【作者】 郝继辉；

【导师】 郝希山； 李强； 史玉荣；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2002， 博士

【摘要】 原发性肝癌是常见的消化系统恶性肿瘤，约占消化系统肿瘤的20％以上，其中肝细胞肝癌约占90％，其生物学特征为易复发、转移、预后差、死亡率高。尽管目前原发性肝癌的治疗方案(手术切除、介入治疗、生物治疗、激光及微波治疗等)日趋完善，但仍难以取得令人满意的疗效，患者生存期极短。因此研究肝癌的发病机理以及探讨新的可行的治疗方案具有极其重要的临床意义。 随着肿瘤学领域中基础研究的不断深入，人们发现在肿瘤组织中存在一些与肿瘤血管生成有关的生长因子及其受体过表达的现象；并且提出了肿瘤生长的血管依赖性学说，为探讨肿瘤的发生机制奠定了基础。血管内皮生长因子(VEGF)是一种重要的促血管生长因子，它参与肿瘤细胞的生长、分化、转移、增殖等多方面的过程。肝癌是富含血管的实体性肿瘤，因此VEGF可能与肝癌的发生、发展有重要的相关性联系。 目前，国内外针对VEGF及其受体基因表达与肝细胞癌分化程度、生长方式、临床特点及转移活性的关系尚未进行深入系统的研究。针对肝癌的血管内皮生成因子(VEGF)反义基因治疗尚无报道。本课题从三个角度进行了系统研究：(1)VEGF基因表达与肝癌恶性程度、临床特点、转移活性、微血管生成及其受体之间的相关性。(2)采用脂质体体外转染VEGF反义RNA，观察转染后肿瘤细胞在体外的生长情况及VEGF表达情况。(3)VEGF反义RNA转染后肿瘤细胞体内生长情况观 天津医科大学博士学位论文 察。结果将为探讨以VEGF为优选靶进行基因治疗，阻止肿瘤的发生 和发展，提高生存率，延长生存期奠定理论基础。 第一部分 人肝细胞癌VEGF及其受体基因表达与肝癌转移活性 和血管形成的相关性研究 本研究对 60例肝细胞癌（HCC）、10例正常肝组织采用逆转录－ 聚合酶链反应、免疫组化法分别检测VEGFmRNA表达及其与相应受 体＊hi lflnl蛋白表达情况；采用＂m23评价肝细胞癌的转移活性；采 用Vlll因子相关抗原兔疫染色确定肿瘤微血管密度，从而分析肝细胞癌 VEGF基因表达与肿瘤血管生成、转移之间的关系。 1．人肝细胞癌VEGFmRNA表达的定量检测 通过RT－PCR方法检测60例人肝细胞癌和10例正常肝脏组织 VEGFmRNA 表达水平：发现正常肝脏组织和肝细胞肝癌的 VEGFmRNA阳性表达率分别为 30％和 78．3％，后者显著高于前者（P ＜0刀 1）：以 p．actin为内参照检测正常肝组织 VEGFmRNA表达水平明 显低于肝癌组织：W级肝细胞癌＊＊＊＊m＊＊A的表达水平为1．36土0．19， 而＊、11级肝细胞癌VEGFmRNA的表达水平分别为1．1210．25、0．79 士0．26，三者之间有明显差异（Pwto刀5人 因此提示，VEGFmRNA表 达水平与肿瘤病理分级呈正相关。此外发现：瘤栓组VEGFmRNA表 达水平为 1．31士0．13，无瘤栓组为 0．89土0．23；无包膜组 VEGFmRNA 表达水平为 1．28士 0．15，有包膜组为 0．96上 0．18，相应的两组之间有显 著性差异。而VEGFmRNA表达水平在不同大小肿瘤之间差别则无显 著性（P＞0．05）。此结果表明＊＊＊FmR＊A表达水平增高与临床上肿瘤 的侵袭特牲有关。 2 天津医科人学博土学位论文 2．VEGF及其受体 fit－llflk－l的蛋白表达 采用免疫组化法检测VEGF蛋白表达。正常肝组织和肝癌组织的 VEGF阳性表达率分别为40％、sl．3％，后者明显高于前者（Prto刀1）。 而不同分化程度的肝细胞肝癌的VEGF蛋白表达强度有显著差异（P t 0．05）。表明：肝癌组织 VEGF蛋白表达水平与肝癌的病理分级呈正 相关。比较不同形态特征的肿瘤之间VEGF蛋白表达水平发现：瘤栓 组VEGF蛋白表达强度大于无瘤栓组，无包膜组VEGF蛋白表达强度 大于有包膜组，对应的两组之间有显著性差异。而不同直径的肿瘤之 间＊＊＊F蛋白表达水平无显著性差异（P＞0刀5）。 采用免疫组化法检测fi t－llflkl 蛋白表达。正常肝组织和肝癌组 织的月卜1什！卜1阳性表达率分别为40％历0％、80畅侣8．3用，后者明显高 于前者（P＜0刀5）。而不同分化程度的肝细胞肝癌的门卜lin卜1表达强 度也有显著差异，并与肝癌的病理分级呈正相关（P t 0刀5）。比较不 同形态肿瘤之间＊－llflkJ蛋白表达水平发现：fltllflki蛋白表达水 平也与瘤栓、包膜有关、与肿瘤直径无关。瘤栓组n卜1用卜1蛋白表达 强度大于无瘤栓组，无包膜组＊卜1厅！卜1蛋白表达强度大于有包膜组， 对应的两组之间有显著性差异。（P＜0乃5） 3．肝癌转移?更多还原

