肝癌血管生成机理探讨及EGCG抑制血管生成的实验研究

【作者】 赵志成；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2004， 博士

【摘要】 目的： 在肿瘤的生长过程中，血管生成起着关键的作用，它提供丰富的养分和营养物质；并且为肿瘤的浸润及转移创造条件。肿瘤细胞释放的促血管生成因子作用于宿主内皮细胞，促进新血管的生成。血管内皮生长因子(VEGF)和酪氨酸激酶受体(KDR)在血管生成中起着重要作用。而血管的成熟和稳定又主要靠酪氨酸受体另一种亚家族Tie2和其配体血管生成素(Angiopioetins)维持。这两条信号传导途径是目前认为最直接参与新血管生成的血管内皮细胞特异的途径。肝癌具有生长快、转移早和高死亡率的特性；也是一种强的血管依赖性肿瘤。血管生成是其恶性化的第一步，而众多的促血管生成因子和抗血管生成因子参与了血管生成的过程。因此，以血管生成信号传导通路为靶向的抗血管治疗可能是一种很有潜力的抗肿瘤浸润、转移和术后复发的治疗方法。但是，在血管生成过程中，许多信号传导分子机制目前还不清楚，本研究采用逆转录聚合酶联方法和蛋白印记方法评估VEGF／KDR和Angiopoietins／Tie2在肝癌中的表达，目的是探索在肝癌生长及转移中这些促血管生成细胞因子及受体作用的分子机理。 方法： 1，对象：我院2002年5月至2002年9月切除的23例肝癌标本，分别切取癌组织23例、癌边缘(距癌组织1cm以内)23例和远离癌的肝组织(距癌组织5cm以上)23例，肝硬化标本8例，正常肝组织4例(均为肝移植手术中切除的供体肝脏)。切取的标本即刻放置液氮中冷冻并移置-80℃冰箱保存备用。本组病人年龄28-68(平均50.86±12.78)。所有标本均经H-E染色，病理证实临床均为肝细胞性肝癌诊断，其中，包膜不完整或无包膜9例，镜下脉管浸润7例，同时伴有卫星结节7例。按Edmonson方法分级Ⅱ 18例，Ⅲ 5例。 2，逆转录聚合酶联反应：用Trizol试剂从切除标本分离RNA。标本称重、冰冻后用研浙江大学专业学位博士论文 钵在Trizol试剂中研磨，分光光度仪测定总RNA浓度。加1闪DNase取出污染的DNA 分子。1陀总RNA用于逆转录。产物cDNAZ川和特异引物Ang一1、Ang一2、VEGF、 KoR、TieZ和内参照引物G3PDH进行聚合酶联反应。1 .5%琼脂糖胶分离13协1 PeR 产物，滨化乙旋染色。KodaDigital Scienee 1 5 2.0 DNA分析软件系统对进行pCR产物 半定量分析。3，蛋白印迹:样本在裂解液中匀浆。加入SDS上样缓冲液煮沸5分钟。每个加样孔上样 量为50林g，sDs一PAGE电泳分离蛋白(6%胶分离TieZ和KDR，8%胶分离Angl、AngZ 和vEGF)。之后将分离的蛋白转移至PVDF膜(l mA/c m 1 .5小时)上。丽春红染色确定 蛋白转移的情况，5%脱脂牛奶(T BST配制)封闭PVDF膜1.5小时，加入一抗4℃过夜; TBST洗膜3又15min，加入二抗(l:1000)孵育l小时，按上述方法洗膜。加显影 剂ECL，压片曝光显影。4，免疫组织化学:甲醛固定石蜡包埋标本连续切片，厚约4一6户m，经二甲苯脱蜡、 梯度酒精脱水，抗原修复。3%过氧化氢5分钟消除内源性过氧化酶活性，10%羊血 清封闭非特异抗原结合位点后，加入鼠抗CD34单克隆抗体(l:25)37.4℃孵育l小 时，按HRP标记手册操作。选取5个200、视野进行CD34阳性细胞计数。5，统计学处理:所有计量数据均以平均值士标准差(又始)表示，RT一PCR吸光度分析 和血管密度分析均采用方差分析。SPSSll.O分析软件进行数据分析。p<0.05表示存 在差异。结果1，两条通路的表达与肿瘤血管密度的关系:本实验中证实vEGF/KDR和AngioPoietinS/TieZ两条信号传导通路在所有的肝癌标本中均有激活。Angl在所有组中均有表达，且无明显的差异，但在肝癌组织及癌旁肝组织中的表达存在升高趋势。与远离癌的肝组织、阳性对照和阴性对照组比较，AngZ在癌组织和癌旁肝组织中的表达明显增强，p<0.01;VEGF的表达在癌组织中最高，与其他组比较p<0.05。KDR和TieZ的表达在各组中不存在明显的差异，但是，在癌组织和癌旁组织中还是存在明显高的吸光条带。浙江大学专业学位博士论文 在肝癌组织中的血管生成因子及其受体的蛋白合成也是明显活跃的，这与他们在基 因转录水平的高低是一致的。在癌组织中，VEGF和Angl的蛋白含量明显高于正 常肝组织和肝硬化组织，而其相应的受体KI〕R和TieZ表达随之升高;而在癌旁肝 组织中，KDR和An沙的表达明显升高。 免疫组化方法检测微血管密度的结果表明，在癌组织及癌旁肝组织中的血管密度明 显高于正常肝和阳性及阴性对照组，p<0.01。本实验中也发现，vEGF和AngZ的表 达与微血管的密度有密切的关系，同时其受体也有不同程度的表达。说明在肝癌血 管生成中，vEGF服nR和Angiopoietins/TieZ两条通路的活化起着关键的作用。 2，肝癌的浸润性及转移与血管生成的信号传导通路活化的关系: 血管生成是肝癌侵袭和转移所必需的。与肝癌侵袭及转移相关的因素包括肿瘤包膜 的完整性、肝脏脉管是否存在癌栓、卫星结节的形成和肿瘤的病理分级。本研究分 析了vEGF/KDR和Angi叩ofetins/TieZ两条信号传导通路与肿瘤侵袭和转移相关因 素的关系，发现在有脉管癌栓的标本中，更多还原

