PBMC中TLR1、TLR2、TLR6在卵巢癌微环境中的作用研究  

Effect of TLR1,TLR2,TLR6 in PBMC from patients with ovarian cancer on tumor microenvironment

作　　者：徐娟[1] 娄鉴芳 史新惠 柯星 张淑平 吴梦 孙瑞红 黄蕾 王芳[2,3] 

机构地区：[1]江苏省泰州市人民医院检验科,江苏泰州225300 [2]南京医科大学第一附属医院检验学部,江苏南京210029 [3]国家临床检验重点专科建设单位,江苏南京210029

出　　处：《国际检验医学杂志》2018年第5期537-542,共6页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(81371894;81272324);国家临床检验重点专科建设项目基金资助;江苏高校优势学科建设工程基金项目资助课题;江苏省实验诊断学重点实验室基金项目(XK201114);2017年泰州市科技支撑计划(社会发展)项目(SSF20170218);南通大学校级自然科学类科研基金项目(16ZY36);泰州市人民医院院级课题(ZL201613)

摘　　要：目的观察卵巢癌患者外周血单个核细胞(PBMC)与卵巢癌细胞共生长环境中,PBMC中Toll样受体(TLR)信号通路活化情况及前炎症细胞因子表达水平,研究TLR1、TLR2及TLR6在卵巢癌发生发展中的可能机制。方法采用密度梯度离心法分离PBMC,利用PBMC和卵巢癌细胞系SK-OV-3共培养体系及抗TLR1、TLR2、TLR6抗体中和实验,RT-PCR法检测细胞中前炎症细胞因子白细胞介素(IL)-1β、IL-6、IL-8,肿瘤坏死因子(TNF)-αmRNA表达水平;利用免疫印迹试验检测MyD88、TRAF6、TANK、核因子(NF)-κB和P-NF-κB的表达水平。结果卵巢癌患者PBMC+SK-OV-3Transwell共培养组较PBMC单独培养组,TLR信号通路蛋白MyD88、TRAF6、TANK及P-NF-κB表达上调,IL-1β、IL-6及IL-8前炎症细胞因子的mRNA表达水平明显增加(Fold=1.74,Fold=1.92,Fold=1.65,P<0.05),而TNF-a mRNA表达水平无明显改变;与PBMC单独培养组比较,良性疾病患者及健康对照者IL-1βmRNA表达水平明显下调(Fold=0.71,P<0.05;Fold=0.72,P<0.05),TNF-α、IL-6、IL-8mRNA及TLR信号通路蛋白MyD88、TRAF6、TANK及P-NF-κB均无明显改变。抗体中和实验显示,卵巢癌患者PBMC+SK-OV-3+Anti-hTLR1-IgG Transwell共培养组、PBMC+SK-OV-3+Anti-hTLR2-IgG Transwell共培养组、PBMC+SK-OV-3+Anti-hTLR6-IgG Transwell共培养组中前炎症细胞因子IL-1β(Fold=0.16,Fold=0.31,Fold=0.29,P<0.05)、IL-6(Fold=0.14,Fold=0.20,Fold=0.28,P<0.05)mRNA表达水平较PBMC+SK-OV-3Transwell共培养组明显下调。结论卵巢癌生长微环境中,TLR1/TLR2/TLR6介导的信号通路活化是诱导卵巢癌患者PBMC中前炎症细胞因子表达上调的主要途径,在卵巢癌的发生、发展中发挥一定作用。Objective To explore the pathogenesis of ovarian cancer by investigating the function of Tolllike receptor 1（TLR1）,TLR2 and TLR6 in peripheral blood mononuclear cell（PBMC）from patients with ovarian cancer.Methods PBMC,SK-OV-3 co-culture system and anti-TLR1,anti-TLR2,anti-TLR6 mAb blocking experiment were used to explore the relationship between TLR1,TLR2 or TLR6 signaling and inflammation in ovarian cancer.Quantitative real-time PCR was used to measure interleukin（IL）-1β,IL-6,IL-8,and tumor necrosises factor（TNF）-αin the PBMC.MyD88,TRAF6,TANK,NF-κB and P-NFκB were observed by Western blotting.Results In the PBMC and SK-OV-3 coculture system,we found the activation of TLR signaling pathways,including significantly increased MyD88,TRAF6,TANK and P-NF-κB levels following cocultured with SK-OV-3 in PBMC from ovarian cancer patients.PBMC derived from ovarian cancer patients led to a increase in IL-1β,IL-6 and IL-8 mRNA levels after 24 hours of co-incubation with SK-OV-3（Fold=1.74,Fold=1.92,Fold=1.65,P〈0.05）,though there was no difference of TNF-a mRNA expression.In contrast to the ovarian cancer patients,coculture of PBMC derived from benign diseases controls and healthy normal controls decreased IL-1βat the mRNA level（Fold=0.71,P〈0.05;Fold=0.72,P〈0.05）,furthermore the expression of MyD88,TRAF6,TANK,P-NF-κB,and NF-κB showed no changes.PBMC which treated with anti-TLR1,anti-TLR2 or-TLR6 mAb could inhibite inflammatory IL-1β（Fold=0.16,Fold=0.31,Fold=0.29,P〈0.05）and IL-6（Fold=0.14,Fold=0.20,Fold=0.28,P〈0.05）.Conclusion TLR1/TLR2/TLR6 in PBMC of ovarian cancer patients participate in the recognition of the factors.

关 键 词：卵巢癌 TOLL样受体 外周血单个核细胞 肿瘤微环境 

分 类 号：Q786[生物学—分子生物学] R734.2[医药卫生—肿瘤]