【作者】 陈本栋；

【导师】 杨广顺；

【作者基本信息】 第二军医大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 肝内胆管癌（Intrahepaticcholangiocarcinoma）是起源于胆管内皮细胞的恶性肿瘤。在肝胆外科肿瘤中发病率仅次于肝细胞癌。随着生活习惯的改变，胆管癌的发病率逐年升高。在1974年至1994年的20年间，胆管癌成为上海地区发病率增长最快的肿瘤，男、女发病率分别升高了119%和124%。【1】再加上胆管癌的恶性程度高，治疗难度大，成为危害人群健康的重要因素。目前，对于胆管癌的发病机理尚不清楚。大量研究表明一些肝胆系统疾病与胆管癌的发生关系密切，如原发性硬化性胆管炎（PCS）、肝内外胆管结石、胆管腺瘤、肝脏吸虫病、先天性胆管畸形如卡洛里病（Calori’sdisease）等，ICC的其他几个重要的危险因素，如丙肝及非酒精性脂肪肝有了明显的升高。国际学术界公认长期的胆管病变，导致各种因子刺激胆管上皮引发胆管上皮细胞的过度增生继而癌变是胆管癌形成的主要原因。【2】已有的研究表明胆管细胞增生调节与内分泌系统有着密切的联系。内分泌系统通过多种不同种类的激素对胆管细胞的增生起到很重要的调节作用，而胆管细胞增生和凋亡的失衡、恶性转变及胆管癌的产生也与内分泌系统的调节障碍有重要关系。与此相关，一些临床研究发现内分泌系统疾病特别是糖尿病亦参与胆管肿瘤的发生发展，并逐渐受到重视,近来，胡先贵等关于糖尿病是否增加胆管癌罹患的风险的Meta分析指出，肥胖、糖尿病可能是胆管癌（包括肝内胆管癌和肝外胆管癌）发病的危险因素。这些研究都表明，内分泌系统疾病与胆管系统疾病和胆管肿瘤有着重要的联系。而糖尿病作为我国的常见病和多发病，也势必对胆管肿瘤的发病率和临床治疗产生影响。然而，糖尿病和胆管癌之间关系的机理还不清楚。胰高血糖素样肽-1（Glucagon-likepeptide-1，GLP-1）是机体神经内分泌系统中重要的一员，而且在糖尿病的治疗领域也备受关注。GLP-1是由肠道上皮L型内分泌细胞所分泌的肽类激素。由于胰腺与胆管系统的胚胎起源相近，【18】近年来，GLP-1对胆管系统的作用也逐渐受到重视。然而，目前尚没有GLP-1对于肝内胆管癌的发生、发展的研究见于报道。胆管细胞的过度增生与神经内分泌系统有着紧密的联系，而临床上肝内胆管癌的发生、发展也与糖尿病等内分泌系统疾病密不可分。而GLP-1作为在两个领域的交点，对解答肝内胆管癌发生发展的问题意义重大。目前，对于GLP-1及其类似物Exendin-4的研究主要集中在糖尿病的治疗领域，而关于其对胆管系统疾病的作用尚未引起重视，在国内外诸多文献证实神经内分泌系统和内分泌疾病对于胆管癌发生发展的影响下，我们希望探讨神经内分泌激素GLP-1及其受体在人胆管癌组织中的表达，GLP-1及其类似物Exendin-4对胆管癌细胞的作用及细胞内信号传导路线，同时利用动物模型来评价GLP-1极其类似物Exendin-4在胆管癌的治疗领域中的意义。研究结果将有助于回答：糖尿病与胆管癌之间究竟通过怎样的机理相互联系？GLP-1在胆管系统疾病中起到了什么样的作用，是否和对胰岛β细胞的作用相似？GLP-1类似物Exendin-4在治疗糖尿病的同时对胆管系统有什么影响？能否为胆管癌的治疗提供新的思路？随着糖尿病和胆管癌发病率持续升高，该课题的开展具有重要的实际意义。因此，本研究从临床资料入手，收集63例肝内胆管癌细胞的临床病例资料，分析其病历资料特点，63例患者全部接受手术切除肝内胆管癌，其平均存活时间为21.6月，中位生存时间为18个月，63例患者中糖尿病的发生率为20.6%（13/63），是胆管癌发病的独立危险因素，单因素分析结果提示，影响预后的整体因素包括肿瘤的TNM分期，肿瘤的数目，肿瘤大小，是否有门静脉癌栓，是否有血管侵犯。多因素分析提示影响预后的独立危险因素为TNM分期，以及门静脉癌栓。同时选取肝细胞癌组织、正常肝组织作为对照，分析GLP-1R在各种组织中的表达，在63例胆管癌组织中，阳性表达率为55.6%肝细胞癌中的阳性表达率为19.2%，说明，GLP-1R在胆管细胞癌中的表达明显高于肝细胞癌和正常肝组织，我们也可能得出这样的结论：GLP-1可能为肝内胆管癌的一个潜在的Mark，或者一个治疗的新靶点。基于上述情况，我们选取人肝内胆管癌细胞系HuCCT1进行进一步的实验，实验中我们发现，GLP-1及其类似物Exendin-4对于肿瘤细胞具有一定的抑制增殖、促进凋亡的作用，进一步联合化疗药物奥沙利铂共同处理肿瘤细胞，发现Exendin-4具有对化疗药物的增敏作用，其肿瘤形成减少，凋亡增多。肿瘤侵袭迁移能力下降。裸鼠种植肝内胆管癌皮下瘤，荷瘤成功后，予以Exendin-4及奥沙利铂共同处理，发现，共同处理的荷瘤小鼠肿瘤体积有明显的缩小，且其组织表达肿瘤增殖的指标也有所下降。在裸鼠肝脏种植肝内胆肝癌移植瘤，分组进行药物调控，发现经过Exendin4处理过的肝内移植瘤，其增殖能力下降，而肿瘤凋亡能力上升。随着肥胖以及2型糖尿病患者的增多，GLP-1在这一领域的应用也逐渐增多。然而，这类药物对恶性肿瘤的影响还没有被研究透彻，受限于样本量和研究时间，我们还需要更长的时间来拿出更令人信服的证据。总之，我们的研究表明，肠促胰岛素GLP-1作为一个强有力的cAMP诱导剂，能够抑制肝内胆管癌细胞的增殖。GLPL-1水平下调也可能是引起肥胖、糖尿病患者发生肝内胆管癌的一个新的机制。更多还原