【Abstract】 Object: Angiogenesis plays a key role in the process of tumor growth by providing abundant oxygen and nutrients to neoplasm and making a channel for the tumor invasion and metastasis. Pro-angiogenic factors released from tumor cells can promote the formation of new blood vessels by functioning on host endothelial cells. Vascular endothelial growth factor (VEGF) and its kinase insert domain containing receptor KDR take part in the process of angiogenesis. The mature and stabilization of neovascular are dependent on another subfamily of tyrosine kinase receptor Tie2 and its ligand angiopoietins. Hepatocellular carcinoma (HCC) characterized by rapid growth, early metastasis and high mortality is an intensive vascular-dependent marlignant tumor. Angiogenesis is an initial step for these malignant features, and lots of pro- or anti-angiogenic factors and receptors take part in this process. It is suggested that targeting at angiogenic signaling pathways may be a potent treatment for the prohibition of tumor invasion and metastasis. The molecular mechanism of these pathways during angiogenesis is still unclear. In this study the method of RT-PCR and Western blot were employed to evaluate the expression patterns of VEGF/KDR and Angiopoietins/Tie2, which are considered as two important signal conductive pathways, and the molecular mechanism underlying these factors and receptors in HCC growth and metastasis. Method1, Patients: Samples were obtained from 23 patients with primary HCC (mean age 50.86 ?12.78 years) who had undergone partial hepatectomy. The Samples were divided into 3 groups: group A (malignant tissue of HCC), group B (liver tissue within 1.0cm around tumor), and group C (liver tissues 5cm beyond the tumor) respectively. Meanwhile group D (8 cases cirrhosis) and group E (4 donors liver) served as positive and negative control groups. The samples collected during operation were put in liquid nitrogen and kept at-80C in a deep refrigerator. They were diagnosed pathologically by H-E staining and graded by Edmonson’ s method. The samples were graded II in 18 patients and III in 5. Incomplete capsule or no capsule was seen in 9 patients, vessel invasion in 7, and satellite lesion in 7.2, Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): RNA was isolated from the specimen by using Trizol reagent (Gibco,USA). Briefly, the samples were weighed, snap frozen, and ground with a mortar and pestle in Trizol reagent, then allowed warming to room temperature. RNA was then treated with RNase-free lul DNase to remove any contaminating DNA. One microgram of total RNA was reverse transcribed using Revert-Aid? M-MuLV reverse transcriptase and Random primer (Sangon, Shanghai,China). PCR was performed using 2ul of the cDNA and primers specific for Ang-1, Ang-2, VEGF, KDR, Tie2 and the housekeeping gene G3PDH. 13(0.1 PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. Images were captured using Kodak DNA Analysis (Gibco BRL, USA) and density values assessed using Kodak digital science 1S 2.0 software. The identity of PCR products was confirmed by sequencing.3, Western Blot: Samples were homogenized in lysis buffer. Proteins were dissolved in SDS-Laemmli buffer by boiling for 5 min. In each lane 50ug of total proteins were loaded and separated by SDS-PAGE (6% gel for Tie2 and KDR, 8% gel for Angl, Ang2 and VEGF). The electrophoresed proteins were then transferred to a PVDF membrane by a semi-dry electrophoretic transfer procedure for 1.5 h at 1 mA/cm2. Ponceau staining was done to check if all proteins were transferred to a comparable extent in all lanes. The membranes were blocked with 5% defatted milk prepared in TBS buffer, pH 7.5. Then, primary antibodies were added to bind specially with the membrane overnight. After washing 3xl5min with TBST, the corresponding second antibodies were added and incubated for 1 hour. Washing the membrane as described above. The immuno-complexwas visualised by enhanced chemiluminescence detection b更多还原