【作者】 陈亚侠；

【导师】 谢幸；

【作者基本信息】 浙江大学 ， 妇产科学， 2004， 博士

【摘要】 绒毛膜癌是一种对化疗敏感的实体肿瘤。甲氨蝶呤(Methotrexate，MTX)是治疗绒癌的首选药物，也是在绝大多数联合化疗方案中必需的药物，但MTX获得性耐药严重，单一MTX约30％患者将出现耐药。耐药是治疗失败患者死亡的首要原因。肿瘤对MTX产生耐药的机制涉及多种因素，在对MTX耐药的多种不同细胞系的研究中，认为通过减少叶酸载体而减少MTX的摄入、靶酶二氢叶酸还原酶(DHFR)活性增加及DHFR与MTX的亲和力降低是MTX耐药的重要机制。但仅用这些机制不能解释在研究中发现的当对MTX出现耐药后也会对其它抗癌机理不同于MTX的其它化疗药物耐药。随着对细胞凋亡研究的深入，发现细胞凋亡是众多化疗药物杀伤肿瘤细胞的共同通路之一，推测凋亡的抑制可能导致对多种药物的耐药。凋亡的基本通路包括死亡受体通路、线粒体通路。对MTX是否通过上述途径诱导绒癌细胞凋亡及凋亡抑制与耐药的关系文献报道甚少。本研究首先采用剂量递增间歇诱导的方法，建立体外获得性耐MTX的绒癌细胞株，并对其生物学特性进行研究，为绒癌耐药机制的研究提供细胞模型；然后对耐药诱导前后的绒癌细胞在MTX作用后凋亡率及凋亡相关蛋白表达的改变，探讨MTX是否也通过诱导绒癌细胞凋亡起作用及其与耐药的关系；最后通过凋亡途径中Caspases酶活性的测定及线粒体膜电位改变的检测，探讨MTX诱导绒癌细胞凋亡的通路。 第一部分 人绒毛膜癌甲氨蝶呤耐药细胞株(JAR／MTX)的建立及其生物学特性 目的：建立耐甲氨蝶呤(MTX)的人绒毛膜癌细胞株，并检测其生物学特性。方法：采用剂量递增间歇诱导的方法，对人绒毛膜癌细胞株JAR进行长期诱导培浙江大学医学院2004届博士学位论文养建立耐药细胞株JA侧MTX。比较两种细胞倍增时间、绒毛膜促性腺激素(hCG)分泌量、对MTX和一些常用化疗药的敏感性，P一糖蛋白、谷眺甘肤S转移酶、核增殖抗原的表达及凋亡特征。结果:历时1年建成JA侧MTX细胞株，对MTX耐药性稳定，耐药指数7.35，对紫杉醇、长春新碱有交叉耐药。与其母系JAR细胞相比JA卿MTX细胞增殖减慢、核增殖抗原表达降低、异倍体出现率增加、谷肤甘肤S转移酶表达增高、HCG分泌量升高、自发凋亡率减少，但P一糖蛋白的表达无改变。结论:JA侧MTx细胞对MTx耐一药性稳定，具有独特的细胞形态、染色体核型及增殖、凋亡等特征，为进一步研究绒癌的耐药机制提供理想的细胞模型。 第二部分甲氨蝶吟诱导JAR细胞及JAR/加ITX细胞凋亡及相关蛋白表达 目的:观察不同浓度MTX作用不同时间诱导JAR细胞及JAR爪ITX细胞凋亡，及 Bd一2、Bax基因在其诱导凋亡中作用。方法:用流式细胞仪检测不同浓度MTX不同作用时间后JAR细胞及JA树MTX细胞凋亡指数及细胞周期变化，同时用免疫细胞化学半定量法检测在JAR细胞凋亡中Bcl一2、Bax基因表达的变化。结果:MTX浓度为0.1一1 .0雌/ml时作用24小时以后可诱导JAR细胞凋亡，当MTX浓度为5.0协g/ml时主要导致细胞坏死。上述浓度MTx都可导致JAR细胞周期阻滞，GO/GI期细胞增加。而MTX浓度为0.1一1 .0卜g/ml时对JAR/MTX细胞凋亡及细胞周期无明显作用。当MTX浓度为5 .0协g/ml时可使JA侧MTX细胞凋亡，细胞周期阻滞，并伴有坏死。JAR细胞及JAR/MTX细胞凋亡时伴有Bax表达增加及Bcl一2表达减少。结论:低浓度MTX可诱导JAR细胞凋亡，而高浓度时导致JAR细胞坏死。凋亡抑制是JA凡MTX细胞对MTX耐药的重要途径，Bd一2/Bax参与及JA卿MTX细胞耐药。 第三部分甲氨蝶吟诱导JAR细胞及JAF口MTX细胞凋亡通路 目的:通过检测在MTX诱导凋亡过程中Caspase一8、Caspase一9活性的变化及线粒体膜电位的改变，探讨MTX诱导JAR细胞及J入田MTX细胞凋亡的通路。方法:用2.5林留ml MTX加入JAR细胞及J戌胃M饮细胞中培养48小时诱导凋亡;采用MitocaptureTM试剂盒在荧光显微镜下观察及采用流式细胞仪定量检测线粒体通路的细胞凋亡率;用FLICE/Caspase一8比色法蛋白酶分析试剂盒及Caspase一9/Mch6比色法蛋白酶分析试剂盒在酶标仪上分别检测凋亡诱导前后浙江大学医学院2004届博士学位论文CasPase一8和Caspase一9酶活性的改变。结果:荧光显微镜观察JAR细胞及JA侧MTX细胞在MTX作用后均可见发绿色荧光的凋亡细胞，但JAR细胞明显多于JA卿MTX细胞。JAR细胞无药培养48h后经流式细胞仪检测线粒体途径的凋亡/丈门目况.七月‘U.率为2.5%士0.25%，在2.5林g/ml MTx作用48小时后线粒体途径凋亡率为28士3.1%。而JA侧MTX细胞不加MTX时线粒体途径凋亡率为0.5%士0.01%，2.5雌/m 1 MTx作用48小时后为1 .0%士0.01%，JA侧MTX细胞在无药和加药后线粒体途径的凋亡率均显著低于JAR细胞。JAR细胞和JA侧MTX细胞加2.5陀/mlMTX作用48小时后Caspase一9的活性分别是不加MTX的4.35士0.76倍和1.15士0.09倍，CasPase一8活性无显著变化。结论:MTX通过线粒体途径诱导JAR细胞及JAR/MTX细胞凋亡，线粒体途径凋亡域值升高是JA刃MTX细胞对MTX耐药的重要机制。更多还原

