【作者】 王丹；

【导师】 惠宁；

【作者基本信息】 第二军医大学 ， 妇产科学， 2008， 硕士

【摘要】 研究背景化疗是临床上晚期卵巢癌的主要治疗手段之一,目前卵巢癌化疗的一线药物以铂类为主,但是肿瘤细胞对铂类药物表现出的耐药性的逐渐增强已经显著限制了其临床疗效。因此,为解决晚期卵巢癌对铂类药物耐药的难题,寻找有效的化疗增敏药物以制定更佳的化疗方案,改善患者的生活质量,国内外学者做了许多的研究。流行病学研究显示,甾体激素在卵巢癌的发生中起到一定的作用,促性腺激素、雌激素以及雄激素可能是致病因子,而促性腺激素释放激素（gonadotropinreleasing hormone,GnRH）可能是保护因子。GnRH是下丘脑肽能神经元分泌的一种十肽激素,其在体内的重要功能是由GnRH受体介导完成的。有研究发现,除垂体外,在人类的一些恶性肿瘤细胞中也有GnRH及其受体的表达。目前临床应用人工合成的GnRH类似物（gonadotropin releasing hormone analog,GnRHa）治疗多种性激素依赖性肿瘤（如乳腺癌,前列腺癌等）,机制在于此类药物和GnRH受体结合,通过垂体的“去势作用”而抑制性激素的分泌,或直接对肿瘤细胞产生抗增殖作用。许多研究表明,约80%的卵巢癌组织中有GnRH及其受体的表达,GnRHa对卵巢癌细胞系的抗增殖作用与抑制表皮生长因子受体（epidermal growth factor recaptor,EGFR）信号转导通路密切相关。正常卵巢上皮组织中EGFR表达量很低或缺如,但70%以上的卵巢癌组织中有EGFR高水平表达。GnRHa与受体结合后激活磷酸酪氨酸磷酸酶（Phosphotyrosine phosphatase,PTP）,抵抗表皮生长因子（epidermal growthfactor,EGF）诱导的EGFR酪氨酸自磷酸化作用,从而干扰表皮生长因子受体上的有丝分裂信号转导,抑制癌细胞的增殖。最近,有研究指出卵巢癌细胞通过EGFR介导的信号转导效应的增强,导致卵巢癌细胞对铂类药物耐药性的增加,而下调或抑制EGFR表达可以增加癌细胞对化疗的敏感性。有关GnRH类似物作为化疗逆转剂逆转人类乳腺癌细胞的多药耐药性研究已被证实,而GnRH类似物对卵巢癌耐药细胞的化疗逆转作用,目前较少有文献报道。本实验建立了卵巢癌顺铂耐药的体外细胞模型,观察GnRH类似物曲谱瑞林（Triptorelin）对逆转卵巢癌细胞顺铂耐药的效果,为卵巢癌临床联合化疗提供新的思路。研究目的观察体外实验中曲谱瑞林逆转卵巢癌细胞顺铂耐药的效果,并初步探讨其作用相关机制。方法和结果第一部分实验方法和结果实验方法体外建立卵巢癌顺铂耐药亚细胞系OVCAR-3/DDP:1、采用浓度梯度递增法,基于人类卵巢癌OVCAR-3细胞系诱导建立卵巢癌顺铂耐药细胞系模型,命名为OVCAR-3/DDP;2、MTT法测定IC₅₀、耐药指数（RI）;3、流式细胞技术检测细胞周期分布情况。结果1、采用浓度梯度递增法对OVCAR-3细胞进行体外诱导,历时10个月获得其耐药细胞亚系,在浓度为3μmol/L的顺铂环境下能生长良好,于无药培养基中培养2个月耐药性稳定,命名为OVCAR-3/DDP细胞;2、细胞周期FCM分析:相对于亲本OVCAR-3细胞,其顺铂耐药株OVCAR-3/DDP细胞对数生长期的增殖速度减慢,细胞多数分布于G₀/G₁期,细胞主要处于潜伏期,增殖缓慢,S期细胞数目明显减少,从而避开顺铂主要作用的细胞周期S期;3、MTT增殖实验结果分析:OVCAR-3细胞对顺铂的IC₅₀为6.22μM,OVCAR-3/DDP对顺铂的IC₅₀为83.48μM,耐药指数（RI）为13.42。第二部分实验方法和结果实验方法研究曲谱瑞林对OVCAR-3/DP细胞的化疗逆转作用及耐药细胞表面EGFR表达改变1、实验分组:选取两药的血药峰值浓度（Cmax）作为联合作用的基础。设计0.01、0.1、1、10、100倍Cmax共5个浓度梯度,分为:Ⅰ、耐药细胞顺铂处理组;Ⅱ、耐药细胞曲谱瑞林处理组;Ⅲ、耐药细胞两药联合处理组（处理浓度为相同梯度倍数的浓度值相加）;Ⅳ、耐药细胞未处理组;Ⅴ、亲本细胞未处理组。2、比较Ⅰ-Ⅳ不同加药情况下细胞抑制率的差异;3、MTT法检测IC₅₀、化疗逆转倍数（RR）:RR=IC₅₀（顺铂处理组）/IC₅₀（顺铂+曲谱瑞林联合处理组）;金式公式求q值分析两药是否具有协同作用;4、EGFR流式免疫荧光测定:采用流式细胞仪将各组别EGFR（RPE）标记好的细胞应用Cell quest软件程序进行资料处理。计算分别以Cmax-T、Cmax-D、Cmax-（T+D）作用和不加药处理四种情况下各组耐药细胞样品中表达EGFR的细胞阳性率及单个细胞表达EGFR的平均荧光强度,以此表示其相对含量。结果曲谱瑞林对OVCAR-3/DDP细胞的化疗逆转作用及耐药细胞表面EGFR表达改变:1、各组细胞生长情况比较:顺铂组、曲谱瑞林组的耐药细胞生长状况与耐药细胞未加药处理组接近,两组间各时间点的生长抑制率比较,差异均无统计学意义（P＞0.05）;而两药联合处理组的耐药细胞生长受到较明显抑制,与单用顺铂处理组比较差异有统计学意义（P＜0.01）;2、采用MTT法及Excel软件加权线性回归法计算Ⅰ-Ⅲ不同加药组别耐药细胞的IC₅₀,化疗逆转倍数（RR）为3.91;根据金氏公式求q值为1.75,q值＞1说明曲谱瑞林和顺铂对耐药细胞的联合作用为协同作用。3、耐药细胞的EGFR表达改变:与单药处理组及未加药处理组细胞比较,在两药联合处理组中表达EGFR的细胞阳性率明显下降（P＜0.01）,且单个细胞表达EGFR的荧光强度明显降低（P＜0.01）。提示联合用药时,耐药细胞表面EGFR的表达显著下调。结论1、成功建立了卵巢癌顺铂耐药细胞亚系OVCAR-3/DDP,为研究逆转卵巢癌顺铂耐药提供了体外实验模型;2、细胞增殖实验结果显示,曲普瑞林与顺铂联合应用可部分逆转卵巢癌顺铂耐药细胞OVCAR-3/DDP对顺铂的耐药性,且两药联合对耐药细胞的杀伤作用为协同作用;3、和单药作用比较,两药联合应用时耐药细胞EGFR的表达降低明显,提示曲普瑞林对顺铂耐药卵巢癌细胞的化疗逆转作用可能与其下调细胞表面EGFR表达有关。更多还原

