【作者】 王举；

【导师】 王有德；

【作者基本信息】 吉林大学 ， 外科学， 2004， 博士

【摘要】 研究背景 原发性肝癌是我国常见的恶性肿瘤，死亡率很高，占恶性肿瘤死亡的第二位，在部分农村中占第一位，而且呈上升趋势。手术切除仍是治疗肝癌的首选方法，但由于切除率低，术后复发率高，所以其总体疗效不太理想，而放疗和化疗常难彻底消灭肿瘤细胞，又易损伤正常组织，因此目前迫切需要研究和发展新的治疗方法。近年来生物治疗已成为肿瘤治疗的第四种模式。肝癌的生物治疗策略主要包括抑制细胞增殖和促进细胞凋亡两个方面，其中反义技术因其靶向性强、易操作、可控性等优势，在肝癌的生物治疗中具有重要意义。目前肝癌反义基因治疗主要是采用反义核苷酸技术。由于肝细胞癌的发生具有多因素、多阶段、多基因、多突变的特点，所以选择反义治疗的靶基因至关重要。Altieri等于1997年首先克隆出Survivin基因，其编码的蛋白是目前发现的作用最强的凋亡抑制因子，被称为生存素或生存蛋白。目前发现其生物学效应为：①抑制细胞凋亡，②调节细胞增殖，③诱导血管生成。在人的生长发育中，Survivin在胚胎组织中表达，而在分化成熟的组织中表达丧失。Survivin广泛表达于人类多种肿瘤组织，在100多种肿瘤特异性表达基因中，其表达的特异性列前4位。由于Survivin在正常成熟组织中几乎不表达，而特异性的在肿瘤组织表达，因而被认为是“肿瘤的理想反义靶向治疗标志”，具有良好的靶向性、特异性和安全性。 <WP=102> 目前针对Survivin与原发性肝癌的发生及生物学行为关系的研究为数不多，尤其Survivin和Caspase-9的关系及其在肝癌细胞凋亡和新生血管中作用的研究未见报道，以Survivin作为反义靶基因行肝癌反义治疗的研究也很少见。本实验在证实“Survivin在肝癌中稳定高效表达可抑制细胞凋亡和诱导肝癌新生血管生成，并可作为肝癌独立的不良预后因子”的前提下，以Survivin为靶基因，针对Survivin mRNA翻译起始密码子区设计Survivin反义寡核苷酸片断，转染SMMC-7721细胞，研究其对肝癌细胞增殖和凋亡的影响，为肝癌综合评估和生物治疗开辟一条新的途径。研究目的 1、研究Survivin和Caspase-9在原发性肝癌中的表达及其与细胞凋亡和新生血管的关系，探讨Survivin和Caspase-9在肝癌发生及生物学行为中的作用，为肝癌综合评估提供一个新的指标。 2、以Survivin为靶基因，设计合成针对Survivin的反义寡核苷酸片断，在阳离子脂质体介导下转染SMMC-7721细胞，研究其对肝癌细胞增殖和凋亡的影响，为肝癌生物治疗提供一条新的途径。主要材料和方法 一、主要材料 1、研究对象 （1）组织标本 42例原发性HCC均为吉林大学第一医院普外科1998-2002年手术切除的标本，其中男33例，女9例，≤60岁16例，>60岁26例。按照Edmondson分级标准，Ⅰ级8例，Ⅱ级7例，Ⅲ级17例，Ⅳ级10。肿瘤直径≤5cm16例，>5cm26例。有肝内外转移倾向或转移者（肝门淋巴结转移、门静脉癌栓形成、远处转移、肿瘤数目>2个、癌周有卫星结节）19例。42例癌旁组织均为相应原发性肝癌病人的距癌肿病灶≤2cm的组织，<WP=103>其中肝硬化组织28例，肝炎组织10例，4例为正常肝组织。12例正常肝组织均为吉林大学基础医学院尸检标本。以上所有标本均经10%中性甲醛溶液固定，4μm石蜡连续切片后贴在涂有切片粘合剂的载玻片上备染。 （2）细胞株 人肝癌细胞株SMMC7721购置中科院上海细胞研究所。 2、主要器材 切片机、孵育箱、超净工作台、紫外光光度仪、PCR扩增仪、流式细胞仪、酶标仪、透射电子显微镜等。 3、主要试剂 兔抗人Survivin多克隆抗体、鼠抗人Caspase-9单克隆抗体、鼠抗人CD34mAb试剂盒、TUNEL检测试剂盒、S-P免疫组化试剂盒、阳离子脂质体、RPMI1640培养液、噻唑蓝、RT-PCR 相关试剂盒等。 二、主要方法 （一）免疫组织化学染色和细胞凋亡的检测 S-P法检测Survivin和Caspase-9表达和微血管密度。肝癌细胞浆或细胞核中出现棕黄色颗粒者为免疫组化染色阳性，参照Caide等的计数方法定量Survivin和Caspase-9表达的阳性程度；参照Weidner等报道的方法判定微血管密，统计学处理MVD采用X±S表示；原位末端标记法检测细胞凋亡。 （二）反义核苷酸的设计合成 应用RNA structure3.5对Survivin的二级结构进行模拟，针对Survivin mRNA翻译起始密码子区设计Survivin反义寡核苷酸（ASODN），其序列为：5/-GGCAACGTCGGGGCACCCAT-3/。正义寡核苷酸（SODN）序列：5/-GCCATCGTAGGGGATCGCTT-3/。两条核苷酸序列各有5个硫代硷基修饰，由上海生工公司合成，-20℃保存备用。 （三）实验分组和细胞转染 实验分3组：空白对照组(control)：以无血清的RPMI1640培养液代替转染复合物作为空白对照组。正义对照组（SODN）：SODN转染作为正义对照。实验组（ASODN）：根据ASODN浓度分为100、200、400、800nmol/L转染组。SMMC-7721细胞用含10%小牛血清的RPMI1640培养液在37℃、5%CO2培养箱中培养，在阳离子脂质体介导下以100、200、400、800nmol/L ASODN、SODN分别转染肝癌细胞，每组设4个复孔，转染24、48、72h后收集各组细<WP=104>胞用于后续实验。 （四）反义核苷酸转?更多还原

