布鲁氏菌OMP10介导的小鼠骨髓源树突状细胞活化及其对小鼠T细胞增殖影响的研究  

Effect of Brucella abortus outer membrane protein OMP10-mediated activation of mouse bone marrow-derived dendritic cells on their T cell proliferation

作　　者：徐朕宇 王月丽[1,2] 易继海 童志霞 邓肖玉 杨宁宁 徐明国 王勇 孟闯[3] 苗玉和 陈创夫[1,2] XU Zhen-yu;WANG Yue-li;YI Ji-hai;TONG Zhi-xia;DENG Xiao-yu;YANG Ning-ning;XU Ming-guo;WANG Yong;MENG Chuang;MIAO Yu-he;CHEN Chuang-fu(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;Collaborative Innovation Center for Healthy Breeding and Prevention and Control of Human-Animal Diseases in Sheep,Shihezi 832000,China;Jiangsu Key Laboratory of Zoonoses,Yangzhou 225009,China;Fujian Biotechnology Co.,Ltd.,Nanping 350000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]绵羊健康养殖与人兽共患病防控协同创新中心,新疆石河子832000 [3]江苏省人兽共患病学重点实验室,江苏扬州225009 [4]福建省圣维生物科技有限公司,福建南平350000

出　　处：《中国预防兽医学报》2022年第10期1084-1090,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(U1803236、32002245);江苏省人兽共患病学重点实验室资助项目(R2104)。

摘　　要：为探究布鲁氏菌外膜蛋白10(OMP10)介导小鼠骨髓源性树突状细胞(BMDC)的活化机制及其对小鼠T淋巴细胞增殖的影响。本研究通过体外分离培养BMDC细胞6 d后,将重组OMP10(rOMP10)与BMDC共孵育24 h,经流式细胞术分析BMDC表面共刺激分子(CD40、CD80、CD86)的表达情况,结果显示,与PBS组相比,rOMP10孵育BMDC能够诱导细胞表面共刺激分子CD40、CD80、CD86的表达量显著升高(P<0.001)。同时采用ELISA检测共孵育后BMDC细胞因子(TNF-α、IFN-γ、IL-6、IL-12、IL-10、IL-4)的表达情况,结果显示,与PBS组相比,rOMP10孵育BMDC能够诱导其细胞因子(IL-6、IL-12、TNF-α、IFN-γ)表达量极显著升高(P<0.01),但IL-4和IL-10的表达量极显著降低(P<0.001)。进一步采用荧光定量PCR(qRT-PCR)检测共孵育后BMDC的Toll样受体(TLRs)和MHC-I、MHC-II类分子的mRNA转录水平,结果显示,与PBS组相比,rOMP10能够诱导BMDC的TLR2和TLR4(P<0.05),以及抗原递呈相关分子MHC-I和MHC-II的mRNA转录水平极显著升高(P<0.01)。另外,将小鼠脾脏T淋巴细胞与rOMP10预处理的BMDC共孵育后,经MTT试验检测T淋巴细胞增殖效率,结果显示,rOMP10处理组比PBS组T淋巴细胞的刺激指数增加2倍以上,且在树突状细胞和淋巴细胞比值(DCs:T)为1:50时,rOMP10处理组的T淋巴细胞增殖效率最高。综上所述,本研究首次证实布鲁氏菌OMP10能够诱导BMDC的活化,促进抗原递呈分子的转录,从而介导T淋巴细胞的高效增殖,该结果为解析布鲁氏菌感染与宿主免疫机制奠定实验基础,同时也为布鲁氏菌新型亚单位疫苗研发提供数据支持。To investigate the activation of mouse bone marrow-derived dendritic cells(BMDC)mediated by the Brucella abortus outer membrane protein OMP10 and its effect on the proliferation of mouse T lymphocytes.In this study,BMDCwere isolated and cultured in vitro for 6 days.After incubation with recombinant OMP10(rOMP10)for 24 hours,the expression of co-stimulatory molecules(CD40,CD80,CD86)on the surface of BMDC cells was analyzed by flow cytometry;the cytokine secretion including TNF-α,IFN-γ,IL-6,IL-12 IL-10,and IL-4)was determined by ELISA;the mRNA transcript levels of Toll-like receptors(TLRs),and MHCI/MHCII-like molecules were quantified by quantitative real-time fluorescence PCR(qRT-PCR).The results showed that rOMP10 could significantly induce the expression of surface co-stimulatory molecules CD40,CD80 and CD86 in BMDC,compared to PBS treatment(P<0.001).r OMP10 treatment also induced a significant increase in cytokines(IL-6,IL-12,TNF-α,IFN-γ)compared to the PBS group(P<0.01),However,secretion of IL-4 and IL-10 was significantly reduced(P<0.001).Moreover,rOMP10 was able to significantly enhance the transcription of TLR2,TLR4,MHC-I and MHC-II molecules(P<0.01).After co-incubation of mouse spleen T lymphocytes with rOMP10-pretreated BMDC,the proliferation efficiency of T lymphocytes was examined by MTT assay.The results showed that the stimulation index of the rOMP10-treated group increased more than 2-fold compared with that of the PBS group,and the T lymphocyte proliferation efficiency of the rOMP10-treated group was highest at a dendritic cell to lymphocyte ratio(DCs:T)of 1:50.This study demonstrated that Brucella rOMP10 can induce BMDC activation and promote the expression of antigen-presenting molecules,thereby mediating the efficient proliferation of T lymphocytes.The results provided a theoretical basis for understanding the interplay between Brucella infection and host immunity,and also provided data to support the development of a new subunit vaccine for Brucella.

关 键 词：布鲁氏菌 外膜蛋白OMP10 小鼠骨髓源树突状细胞 抗原递呈 T淋巴细胞增殖 

分 类 号：S852.61[农业科学—基础兽医学]