胃癌细胞MGC-803耐药细胞株的建立及分泌蛋白差异分析  

Establishment of drug resistant cell line of MGC-803 and analysis of differential secretome

作　　者：黄昊[1] 韩勇[1] 吴健[1] 田志华[1] 曲立科[1] 寿成超[1] 

机构地区：[1]北京大学肿瘤医院暨北京市肿瘤防治研究所,恶性肿瘤发生机制及转化研究教育部重点实验室

出　　处：《北京大学学报（医学版）》2014年第2期183-189,共7页Journal of Peking University:Health Sciences

基　　金：国家重点基础研究发展计划(973计划)(2009CB521805)资助~~

摘　　要：目的:应用蛋白质组学技术系统比较5-氟尿嘧啶、紫杉醇及顺铂耐药细胞与敏感细胞的分泌蛋白表达谱,以期发现胃癌耐药相关分泌蛋白标志物,为临床选择有针对性的化疗药物提供依据。方法:通过药物梯度诱导建立胃癌MGC-803耐药细胞株。从培养上清中收集MGC-803亲代及耐药细胞的分泌蛋白,应用双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)进行分离,通过PD Quest 7.1.0软件分析获得差异蛋白,基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry,MALDI-TOF-MS)进行鉴定,并用实时定量PCR检测各差异蛋白在耐药细胞株和亲本细胞间mRNA水平上的表达差异。结果:分别建立了对5-氟尿嘧啶、紫杉醇及顺铂耐药指数为110.6、70.0和13.3的耐药细胞株。顺铂耐药细胞对5-氟尿嘧啶和紫杉醇、5-氟尿嘧啶耐药细胞对紫杉醇均有较强的交叉耐药,前者耐药指数分别为23.5和114.0,后者为70.0。制备获得了耐药细胞培养上清的双向凝胶电泳蛋白组分图谱;经PD Quest软件分析获得18个差异在2倍以上、3株耐药细胞培养上清中丰度均增高的蛋白点,其中13个得到了质谱鉴定,它们分别属于蛋白酶类、信号转导分子等。与亲本细胞相比,在mRNA水平上,SLMAP、TOP3A、DYNC1H1、RHPN1、PUF60和SIAH1在3种耐药细胞中皆有明显上调;IFT172、FILIP在耐5-氟尿嘧啶和紫杉醇细胞中上调显著;PLVAP、LMNA在耐紫杉醇及顺铂细胞中表达有所升高,但在耐5-氟尿嘧啶细胞中升高不显著。在蛋白水平上的进一步验证发现,SIAH1在3种耐药细胞中的表达量及培养上清中的含量皆有升高。结论:本研究以胃癌细胞MGC-803为亲本细胞分别建立了针对5-氟尿嘧啶、紫杉醇及顺铂的耐药细胞株,在此基础上应用双向电泳及蛋白质谱技术,整体展示了耐药细胞与亲本细胞的差异分泌蛋白,并发现SIAH1蛋白在耐�Objective：To identify chemoresistance-associated secretory proteins by proteomic approaches, and to provide the basis for selecting suitable chemotherapy in gastric cancer treatment. Methods： Drug resistant cell lines were established by gradient drug treatment with MGC-803 gastric cancer cells. The secreted proteins of MGC-803 parental and resistant cells were collected from the conditional medium without serum and separated by two-dimensional gel electrophoresis （2-DE）. The proteins were analyzed by PD Quest 7.1.0 software and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. Real-time RT-PCR was performed to confirm the difference of ex- pression on the mRNA level. Results： The 5-fluorouracil （SFU）, paclitaxel （TA） and eisplatin （DDP）-resistant gastric cancer cell lines with the resistance indexes of 110.6, 70.0 and 13.3 respectively, were established successfully. DDP-resistant cells had strong cross-resistance to 5FU and TA, and the resistance indexes were 23.5 and 114.0. 5FU-resistant cells also had strong cross-resistance to TA with the resistance index 70.0. The 2-DE patterns of protein component spectra from the conditional medium were obtained with 18 proteins whose abundances were increased in all chemoresistance cells for more than 2-fold, 13 of which were identified by mass spectrometry, including protease and proteins in- volved in signal transduction. Compared with the parental cell MGC-803, SLMAP, TOP3A, DYNCIH1, RHPN1, PUF60 and SIAH1 were significantly up-regulated in three drug resistant cells, IFT172 and FILIP were up-regulated in 5FU-resistant and TA-resistant cells, PLVAP and LMNA were up-regulated in TA- and DDP-resistant cells. Further validation revealed that SIAHI protein was enriched in cell lysates and the conditional medium from all the drug resistant cells. Conclusion： By establishing the 5FU-,TA- and DDP- resistant gastric cancer cell lines and assisted by 2-DE and mass spectrometry, we demonstra- t

关 键 词：胃肿瘤 抗药性 多药 泛素蛋白连接酶 蛋白质组学 

分 类 号：R735.2[医药卫生—肿瘤]