【作者】 杨平；

【导师】 于成国；

【作者基本信息】 中国医科大学 ， 生物化学， 2003， 硕士

【摘要】 前言 端粒是真核细胞染色体末端的特殊结构，它由串联排列TTAGGG碱基的重复序列组成，具有维持染色体稳定性和完整性、固定染色体在细胞内位置的作用。端粒的重复序列在每次细胞分裂后缩短，当长度短到一定程度时，细胞停止分裂进入衰老和死亡。端粒的合成依赖于端粒酶，它能利用自身RNA为模板逆转录合成染色体末端的端粒DNA，从而维持端粒一定的长度。多数正常体细胞不表达端粒酶活性，多数永生化细胞、恶性肿瘤细胞表达端粒酶活性，端粒酶的重新表达在细胞永生化及癌变过程中起着重要的作用。 卵巢癌是妇科恶性肿瘤中死亡率最高的肿瘤，卵巢癌的发展及转移也与端粒酶活性相关。顺铂(DDP)是卵巢癌化疗中最有效的基础药物之一，它是一种类烷化剂的抗肿瘤药物，为细胞周期非特异性药物，可作用细胞周期的任一时相，利于临床的联合用药，但由于DDP的诸多毒副作用及卵巢癌细胞易产生对其耐药性，常使化疗效果不满意。研究发现，人体内必须的营养物质维生素C(VC)可降低抗肿瘤药的毒副作用，并具有抗肿瘤的能力，给各类晚期癌症患者大剂量注射VC，具有显著缓解肿瘤患者症状并延长寿命的作用，但目前对VC抗肿瘤作用机制还不清楚。 本文采用DDP、VC作用于卵巢癌细胞，以细胞端粒酶为靶向研究DDP、VC二者抗肿瘤的作用机制，以及VC对DDP抗肿瘤作用的影响，为肿瘤的临床治疗提供科学依据。 材料与方法 1．材料 1．1材料：CAOV3细胞(卵巢癌细胞株) 互．2试剂和仪器：Rnase、MTI’、SDS、TEMED、Acrylamide、Bi－sacrylamide、CHAPS、AEBSFB、Tas DNA聚合酶、dNTP＊s和 Cx弓物＊厕铂oBR322 DNA／Hae Ill Markers，其它试剂为国产分析纯。 752C型紫外可见光光度仪、相差显微镜3CR－4型高压电泳仪JHZ－95台式恒温震荡箱JLX－800酶标仪、HRSPO520型PCR扩增仪SORVALL高速离心机人O。孵箱、超净台等。 2．方法 2·ICAOV3细胞的培养 2．2 DDP、VC诱导 CAOV3细胞 2．3 Mrt法测定细胞活力 2．斗 蛋白定量：采用酚试剂法测定蛋白。 2．5 端粒酶纯化 2．6 TMP法测定端粒酶活性 结 果 1．细胞毒性试验（M’IT法）DDP、AA—DDP对肿瘤细胞株CAOV3有不同程度的毒性作用，MTh测得IC。。分别是：DDP为8 pmoVL、VC（10 pmol／L）－DDP为 7 pmoUL、VC（50卜moUL）－DDP为4pmol／L；致死剂量分别是：DDP为200pmo＊L、VC（10pmoVL）－DDP为160pmoVL、VC（50pmol／L）－DDP为120pmoVL。DDP浓度为30p moVL时，死亡率是72．9助JC（10pmoVL）－DDP为73．8％、VC（50卜moVL）－DDP为90．3％。 2．DDP对端粒酶活性的影向：当200mol／L DDP作用 CAOV3细胞4小时，即刻收集细胞，药物未对细胞端粒酶活性产生抑制；在细胞经过4小时DDP处理，去除药物再培养20小时，端粒酶活性完全抑制；8 rmol／L DDP作用细胞 24 ／J’时，端粒酶活性部分抑制；当sumol／L DDP作用细胞120／J’时，对端粒酶活性显著抑制 ·2·作用。 3．VC和 VC－DDP对端粒酶活性的改变：10 pmOUL和50pmoUL VC作用 CAOV3细胞60／J’时，都不改变细胞端粒酶活性，但二者可加强对端粒酶活性的抑制作用，且50pmol／L VC加强＊＊P对端粒酶活性的抑制作用比10卜mo＊L＊C更显着。 讨 论 由于抑制端粒酶活性或人为地缩短端粒的长度，都可使细胞活力及不死性受到损伤，耐药的肿瘤细胞端粒酶不发生改变等原因，以端粒酶做为肿瘤治疗的目标已成为研究的热门。化疗是卵巢癌最主要治疗方法，而DDP是卵巢癌诸多化疗药物中最有效的药物之一，其细胞毒性的分子机制为：DDP分子中的一个氯原子与鸟膘吟的第7位氮原子（G－·N＊结合，另一个氯原子可以和蛋白质或一些小分子的亲核基团如－NH卜－SH结合，造成DNA链内或链间交联，或DNA一蛋白交联，形成了铂－DNA络合物，导致＊＊A致命损伤而发挥作用。本实验通过用12O卜mo＊L和speVL DDP处理人CAOV3细胞，结果说明两种浓度的 DDP对端粒酶活性都有抑制作用。细胞经过120卜mol／L DDP处理4／J’时，直接检测无端粒酶活性的变化；但去除DDP经过20小时再培养后端粒酶活性受到显著抑制，这说明＊＊P对端粒酶活性抑制可能是一个时间依赖性过程，即细胞需要经过一定的时间才表现出端粒酶活性受到抑制。实验还发现，用 120pmol／L和spmoVL DDP分别作用于CAOV3细胞，24小时端粒酶活性分别下降88％和48％；而用spmoUL DDP作用细胞 120／J’时后，端粒酶活性也可下降达84％。可见，DDP能有效抑制端粒酶活性，随浓度递增和诱导时间的延长，对端粒酶活性抑制越显著。在晕丸癌的研究中发现：DDP的抑制作用是由于此药物进人细胞后和DNA连接，形成G－Pt－G的连接方式。而端粒和端粒酶RNA复合物（hTR）也富含 ·3·G，因此DDP对端粒酶活性的抑制亦可能是通?更多还原