【Abstract】 Background Primary liver cancer is a common malignant tumor in China. It is the second cause which leads to death, especially in countryside. At present its prevalence has an increase tendency. Now the surgical operation is still the first choice, however, the effect isn’t very satisfying because the rate of postoperation relapse is very high. On the other hand, radiotherapy and chemotherapy can’t kill all tumor cells, and they are easy to impair the normal tissues. So we need to seek new treatments. Recently biological therapy has become the fouth model in treating tumors which mainly includes inhibiting cell proliferation and promoting cell apoptosis. Anyway, anti-sense gene therapy plays an important role because it is easy to operate and control. At present it means anti-sense oligonucleotides technology. Because the pathogenesis of hepatocellular cancer is characterized as multi-factor, multi-stage, multi-gene and multi-mutation.The choice of exact targeting gene is very crucial. Altieri et al first clones the gene of Survivin in 1997. The protein is the strongest apoptosis inhibitor at present called survivin protein. Its biological effects includ: ①inhibit cell apopt, ②regulate cell proliferation, ③induce angiogenesis. Survivin can express in embryonic tissues, but undetectable in differentiated adult tissues It can be detected in many human tumors. And it is the fouth of over 100 tumor-specific genes. Survivin can’t express in normal tissues, so it is regarded as <WP=109>the ideal anti-sense therapeutic target. At present the research on the relationship of Survivin and the pathogenesis of liver cancer is few. Especially there is no report about the relationgship about Survivin, Caspase-9 and its effect in inhibiting liver cell apoptosis and angiogenesis. Anti-sense Survivin gene therapy in the field of liver cancer is very limited. This study proves that Survivin can stably express in liver cancer to inhibit cell apoptosis, and induce angiogenesis. It can function as a kind of poor prognosis index. We design anti-sense oligonucleotides sequences targeting Survivin mRNA translation initiation coden which can transfect SMMC-7721 cells to study the proliferation and apoptosis of liver cancer. This can open a new way for the comprehensive evaluation and biological therapy.Objective 1. We study the expression of Survivin and Caspase-9 in primary live cancer and their effect on cell apoptosis and angiogenesis, and discuss their effect on the pathogenesis of liver caner and biological action. Finally, we offer a new index for liver cancer comprehensive evaluation. 2.Anti-sense oligonucleotides sequences targeting Survivin can transfect SMMC-7721 with cation liposome to examine its effect on liver cancer cell proliferation and apoptosis, this offer a new way for biological therapy of liver cancer finally.Materials and Methods Materials 1.Target (1) tissues specimen: 42 primary liver cancer tissues were obtained by the surgical resection from the department of general surgery, the first hospital of Jilin University from1998 to 2000: 33 male and 9 female, 16 (<60yr) and 26 (>0yr). According to Edmondson Grade Criteria, Grade I is 8 cases, Grade II is 10, Grade III is 16, Grade IV is 8. The diameter of tumor (<5cm) is 16 cases, >cm is <WP=110>26.Metastasis tendency (lymph node metastasis, portal vein cancer thrombosis, distant metastasis, the tumor amounts >2, satellite node around cancer) is 19 cases. 42 nontumor tissues were those which were 2cm away from the lesion, including 28 cirrotic tissues, 10 hepatitis tissues and 4 normal liver tissues. 12 normal liver tissues were obtained by autopsy from Faculty of medicine of Jilin University. All tissues were fixed in 10% formalin for slices staining. (2) Cell lines: SMMC-7721 is purchased from Shanghai cell research institute of Chinese Academy of science. 2.Equipment: Microtome, Incubator, Hyperclean Working platform, Ultraviolet Spectrophotometer, PCR Flycyte Meter, Enzyme Labelling Meter, Transmission Electron Mic更多还原