【Abstract】 Introduction At present,chemotherapy is one of the primary modalities for the treatment of ovarian carcinoma.The cisplatin is one of the most effective anticancer drugs.However, the acquired resistance to cisplatin is one of the most significant reasons for relapsing and recurrence of the ovarian cancer.Various studies demonstrated that the epidermal growth factor receptor-mediated signal transduction in human ovarian cancers plays an important role in celluar reproduction and development of acquired resistance to drugs.About 70%-100%of ovarian cells overexpress receptors for EGF.The ovarian cells with expression of EGFR have more ability of metastasis,invasion,insensitivity to chemotherapy or radiotherapy,poor prognosis,high recurrence rate and short life span.Other studies have shown that antiproliferative effet of GnRH and its analogs（GnRHa） in ovarian cancer cells may probably mediated by down-regulation expression of EGFR.The antiproliferative effects of GnRHa and the resistance to cisplatin in ovarian cancer cells are both mediated by the EGFR signal pathway,so we presume that GnRHa（Triptorelin） canreverse acquired resistance to cisplatin.The purpose of our study is to explore the potentiation effect of Triptorelin for chemotherapy.Objective To show down-regulation of expression of EGFR of Triptorelin contributes to reversion effects in OVCAR-3/DDP cells,which are resistant to cisplatin.MethodsPartⅠGeneration and maintenance of resistant subclone:1、The cisplatin-resistant subclone（OVCAR-3/DDP） was established by maintaining the cells with stepwise increases in the concentration of cisplatin;2、The changes of cellular morphology were observed by phase-contrast microscope;3、IC₅₀（50%inhibiting concentration） and RI（resistance index） were detected by MTT assay;4、The cell cycle and the cell apoptosis were detected by flow cytometry.PartⅡExplore the potentiation effect of triptorelin forchemotherapy:1、Experiment groups:we divided five groups asⅠcisplatin、ⅡTriptorelin、Ⅲcombination（cisplatin and Triptorelin）、Ⅳnon-drug、Ⅴparental cells all based on concentrations of C_max of both drugs.Ⅰ-Ⅳgroups use OVCAR-3/DDP cells andⅤgroup uses OVCAR-3 cells.2、 The doubling time（Td）、RI and RR were detected by MTT assay;3、After treated with cisplatin,Triptorelin,and combination of cisplatin and Triptorelin in different concentration respectively,the proliferation and EGFR expression of cells of every group were detected by MTT assay and flow cytometry.ResultsPartⅠEstablishment of OVCAR-3/DDP 1、The resistant cell lines were named OVCAR-3/DDP.Even when OVCAR-3/DDP cells were grown in the absence of cisplatin for further 2 months,their resistant properties were sustained;2、Cellular morphology. compared with parental cells,OVCAR-3/DDP cells were larger in volume and intercellular space;3、OVCAR-3/DDP cells were 13.42-fold more resistant to cisplatin than the parent cisplatin sensitive OVCAR-3 cells;IC₅₀ values were 6.22 and 83.48μM for OVCAR-3 and OVCAR-3/DDP cells,respectively;4、A clear G₀/G₁ phase block in OVCAR-3/DDP cells was shown.PartⅡ1、Direct antiproliferative effects of Triptorelin:the reversal reaction（RR） induced by the combination was 3.91;2、Effects of Triptorelin on EGFR: overexpression of EGFR is in both OVCAR-3/DDP and OVCAR-3 cells.Fluorescence intensity of EGFR of OVCAR-3/DDP cells was down-regulated obviously by the combination（cisplatin and Triptorelin）.Conlusions:1、Cisplatin-resistant cell subline OVCAR-3/DDP was an essentially experimental model in vitro for the study on reversion effects of cisplatin resistance;2、Triptorelin probably resensitizes resistant ovarian cancer cells to cisplatin in vitro by modulating the expression of EGFR.更多还原