三基序蛋白23在树突状细胞分化成熟中的调控作用  

Role of tripartite motif-containing 23 in the differentiation and maturation of dendritic cells

作　　者：丁骏[1] 段雪含 谢叶文 潘洁 夏蕾[1] 宁永玲[1] 何树燕 戚春建[1] Ding Jun;Duan Xuehan;Xie Yewen;Pan Jie;Xia Lei;Ning Yongling;He Shuyan;Qi Chunjian(Central Laboratory,Changzhou No.2 People′s Hospital Affiliated to Nanjing Medical University,Changzhou 213614,China;Department of Oncology,Jiangyin Hospital Affiliated to Nantong University,Wuxi 214400,China)

机构地区：[1]南京医科大学附属常州市第二人民医院中心实验室,常州213614 [2]南通大学附属江阴医院肿瘤科,无锡214400

出　　处：《中华微生物学和免疫学杂志》2023年第4期285-293,共9页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(32270954);江苏省自然科学基金(BK20200178);常州市科技支撑计划(CE20215054);江阴市卫健委面上项目(S202101)。

摘　　要：目的探讨三基序蛋白23(tripartite motif-containing 23, Trim23)对树突状细胞分化成熟的影响及相关作用机制。方法采用FMS样酪氨酸激酶3配体(FMS-like tyrosine kinase 3 ligand, Flt3L)诱导小鼠骨髓来源树突状细胞(bone marrow-derived dendritic cells, BMDCs), 经脂多糖(lipopolysaccharide, LPS)激发后, 荧光定量PCR及Western blot检测BMDCs中Trim23的表达。构建Trim23过表达载体并转染BMDCs, pcDNA3.1空载体作为对照组。经LPS激发后, 流式细胞术检测BMDCs表面分子CD80、CD86、CD40以及MHCⅡ的表达, ELISA检测培养上清中细胞因子IL-12p40、TNF-α、IL-6、IL-10的含量;磁珠分选出OT-Ⅰ和OT-Ⅱ小鼠脾脏和淋巴结中的CD8^(+)和CD4^(+)T细胞, 加入特异性抗原卵清蛋白(ovalbumin, OVA), 与LPS激发的BMDCs共培养。流式细胞术检测不同转染组BMDCs诱导CD8^(+)或CD4^(+)T细胞增殖分化的方向。Western blot检测BMDCs内p38、ERK1/2、AKT蛋白的磷酸化水平。构建缺失RING结构域或ARF结构域的两个Trim23短截体(Trim23 ΔRING和Trim23 ΔARF), 将Trim23、Trim23 ΔRING、Trim23 ΔARF载体分别转染BMDCs, 经LPS激发后, 流式细胞术及ELISA检测BMDCs表面分子及细胞因子的表达水平。结果经LPS激发后, BMDCs中Trim23表达水平显著下调;过表达Trim23后, 其表面分子MHCⅡ、CD86、CD80的表达及细胞因子TNF-α、IL-6的分泌均显著下降;与T细胞共培养结果显示, 过表达Trim23可显著抑制BMDCs诱导CD4^(+)T细胞增殖分化以及诱导CD8^(+)T细胞增殖的能力;Western blot结果显示, 过表达Trim23的BMDCs中p38和ERK1/2的磷酸化水平明显降低。与Trim23组比较, RING结构域的缺失显著逆转了过表达Trim23对LPS诱导的BMDCs表面分子MHCⅡ、CD86及细胞因子TNF-α、IL-6表达水平的抑制作用。结论过表达Trim23能够抑制BMDCs的分化成熟及免疫活化功能, 其作用机制可能依赖于MAPK信号通路及Trim23的RING功能域, 本研究将为靶向Trim23提高肿瘤树突状细Objective To investigate the effect of tripartite motif-containing 23(Trim23)on the differentiation and maturation of dendritic cells and the possible mechanism.Methods Mouse bone marrow-derived dendritic cells(BMDCs)were prepared from bone marrow cells of C57BL/6 mice with the presence of Flt3L.Real-time quantitative PCR and Western blot were used to detect the expression of Trim23 in BMDCs after LPS stimulation.An overexpression vector for full-length Trim23(Trim23 OE)was constructed and transfected into BMDCs,and the pcDNA3.1 empty vector was used as control.Flow cytometry was used to detect the expression of CD80,CD86,CD40 and MHCⅡon the surface of vector-transfected BMDCs after LPS stimulation and ELISA was used to detect the secretion of IL-12p40,TNF-α,IL-6 and IL-10 by these cells.CD8^(+)and CD4^(+)T cells were isolated from spleen and lymph nodes of OT-Ⅰand OT-Ⅱmice by magnetic beads and co-cultured with LPS-treated BMDCs in the presence of ovalbumin(OVA).Flow cytometry was used to detect the proliferation and differentiation of CD8^(+)and CD4^(+)T cells.Western blot was performed to analyze the phosphorylation of p38,ERK1/2 and AKT in BMDCs.Two overexpression vectors for Trim23 mutants lacking RING or ARF domain(Trim23ΔRING and Trim23ΔARF)were constructed and transfected into BMDCs.Then flow cytometry and ELISA were used to detect the expression of surface molecules and cytokines.Results The expression of Trim23 in BMDCs was significantly down-regulated after LPS stimulation.The expression of MHCⅡ,CD86 and CD80 and the secretion of TNF-αand IL-6 decreased significantly in BMDCs overexpressing Trim23.Furthermore,overexpression of Trim23 inhibited the ability of BMDCs to induce the proliferation and differentiation of CD4^(+)T cells and the proliferation of CD8^(+)T cells.Western blot showed that the phosphorylation of p38 and ERK1/2 decreased significantly in Trim23-overexpressing BMDCs.Compared with wildtype Trim23,overexpression of Trim23ΔRING had no significant influence on the expression

关 键 词：树突状细胞 Trim23 功能域 免疫功能 

分 类 号：R392.12[医药卫生—免疫学]