【Abstract】 Choriocarcinoma is one of solid tumors which are sensitive to chemotherapy. Methotrexate (MTX) is a first-line and essential component in combined chemotherapy in the treatment of choriocarcinoma. However acquired drug-resistance is common and serious in choriocarcinoma. It is estimated that about 30% of patients will develop drug-resistance if single MTX is used. Drug-resistance is an important reason that causes failure of treatment and results in death of patients .The mechanisms of MTX-resistance are complicated, including decrease of MTX intake, increase of DHFR activity and decrease of the infinity of MTX to DHFR. But these mechanisms can not explain cross-resistance to other chemotherapy drugs. Following the advance of studies on cell apoptosis, it is found that cell apoptosis is the common pathway of cell toxity of chemotherapy drugs. Therefore , we suposed that suppression of apoptosis maybe one of mechanisms of drug-resistance. The basic apoptotic pathway includes death receptor pathway and mitocondrial pathway. Little reports have been found on the roles of apoptosis in cytotoxicity of MTX and it’s relationship to drug reisitance. In the part one of the present study , we established MTX-resistant cell line (JAR/MTX) which was derived from JAR cell line by exposed to MTX with intervally and progressively increasing concentration , and observed it’s biological characters . In the part two of the study , we detected the rate of methotrexate-induced apoptosis and apoptotic relatedproteins .In the part three of the study . we investigated the apoptotic pathway involved in methotrexate-induced apoptosis by detecting the activity of Caspases-8, Caspases-9, and the change of mitochondrial membrane potential.Part I Establishment and biological characters of methotrexate-resistant human choriocarcinoma cell line(JAR/MTX)Objective: To establish a MTX-resistant choriocarcinoma cell line (JAR/MTX) and determine its biologic characteristics. Methods: JAR/MTX was derived from JAR cell line by exposed to MTX with intervally and progressively increasing concentration . Sensitivity to some chemotherapy drugs was detected by MTT. P-gp> GST-7I and PCNA expressions were detected by immunohistochemistry. Cells apoptosis was detected with flow cytometry (FCM) assessment of PI/Annexin V stain. Growth rates and human chorionic gonadotropin (hCG) were also detected. Results: JAR/MTX with stable drug resistance was established after one year. Its resistance index to MTX was 7.35. Besides resistance to MTX, it exhibited cross-resistant to TAX and VCR. Growth rates of JAR/MTX were slower than that of JAR. Expression level of PCNA in JAR/MTX was lower and GST-it expression level in JAR/MTX was higher than that in JAR. No statistical difference of expression level of P-gp existed between in the two cell lines. JAR/MTX secreted high hCG than JAR. The flow cytometry showed that the spontaneous apoptosis in JAR/MTX was significantly lower than that in JAR. Conclusions: JAR/MTX cell line presented stable resistant to MTX with different characteristics, including morphology, proliferation, apoptosis rate from it’s parental line JAR. It is indicated that JAR/MTX may serve as an ideal model for studying the mechanisms of drug resistance in choriocarcinoma.Part II Methotrexate-induced apoptosis and expression of apoptotic-related proteins in JAR and JAR/MTX.Objective: To investigate MTX-induced apoptosis in human choriocarcinoma JAR and JAR/MTX cell line and the change of Bax/Bcl-2 expression during apoptosis. Methods: Cells apoptosis was detected by assessment of PI/Annexin V stain with FCM.Bcl-2 and Bax expressions were detected by immunocellchemistry. Results: JAR cells underwent apoptosis and cycle retardation when exposed to 0.1-1.0ug/ml MTX after 24 hours but not JAR/MTX cells. JAR cells presented necrotic death when exposed to 5.0ug/ml MTX,while JAR/MTX cells just started to present apoptosis. The H-score of Bcl-2 was decreased while H-score of Bax was increased when both kind of cells underwent apo更多还原