北京大学学报(医学版) ›› 2014, Vol. 46 ›› Issue (2): 183-189.

• 论著 • 上一篇    下一篇

胃癌细胞MGC-803耐药细胞株的建立及分泌蛋白差异分析

黄昊,韩勇,吴健,田志华,曲立科,寿成超△   

  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, 北京100142)
  • 出版日期:2014-04-18 发布日期:2014-04-18

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

HUANG Hao, HAN Yong, WU Jian, TIAN Zhi-hua, QU Li-ke, SHOU Cheng-chao△   

  1. (Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China)
  • Online:2014-04-18 Published:2014-04-18

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蛋白在耐药细胞总蛋白及培养上清中的含量均明显升高,其有可能成为研发胃癌血清耐药标志物的候选分子。

关键词: 胃肿瘤, 抗药性, 多药, 泛素蛋白连接酶, 蛋白质组学

Abstract: 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 expression on the mRNA level. Results: The 5-fluorouracil (5FU), paclitaxel (TA) and cisplatin (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 involved in signal transduction. Compared with the parental cell MGC-803, SLMAP, TOP3A, DYNC1H1, 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 SIAH1 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 demonstrated the different secretory protein profiling and found that SIAH1 had significantly increased in both cell lysates and the conditional medium of the drug-resistant cells, which are potential candidates for developing chemoresistance markers in sera from gastric cancer patients.

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