【作者】 蔡磊；

【导师】 董家鸿；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 研究背景与目的:肝细胞性肝癌(Hepatocellular carcinoma,HCC)是目前最常见的恶性肿瘤之一,为当今世界第三位致死性肿瘤,占我国肿瘤死因的第二位,全世界约有100万患者,每年约有30万新增病例。在新增的病例中,45%发生在中国,主要分布在东南及沿海流域。近年来,随着乙型肝炎病毒的流行,我国肝癌的地域分布及发病趋势发生明显变化。其发病年龄高峰移向青壮年,死亡率也向小年龄组推移。目前对肝细胞癌的治疗主要基于外科手术切除,切除后使用相关的化疗药物对肝细胞癌的作用非常有限,因此研究原癌基因对肝细胞癌增殖及侵袭的影响,对进一步干预治疗,有非常重要的意义。c-Kit是编码受体酪氨酸激酶家族的原癌基因。目前研究表明,c-Kit与多种肿瘤的发生、发展、预后都有着密切的联系,如胃肠道间质瘤、急性髓细胞白血病、肺癌等多种人类肿瘤组织中均存在c-Kit受体的异常表达。针对这些高表达c-Kit受体的肿瘤,应用酪氨酸激酶抑制剂治疗恶性肿瘤已经取得了一定的临床疗效。目前,肝细胞癌中c-Kit受体表达的研究,国内外鲜有报道,KIT/SCF信号通路究竟在肝细胞癌中起怎样的作用,需要深入研究。通过查阅文献,我们收集到以下信息:首先,c-Kit原癌基因的表达上调,在多种肿瘤中都提示患者预后较差;其次,c-Kit在正常肝脏中,仅出现在汇管区的部分卵圆细胞中,而在肿瘤中的分布并不均匀,有研究报道其表达可能在肝硬化-肝癌病程进程中发生变化;第三,KIT/SCF信号的下游的部分信号通路中的PI3K/AKT信号通路,与经典的Wnt通路有密切联系,而Wnt通路是肝细胞癌侵袭转移的重要途径之一。基于以上理论,我们得出假设:KIT/SCF信号通路被激活后,可能引起下游的信号通路发生相应变化,从而引起肝细胞癌的增殖及侵袭转移特性增强。本文重点是验证上述假设,探讨c-Kit对肝细胞癌增殖及侵袭性影响及其分子机制。材料与方法:1.通过免疫组化方法在89例临床肝细胞癌组织中检测c-Kit的表达,探讨其与各临床病理因素间的关联;在不同侵袭、转移潜能的同系肝癌细胞株MHCC-97L和MHCC-97H中,利用Realtime-PCR及Western-blot方法检测其c-Kit及Kit的基因和蛋白表达,以明确其与侵袭、转移能力的关系;2.利用Adeasy系统,构建c-Kit重组腺病毒载体,用其转染Hep-G2肝癌细胞,通过Western-blot、侵袭实验、运动实验、裸鼠成瘤实验等方法,观察过表达c-Kit对肝癌细胞侵袭、运动能力的影响;3.利用外源性的c-Kit受体的配体SCF添加入SMMC-7721肝癌细胞,观察c-Kit/SCF信号系统对对β-catenin分布情况的影响;并研究c-Kit/SCF信号系统对β-catenin分布情况的影响是否是通过PI3K/AKT信号系统实现的,以及研究在此过程中PI3K/AKT信号系统中关键因子AKT,GSK-3β磷酸化情况;4.通过免疫组化方法在89例临床肝细胞癌组织中检测FAK的表达,探讨其与各临床病理因素间的关联,并通过统计学分析明确FAK与c-Kit的相关性;利用免疫共沉淀的方法,检测SMMC-7721和Hep-G2肝癌细胞中FAK与KIT蛋白的结合情况。结果及结论:1.在临床HCC组织标本及不同侵袭、转移潜能的肝癌细胞中检测c-Kit表达,我们得出如下结果:1.1. c-Kit的表达在肝细胞癌中显著增高;1.2. c-Kit的表达程度与肿瘤TNM分期、血管侵袭、HBsAg和HBV DNA等临床病理因素均呈显著正相关,提示c-Kit表达与HCC的进展可能有密切关系,其表达随着肿瘤的进展而上调;1.3.在同系中分离出的不同侵袭、转移潜能的细胞株中,高侵袭、转移潜能的肝癌细胞株MHCC-97H中c-Kit的表达明显高于低侵袭、转移潜能的MHCC-97L,提示了c-Kit的表达随着肿瘤侵袭、转移能力的增强而上调;2.成功构建了携带c-Kit基因的重组腺病毒载体,并能够高效转染Hep-G2肝癌细胞,目的基因得到高表达;外源性转染c-Kit基因,可以提高Hep-G2肝癌细胞的侵袭、运动能力。3.当外源性的c-Kit受体的配体SCF添加入SMMC-7721肝癌细胞后,PI3K/AKT信号系统被激活,其关键因子AKT及GSK-3β发生明显的磷酸化,β-catenin分布明显向细胞核内转移,加入KIT/SCF信号通路的特异性抑制剂Imatinib后,β-catenin无法向细胞核内转移;4.在临床HCC组织标本及不同侵袭、转移潜能的肝癌细胞中检测FAK表达,并分析FAK与c-Kit的相关性;利用免疫共沉淀的方法,检测SMMC-7721肝癌细胞中FAK与KIT蛋白的结合情况。4.1. FAK的表达在肝细胞癌中明显增高;4.2. FAK的表达与血管侵袭、肿瘤TNM分期、HBsAg和HBV DNA水平等临床病理因素均呈显著正相关。提示FAK表达与HCC的进展有密切关系,其表达随着肿瘤的进展而上调;同时FAK与c-Kit的在肝癌细胞中的表达密切相关;综上所述,本研究主要结论如下:1. c-Kit基因在HCC中的表达与肿瘤的进展密切相关,其表达上调,暗示着肿瘤的预后不良;外源性转染c-Kit基因可增强HCC的侵袭、运动、成瘤能力。2. c-Kit基因是肝细胞癌中的重要基因,它可以通过激活PI3K/AKT信号通路,以及与FAK发生协同作用,增加HCC的侵袭性。3. KIT/SCF信号通路的抑制剂Imatinib可能对HCC的发生发展起到保护性的作用。4.KIT蛋白在肝癌细胞中,可以特异性结合FAK。更多还原

【Abstract】 Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumor that has been claimed as the third most lethal tumor in the world, accounting for the second death cause of china’s tumor patients. There are now one million HCC patients in the globe, with an increase of 300 thousands each year, 45% of which are Chinese who lives in south eastern and coastal area of china. With recent years’prevalence of hepatitis B virus, the geographical distribution and incidence of liver cancer has significantly changed. Onset peak trends and mortality rate also goes to the young