SPINK1调控肝细胞癌复发和转移的机制研究

发布时间：2018-06-08 03:20

本文选题：丝氨酸蛋白酶抑制因子Kazal + 1型　；参考：《南京医科大学》2017年博士论文

【摘要】：背景:原发性肝癌是世界上常见且死亡率较高的肿瘤之一。根据世界卫生组织GLOBOCAN 2008数据统计,每年有748,300人次被检测出罹患肝癌,男性的发病率超过女性的二倍以上,每年有695,900人次死于肝癌。中国每年的新发病例和死亡病例占到全球的一半以上,原发性肝癌是中国第二高发生率和死亡率的肿瘤。肝细胞癌(hepatocellular carcinoma,HCC)是原发性肝癌最主要的病理学类型,占到所有肝癌的75-80%。尽管外科手术切除和肝脏移植是治疗肝癌的主要方法,但是仍然有75%的患者在术后5年内复发和/或转移。这往往是导致患者死亡的主要因素。因此,开展研究肝细胞癌的复查和转移机制显得十分重要。既往研究发现,正常生理情况下,丝氨酸蛋白酶抑制因子Kazal1型(serine protease inhibitor Kazal type 1,SPINK1)由胰腺的腺泡细胞分泌,起到抑制胰蛋白酶原过度激活,保护胰腺组织的作用。另一方面,SPINK1也是一种潜在的肿瘤标志物,在卵巢癌、前列腺癌、结直肠癌等肿瘤中表达。血清中SPINK1过表达和肿瘤组织中SPINK1过表达预示着不良预后。基因组学研究发现在遗传性血色素沉着相关的肝癌中SPINK1过度表达。SPINK1的结构较表皮生长因子(EGF)相似,有50%的同源性,与表皮生长因子受体(EGFR)亲和力是EGF的一半。因此,SPINK1被认为在肿瘤的发生发展中可能起到生长因子的作用。所以,本文就SPINK1在肝癌进程中所扮演的角色以及机制展开进一步研究。目的:研究人肝细胞癌中SPINK1的表达与预后的关系,探索SPINK1调控肝癌细胞转移侵袭的分子机制。方法:1、临床收集肝癌根治术患者的组织标本80例,构建组织芯片,免疫组化检测人肝癌组织中SPINK1表达含量,根据染色强度以及染色面积进行评分。结合临床资料以及随访资料,用统计学方法分析肝癌患者肝癌组织中SPINK1表达量、血管侵犯、包膜完整度、肿瘤分化程度、TNM分期、单发/多发病灶间等之间的关系,探究SPINK1与肝细胞癌预后的相关性。2、构建siRNA-SPINK1基因慢病毒载体,鉴定测序正确后,进行病毒的包装和滴度检测并转染MHCC97-H,MHCC97-L细胞,荧光显微镜观察细胞荧光含量,Western blotting鉴定转染前后肝癌细胞中SPINK1蛋白表达以及EMT标志蛋白E-caherin、vimentin表达量。应用Transwell(体外小室迁徙+侵袭)、划痕实验方法检测各组细胞细胞迁移和侵袭能力。3、将人重组SPINK1 蛋白(rSPINK1)10ng/ml加入MHCC97-H,MHCC97-L的培养基中,培养24小时,观察细胞形态变化,Western blotting检测各组细胞SPINK1蛋白表达以及EMT标志蛋白E-caherin及vimentin表达量。应用Transwell(体外小室迁徙+侵袭)、划痕实验方法检测各组细胞细胞迁移和侵袭能力。4、检测筛选信号通路:Western blotting检测MEK/ERK信号通路相关蛋白MEK,p-MEK,ERK,p-ERK在以上各组细胞中表达的变化。5、信号通路抑制剂实验:将U0126(MEK/ERK通路抑制剂)加入rSPINK1处理过的细胞中,Transwell、划痕实验检测再次检测细胞的侵袭力和转移力,观察U0126是否可以抑制了rSPINK1带来的细胞运动能力的增强作用;Western blotting检测相关通路的蛋白水平变化;结合以上实验结果,进一步阐明SPINK1调节肝癌细胞迁移的机制。结果:1、肝癌组织中SPINK1的表达高低与肝癌病理分级、TNM分期、是否存在门脉癌栓的差异有统计学意义。SPINK1高表达组的患者总生存时间低于低表达组,术后复发率高于低表达组。SPINK1表达高低是肝癌患者总生存时间和术后复发率是一个独立风险因素。2、上调肝癌细胞SPINK1蛋白含量可以促进肝癌细胞由上皮细胞表型向间质细胞表型转换(EMT),上皮标志物E-Cadherin表达水平下调,间质标志物vimentin表达上调,同时伴有肝癌细胞转移能力和侵袭能力的增强。抑制肝癌细胞中SPINK1的表达可以抑制肝癌细胞的转移和侵袭能力,同时伴有E-Cadherin上调,vimentin 下调。3、上调肝癌细胞中SPINK1含量激活MEK/ERK信号通路,抑制肝癌细胞中SPINK1的表达可以抑制MEK/ERK信号通路;加入U0126抑制MEK/ERK通路激活,可以抑制SPINK1引起的EMT的相关标志物蛋白含量改变以及对细胞侵袭迁移增强作用。结论:SPINK1高表达是影响肝癌预后的独立危险因素;SPINK1通过MEK/ERK信号转导途径诱导肝癌细胞发生EMT,促进肝癌细胞转移和侵袭。
[Abstract]:Background: primary liver cancer is one of the most common and high mortality tumors in the world. According to the data of the WHO GLOBOCAN 2008, 748300 people have been detected each year with liver cancer, the incidence of men is more than two times more than that of women, and 695900 people die of liver cancer each year. New cases and deaths in China each year are accounted for. To more than half of the world, primary liver cancer is the second high incidence and mortality rate in China. Hepatocellular carcinoma (HCC) is the most important pathological type of primary liver cancer, which accounts for the 75-80%. of all liver cancer, although surgical resection and liver transplantation are the main methods for the treatment of liver cancer, but there are still 75% patients. The recurrence and / or metastasis of the patients within 5 years after the operation is a major factor leading to the death of the patients. Therefore, it is important to conduct a review and transfer mechanism of hepatocellular carcinoma. Previous studies have found that the serine protease inhibitor Kazal1 (serine protease inhibitor Kazal type 1, SPINK1) is from the pancreas under normal physiological conditions. Acinar cells are secreted to inhibit the overactivation of trypsinogen and protect pancreatic tissue. On the other hand, SPINK1 is also a potential tumor marker, expressed in ovarian, prostate, and colorectal cancer. SPINK1 overexpression in serum and SPINK1 overexpression in tumor tissues indicate poor prognosis. Genomics study The structure of SPINK1 overexpression of.SPINK1 in hereditary hemochromatosis related liver cancer was found to be similar to that of epidermal growth factor (EGF), with 50% homology and half of the epidermal growth factor receptor (EGFR) affinity as EGF. Therefore, SPINK1 was considered to play a role in the growth factor in the development of the tumor. Therefore, this article is on SPINK 1 the role and mechanism of the liver cancer process to be further studied. Objective: To study the relationship between the expression of SPINK1 and the