【目的】 胆囊癌是世界上最常见的胆道恶性肿瘤，胆囊癌预后差，易转移，但病因尚不明确。胆囊的慢性炎症，尤其是胆囊结石引起的慢性炎症是胆囊癌发生的重要原因。其致病的机制很可能是由于慢性炎症的持续影响。慢性炎症肿瘤微环境中肿瘤相关巨噬细胞(TAMs)在肿瘤的发生发展过程有重要的作用，而肿瘤相关巨噬细胞与胆囊癌发生的致癌机制仍有待阐明。本研究欲进一步探索肿瘤相关巨噬细胞在胆囊癌发生发展中的作用及其可能机制，以求为临床诊治提供新的思路和方向。 【方法】 1.为了研究M2型巨噬细胞浸润是否与胆囊癌的发生和进展有关，我们将2008年1月至2015年12月期间在中山大学孙逸仙纪念医院接受根治性切除术，术前未行放疗或化疗的78例胆囊癌患者组织标本和16例同期行胆囊切除术的胆囊息肉患者的正常胆囊组织行免疫组化染色，对标本中M2型巨噬细胞的数量计数。然后分析M2型巨噬细胞数量与胆囊癌患者的临床病理资料之间的关系。 2.将人单核细胞系THP-1并诱导分化为M0型和M2型巨噬细胞。然后将THP-1衍生的巨噬细胞在Boyden小室中与胆囊癌细胞株NOZ和GBC-SD细胞共培养以确定M2巨噬细胞是否能够促进胆囊癌细胞迁移和侵袭。进一步检测共培养后胆囊癌细胞中EMT标志物的表达情况明确M2型巨噬细胞的促进EMT作用。同时，我们检测了M2型巨噬细胞分泌的六种常见细胞因子，发现最丰富表达的因子是CCL18。再比较CCL18处理与M2型巨噬细胞共培养的胆囊癌细胞迁移和侵袭能力及EMT变化。 3.通过在共培养体系中添加CCL18中和抗体观察CCL18是否就是M2型巨噬细胞的促进胆囊癌EMT效应的原因。进一步检测共培养体系EMT常见通路PI3K／Akt通路的激活状态，并通过添加CCL18中和抗体和PI3K／Akt通路抑制剂LY294002以观察胆囊癌细胞的迁移和侵袭能力及EMT转录因子的变化。 【结果】 1.胆囊癌组织中M2型巨噬细胞的数量显著多于正常胆囊组织中的。结合患者的临床数据分析，M2型巨噬细胞高记数的胆囊癌患者与淋巴结转移数目更高，病理组织学分级较差，总体生存时间更短，提示肿瘤的侵袭性生物学特性更高。 2.与M2型巨噬细胞共培养可显著增强胆囊癌细胞NOZ和GBC-SD的迁移和侵袭能力，促进胆囊癌细胞的EMT。CCL18在M0型、M2型巨噬细胞分泌的六种常见促炎性细胞因子中的表达含量最高，而且M2型巨噬细胞分泌的CCL18水平较M0型更高。在M0型巨噬细胞与NOZ和GBC-SD细胞共培养体系中加入CCL18处理后可显著增强胆囊癌细胞NOZ和GBC-SD的迁移和侵袭能力。通过检测EMT下游蛋白表达情况，我们发现添加CCL18与M2型巨噬细胞共培养均可导致胆囊细胞NOZ和GBC-SD细胞中E-cadherin显著减少，N-cadherin、Vimentin和Fibronectin同时上调。与M2型巨噬细胞共培养可显著增强胆囊癌细胞NOZ和GBC-SD的迁移和侵袭能力，促进胆囊癌细胞EMT，而通过添加CCL18可起到近似的作用。 3.在M2型巨噬细胞与胆囊癌细胞共培养系统中添加CCL18中和抗体后，迁移和侵袭性胆囊癌细胞的数目显著减少，且EMT标志物E-cadherin表达减少， N-cadherin、Vimentin和Fibronectin的表达升高情况被显著逆转。提示M2型巨噬细胞分泌的CCL18可能介导了其促进胆囊癌细胞的EMT效应。加入CCL18抗体后还可以显著减少胆囊癌细胞EMT转录因子Snail和Slug表达，提示M2型巨噬细胞促进胆囊癌细胞EMT的作用可被CCL18中和抗体拮抗。PI3K／Akt通路的激活状态检测结果表明，与M2型巨噬细胞共培养的胆囊癌细胞Akt和GSK3 β的磷酸化显著增强，而在该共培养系统中添加CCL18中和抗体后可显著减弱Akt和GSK3 β的磷酸化，而且，添加LY294002后，与M2型巨噬细胞共培养的胆囊癌细胞迁移和侵袭能力均明显减弱。提示M2型巨噬细胞可通过PI3K／Akt通路影响胆囊癌细胞EMT。 【结论】 在本研究中，我们研究了M2型巨噬细胞在胆囊癌中的促进迁移和侵袭的作用。我们发现(1)胆囊癌组织中M2型巨噬细胞的数量显著多于正常胆囊组织中的。高数量的M2型巨噬细胞胆囊癌患者与淋巴结转移数目更高，病理组织学分级较差，总体生存时间更短。(2)胆囊癌细胞与M2型巨噬细胞共培养可显著促进癌细胞的迁移，侵袭和EMT。M2型巨噬细胞分泌的CCL18对胆囊癌细胞具有与M2型巨噬细胞相同的效果。(3)在共培养系统中阻断CCL18的功能可减轻M2型巨噬细胞的胆囊癌的细胞的迁移，侵袭和EMT。M2型巨噬细胞可通过分泌的CCL18活化胆囊癌细胞中的PI3K／Akt信号通路传导，从而导致胆囊癌细胞的迁移，侵袭和EMT。 我们的实验结果突显了M2型巨噬细胞在胆囊癌进展中的重要作用，阐明了M2型巨噬细胞分泌的CCL18可通过PI3K／Akt信号通路促进胆囊癌细胞的迁移，侵袭和EMT。这可能有助于发现针对胆囊癌治疗的新靶标。 关键词：胆囊癌，M2型巨噬细胞，CCL18，上皮间质转化，慢性炎症

 Aims Gallbladder cancer (GBC) is the most frequent malignancy of the biliary tract and the fifth most common gastrointestinal cancer worldwide.Generally, prognosis of GBC is extremely poor, However, the accurate pathogenesis of GBC remains unclear. It has been purposed that chronic cholecystitis especially which caused by cholelithiasis predisposes to GBC. The pathogenic mechanism is probably due to the persistent effects of chronic inflammation. It has been proposed that tumor associated macrophages(TAMs) in chronic inflammation microenvironment plays an important role in tumor initiation, progression and the metastatic process. However, to date, seldom study have explored the role of macrophages in GBC. In this study,we aim to explore the role of TAMs in the development of GBC and find out the possible mechanisms ,which may help to find new therapeutic target against