缺氧微环境中胰腺星状细胞通过CCL7/CCR5轴促进胰腺癌侵袭  

Hypoxic stellate cells of pancreatic cancer stroma induce cancer cell migration and invasion via CCL7/CCR5 axis

作　　者：吴飏[1,2,3] 刘东方[1,3] 张纯[4] 陆子鹏[1,2,3] 彭云鹏[1,3] 蔡宝宝 吴鹏飞[1,2,3] 田蕾[1,2,3] 钱栋[1,3] 袁昊[1,3] 时国东 朱毅[1,2,3] 张静静[1,2,3] 蒋奎荣[1,2,3] 

机构地区：[1]南京医科大学胰腺研究所,江苏南京210029 [2]南京医科大学第一附属医院胰腺中心,江苏南京210029 [3]南京医科大学第一附属医院普外科肿瘤实验室,江苏南京210029 [4]南京医科大学附属上海松江中心医院消化内科,上海201600

出　　处：《南京医科大学学报（自然科学版）》2017年第5期521-525,543,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金面上项目(81272382);江苏省六大人才高峰(2014-WSW-006)

摘　　要：目的:探讨缺氧状态下胰腺星状细胞的单核细胞趋化因子7(C-C motif chemokine ligand 7,CCL7)表达情况及其对胰腺癌侵袭的影响。方法:通过组织块外植法获得人肿瘤相关胰腺星状细胞(pancreatic stellate cells,PSCs),于常氧(20%O_2)、缺氧(1%O_2)下培养,并通过人趋化因子抗体芯片技术分析其上清液中多种趋化因子的表达差异;通过加入不同浓度外源性人重组蛋白CCL7(0、1、10、100 ng/mL)研究趋化因子CCL7与胰腺癌细胞侵袭之间的浓度依赖关系;使用慢病毒转染、Transwell等技术着重分析缺氧PSCs过表达的CCL7促胰腺癌侵袭的作用。结果:相较于常氧培养PSCs,缺氧培养PSCs的条件培养基可显著增强胰腺癌侵袭能力(P<0.05);缺氧培养下PSCs上清液中CCL7相较于常氧培养显著高表达(fold change=2.38);外源性重组CCL7蛋白(0、1、10、100 ng/mL)可显著增加胰腺癌细胞(Miapaca-2、Colo357)侵袭能力,且呈浓度依赖关系。流式细胞术检测CCL7受体(CCR1、CCR2、CCR3、CCR5)表达,显示胰腺癌细胞(Miapaca-2、Colo357)仅有CCR5高表达。Western blot印迹显示,缺氧诱导PSCs高表达CCL7并促胰腺癌侵袭的过程中存在EMT改变,即E-cadherin表达下降,Vimentin、N-cadherin表达上升。结论:缺氧可诱导PSCs高表达CCL7,并通过CCL7/CCR5轴促进胰腺癌细胞迁移和侵袭,其机制可能与诱导胰腺癌上皮间质化有关。Objective：To explore the influence of motif chemokine ligand 7（CCL7） on pancreatic cell migration and invasion under hypoxic condition.Methods：We isolated cancer-associated pancreatic stellate cells（PSCs） by an outgrowth method,and analyzed the difference of chemokine expression in PSCs between hypoxia （1%） and normoxia （21%） by conducting a chemokine antibody array.By adding different concentration of recombinant protein CCL7（0 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL） to the lower chamber containing 0.1% FBS DMEM,cell invasion assays were performed to determine the dose-dependent manner between CCL7 and invasion of pancreatic ancer cell（PCCs）.By conducting Transwell invasion assays and lentiviral transfection,we were able to show that hypoxia induced secretion of CCL7 in PSCs,leading to increased invasion of PSCs.Results：Conditioned media from hypoxic PSCs enhanced PCCs invasiveness more intensely than that from normoxic PSCs（P〈0.05）.Among various chemokiness,which were related to invasiveness,CCL7 was one of the overexpressed molecules in supernatant of hypoxic PSCs.CCL7 recombinant protein（0 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL） induced pancreatic cells migration in dose-dependent manner.Pancreatic cells were shown to express CCR5 receptors for CCL7 detected by flow cytometry（P〈0.05）.PSCs（Miapaca-2,colo357）,which were chultured with PSCs supernatant,were involved an epithelial mesenchymal transition（EMT）cascade.Furthermore,the inbition of CCL7 by lentiviral transfecton suppressed the EMT.Conclusion：Hypoxic stellate cells of pancreatic cancer stroma induce PCCs invasion vis CCL7/CCR5 axis,of which the mechanism might inclued an epithelial mesenchymal transition （EMT） process.

关 键 词：缺氧 胰腺星状细胞 胰腺癌 CCL7 CCR5 上皮间质化 

分 类 号：R735.9[医药卫生—肿瘤]