卵巢癌细胞外泌体抑制外周单个核细胞源性树突状细胞成熟的机制研究  

Mechanism of ovarian cancer cell secretions inhibiting the maturation of peripheral mononuclear cell-derived dendritic cells

作　　者：董育清 王怡 李丽华 司胜斌 汤业珍 董晓微[2] 盛青松 赵薇 DONG Yuqing;WANG Yi;LI Lihua;SI Shengbin;TANG Yezhen;DONG Xiaowei;SHENG Qingsong;ZHAO Wei(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750001,China;Central Laboratory of People's Hospital of Ningxia Hui Autonomous Region,Yinchuan 750011,China;Department of Obstetrics and Gynecology,No.900 Hospital of the Joint Service Support Force of the People's Liberation Army of China,Fuzhou350001,China)

机构地区：[1]宁夏医科大学基础医学院生物化学与分子生物学系,宁夏银川750001 [2]宁夏回族自治区人民医院中心实验室,宁夏银川750011 [3]中国人民解放军联勤保障部队第九〇〇医院妇产科,福建福州350001

出　　处：《中华肿瘤防治杂志》2023年第9期520-526,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(31860328);宁夏自然科学基金(2022AAC02057,2022AAC03366);福建省自然科学基金(2021J011272)。

摘　　要：目的探究卵巢癌细胞源性外泌体对人外周血树突状细胞(DCs)成熟的影响。方法从健康人外周血中分离单个核细胞(PBMC),利用免疫磁珠从中分选出CD14^(+)的细胞;采用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和重组人白介素-4(rhIL-4)联合刺激的办法诱导CD14^(+)/PBMC分化为未成熟树突状细胞(iDCs),光镜确认细胞形态;超速离心提取卵巢癌顺铂耐药细胞外泌体(A2780cis-外泌体),蛋白质印迹法检测其特征性蛋白标志物,透射电镜(TEM)观察外泌体形态,纳米颗粒跟踪分析仪(NTA)检测外泌体粒径分布;iDCs与A2780cis-外泌体共培养后给予重组人肿瘤坏死因子-α(rhTNF-α)诱导,流式细胞术检测CD83^(+)/HLA-DR^(+)/CD1a^(+)细胞比例变化。结果超速离心提取的卵巢癌顺铂耐药细胞外泌体,TEM下可检测到直径约为60 nm的“杯盏状”外泌体颗粒。蛋白质印迹法结果显示A2780cis细胞各蛋白表达均为1,A2780cis-外泌体中CD9、CD81和CD63相对表达量分别为1.08±0.07、0.67±0.04和1.78±0.19,t值分别为2.928、13.760和7.900,P值分别为0.043、<0.001和0.001;但A2780cis-外泌体无Calnexin表达。NTA检测显示,外泌体粒径范围主要集中于(56.80±1.60)nm。经流式细胞术检测CD14磁珠分选前后CD14^(+)/PBMC比例分别为(10.20±0.53)%和(97.60±0.26)%,t=255.900,P<0.001。rhGM-CSF和rhIL-4联合诱导的CD14^(+)/PBMC,细胞胞体变大,伸出半圆形、梭形、星形等毛刺状突起,形态不规则,表现为iDCs形态特征;rhTNF-α刺激iDCs后,细胞毛刺状突起明显增多,细胞贴壁能力增强,而A2780cis-外泌体与iDCs共培养组接受rhTNF-α刺激后,细胞毛刺状突起较少,细胞贴壁能力减弱。流式细胞术检测结果显示,rhTNF-α诱导前细胞CD86、CD40、HLA-DR、CD1a和CD83比例分别为(86.27±2.25)%、(67.43±0.64)%、(69.50±0.46)%、(68.20±0.46)%和(5.87±0.55)%,符合iDCs抗原表型特征;TNF-α诱导48 h后,CD86、CD40、HLA-DR、CD1a和CD83比例分别为(9Objective To investigate the effect of ovarian cancer cell derived exosomes on the maturation of human peripheral blood dendritic cells(DCs).Methods Mononuclear cells(PBMC)were isolated from peripheral blood of healthy people,and CD14^(+)cells were isolated from PBMC by immunomagnetic beads;CD14^(+)/PBMC was induced to differentiate into immature dendritic cells(iDCs)by the combined stimulation of recombinant human granulocyte-macrophage colony stimulating factor(rhGM-CSF)and recombinant human interleukin-4(rhIL-4),and the cell morphology was confirmed by light microscopy;The exosomes of cisplatin-resistant ovarian cancer cells(A2780cis exosomes)were extracted by ultracentrifugation.The characteristic protein markers were detected by western blot,the morphology of exosomes was observed by transmission electron microscopy(TEM),and the size distribution of exosomes was detected by nanoparticle tracking analyzer(NTA);After co-culture of iDCs with A2780cis exocrine body,recombinant human tumor necrosis factor was given-α(rhTNF-α);After induction,the ratio of CD83^(+)/HLA-DR^(+)/CD1a^(+)cells was detected by flow cytometry.Results The exosomes of cisplatin-resistant ovarian cancer cells extracted by ultracentrifugation could be detected by TEM with a diameter of about 60nm.The results of western blot showed that the expression of each protein in A2780cis cells was 1,and the relative expressions of CD9,CD81and CD63in A2780cis-exosome were 1.08±0.07,0.67±0.04and 1.78±0.19,respectively,with t values of 2.928,13.760and 7.900,Pvalues of 0.043,<0.001and 0.001,respectively;However,there was no expression of Calnexin in A2780cis-exosome.NTA detection showed that the size range of exosomes was mainly(56.80±1.60)nm.The ratio of CD14^(+)/PBMC before and after CD14magnetic bead sorting was(10.20±0.53)%and(97.60±0.26)%respectively by flow cytometry,t=255.900,P<0.001.The cell body of CD14^(+)/PBMC induced by rh-GM-CSF and rhIL-4became larger,and extended semicircular,fusiform,star-shaped and other burr-like processes with i

关 键 词：顺铂耐药卵巢癌细胞株 外泌体 体外培养 树突状细胞 流式细胞术 

分 类 号：R737.31[医药卫生—肿瘤]