【Abstract】 Intrahepatic cholangiocarcinoma (ICC) is a rare malignant tumor which arises from the epithelial cells of intrahepatic bile ducts (beyond the second order bile ducts), and it has a global increasing trend in recent years. ICC is the second most common primary liver malignancy after hepatocellular carcinoma (HCC), accounting for10%to15%of all primary liver cancers. From1972to1994, cholangiocarcinoma was the most rapidly increasing malignancy in Shanghai, with an increase in incidence of119%in men and124%in women. The reason for the increasing incidence of ICC is not entirely understood. Several risk factors can predispose an individual to ICC, include primary biliary cirrhosis, primary sclerosing cholangitis, hepatolithiasis, choledochal cyst disease, the use of the radiological contrast agent thorotrast, parasitic biliary infestation (Clonorchis sinensis and Opisthorchis viverrini), Calori’s disease and hepatitis B. Other important risk factors for ICC, hepatitis C and nonalcoholic fatty liver disease (NAFLD), have been increasing in incidence. Recognized by the world that long-term bile duct lesions may be the main reason for the formation of cholangiocarcinoma.Previous studies have shown that the proliferation of cholangiocytes have close link with endocrine system. Endocrine system through a variety of different hormones on the proliferation of cholangiocytes, and the imbalance between proliferation and apoptosis, malignant transformation, formation of cholangiocarcinoma has closely relationship with endocrine system. Related to this, a number of clinical studies have found endocrine system diseases, especially diabetes also participate in the development of the cholangiocarcinoma. Recently, a mata anaysis from Hu et. about whether diabetes increase the risk of choalangiocarcinoma, shows that DM may be the risk factors of cholangiocarcinoma. These studies have shown that the endocrine system disease and bile duct disease and cholangiocarcinoma has closely link. Although diabetes mellitus (DM) has been shown to be risk factors for many cancers, the associations remain inconclusive for ICC.Glucagon like peptide1(GLP-1) is a gut-derived peptide secreted in a nutrient-dependent manner from the small intestine and stimulates glucose-dependent insulin production and secretion via specific receptors expressed on islet b cells. Similar embryonic origin of the pancreas and bile duct system, in recent years, GLP-1on the role of the bile duct system is also gradually be taken seriously. we demonstrated that activation of the GLP-1R in cholangiocytes by its selective agonist exendin-4prevents cholangiocyte apoptosis. Overall, our findings suggest that exendin-4is a molecule that is able to correct the dysregulated balance between cholangiocyte proliferation and apoptosis. Besides being relevant for the understanding of the pathophysiology of chronic cholestasis. While there is no recent study on relationship between GLP-1and intrahepatic cholangiocarcinoma. GLP-1as the intersection of the two areas, has significant