GBC. Materials and methods l.To investigate whether M2 macrophage infiltration is associated with tumorigenesis and progression of GBC, we first assessed the number of M2 macrophages in 78 GBC and 16 normal gallbladder tissues obtained from gallbladder polyps patients. A total of 78 patients with GBC who underwent curative resection without prior radiotherapy or chemotherapy between January 2008 and December 2015 at the Sun Yat-Sen Memorial Hospital were enrolled in this study. In addition, 16 normal gallbladder tissues obtained from the gallbladders of 16 patients with gallbladder polyps who underwent cholecystectomy at the same period were selected. Immunohistochemical staining was performed in pathological section to count the number of M2 macrophages. The relationship between M2 macrophage counts and clinicopathological data in patients were analyzed. 2.Then the human monocyte cell THP-1 was induced differentiation into MO and M2 macrophages. THP-l-derived macrophages were then cocultured with NOZ and GBC-SD cells in Boyden chambers to determine whether M2 macrophages could promote GBC cells migration and invasion. The EMT markers expression of NOZ and GBC-SD cells coculturing with M2 macrophages to confirmed the role of M2 macrophages in promoting EMT. We therefore detected the expression of six common proinflammatory cytokines, secreted by M2 macrophages, in THP-l-derived M2 macrophages,CCL18 was the most abundantly expressed cytokine. The level of secreted CCL18 was significantly higher in M2 macrophage-conditioned media.The GBC cells migration,invasionand the expression of EMT marker in GBC cells were comparing from CCL18 treatment to M2 macrophage-conditioned media. 3.Anti-CCL18 antibody were employed to neutralize CCL18 function in the coculture supernatants toassessthat whether CCL18 was necessary for the pro-EMT effect of M2 macrophages on GBC cells. Examine the activation status of the PI3K/Akt pathway in the media after Anti-CCL18 antibody and PI3K inhibitor, LY294002employed to media. Measure the GBC cells migration,invasionand the expression of EMT transcription factorsin GBC cells. Results l.The counts of M2 macrophages in GBC tissues was significantly more than normal gallbladder tissues. Higher M2 macrophage level was associated with more aggressive tumor biology. Moreover, patients with higher M2 macrophages counts had poorer overall survival. Therefore, our histological results suggest an important role of M2 macrophages during GBC tumorigenesis and progression. 2.Coculturing with M2 macrophages significantly enhanced the migration and invasion capacities of NOZ and GBC-SD cells.A significant reduction of E-cadherin and a concurrent upregulation of N-cadherin, vimentin and fibronectin in NOZ and GBC-SD cells. Previous data suggest that M2 macrophages may promote migration and invasion of GBC cells through EMT. The level of secreted CCL18 was significantly higher in M2 macrophage-conditioned media, the most abundantly expressed cytokine secreted byTHP-1-derived M2 macrophages,.The GBC cells migration,invasionand the expression of EMT marker in GBC cells were mediate in CCL18 treatment andin M2 macrophage-conditioned media. 