【作者】 沈建国；

【导师】 王林波； 吴金民；

【作者基本信息】 浙江大学 ， 肿瘤学， 2006， 博士

【摘要】 胃癌是消化系统常见恶性肿瘤之一,也是在我国造成死亡人数最多的恶性肿瘤。提高胃癌的综合诊治能力对改善胃癌的预后,提高人们的生活水平具有重要意义。纵观胃癌治疗的现状,随着医疗水平的提高以及新技术的不断开发利用,胃癌治疗有了很大的进步,尤其是早期胃癌,通过早期发现,及早治疗,预后较好。据报道早期胃癌五年生存率可达90%以上。尽管如此,目前仍然有大部分病人就诊时已是胃癌进展期或晚期,在治疗上存在较大的困难。化疗作为胃癌辅助治疗的一个重要手段,在临床上有着普遍的应用,但疗效仍然不甚满意,50%～90%胃癌病人仍将发生复发或转移并因此而死亡。 肿瘤与宿主之间的相互作用在肿瘤发生、发展及转移过程中起着重要作用。宿主间质细胞不但可提供肿瘤细胞生长的营养,而且也为癌细胞进入间质提供空间与支架,有利于癌细胞的浸润活动。目前对间质细胞在肿瘤中的作用研究已成为肿瘤研究的热点和难点。 肝细胞生长因子(HGF)是一种多功能细胞因子,主要来源于间质细胞(如成纤维细胞,巨噬细胞等),在某些上皮细胞、内皮细胞、肝脏的枯否氏细胞和脂肪储存细胞,甚至一些肿瘤细胞中也可产生。HGF可通过其酪氨酸激酶受体c-Met结合发挥其生物学效应,刺激多种类型细胞分化、增殖、再生、运动、迁移及形态的发生,在细胞生长调控,肿瘤转移,新生血管形成等方面发挥重要作用。 研究表明,HGF能够抑制正常细胞如肝细胞,肾上皮细胞,角膜上皮细胞等凋亡,维持细胞存活而发挥正常作用。在部分肿瘤细胞中,HGF同样可抑制肿瘤细胞的凋亡而发挥其抗凋亡的作用。近期研究还发现,HGF可抑制一些DNA损伤剂诱导的肿瘤细胞如乳腺癌,更多还原