age group. At present, the treatment of hepatocellular carcinoma is mainly based on surgical resection, after which the use of chemotherapy-related drugs in hepatocellular carcinoma will be largely limited. Therefore, the research on the impact of proto-oncogene on the proliferation and invasion of HCC is of great significance for further treatment.c-Kit is a proto-oncogene encoding receptor tyrosine kinase family. The current study shows that c-Kit is closely linked with the development, and prognosis of a variety of tumors. Many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia and lung cancer were proved with the abnormal expression of c-Kit. Aimed directly at these c-Kit high expression tumors, tyrosine kinase inhibitors have been successfully used in clinical patient. At present, research on expression of c-Kit receptor in HCC was rarely reported. The detailed mechanisms of KIT/SCF signaling pathway in HCC need to be further elucidated.Our extensive literature review showed the following information: First, patients with increased c-Kit proto-oncogene expression implies poor prognosis. Second, in normal liver, c-Kit exists only in oval cells that have uneven distribution in tumor. Research indicates its changment during the process of liver cirrhosis-liver cancer. Third, the PI3K/AKT signaling pathway, a part of the down stream of KIT/SCF signaling pathway, is closely linked with Wnt pathway that is one of the most important invasion and metastasis path of HCC. On the basis of the above theories, we hypothesized that the downstream of KIT/SCF pathway develops corresponding alteration, promoting the invasion and metastasis of HCC after its activation. This study aimed to verify these assumptions and elucidate the possible underlying molecular mechanisms of c-Kit in the proliferation and invasion of HCC.Materials and methods1. 89 clinic HCC patients were selected and examined for c-Kit expression by immunohistochemistry. Results were analyzed for the correlation between c-Kit expression and clinical pathological factors. Two MHCC-97 sub cell lines with different invasion and metastasis potential, MHCC-97L and HCC-97H, were chosen and examined for c-Kit expression by Immunofluorescence, Realtime-PCR and Western-blot, and were analyzed for the correlation between c-Kit expression and invasive potential.2. An adenovirus vector system, Adeasy, was adopted for the construction of c-Kit containing vector. The vector was then transfected into cell line Hep-G2. Western-blot, invasion test and motility test were carried out for the contrast study of c-Kit’s role in invasion and metastasis process of HCC. Nude mice were injected with Hep-G2 that had been infected by c-Kit gene-contained recombinant adenovirus vector and its blank control. The tumor formation potential was observed and the role of c-Kit in the process of tumor forming and invasion was studied.3. An exogenous ligand of c-Kit receptor was introduced into SMMC-7721. the impact