论文部分内容阅读
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 investigate the expression of C-C motif chemokine ligand 7 (CCL7) in pancreatic stellate cells and its effect on pancreatic cancer invasion in hypoxia. Methods: Human pancreatic stellate cells (PSCs) were obtained by tissue explant explant culture and cultured under normoxia (20% O 2) and hypoxia (1% O 2). Human pancreatic stellate cells ChIP technique was used to analyze the expression of various chemokines in the supernatant. CCL7 (0, 1, 10, 100 ng / mL) was used to study the chemotaxis of CCL7 and pancreatic cancer cells by adding different concentrations of exogenous human recombinant protein CCL7 Using a lentiviral transfection, Transwell and other technologies focused on the analysis of hypoxic PSCs overexpression of CCL7 promote pancreatic cancer invasion. Results: Compared with normoxia-cultured PSCs, hypoxia-conditioned medium of PSCs significantly enhanced the invasiveness of pancreatic cancer (P <0.05). CCL7 in PSCs supernatant under hypoxia was significantly higher than that in normoxic culture (fold change = 2.38). Exogenous recombinant CCL7 protein (0, 1, 10 and 100 ng / mL) significantly increased the invasion ability of pancreatic cancer cells (Miapaca-2 and Colo357) in a concentration- The expression of CCR7 receptor (CCR1, CCR2, CCR3, CCR5) was detected by flow cytometry. Only CCR5 overexpression was found in pancreatic cancer cells (Miapaca-2, Colo357). Western blot showed that hypoxia induced the expression of CCL7 and promote the invasion of pancreatic ductal adenocarcinoma. The expression of E-cadherin decreased and the expression of Vimentin and N-cadherin increased. CONCLUSION: Hypoxia induces the high expression of CCL7 in PSCs and promotes the migration and invasion of pancreatic cancer cells through the CCL7 / CCR5 axis, which may be related to the induction of epithelialization of pancreatic cancer.