文献类型：期刊文献

中文题名：人视网膜母细胞瘤Y79细胞依托泊苷耐药株的建立及其耐药机制初探

英文题名：Generation of etoposide-resistant subline of human retinoblastoma Y79 cells and preliminary study on the mechanism of drug resistance

作者：宋文凭[1];张诚玥[2];张燕[2];李毅[1];曹睿[1];叶程[1];张琳[3];邵荣光[1];李亮[1];赵军阳[2]

第一作者：宋文凭

机构：[1]中国医学科学院北京协和医学院医药生物技术研究所肿瘤室;[2]首都医科大学附属北京儿童医院眼科;[3]北京联合大学特殊教育学院

第一机构：中国医学科学院北京协和医学院医药生物技术研究所肿瘤室,北京100050

年份：2017

卷号：12

期号：4

起止页码：297-302

中文期刊名：中国医药生物技术

外文期刊名：Chinese Medicinal Biotechnology

收录：CSTPCD

基金：国家自然科学基金(81472787)

语种：中文

中文关键词：视网膜母细胞瘤;依托泊苷;化疗耐药细胞株;Y79细胞系

外文关键词：Retinoblastoma; Etoposide; Chemoresistance resistant cell line; Y79 cell line

摘要：目的通过构建人视网膜母细胞瘤Y79细胞的依托泊苷耐药株,比较耐药株的药物敏感性和细胞生长情况,观察耐药相关基因及细胞信号通路的变化,从而阐释人视网膜母细胞瘤对依托泊苷的耐药机制。方法以不同浓度梯度的依托泊苷处理Y79细胞,采用浓度递增不间断刺激法构建依托泊苷耐药株,观察耐药株细胞的形态学变化和细胞生长增殖情况以及对依托泊苷细胞毒作用的敏感性;以caspase 3/7酶活性来评估耐药株的细胞凋亡程度,并通过Western blot方法研究与细胞增殖和耐药相关蛋白的表达变化。结果成功构建耐受依托泊苷的人视网膜母细胞瘤耐药株Y79/EDR,最大耐药浓度为500 nmol/L。和亲本细胞相比,耐药细胞具有较强的生长增殖能力,倍增时间缩短14.5 h(P<0.001);且对依托泊苷的敏感性显著下降,耐药指数达29.47。Caspase 3/7活性检测结果显示,给予亲本及耐药细胞不同浓度的依托泊苷后,耐药细胞中caspase 3/7活性明显低于亲本细胞,凋亡减少,且呈药物浓度依赖性。Western blot结果表明,耐药细胞中磷酸化AKT(p-AKT)表达明显高于亲本细胞,提示其p-AKT的活性明显增强,p-AKT促进其下游蛋白MDM2的磷酸化从而抑制p53的磷酸化,而凋亡相关蛋白Bax/Bcl-2的比值明显降低。多药耐药性蛋白P-糖蛋白(P-gp)的表达下调,Rb1蛋白表达水平无明显变化,提示耐药株Y79/EDR的耐药并不依赖于P-gp而产生,且与Rb1蛋白无关,而可能通过增加肿瘤驱动蛋白AKT的磷酸化,激活其所介导的下游信号通路,增加靶蛋白MDM2的表达,从而抑制抑癌蛋白p53的表达;同时增加抗凋亡蛋白Bcl-2和降低抑凋亡蛋白Bax的表达水平;最终导致细胞通过增殖显著增强和凋亡明显减少而产生耐药。结论成功构建依托泊苷耐药株Y79/EDR,初步结果表明,其耐药细胞株可能通过促进细胞生长与增殖,同时抑制细胞凋亡而导致耐药,这将为深入研究人视网膜母细胞瘤的病变机制,阐释其对依托泊苷的耐药机制,寻找有效的耐药逆转方案提供一定的前期实验基础。
Objective To generate etoposide-resistant cell line of human retinoblastoma Y79 cells （Y79 etoposide-drug-resistant, Y79/EDR） and explore the underlying mechanism of etoposide resistance. Methods The etoposide-resistant subline of human retinoblastoma Y79 cells were selected in vitro by prolonged exposure to 3-fold stepwisely increasing concentrations of etoposide. Y79/EDR cells were finally maintained in culture with 500 nmol/L of etoposide, followed by cell counting kit （CCK-8） and caspase-glo 3/7 assays for testing the growth rate, cytotoxicity and apoptosis induced by etoposide in both Y79 and Y79/EDR cells, respectively. Western blot assay was performed to determine if any cell signaling pathways might be involved in etoposide resistance in Y79/EDR cells. Results Y79/EDR cells were obtained after 6 months constitutive treatment at the concentration of 500 nmol/L. The growth rate of Y79/EDR significantly increased and its doubling time was shortened by 14.5 h as compared to Y79 parental cells （P 〈 0.001） with the resistance index of 29.47. The activity of caspase 3/7 in Y79/EDR cells was significantly decreased as compared to Y79 cells in a manner of etoposide concentration dependent （P 〈 0.01）. The results from Western blot assay indicated that AKT and p53 mediated cellular proliferation signaling pathways might be involved in etoposide resistance of Y79/EDR cells, whereas the expression levels of Rbl and P-glycoprotein （P-gp） genes had no alteration. Conclusion Our preliminary results suggested that the mechanisms of acquired resistance to etoposide in human retinoblastoma Y79 cells might be involved in the promotion of cellular proliferation and inhibition of cell apoptosis mediated by AKT signaling pathway.