树突状细胞来源胞外体转运丁型肝炎抗原诱导特异性细胞毒性T淋巴细胞反应的体外研究  

In vitro study of dendritic cell derived exosomes transporting hepatitis D antigen induced specific cytotoxic T lymphocyte response

作　　者：姚婷 吕梦娇 陈金梅 张毅[1] 马思远 余永胜[1] 臧国庆[1] 陈小华[1] Yao Ting;Lyu Mengjiao;Chen Jinmei;Zhang Yi;Ma Siyuan;Yu Yongsheng;Zang Guoqing;Chen Xiaohua(Department of Infectious Diseases,Shanghai Jiao Tong University Affiliated Sixth People′s Hospital,Shanghai 200233,China)

机构地区：[1]上海交通大学附属第六人民医院感染病科,上海200233

出　　处：《中华传染病杂志》2022年第4期234-240,共7页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金(81770589);上海市自然科学基金(17ZR1421500)。

摘　　要：目的探讨携带泛素化修饰丁型肝炎抗原(hepatitis D antigen, HDAg)的树突状细胞来源的胞外体(dendritic cell derived exosomes, Dexs)在诱导特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)反应中的作用及其分子机制。方法提取并诱导C57BL/6小鼠髓源性树突状细胞(dendritic cell, DC)后与Ub-S-HDAg-Dexs共孵育48 h, 流式细胞仪检测表面分子[CD86、CD80、主要组织相容性复合体(major histocompatibility complex, MHC)Ⅱ]的表达水平。提取C57BL/6小鼠脾源性T淋巴细胞与经胞外体刺激的DC共孵育后共培养72 h, 分为Ub-S-HDAg-Dexs组(加入50 μg/mL Ub-S-HDAg-Dexs)、Blank-Dexs组(加入50 μg/mL无质粒转染的DC来源胞外体)、Con-Dexs组(加入50 μg/mL经空白慢病毒转染的DC来源胞外体)、PBS组[加入50 μL/mL磷酸盐缓冲液(phosphate-buffered saline, PBS)]、Ub-S-HDAg-Dexs+AG490组(加入50 μg/mL Ub-S-HDAg-Dexs, 经胞外体刺激的DC与T淋巴细胞混合时同时加入50 μmol/L AG490), 流式细胞术检测CD8与γ干扰素双阳性T细胞, 非放射性乳酸脱氢酶检测试剂盒检测CTL细胞的杀伤活性。采用实时定量聚合酶链反应和蛋白质印迹法分别检测T淋巴细胞中Jak激酶(Janus kinase, JAK)2、GATA结合蛋白3(GATA-binding protein 3, GATA3)、T-bet、信号转导及转录激活因子(signal transduction and activator of transcription, STAT)1、STAT4的mRNA和蛋白质表达水平。统计学分析采用独立样本t检验。结果经Ub-S-HDAg-Dexs刺激, DC表面分子CD80、CD86、MHCⅡ阳性率为83.850%±0.219%、68.910%±0.134%、84.320%±0.445%。在Ub-S-HDAg-Dexs组, γ干扰素和CD8双阳性率为6.420%±0.028%, 高于PBS组、Blank-Dexs组、Con-Dexs组、Ub-S-HDAg-Dexs+AG490组(t=90.78、30.32、63.06、85.42, 均P<0.001);细胞杀伤率为82.4%±3.9%, 高于PBS组、Blank-Dexs组、Con-Dexs组、Ub-S-HDAg-Dexs+AG490组(t=17.28、9.74、3.95、15.89, 均P<0.050)。Ub-S-HDAg-Dexs组JAK2、T-bet、STAT1、STAT4 mRNA相对表达量分别与Con-DexsObjective To explore the effect and mechanism of dendritic cell derived exosomes(Dexs)loading ubiquitinated(Ub)hepatitis D antigen(HDAg)on activating specific cytotoxic T lymphocytes(CTL).Methods Ub-S-HDAg-Dexs were co-cultured with dendritic cells(DC)which were from the femora of C57BL/6 mice for 48 h,then flow cytometry was used to detect the maturity of DC(CD86,CD80 and major histocompatibility complex(MHC)Ⅱ).The spleen-derived T lymphocytes from C57BL/6 mice were added in vitro to activate DC and co-cultivated for 72 h.The T cells were divided into Ub-S-HDAg-Dexs group(add 50μg/mL Ub-S-HDAg-Dexs),Blank-Dexs group(add 50μg/mL DC derived exosomes without plasmid transfection),Con-Dexs group(add 50μg/mL DC derived exosomes transfected by cantrol plasmid),PBS group(add 50μL/mL phosphate buffered saline),and Ub-S-HDAg-Dexs+AG490 group(add 50μg/mL Ub-S-HDAg-Dexs,DC and T lymphocytes stimulated by exosomes,and 50μmol/L AG490 was also added to the cell mix).Flow cytometry was used to detect CD8+T cells secreting interferon-gamma,non-radioactive lactate dehydrogenase release test to detect the killing activity of specific CTL.Real-time quantitative polymerase chain reaction(PCR)and Western blotting were used to detect the mRNA and protein expressions of JAK kinase(JAK)2,GATA-binding protein 3(GATA3),T-bet,signal transduction and activator of transcription(STAT)1 and STAT4.Independent sample t test were used for statistical analysis.Results The positive rates of the surface molecules CD80,CD86,MHCⅡof DC stimulated by Ub-S-HDAg-Dexs were 83.850%±0.219%、68.910%±0.134%、84.320%±0.445%,respectively.In the Ub-S-HDAg-Dexs group,the rate of CD8+T cells secreting interferon-gamma was 6.420%±0.028%,which was higher than those of other groups,including PBS group,Blank-Dexs group,Con-Dexs group and Ub-S-HDAg-Dexs+AG490 group(t=90.78,30.32,63.06 and 85.42,respectively,all P<0.001).The cytotoxicity of T cells in the Ub-S-HDAg-Dexs group was 82.4%±3.9%,which was higher than those of other groups(t=17.28,9.74,3.95 a

关 键 词：丁型肝炎 外泌体 适应性免疫 JAK-STAT信号通路 

分 类 号：R512.64[医药卫生—内科学]