【作者】 郭玉忠；

【导师】 薛乐勋；

【作者基本信息】 郑州大学 ， 病理学和病理生理学， 2004， 博士

【摘要】 卵巢癌是死亡率最高的妇科恶性肿瘤，因为卵巢癌缺乏完善的早期诊断方法，多数患者就诊时已属晚期，传统手术切除和联合化疗的总体疗效不理想，化疗还容易产生耐药性；而且易复发，晚期卵巢癌的5年存活率低于30％，因此有必要探索新的有效治疗方法。随着分子生物学的发展，人们希望从分子水平上干预肿瘤生长达到治疗目的。由于卵巢癌具有非常独特的生物学特性，即使在病变晚期，肿瘤细胞也主要局限在腹腔内，为局部基因治疗提供了便利条件，不仅可以提高原位基因转染的载体浓度，而且可以防止转染基因的腹膜外全身扩散，既提高了基因治疗的效率，又可最大限度地减少基因治疗的不良反应。 分子化学治疗又称自杀基因治疗，他是将编码某些特定酶的基因导入肿瘤细胞，这些酶可将无活性或低毒的药物前体转化为具有较强细胞毒性的药物，从而杀死肿瘤细胞。单纯疱疹病毒胸苷激酶基因(herpes simplex virus thymidine kinase，HSV-tk)／更昔洛韦(ganciclovir，GCV)系统是分子化疗中研究最为深入的一种。HSV-TK酶可将GCV磷酸化为GCV一磷酸，继而在哺乳动物细胞TK酶的催化下最终转变为GCV三磷酸，抑制DNA聚合酶的活性，掺入DNA合成，产生细胞毒性。而且郑州大学2004年博}:论文hTERT启动了调控的HSV-tk从因治疗系统联合拓普替康对卵巢癌细胞的作用刁}究发现该方法还具有“旁现者效应”，即转染细胞可以杀死附近未转染细胞的特性，从而弥补转染效率低的缺陷，发挥更强大的治疗作用。在分子化学治疗，I，，要实现选择性地消除肿瘤细胞，必须将基因的表达限定于肿瘤细胞，即实现肿瘤基因治疗的靶向性。它一可以采用下列手段:(1)将日的基因特异地导入靶细胞，即基因转移的靶向性;(2)采用组织或肿瘤特异性启动子调控己导入靶细胞的目的基因，使之在特定的组织器官中表达，达到基因表达的靶向性。日前在}}中瘤基因治疗临床试验中应月J的是组成性原核启动子，在细胞中可以大量表达但缺乏特异性或选择性。最常用的启动子来源于巨细胞病毒和SV40病毒启动子，他们缺少肿瘤特异性，对正常细胞有潜在毒性。山于它们来源于病毒，在体内其活性一可能下调，而且巨细胞病毒启动子还可以通过甲基化失活。因此选择一个和正常细胞相比在卵巢肿瘤细胞中特异上调的启动子，有可能解决上述问题。现已有儿个肿瘤特异性或卵巢癌特异性启动子正处于临床前研究中，但他们同样存在转录活性低和卵巢癌特异性低的问题，因此仍然有必要寻找转录活性高、肿瘤特异性强的启动子。 人端粒酶是细胞的逆转录酶，由一条单链RNA与多个蛋白质组成的核糖核蛋白复合物。也是至今发现的特异性最高的肿瘤标志物。其功能是以自身的RNA为模板，逆转录合成端粒重复序列单元，加到染色体末端，维持分裂细胞的端粒长度，防止染色体缩短，使细胞具有继续增娘的能力。立尚粒酶的主要成分之一是人端粒酶逆转录酶(human tefomeraserevcrse transcriptase，hTERT)，hTERT仅在端粒酶l‘l二{性的)J中瘤细胞和永生化细胞中表达，hTERT的转录伴随着端粒酶的活性，提示它是端粒酶活性的限速囚索之·，他的启动子可能是一个好的肿瘤特异性启动子。}川此我们克隆厂hTEltrr启动一子，构建了hTERrr启动子一调控的HS丫tk逆转郑州大学2004年博士论文hTERT启动子调控的HSV-tk基因治疗系统联合拓普替康对卵巢癌细胞的作用录病毒载体，将其导入人卵巢癌细胞，观察了它的体内和体外对卵巢癌细胞的治疗作用。并将该系统和常规化疗药物拓普替康(t叩otecan，TPT)合用，研究合用是否能增强肿瘤治疗效果。第一部分hTERI，启动子的克隆及鉴定方法1.细胞端粒酶蛋白的含量测定用考马斯亮蓝G一250溶液和牛血清白蛋 白在石英比色管中稀释成不同浓度的牛血清白蛋白，测定595nm处的 光吸收值，制备蛋白标准曲线。将常规培养的人卵巢癌SKOv3和人胚 肺WI38细胞用0.25%胰酶和0.02%EDTA消化，冰预冷的PBS洗涤一 次，加入200协l冰预冷的裂解液，裂解后离心30min，吸取上清10 pl， 用蛋白标准曲线测定细胞端粒酶蛋白的含量。2.细胞端粒酶活性的测定首先应用PCR方法扩增端粒重复序列，然后 用ELISA方法显色，使用酶标仪以69Onm为参照波长测标本450nm 的吸光值(A)。同时设阴性对照和阳性对照。3.hTERT启动子的克隆取正常人外周血单核细胞基因组DNA，以此 DNA为模板，根据设计好的引物，用PCR方法克隆hTERT启动子， 1%琼脂糖电泳检测，产物片段大小正确后，PCR反应液进行1.0%琼 脂糖凝胶电泳，回收纯化PCR产物后，将该片段与pMD18一T载体连 接，转化大肠杆菌，提取质粒并用酶切鉴定正确后进行测序分析。4.表达载体的构建用Notl和KPnl酶切pEGFP一NZ质粒，回收约800 bp的绿色荧光蛋白(green fluoreseent protein，GFp)片段，插入 peDNA3.1(+)载体的多克隆位点，阳性重组子命名为pcDNA3.1(+)一GFp (PG)。用Bgl 11和Kpnl酶切pMD18一T载体，回收733 bp的片段，郑州人学2004年博卜论文hTERT启动了调控的HS丫tk从l川治疗系统联合拓普替康对卵巢痴细胞的作用 与预先经Bglll和助nl?更多还原

