HPV16整合感染人宫颈上皮细胞分泌外泌体介导宫颈成纤维细胞转化体外研究  

Exosomes derived from HPV16-integrated human cervical epithelial cells mediate cervical fibroblast transition in vitro

作　　者：梁罗娇 魏文斐 王筱玥 周琛斐 陈晓静 吴湘光 杨杨 王梓慈 吴砂 王薇[1,2] LIANG Luo-jiao;WEI Wen-fei;WANG Xiao-yue;ZHOU Chen-fei;CHEN Xiao-jing;WU Xiang-guang;YANG Yang;WANG Zi-ci;WU Sha;WANG Wei(Southern Medical University,Guangzhou 510515,P.R.China;Department of Obstetrics and Gynecology,First Affiliated Hospital of Guangzhou Medical University,Guangzhou 510120,P.R.China)

机构地区：[1]南方医科大学南方医院妇产科,广东广州510515 [2]广州医科大学附属第一医院妇产科,广东广州510120 [3]南方医科大学基础医学院免疫学教研室,广东广州510515

出　　处：《中华肿瘤防治杂志》2020年第9期698-705,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81672589;81971341);国家自然科学基金青年科学基金(81902621)。

摘　　要：目的高危人乳头瘤病毒(high-risk human papillomavirus,hr-HPV)整合感染的上皮细胞与宫颈间质微环境可能互为"同谋",在宫颈癌病变过程中发挥重要作用。本研究探讨HPV16整合感染的人宫颈上皮细胞通过分泌外泌体(exosome,Exo)对正常宫颈间质中成纤维细胞(normal fibroblasts,NFs)功能的影响。方法选取2018-01-28-2018-03-20南方医科大学南方医院妇产科收治的患者3例,提取2株人宫颈原代正常NFs并进行鉴定,分别命名为NFs1和NFs2。选取HPV16整合感染的永生化人宫颈上皮细胞ECT-1和S12为实验组,并以无HPV感染的正常人宫颈上皮细胞HCerEpiC为对照组,差速超速离心法提取上皮细胞分泌的Exo,经蛋白定量后,透射电镜和蛋白质印迹法分别检测Exo的形态和标志性蛋白。每组使用10μg Exo进行体外刺激NFs,孵育48h。收集上皮细胞的条件培养基(conditioned medium,CM)及Exo,分别处理NFs,通过细胞骨架染色观察CM/Exo对NFs细胞形态的影响,Transwell迁移实验、划痕实验和细胞计数盒8实验观察Exo对NFs迁移和增殖能力的影响,ELISA实验检测NFs/CM中人转化生长因子β1(transforming growth factor betal 1,TGF-β1)的表达量。结果从正常宫颈组织分离出NFs,并成功提取宫颈上皮细胞来源的Exo。与对照组比较,NFs经实验组处理后,细胞形态多变不均一,肌动蛋白微丝丰富,并伸出丝状伪足,且细胞增殖速度增加,差异有统计学意义(NFs1:tECT-1=2.452,PECT-1=0.0496;tS12=3.185,PS12=0.0190。NFs2:tECT-1=3.295,PECT-1=0.0165;tS12=2.817,PS12=0.0305)。实验组和对照组NFs1穿透聚碳酯膜的细胞数如下:ECT-1/Exo 98.333±3.055,S12/Exo 121.000±5.292,HCerEpiC/Exo 31.333±4.041(F=364.6,P<0.0001);划痕实验显示,NFs1的相对迁移率分别为ECT-1/Exo 3.942±0.065,S12/Exo 3.858±0.199,HCerEpiC/Exo 1.355±0.160(F=279.6,P<0.0001),表明NFs经ECT-1/Exo和S12/Exo处理后迁移能力均显著增强。实验组NFs1分泌TGF-β1的水平ECT-1/Exo(1.195±0.004)ng/mL,SOBJECTIVE High-risk human papillomavirus(hr-HPV)integrated infection of epithelial cells and cervical microenvironment may be’conspirator’with each other,which plays an important role in the progression of cervical cancer.This study investigates the effects of exosomes(Exo)secreted by HPV16-integrated human cervical epithelial cells on biological behavior of normal fibroblasts(NFs)in cervical interstitium.METHODS Three patients who were admitted to the Department of Obstetrics and Gynecology,Nanfang Hospital,Southern Medical University from January 28 th,2018 to March 20 th,2018,were selected,and two human cervical primary NFs were isolated and then identified,which were named as NFs1 and NFs2 respectively.HPV16 integrated immortalized human cervical epithelial cells ECT-1 and S12 was selected as the experimental group,and normal human cervical epithelial cells HCerEpiC without HPV infection were used as the control group.Exo secreted by the above cells were extracted by different ultracentrifugation,morphology and marker protein of Exo were detected by transmission electron microscopy and western blot after quantification of the protein.10μg Exo were used to stimulate NFs in each group for 48 hours of incubation in vitro treatment.The conditioned medium(CM)and Exo of epithelial cells were collected to treat NFs,the effects of CM/Exo on NFs morphology were observed by cytoskeleton staining.Then,transwell migration assay,scratch assay and CCK8 assay were used to observe the effects of Exo on the migration and proliferation of NFs,respectively.The concentration of TGF-β1 in NFs/CM was detected by ELISA assay.RESULTS NFs were isolated from normal cervical tissues,and Exo derived from cervical epithelial cells was successfully extracted.Compared with the control group,NFs were pretreated by the experimental group showed the morphology was variable and uneven with more actin microfilaments and pseudopods,and the cell proliferation rate was significantly increased(NFs1:tECT-1=2.452,PECT-1=0.0496;tS12=3.185,PS12=0

关 键 词：人乳头瘤病毒16 外泌体 成纤维细胞 增殖 迁移 转化生长因子β1 

分 类 号：R737.33[医药卫生—肿瘤]