【Abstract】 IntroductionHuman ovarian cancer is the hightest death - rate disease in Gynecologic cancer , drug - treat is the important therapy. Telomerase can be dective in most of gynecologic cancer cells . Maintenance of te-lomere length is crucial for survival of cells. Telomerase, an enzyme that is responsible for elongation of shortened telomeres, is active in human ovarian cancer cells as well as most tumor tissues and experimentally immortalized cells. In contrast, most mature somatic cells in human tissues express undetectable or low telomerase activity, implying the existence of a stringent and negative regulatory mechanism. In this study, we report the effects of cisplatin on telomerase activity in human ovarian cancer cells. The special relationship between activity and cancer has a important application value. Vitamin C can remission the symptom of cancer patien and lengthen life span, but the relationship of it and telomerase was not reported. Cisplatin is widely administrated in cancer therapy, it is cell cycle non - specific agent and is combined with other drugs. The caov3 cells were cultured in medium containing different concentrations vitamin C and for different time. Cisplatin and vitamin C colture caov3 cells, then the telomerase activity was observed in this text.In this study, we investigated the telomerase activity of vitamin C with or without cisplatin on cultured human ovarian cancer. At thesame time , investigated whether inhibitory effects induced by cispln tin resulted in tumor growth inhibition.Material and Methodsmaterial . caov3 cells (human ovarian cancer cell) Reagent and equipment . Rnase,MTT,TEMED,Acrylamide,Bi-sacrylamide,CHAPS,AEBSFB,Tag DNA polymerase,dNTP,Ts and Cxprimer,cispiatin,pBR322 DNA/Hae III Markers, all other chemicals were of resgent grade.752 Cultraviolet - visible spectrophotometer, phase contrast microscope,SCR-4 Hyperbaric electrophresis apparatus, THZ-95set homoiothermyshake box; ELX-800 enzyme-labelled equipment, HRSP0520 PCR instrument, SORVALL centrifugal machine,CO2 incubation equipmen, super clean benchMethods2. 1 Cultivation the caov3 cells.2. 2 Induction of the cisplatin and vitamin C.2. 3 The MTT assay.2.4 Protion determination . Pritein was determined by the method of lowry et al.2. 5 Purify the telomerase in human ovarian cancer cells2. 6 The telomerase activity in cells was detected by telomeric repeat amplification protocol ( TRAP) - silver straining and was analyzed by the specific software.ResultsBoth 50mol/L and 10mol/L vitamin C were not due to thechange of telomerase activity , however they can enhance the inhibition of telomerase activity by cisplatin . The suppressing effect was enhanced with the concentration increasing of vitamin C .In 200mol/L cisplatin, no telomerase inhibition was found when cells were collected only after 4 hours of drugs treatment, but the telomerase activity of cells was completely inhibited by cisplatin when removed the drug and recultured for 20 hours. However, vitamin C had no effect. In 8mol/L cisplatin , it was similar with 200mol/L cisplatin to uppress the telomerase acting of caov3.DiscussionTelomeres, which are important for maintaining chromosome integrity and functions, shorten with each cell division. Telomerase,responsible for telomere synthesis, is expressed in most of human tumor cells but seldom in normal somatic cells. Telomerase inhibition has, therefore, been proposed as a novel and potentially selective target for therapeutic intervention . A down - regulation of telomerase activity was found when cells of a human ovarian cancer cell line, caov3 , were treated with cisplatin. The present results may indicate a positive relationship between anticancer effects and down - regulation of telomerase activity by anticancer drugs. DDP reduced telomerase activity in the cells in a concentration - dependent manner as measured by using the telomere repeat amplification protocol (TRAP) assay. The effect on the telomerase activity of the type of an更多还原