meaning of the development of intrahepatic cholangiocarcinoma. Currently, the GLP-1and its analogs Exendin-4research mainly in the field of diabetes treatment, attention on diseases of the biliary system has not yet, in many domestic and international literature precisely the neuroendocrine system and endocrine diseases play an important role of the development of the cholangiocarcinoma, we hope to explore the role of neuroendocrine hormones GLP-1and its receptor expression in human cholangiocarcinoma, how the GLP-1and its analogs Exendin-4work on cholangiocarcinoma cell and intracellular signaling transmission line, while the use of animal models to evaluate the GLP-1analogue Exendin-4in significance in the field of treatment of cholangiocarcinoma extremely.Therefore, in our study63cases of intrahepatic cholangiocarcinoma who were underwent a surgical resection of intrahepatic cholangiocarcinoma, the average survival time of21.6months, the medianthe survival time of18months, among the63cases,13cases have been diagnosis of DM, diabetes is associated with an increased risk for ICC, for the univariate analysis results suggest that, overall prognostic factors including TNM stage, tumor number, tumor size, and portal vein thrombosis, multivariate analysis prompted an independent risk factor affecting the prognosis for TNM staging, as well as the number of tumors. Select hepatocellular carcinoma, normal liver tissue as a control, GLP-1R expression in various tissues, These results suggest that GLP-1R expression in cholangiocarcinoma was significantly higher than that of hepatocellular carcinoma and normal liver tissue, We may also conclude that:GLP-1may be a potential Mark for intrahepatic cholangiocarcinoma, or a treatment of a new target. We found that GLP-1and its analogs Exendin-4tumor cells with a certain degree of inhibition of proliferation, promote apoptosis, further combination chemotherapy drug oxaliplatin with tumor cells, we found that Exendin-4has a sensitizing effect of chemotherapy drugs, it can make tumor formation decreased, and apoptosis increased.With the increase in obesity and type2diabetes, GLP-1in this field of application is also gradually increasing. However, the impact of these drugs on malignant tumors has not been studied thoroughly, however, due to the limited of the sample size and study time, we also need a longer time to come up with more convincing evidence. In conclusion, our study shows that the incretin GLP-1as a strong cAMP inducer, can inhibit the proliferation of intrahepatic cholangiocarcinoma cells. Down regulation of the GLP-1may cause obesity, diabetes patients with a new mechanism of intrahepatic cholangiocarcinoma.更多还原