【Abstract】 Gastric cancer is one of the most popular digestive tumors in the world that kills a lot of people in china. Improvement of the diagnosis and treatment of gastric cancer results in better prognosis and patients quality of life. During the past decades, the prognosis of gastric cancer was improved especially in early gastric cancer based on early diagnosis and treatment, which confers the five-year survival rate above 90%. However, there still are tremendous numbers of patients who are diagnosed with advanced and/or late stage gastric cancer in the clinic. The prognosis of these patients is quite unsatisfied even after intergrated therapy which including surgery, chemotherapy, radiation as well as traditional medicines. A total of 50% to 90% patients will die because of tumor recurrence and/or metastasis.Numerous reports have demonstrated that the host-tumor interactions play a significant role in the tumor development, process as well as metastasis. The mesenchymal cell adjacent to the tumor may not only stimulate tumor cell growth by supplying nutrition for tumor cells, but also afford tissue space and cell skeleton, which help tumor cell infiltrate to the extracelluar matrix membrane. Several investigations have been focused on the role of mesenchymal cell in tumor development and process.Hepatocyte growth factor is a multifunctional factor, which generates from mesenchymal cell including fibroblast cell, macrophage, epithelium, endothelium, lipid-storage cell as well as several cancer cells. HGF may function as a mitogen, motogen and morphogen after being combined with the tyrosine kinase receptor, c-Met, therefore, it play an important role in cell growth, migration, invasion as well as angiogenesis.Previous studies have reported that HGF may inhibit normal cells such as hepatic cell, kidney cell, corneal cell from apoptosis, its anti-apoptic effect can also be observed in several cancer cells including lung, breast cancer, glioma cells. Recently, HGF was found to suppress DNA-damage agent induced tumor apoptosis, which may result in tumor chemoresistance. However, this anti-apoptotic effect is cell selective. Furthmore, it was reported that the unsatisfied effects of chemotherapy in gastric cancer was possibly due to the chemoresistance and/or drug insensitivation to gastric cancer. It is rarely studied whether HGF may inhibit gastric cancer cell apoptosis that is induced by DNA damage agents, and whether the anti-apoptotic effect of HGF may be associated with chemoresistance in gastric cancer is still unclear.HGF may also stimulate cell motility, migration as well as invasion, which play a significant role in tumor invasion and metastasis. It is reported that HGF can stimulate cancer cell such as breast cancer, lung cancer, prostate cancer, and glioma cell invasion. However, this biological behavior is organ specific, few studies have concentrated on the influence of HGF on cancer invasion in gastric cancer.Based on the previous studies, we selected a gastric cancer cell line, MKN-45, which does not autonomously express HGF to investigate the influence of HGF on cell apoptosis and cancer invasion. Our study tried to evaluate the possible role of HGF on chemoresistance in gastric cancer as well as to demonstrate the possible mechanism of HGF on cancer invasion.This study was composed of four sections.Section I: pIRES2-EGFP-HGF plasmid constructionPUC-SRot/HGF cDNA was bestowed from Prof. Nakamura of Osaka University College of Medicine, Japan. A 2.3kb target of HGF cDNA was inserted using Not I site of thePUC-SRa plasmid. pIRES2-EGFP was purchased from B&D company. PUC-SRct/HGF and pIRES2-EGFP were both digested by Bam HI, after purification and proliferation of HGF cDNA and pIRES2-EGFP, the targeted band were ligated by T4 DNA ligase, the ligation product were then transformed into DH5ot sensitive E coli, the anti-kanamycin E coli clone were selected out. Among them, HGF inserted clone was confirmed by Bam HI digestion. The order of HGF insertion was confirmed by both Kpn I, Pst I digestion and PCR. Our results found three HGF insertion recombinant, both enzyme digestion and PCR reaction confirmed that HGF cDNA was inserted in a correct order. One of the recombinant was amplified and purified for further experiment.Section II: Establish a gastric cancer cell linethat may stably express HGFWe, firstly, selected several gastric cancer cell line including MKN-45, YCC-3, NCI-N87, SNU-16 and AGS to investigate HGF expression by using RT-PCR, we found that MKN-45 itself can not express HGF. Futhermore, MKN-45 is a cell line that expresses high level of tyrosine kinase receptor, c-Met. We then selected MKN-45 as a cell line for HGF transfection. HGF recombinant and null plasmid were transfected and selected by G418. HGF expression was detected by RT-PCR and western blot. The morphology and cell growth and proliferation was compared between HGF transfectant, control vector transfectant and wild type MKN-45 cell, and no significant difference was observed among them. We also check the HGF function in the stably expressed HGF transfectant cells using MDCK as a target cell, we found MDCK was scattered significantly when cultured with the medium of HGF transfectant. Therefore, we established a cell line that may stably and functionally express HGF.Section III, HGF suppresses adriamycin induced apoptosis in gastric cancer ceU line, MKN-45We investigated the influence of HGF on adriamycin induced apoptosis in MKN-45 cell line using MTT proliferation assay, DAPI stain, DNA defragmentation assay and flowcytometry. Our results found that cell proliferation was inhibited more significantly by adriamycin in control vector transfectant and wild type cells than that in HGF transfectant. We also count the apoptosiscell by staining with DAPI, and observed that HGF transfectant showed less cell apoptosis than that of nontransfectant. When cells treated with 0. lug/ml adriamycin, DNA radder was observed in wild type and control vector transfectant cells, However, it is not clear in HGF transfectant cells. Finally, we confirmed the above results by using flowcytometry with PI staining. Therefore, HGF may suppress adriamycin induced apoptosis in gastric cancer cell line, MKN-45.Section IV. Influence of HGF on cell adhesion, migration and invasion in gastric cancer cell line, MKN-45We investigated the influence of HGF on cell adhesion, migration and invasion in MKN-45 cell line using cell adhesion assay, boyden chamber assay, wound assay as well as detecting the tumor metastasis associated genes expression between three group of cells. There were no significant differences of the adhesion and migration rate between the HGF transfectant and control vector transfectant, wild type MKN-45 cell. No difference of cell invasion was found between the three types of cells by using wound assay. We finally assessed the tumor metastatic associated genes expression between the three type of cells, and no different expression was found between them regarding E-cadherin, itergrin, MMP2, MMP-9 as well as MT1-MMP. We therefore may conclude that HGF does not affect cell adhesion, migration and invasion in MKN-45 cell line, implying that HGF is not associated with tumor metastasis for MKN-45 cell.We successfully constructed a GFP expressing plasmid pIRES2-EGFP-HGF and established a gastric cancer cell line that can stably express HGF. Our results observed that HGF could suppress MKN-45 apoptosis that was induced by adriamycin. However, HGF could not enhance MKN-45 cell adhesion, migration and invasion. Our study implicate that HGF may be involved in chemoresitance in gastric cancer, nevertheless, the aggressive and invasive characteristics of MKN-45 may not be dependent on HGF/Met signaling, some other cytokines may play a role in MKN-45 cancer cell invasion.更多还原