【Abstract】 Ovarian cancer remains the leading cause of death from gynecological malignancies. Due to the lack of effective prevention and screening modilities, the majority of patients with epithelial ovarian cancer are at advanced-stage of the disease when they are diagnosed. Despite improved surgical techniques and chemotherapy, the long term prognosis for patients with advanced disease has not markedly improved over the past 10 years. The 5-year survival rate for patients with advanced-stage ovarian cancer is approximately 30% and most patients ultimately succumb to the disease. Hence, better therapies must be investigated. The confinement of ovarian cancer within the peritoneal cavity in the majority of cases suggests the possibility of effective locol gene therapy.Molecular chemotherapy is a gene therapy approach designed to achieve selective eradication of cacinoma cells via a genetically expressed toxin.This method is often named as "suicede gene therapy." One of the strategies utilizes a enzyme/prodrug system which can convert a prodrug into a toxic metabolite leading to cell death. The most frequently utilizedsystem is the thimidine kinase (tk) gene from the herpes simplex virus (HSV), given in combination with ganciclovir (GCV). The HSV-tk product has high affinity for acyclovir and its analogues, including ganciclovir, maimly producing the phosphorylated product. Subsequent modification by the host cell to a triphosphorylated form and incorporation during replication halts growth of the developing DNA strands and inhibits DNA polymerase activity. Mammalian TK has a much lower affinity for the prodrug such that tumor cells once transduced are selectively killed in the presence of ganciclovir. The efficiency of this approach can be amplified by a "bystander effect", killing the nontransduced neigbouring cells. Thus, suicide gene therapy theoretically is not necessarily targeting 100% of tumor cells for effective treatment.However, gene expression in nontarget cells is a central problem in cancer gene therapy. Efficient gene therapy regiments require transgene expression especially in tumors. There are two strategies to achieve this goal. Transductional targeting can be accomplished by modification of vectors. Another method is transcriptional targeting achieved by regulation of transgene expression. Gene regulatory elements that drive transcription of some special proteins in tumors have the potential capacity to control gene expression in a tumor cell-specific manner. Transcriptional targeting is based on the use of these tissue or tumor-specific elements, mainly promoters. There have been several candidate promoters analyzed in gene therapy studies for specific transcriptional control in ovarian cancer cells. Their activity and specificity are still being evaluated. Telomerase is a specialized DNA polymerase responsible for the replication of telomeres. Telomerase is highly active in most immortalized cell lines and more than 85% human cancers but is inactive in most somatic cells. The main components of thehuman telomerase enzyme are the human template RNA (hTR) component and the human telomerase reverse transcriptase(hTERT). Although hTERT and hTR are both necessary for telomerase activity, expression of hTERT is present specifically in tumor cells whereas hTR is present in both normal and tumor cells. Because the hTERT gene is highly active in tumor cells but repressed in most normal cells and because its expression is regulated at the transcription level, the hTERT promoter may be used for tumor-specific expression of transgenes. In the present study, hTERT promoter was used to induce exprssion of the HSV-tk gene, and efficiency was tested in cancer gene therapy combined with topotecan.The first part: Cloning and identification of human telomerasereverse transcriptase gene promoter Methods1. Preparation of standard protein curve: Standard protein curve was prepared with different bovine concentration in Coomassie Brilliant Blue G-250. Afetr abstracting protein from human ovarian cancer更多还原