3.The number of migrated and invasive GBC cells was significantly reduced after addition of the anti-CCL18 antibody in the coculture system of M2 macrophages with GBC cells.Moreover, neutralization of CCL18 significantly reversed the downregulation of epithelial marker and the upregulation of mesenchymal marker induced by coculture of GBC cells with M2 macrophages.Coculture of GBC cells with M2 macrophages and neutralization of CCL18 significantly significant reduction of E-cadherin and a concurrent upregulation of N-cadherin, vimentin and fibronectin. These data indicates that M2 macrophages promote migration, invasionand EMT of GBC cells via CCL18. Coculture of GBC cells with M2 macrophages significantly enhanced the phosphorylation of Akt and GSK3β, while addition of the anti-CCL18 antibody in this coculture system significantly attenuated the phosphorylation of Akt and GSK3β.Thurthermore, the expression of phosphorylated Akt and EMT markers in GBC cells treated with CCL18 or cocultured with M2 macrophages were reversed by LY294002 (Fig. 5b). Furthermore, the migration and invasion capacities of GBC cells cocultured with M2 macrophages was inhibited by LY294002. These results suggest that CCL18 secreted from M2 macrophages could promote migration, invasionand EMT of GBC cells via PI3K/Akt signaling pathway. Conclusion We explored the oncogenic role of M2 macrophages, the most abundant immune-related stromal cells in cancer-related chronic inflammation, in GBC. We found that the average number of M2 macrophages was significantly more than those in normal gallbladder tissues. Higher M2 macrophages counts were associated with higher lymph node metastasis, advanced histological grading and shorter overall survival. Coculturing with M2 macrophages significantly promoted migration, invasion and EMT of GBC cells. Moreover, we found that CCL18 secreted from M2 macrophages had the same effect on GBC cells as M2 macrophages. Blocking the function of CCL18 with neutralizing antibody in coculture system attenuated the oncogenic effect of M2 macrophages. In addition, we found that secreted-CCL18 from M2 macrophages could activate PI3K/Akt signaling in GBC cells, thereby leading to migration, invasion and EMT of GBC cells. We confirmed the oncogenic role of M2 macrophages in human GBC tissue samples by using IHC and subsequently analyzing its clinicopathological significance. We also provided evidences that M2 macrophages promote GBC cells migration, invasion and epithelial-mesenchymal transition (EMT) via CCL18 and its downstream, Akt signaling pathway. These findings highlight the important role of M2 macrophages in GBC, which may help to find new therapeutic targetagainst GBC. Key words Gallbladder cancer, M2 macrophages, CCL18, Epithelial-mesenchymal transition, Chronic inflammation