prognosis in human hepatocellular carcinoma (HCC), and to explore the molecular mechanism of SPINK1 regulating the metastasis and invasion of hepatoma cells. Methods: 1, 80 tissue specimens were collected from the patients with radical hepatectomy, and the tissue microarray was constructed and the immunohistochemistry was used to detect people. The expression of SPINK1 in liver cancer tissue was evaluated according to the intensity and area of dyed. Combined with clinical data and follow-up data, the expression of SPINK1 expression, vascular invasion, envelope integrity, tumor differentiation, TNM phase, single / multiple lesions were analyzed by statistical methods. SPINK1 The correlation with the prognosis of hepatocellular carcinoma (HCC).2, the siRNA-SPINK1 gene lentivirus vector was constructed and the sequencing was correctly identified. The virus packaging and titer detection were carried out and MHCC97-H, MHCC97-L cells, fluorescence microscopy were used to observe the fluorescence content of the cells. Western blotting was used to identify the expression of SPINK1 protein in the liver cancer cells before and after the transfection as well as the EMT marker protein E-ca. Herin, vimentin expression. Use Transwell (in vitro migration + invasion), scratch test method to detect cell cell migration and invasion ability.3, the recombinant SPINK1 protein (rSPINK1) 10ng/ml is added to MHCC97-H, MHCC97-L medium, culture for 24 hours, observe cell morphological changes, Western blotting detection of each group of cell SPINK1 protein Expression and expression of EMT marker protein E-caherin and vimentin. Use Transwell (in vitro migration + invasion), scratch test method to detect cell cell migration and invasion ability.4, and detect the screening signal pathway: Western blotting detection of MEK/ERK signaling pathway related proteins MEK, p-MEK, ERK, p-ERK in the above groups of cells expression of the changes .5, signal pathway inhibitor experiment: adding U0126 (MEK/ERK pathway inhibitor) into rSPINK1 treated cells, Transwell, scratch test to detect the invasiveness and transfer force of cells again, and observe whether U0126 can inhibit the enhancement of cell motility caused by rSPINK1; the protein of the Western blotting detection pathway In combination with the above experimental results, we further elucidate the mechanism of SPINK1 regulating the migration of hepatoma cells. Results: 1, the expression of SPINK1 in the liver cancer tissues and the pathological classification of liver cancer, TNM staging, and whether there is a significant difference between the portal vein tumor thrombus and the portal vein, the total survival time of the.SPINK1 high expression group is lower than that of the low expression group, and the postoperative recurrence rate is lower. Higher than low expression group.SPINK1 expression level is an independent risk factor.2 for the total survival time and postoperative recurrence rate of liver cancer patients. The up-regulation of SPINK1 protein content in hepatoma cells can promote the phenotype transformation of hepatoma cells from epithelial cells to interstitial cells (EMT), the level of E-Cadherin expression of epithelial markers, and the vimentin table of interstitial markers. The expression of SPINK1 in hepatoma cells can inhibit the metastasis and invasion of hepatoma cells, accompanied by up regulation of E-Cadherin, vimentin down.3, up regulation of SPINK1 content in hepatoma cells and the activation of MEK/ ERK signaling pathway to inhibit the expression of SPINK1 in hepatoma cells. The MEK/ERK signaling pathway can be inhibited and the activation of MEK/ERK pathway by adding U0126 can inhibit the changes in the protein content of the related markers of EMT induced by SPINK1 and the enhancement of cell invasion and migration. Conclusion: the high expression of SPINK1 is an independent risk factor affecting the prognosis of liver cancer; SPINK1 can induce hepatoma cells to be induced by MEK/ERK signal transduction pathway. EMT, which promotes the metastasis and invasion of hepatoma cells.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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