma.ObjectiveTo identify the activated phenotypes of TAM in lung adenocarcinoma, and to explore the possible mechanisms by which aaMphi induce lymphangiogenesis in mouse lung adenocarcinoma.Methods1. The expression of CD68, vascular endothelial growth factor (VEGF)-C and vascular endothelial growth factor receptor (VEGFR)-3 was detected by immunohistochemistry (SP method) in human lung adenocarcinoma, respectively, and then CD68 positive TAM counts, VEGF-C positive indexes and VEGFR-3 positive lymphatic microvessel density (LMVD) were counted for the analysis of the correlations among them and their relationships with clinicopathological features.2. Since CD68 being the marker of TAM, co-expression of CD68 and macrophage mannose receptor (MMR) was used as the marker of aaMphi, and co-expression of CD68 and inducible nitric oxide synthase (iNOS) was used as the marker of classically activated macrophages (caMphi). The activated phenotypes of TAM in human lung adenocarcinoma were detected by double-labelled immunofluorescence staining and observed by laser confocal microscopy.3. To establish the models of caMphi or aaMphi, RAW264.7 macrophages were treated by IFN-γ+LPS or IL-4, respectively. Then, whether the reversion of aaMphi phenotype by CpG+IL-10RA or not was investigated. The models of mice LLC transplantation tumors were established via subcutaneous injection of LLC cells and aaMphi. The LYVE-1+ LMVD were detected by immunohistochemistry (SP method), meanwhile, the numbers of metastatic lymph nodes and nodes in distant organs were detected by HE staining. Furthermore, another 50 mice were monitored for survival rates.4. Mice lymphangiomas in abdominal cavity were induced by intraperitoneal injection of incomplete Freund’s adjuvant (IFA). LEC were obtained from the induced lymphangiomas after disruption and digestion, and then were cultured in the flask or plate previously coated with self-made rat-tail collagen. Expressions of VEGFR-3 and LYVE-1 were identified by immunofluorescence. The proliferation activity of LEC was evaluated by 3H-TdR incorporation efficiency. The capability of LEC to form lymphatic vessel-like structures was assessed by the in vitro lymphatic vessel formation assay.5. Based on the co-culture systems of aaMphi and murine LEC, cell growth curve of LEC was drawn, and migration assay and matrigel assay were used to observe the effect of aaMphi on the migration and tube-like structure formation of LEC. Expressions of VEGF-C and VEGF-D mRNA in aaMphi, VEGFR-3 and Prox1 mRNA in LEC were detected by real time quantitative RT-PCR. Then, 100ng/ml VEGF-C was selected for establishing the aaMphi transdifferentiation system. The mRNA expression of VEGFR-3, Prox1 and Fizz1 in aaMphi were detected by real time quantitative RT-PCR on the day 0, 7, 14 and 28 following VEGF-C stimulation, respectively. During the 28 days, tube-like structure formation was observed by inverted phase contrast microscope continuously. Finally, based on the aaMphi-LLC cells co-culture, VEGF-C expressions in LLC cells were detected by immunocytochemsitry. Besides, 3H-TdR incorporation and transwell invasion model were used to observe the effect of aaMphi on the proliferation and invasion of LLC cells, respectively.Results1. TAM counts and VEGF-C positive indexes in lung adenocarcinoma were (103.36±15.31) and (20.60±11.96), which were both higher than those in lung benign lesion (32.15±5.48, 3.82±3.21), respectively (P<0.01). There was no significant difference in VEGFR-3 positive rates between lung adenocarcinoma (54.7%) and lung benign lesion (50.0%) (P>0.05), but VEGFR-3 positive LMVD was significantly higher in the former (11.56±10.73) than in the latter (4.29±4.18) (P<0.01). CD68 positive TAM counts, VEGF-C positive indexes were related to lymph node metastasis and P-TNM staging, but VEGFR-3 positive LMVD was only related to lymph node metastasis. Close correlations were found between TAM counts and VEGF-C positive indexes, VEGFR-3 positive LMVD, respectively (r=0.338, P<0.01; r=0.410, P<0.01), and also found between VEGF-C positive indexes and VEGFR-3 positive LMVD (r=0.653, P<0.01).2. Co-exprssion of CD68 and MMR of TAM were detected in all the 40 lung adenocarcinoma specimens, and the percentages of MMR positive macrophages in TAM were 74.7~98.2%. Among the 40 cases, co-exprssion of CD68 and iNOS of TAM were detected in 3 cases, and the percentages of iNOS positive macrophages in TAM were only 2.4~12.7%. Among the 10 cases with lung benign lesion, 8 cases co-expressed CD68 and iNOS, 2 cases only expressed CD68, and there was no case which co-expressed CD68 and MMR.3. Mouse caMphi and aaMphi models were established successfully, and aaMphi could be reversed into caMphi by CpG+IL-10RA. Compared to blank control group, aaMphi group and RAW264.7 group have increased LMVD in transplantation tumors (14.80±3.64, 10.50±3.17), enhanced number of metastatic lymph nodes (10.80±1.32, 7.30±0.95) (P<0.01), incremented quantity of metastatic nodes in double lungs (32.50±4.91, 29.30±4.42) (P<0.01), and decreased survival rates of tumor-bearing mice (P<0.01). Besides, the number of metastatic lymph nodes and LMVD in aaMphi group were higher than those in RAW264.7 group (P<0.05), but there was no significant difference in the metastatic nodes in double lungs and the survival rates of mice between them (P>0.05). In caMphi group and aaMphi+CpG+IL-10RA group, LYVE-1 expression in transplantation tumors and lymph node metastasis were hardly detected, the metastatic nodes in double lungs were less (5.80±1.15, 5.17±1.28) (P<0.01), and the survival rates of tumor-bearing mice increased (P<0.01).4. Mice lymphangiomas in abdominal cavity were induced successfully by IFA, and more than 98% living LEC were harvested. The expression of VEGFR-3 and LYVE-1 were positive in LEC. In the rat-tail collagen coated flask or plate, LEC grew well, with 3H-TdR incorporation efficiency increasing obviously. In the gel formed by rat-tail collagen, LEC could form lymphatic vessel-like structures.5. After co-culture, the ability of LEC to proliferate, migrate and form tube-like structure in aaMphi group was significantly stronger than that in blank control, caMphi, and RAW264.7 groups. The expression of VEGF-C mRNA in aaMphi, VEGFR-3 and Prox1 mRNA in LEC increased significantly after co-culture (P<0.05~0.01), however, there was no significant change in VEGF-D mRNA level in aaMphi (P>0.05). After the aaMphi transdifferentiation system was established, the expression of VEGFR-3 and Prox1 mRNA in aaMphi incresesd greatly, while the Fizz1 mRNA in aaMphi decreased. On day 14, VEGFR-3 and Prox1 mRNA reached the peak value, and Fizz1 mRNA reduced to the minimum level. There was no significant difference in the expression of VEGFR-3, Prox1 and Fizz1 mRNA between on the day 28 and day 14. With the time passed, aaMphi could form tube-like structure in matrigel. Among the four macrophages-LLC cells co-culture groups, aaMphi group has the highest VEGF-C expression, fastest growth velocity, and strongest invasion capability of LLC cells, with RAW264.7 group ranking the secondary. Compared to blank control group, VEGF-C expression in LLC cells of caMphi group has no change, and the proliferation and invasion of LLC cells were inhibited.Conclusions1. TAM are positively correlated to VEGF-C expression and lymphangiogenesis, and probably contribute to lymph node metastasis in human lung adenocarcinoma.2. TAM in human lung adenocarcinoma is proved to be one kind of aaMphi.3. aaMphi can promote the lymphangiogenesis, lymph node and lung metastasis of LLC transplantation tumors, and reduce the tumor-bearing mice survival. In the progression of transplantation tumors, the function of RAW264.7 cells may be polarized toward that of aaMphi. CpG+IL-10RA can inhibit aaMphi-induced lymphangiogenesis by the reversion of aaMphi phenotype.4. Mouse LEC culture system using rat-tail collagen as adhesives is a convenient, cheap and stable culture system.5. aaMphi can induce lymphangiogenesis in lung adenocarcinoma by the means of promoting the proliferation, migration and tube-like structure formation of LEC, transdifferentiating into LEC, and promoting the expression of VEGF-C in LLC cells.更多还原