胆管细胞癌来源的外泌体通过内质网应激破坏血管内皮细胞紧密连接实现转移

Cholangiocarcinoma derived exosomes break down vascular integrity and cause metastasis by triggering endoplasmic reticulum stress in vascular endothelial cells

目的:探讨胆管细胞癌分泌的外泌体对血管内皮细胞紧密连接的影响及其可能的机制。方法:使用超速离心法分离胆管细胞癌CCLP细胞系外泌体并作用于人脐静脉内皮细胞(HUVEC),Western blot、免疫荧光检测内皮细胞的紧密连接。BALB/c裸鼠随机分为PBS组、exo^(CCLP)组及exo^(HIBEC)组并分别尾静脉注射PBS、exo^(CCLP)或exo^(HIBEC)后尾静脉注射FITC-右旋糖酐或CCLP,收集肺及肝脏观察组织内的FITC-右旋糖酐渗漏或肺组织内的肿瘤转移灶。Western blot及PCR检测CCLP外泌体处理的内皮细胞内质网应激相关基因及蛋白水平。予小干扰RNA抑制血管内皮细胞PERK、ATF6或IRE1α,并进一步接受CCLP外泌体作用,观察紧密连接蛋白变化。结果:①CCLP外泌体在体外明显下调HUVEC的紧密连接蛋白水平,增加血管内皮层体外的通透性,并在体内增加裸鼠肺部及肝脏血管的通透性。②经CCLP外泌体预处理后的裸鼠其肺部的肿瘤转移灶数量明显增加,内皮细胞内质网应激相关的基因及蛋白水平均明显增加,予小干扰RNA下调HUVEC的PERK及IRE1α水平能够逆转CCLP外泌体下调的HUVEC紧密连接蛋白而ATF6的敲低并无此作用。结论:胆管细胞癌来源的外泌体能够抑制血管内皮细胞的紧密连接蛋白并增加血管的通透性,从而实现肿瘤转移。这种作用部分是通过引起血管内皮细胞内质网应激实现的。

Objective:To explore the effects of cholangiocarcinoma exosomes on the endothelial tight junction and the possible underlying mechanism.Methods:Exosomes were isolated from the supernatant of cholangiocarcinoma cell line CCLP and human intrahepatic bile duct epithelial cells(HIBEC)by ultracentrifugation according to the well-established protocol and were administrated to human umbilical vein endothelial cells(HUVEC),PBS was chosen as control.Endothelial monolayer permeability was assessed in vitro and tight junction proteins(TJPs)were evaluated by Western blot and immunofluorescence.BALB/c null mice were divided as three groups(PBS,exoCCLP and exoHIBEC),which were administrated with PBS,CCLP exosomes or HIBEC exosomes via tail vein respectively for indicated durations.FITC-Dextran or CCLP were then injected intravenously and leakage of FITC-Dextran in lung and liver,or pulmonary metastases were assessed.The endoplasmic reticulum stress related genes and proteins were analyzed by real-time PCR and Western blot.Endothelial PERK,ATF6 or IRE1αwere partially knocked down by siRNA before HUVECs were treated with CCLP exosomes,and TJPs were evaluated afterwards.Results:CCLP exosomes downregulated the expression of TJPs in HUVECs,and increased the permeability of endothelial monolayer in vitro.In vivo experiments indicated that compared to PBS and exo^HIBEC,exo^CCLP administration led to an increase of FITC-Dextran leakage both in lung and liver,as well as tumor metastases in lung.ER stress related genes and proteins were elevated in exo^CCLP treated HUVECs.Knocking down of PERK or IRE1α,instead of ATF6,could partially restore the TJPs expression of HUVECs downregulated by exo^CCLP.Conclusion:Cholangiocarcinoma derived exosomes break down endothelial tight junction expression and increase vascular permeability,which further leads to tumor metastasis.Such effect is partially achieved by triggering of ER stress in endothelial cells by exosomes.