【作者】 晋云；

【导师】 董家鸿；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 肝癌是消化系统常见的恶性肿瘤,对肝癌的防治研究具有积极意义。近年来对肿瘤转移机制的研究发现,肿瘤转移是一个复杂过程,在这一过程中有多个因素参与了转移的调控,肝癌细胞的侵袭和转移是恶性肿瘤的生物学本质,也是肝癌治疗失败和患者致死的主要原因,随着生物技术的发展,从基因水平上入手,解决肝癌的转移有了实现的可能。基因芯片技术作为一个有力的工具应运而生,可提供全基因组范围的生物学研究。由于基因芯片高速度、高通量、集约化和低成本的特点,其诞生以来就受到科学界的广泛关注。芯片技术在疾病诊断,特别是肿瘤的判别、比较肿瘤和正常组织、鉴别组织来源、肿瘤的亚分类等基础研究上具有重要作用。以前肿瘤研究方法中只针对某一单个基因进行研究,没有横向比较,高密度基因芯片技术可以提供平台进行多种基因的横向比较,探讨在某一模型中哪些基因起关键作用。本实验以临床转移肝癌标本为研究对象,通过SuperArray基因芯片技术对肝癌转移相关基因进行筛选,并通过免疫组织化学染色、RT-PCR和免疫印迹等技术进行临床标本验证,结合患者临床相关资料,探讨筛选出的基因与患者年龄、病程、肿瘤类型、临床分期及转移之间的关系。再通过肝癌细胞体外培养模型观察其表达及抑制后的转录水平、蛋白水平的变化及细胞侵袭力的变化。通过以上研究,探讨基因芯片筛选出的肝癌转移相关基因在肝癌发生发展中的作用,为肝癌的临床治疗提供研究基础。主要结果及结论如下:第一部分:肝细胞癌肿瘤转移相关基因SuperArray基因芯片表达研究1、选取正常肝组织、未转移肝细胞癌和转移肝细胞癌组织进行肿瘤转移基因芯片检测,结果发现未转移肝癌较正常组织表达上调3倍以上的基因有33个,转移肝癌较正常组织表达上调3倍以上的基因有50个,转移肝癌较未转移肝癌表达上调3倍以上的基因有13个。未转移肝癌较正常组织表达下调3倍以上的基因有5个,转移肝癌较正常组织表达下调3倍以上的基因有9个,转移肝癌较未转移肝癌表达下调3倍以上的基因有3个。2、根据实验结果选择上调基因VEGF和组织蛋白酶L和下调基因Kai-1和Cadherin 11进行验证,通过RT-PCR和WB方法证实了基因芯片的检测结果。第二部分:肝细胞癌Cathepsin L临床表达特征及与血管生成关系选取肝癌组织标本58例,其中高分化18例,中分化18例,低分化22例,临床分期Ⅰ-Ⅱ期26例,Ⅲ-Ⅳ期32例,有转移28例。免疫组化法、RT-PCR和免疫印迹法进行Cathepsin L,VEGF和CD34检测,统计实验结果。结果发现1、肝癌Cathepsin L表达与患者性别和年龄无相关性,与肝癌病理分级有显著相关性,分化越低的肝癌,Cathepsin L表达越强。2、肝癌Cathepsin L表达与肝癌临床分期有显著相关性,晚期肝癌Cathepsin L表达强于早期肝癌。3、临床晚期肝癌中VEGF表达强度高于早期肝癌,低分化肝癌VEGF表达强度高于高中分化肝癌,肝癌Cathepsin L表达强度变化与VEGF表达强度呈相同趋势,呈正相关关系。4、肝癌不同年龄组MVD无显著区别,肝癌临床晚期MVD显著高于早期肝癌,低分化肝癌MVD显著高于高中分化肝癌。统计分析表明,肝癌MVD与VEGF及Cathepsin L表达强度呈正相关。第三部分:RNA干扰沉默Cathepsin L基因表达对肝癌细胞影响实验研究肝癌细胞分为正常对照组(正常组)、转染对照组(对照组)和Cathepsin L siRNA转染实验组(转染组),观察时间为转染后1d,3d和6d。转染组进行Cathepsin L siRNA转染,MTT法及流式细胞技术检测细胞增殖状况,免疫荧光、RT-PCR及WB法进行Cathepsin L检测,transwell小室法进行细胞侵袭力测定。结果发现:1、RNAi可有效沉默肝癌细胞Cathepsin L mRNA转录和蛋白表达,同时抑制细胞增殖,并诱导细胞凋亡,提示Cathepsin L在肝癌细胞的生长和增殖中起重要作用,是肝癌细胞增殖和细胞凋亡通路的关键性生物因子之一。2、Cathepsin L表达受抑导致肝癌细胞侵袭力相对降低说明Cathepsin L是肝癌细胞浸润转移的重要生物学因素,同时提示RNAi技术作为肿瘤治疗的新途径。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is a common malignant tumor of the digestive system, and the studies on the prevention and treatment of HCC are of significance. It has been shown recently that tumor metastasis is a complex process regulated by multiple factors. Invasion and metastasis of cancer cells are the biological properties of malignant tumor, and underlie the failure of HCC treatment and death of HCC patients. With the development of biotechnology, it becomes possible to prevent HCC metastasis at the genetic level.Gene microarrays, as a useful tool, make possible biological research on the full-genome scale. Gene microarrays are characterized by high speed, high throughput, intensification, and low costs; hence, they have drawn much attention from researchers. Gene microarrays play important roles in the diagnosis of diseases, particularly in the differentiation of tumors, tumor and normal tissues, histological origin, and tumor classification. Previously, single tumor-related genes were studied, but transversal comparisons of different genes did not performed. High-density gene microarrays make possible transversal comparison of multiple genes to determine which gene plays a key role in a certain model.In the present study, metastasis related genes were screened in HCC specimens with clinical metastasis using SuperArray microarrays, and verified immunohistochemically. The relationship of the screened genes with the patient’s age, course of disease, tumor classification, clinical staging and metastasis were investigated. The expression of the screened genes and transcripiton and translation changes after suppression of the expression of these genes were analyzed in cultured HCC cells. The aim of the study was to investigate the role of the screened genes related to HCC metastasis