【Abstract】 Liver cancer is one of the common types of gastrointestinal tumors with poor prognosis % metastasis and high mortality , in which hepatocellular carcinoma (HCC) accounts for about 95% . Although the different available multi-modalities of therapies , including radical surgical operation % intervention therapy > laser therapy et al, have currently been used in the treatment for liver cancer , the prognosis of liver cancer has not been markedly improved. Therefore, it has an important clinic significance to study on the mechanism of the development and progression of liver cancer and the new strategies for the treatment of liver cancer with the molecular biology. With the development of the basic research on oncological fields, it was found that many tumors have the overexpression in growth factors & their receptors, and progression of tumors depends on angiogenesis. The vascular endothelial growth factor ( VEGF) is an important factor which elicits the angiogensis in tumors. It takes an important role in the growth, differentiation , metastasis, proliferation of tumors. The HCC is well known to be vascular rich tumors and its growth is highly depended on angiogenesis, so VEGF is considered to have key roles in the development of HCC. By now , the systemic researches on therelationship between the expression of VEGF and histopathologic type, metastasis activity > angiogenesis of HCC have not been reported. And there are also no reports on gene therapy against angiogeneis of liver cancer. This study contains three parts of work: The first, the relationships between the expression of VEGF gene & its receptors ( flk-1 , flt-1 ) in HCC and the tumor grade , tumor metastasis , angiogenesis of liver cancer. The secondi the inhibitory effect of VEGF antisense RNA on the growth of 7721 hepatocellular cell line (7721 cancer hepatocytes)in vivo. The third, the inhibitory effect of VEGF antisense RNA on 7721 cell in nude mouse.Part I : Study on the expression of VEGF and its receptors and its relationship to metastasis activity and angiogenesis of human HCC1. Expression of VEGF gene in human HCC and normal liver tissue The gene expression of VEGF in 60 human HCCs and 10 normalliver were studied by RT-PCR method. The expressive rates of HCCs and normal tissue were 78.3% and 30% respectively. The levels of VEGFmRNA (VEGFmRNA/P -actin mRNA) were 0.79 + 0.26 in grade IK 1.12 + 0.25 in grade IIK 1.36?.19 in grade IV, respectively . The expression levels of VEGFmRNA were positively correlated to pathologic grades^ embolus and envelops of tumors.2. The protein expression of VEGF and its specific receptor in human HCC and normal liver tissueThe protein expression of VEGF in 60 human HCCs and 10 normal liver were studied by immunohistochemistry .The expression rates of HCCs and normal tissue were 81.3% and 40% respectively. The level of VEGF protein was also significantly correlated to pathologic grades>embolus and capsules of the tumors.The protein expression of flk-l/flt-1 in 60 human HCCs and 10 normal liver were studied by immunohistochemical method. The expression rates of flk-1 in HCCs and normal tissue were 88.3% and 50% respectively. The expression rates of flt-1 in HCCs and normal tissue were 80% and 40% respectively. The levels of fik-l/fit-1 were correlated to pathologic grades^ embolus and capsules of the tumors.The overexpressions of VEGFmRNA -. VEGF protein and its fll-l/flk-1 were found in HCC. It suggested that the VEGF gene play an important role in the malignant progression of cancers. The overexpression of VEGF and the correlation between VEGF expression and tumor grade provided a useful parameter for evaluating the degree of malignancy at molecular level and for selecting the target gene in gene therapy.Part II The inhibitory effect of VEGF antisense RNA on the growth of HCC cells in vitroBecause there was no effective treatments on liver cancers, it is very urgent and important to find out new methods to solve this problem. According to the results in part I , w更多还原