of c-Kit/SCF signaling system on the distribution ofβ-catenin was analyzed. Meanwhile, whether this impact is conducted through the PI3K/AKT signaling system was analyzed. And the phosphorylation status of AKT and GSK-3, the key factor of PI3K/AKT signaling system, was examined.4. FAK expression in the 89 clinical HCC patients were examined by immunohistochemistry and the result was evaluated for the correlation between FAK and c-Kit with other pathological factors. Co-immunoprecipitation was adopted to examine the binding between FAK and KIT in cell line SMMC-7721 and Hep-G2. Results and Conclusions1. c-Kit gene expression was detected and the following results in clinical HCC tissue samples and HCC cell lines with varied potential of invasion and metastasis were obtained:1.1. Expression of c-Kit was significantly increased in HCC tumor cells.1.2. c-Kit expression level is positively correlated with vascular invasion, TNM classification, HBV DNA level and HBSAg, which indicates that c-Kit expression was closely correlated with HCC progression and its increased level with the HCC development.1.3. Among the sub-cell lines of MHCC-97 that with various potential of invasion and metastasis, c-Kit was significantly higher in MHCC-97H than in MHCC-97L. This result confirmed that c-Kit was up-regulated as the invasion and metastasis increased.2. Recombinant adenovirus containing c-Kit was successfully constructed and transfected into HCC cell line Hep-G2. After the expression of c-Kit was verified, it was observed that exogenous c-Kit increased the invasion and motility of Hep-G2.3. After the exogenous ligand of c-Kit receptor was introduced into SMMC-7721, PI3K/AKT signaling system was activated and its key factor AKT and GSK-3βunderwent significant phosphorylation,β-catenin showed an intranuclear redistribution. After the addition c-Kit/SCF inhibitor Imatinib,β-catenin intranuclear-redistribution was blocked.4. FAK gene expression was detected and the following results in clinical HCC tissue samples and HCC cell lines with varied potential of invasion and metastasis were obtained.4.1.FAK was significantly up-regulated in HCC cells.4.2.FAK level was significantly correlated with great vessel invasion,TNM classification, HBsAg and HBV DNA level, indicating that FAK expression is up-regulated as the tumor progressing. And FAK and c-Kit expression was closely connected in HCC cells.To sum up, the follow conclusion was obtained form this study:1. c-Kit gene expression was closely correlated with tumor progression and its up-regulation indicates poor prognosis. Exogenous c-Kit gene increases the invasion, motility and tumor forming potential of HCC. 2. As a crucial gene in HCC, c-Kit increases invasion potential of HCC through activation of PI3K/AKT signaling system, showing synergistic effect with FAK.3. Kit/SCF signaling system inhibitor Imatinib may have protective effect on HCC progression.4. KIT and FAK could bind in hepatoma cell line.更多还原