in the incidence and development of HCC and thus provide a basis for the treatment of HCC. Results and conclusions:Part 1: Study on the expression of metastasis related genes using SuperArray microarrays1. Metastasis related genes were screened in normal liver tissues, HCC tissues with metastasis and HCC tissues without metastasis using SuperArray microarrays. The results indicated 33 genes with their expression upregulated more than 3 folds in HCC specimens without metastasis compared with the normal tissue, 50 genes with their expression upregulated more than 3 folds in HCC specimens with metastasis compared with the normal tissue metastasis, and 13 genes with their expression upregulated more than 3 folds in HCC specimens with metastasis compared with HCC specimens without metastasis. The results also indicated 5 genes with their expression downregulated more than 3 times in HCC specimens without metastasis compared with the normal tissue, 9 genes with their expression downregulated more than 3 times in HCC specimens with metastasis compared with the normal tissue, and 3 genes with their expression downregulated more than 3 folds in HCC specimens with metastasis compared with HCC specimens without metastasis.2. The upregulated genes VEGF and cathepsin L and the downregulated genes Kai-1 and cadherin 11 were verified by RT-PCR and Western blotting.Part 2: The characteristics of cathepsin L expression in HCC and its relationship with angiogenesis58 HCC specimens (highly differentiated, 18; moderately differentiated, 18; poorly differentiated, 22; clinical stages I-II, 26; III-IV, 32; metastasis, 28) were detected for the expression of cathepsin L, VEGF and CD34 by immunoblotting and RT-PCR.Results1. Cathepsin L expression did not correlate to the gender and age of HCC patients, but significantly correlated to the pathological classification of HCC. The differentiation level of HCC negatively correlated to the level of cathepsin L expression.2. Cathepsin L expression was significantly related to clinical HCC staging, and cathepsin L expression was higher in advanced HCC than early-stage HCC.3. The VEGF expression level was higher in advanced HCC than early-stage HCC, and was higher in poorly differentiated HCC than in moderately to highly differentiated HCC. The cathepsin L expression level was higher in advanced HCC than early-stage HCC, and was higher in poorly differentiated HCC than in moderately to highly differentiated HCC.4. The microvessel density (MVD) differed insignificantly between different age groups, but was significantly higher in advanced HCC than early-stage HCC, and was significantly higher in poorly differentiated HCC than moderately to highly differentiated HCC. Statistical analysis demonstrated that MVD positively correlated to the level of VEGF and cathepsin L expression.Part 3: Effect of RNA interference of cathepsin L expression on HCC cellsHCC cells were randomized to the blank control group (blank), the transfection control group (control) and the cathepsin L siRNA transfection group (transfection group), and the cells were observed 1d, 3d and 6d after transfection. The transfection group was transfected with cathepsin L siRNA. Cell proliferation was analyzed by MTT assay and flow cytometry, cathepsin L expression was detected by immunofluorescence, RT-PCR and Western blotting, and cell invasiveness was determined by transwell assay. The results indicated that RNAi effectively silenced cathepsin L mRNA and protein expression in HCC, inhibited cell proliferation, induced apoptosis, and significantly reduced cell invasiveness, suggesting that RNAi may be a new treatment option for HCC.更多还原