应用蛋白芯片检测CIK细胞与其培养上清蛋白谱的改变  

Protein Profiling of CIK Cell and Its Supernatant Detected by Protein Chip Technology

作　　者：唐慧[1] 董虹[1] 李丽[1] 王金丽[1] 王林坪[1] 左荣霞[1] 高建梅[1] 华映坤[1] 严新民[1] 

机构地区：[1]云南省第一人民医院(昆明理工大学附属医院)临床基础医学研究所 云南省肿瘤转化医学工程技术研究中心,昆明650032

出　　处：《肿瘤防治研究》2013年第12期1156-1162,共7页Cancer Research on Prevention and Treatment

基　　金：云南省肿瘤转化医学工程技术研究中心基金(2011DH011);云南省中青年学术技术带头人后备人才培养基金(2013HB083)

摘　　要：目的应用蛋白芯片技术检测不同患者来源的细胞因子诱导的杀伤细胞(cytokine induced killer,CIK)细胞裂解液和培养上清的蛋白谱改变。方法将3例不同患者来源的人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)在体外经细胞因子诱导成CIK细胞,经流式细胞术测定细胞表型后,分别收集培养第20天的CIK细胞和细胞培养上清,应用AAH-BLM-1蛋白芯片分别获得CIK细胞裂解液和细胞培养上清中507个蛋白的改变。结果 CIK细胞在培养第20天,CD3+和CD3+CD56+的T细胞分别为(86.43±10.65)%和(38.58±3.94)%。CIK细胞培养上清液中共有6个蛋白明显升高(Signal≥700,FC≥1.8):MIP-1β(FC=21.28)、GzmA(FC=5.54)、IFN-(FC=2.78)、MCP-1(FC=2.22)、TMPO(FC=2.05)、IL-13(FC=1.81);细胞裂解液共有8个蛋白明显升高(Signal≥700):GzmA(Signal=1 968.77)、ET(Signal=1,398.60)、IL-13(Signal=1 333.47)、TFPI(Signal=959.76)、NRG3(Signal=9 4 4.0 9)、I L-7(Signal=8 6 7.1 2)、MIP-1α(Signal=833.43)、MIP-1β(Signal=704.88)。将上述两组结果对比分析后发现GzmA、IL-13和MIP-1β这3个蛋白在两组中均明显升高。结论 CIK细胞在活化过程中合成并分泌如GzmA、IFN-γ、IL-8和IL-13等多种蛋白产物,这些蛋白产物通过直/间接杀伤肿瘤细胞并促进T细胞增殖活化在抗肿瘤治疗中发挥重要作用。Objective Protein profiling was detected both in cells lysates and culture supernatant of cytokine induced killer （CIK） ceils from different patients by protein chip technology. Methods The peripheral blood mononuclear cells （PBMC） of 3 tumor patients were stimulated by different cytokines and induced into CIK cells in vitro. The phenotypes of CIK cells were analyzed by flow cytometry. CIK ceils and culture supernatant was collected respectively after cultured for 20 days. Expression changes of 507 proteins were detected either in cells lysates or in cell culture supernatant by AAH-BLM-1 protein chip technology. Results The percentages of CD3＋ and CD3＋CD56＋ were （ 86.43±10.65 ） % and （ 38.58±3.94 ） % respectively after CIK cells expanding for 20 days. Six proteins were increased significantly in cell culture supernatant （signal ≥700, FC≥ 1.8）： MIP - 1β （FC-21.28）, GzmA （FC=5.54）, IFN- 7 （FC=2.78）, MCP - 1 （FC=2.22）, TMPO （FC=2.05）, IL - 13 （FC=1.81）. Eight proteins were increased significantly in cells lysates （signal≥700）： GzmA （signal=1,968.77）, ET （signal=1,398.60）, IL - 13 （signal=1,333.47）, TFPI （signal=959.76）, NRG3 （signal=944.09）, IL - 7 （signal=867.12）, MIP - 1α （signal=833.43）, MIP - 1β （signal=704.88）. Three proteins of GzmA, IL-13 and MIP-1β were increased significantly both in two groups. Conclusion CIK cells synthesize and release a variety of protein products during the process of activation, such as GzmA, IFN-γ, IL-8, IL-13, etc. These proteins play important roles of anti-tumor by killing tumor cells directly / indirectly and promoting the proliferation and activation of T cells.

关 键 词：细胞因子诱导的杀伤细胞 培养上清液 细胞裂解液 蛋白芯片 

分 类 号：R73-362[医药卫生—肿瘤]