轴突导向因子3A对脂多糖诱导的CD4+CD25+调节性T细胞稳定性的影响  

Effect of semaphorin-3A on the cellular stability of CD4+CD25+regulatory T cells induced by lipopolysaccharide

作　　者：高玉雷[1] 李鎏鑫 刘艳存[1] 李文杰[1] 王紫怡 寿松涛[1] 柴艳芬[1] Gao Yulei;Li Liuxin;Liu Yancun;Li Wenjie;Wang Ziyi;Shou Songtao;Chai Yanfen(Department of Emergency Medicine,Tianjin Medical University General Hospital,Tianjin 300052,China;Department of Endocrinology and Metabolism,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院急诊医学科,天津300052 [2]天津医科大学总医院内分泌代谢科,天津300052

出　　处：《中华危重病急救医学》2020年第12期1454-1460,共7页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81701931,81871593);天津市自然科学基金青年基金(18JCQNJC10500)。

摘　　要：目的探讨轴突导向因子3A(Sema3A)对脂多糖(LPS)诱导的CD4^(+)CD25^(+)调节性T细胞(Tregs)稳定性的作用及机制。方法采用体外免疫磁珠法分离和培养C57BL/6J小鼠脾脏CD4^(+)CD25^(+)Tregs,将分离的细胞按随机数字表法分为对照组(仅给予抗小鼠CD3e和CD28诱导细胞处于激活状态)、LPS组(在对照组的基础上给予LPS 100μg/L)、LPS+核转录因子-κB(NF-κB)抑制剂吡咯烷二硫代氨基甲酸酯(PDTC)组(给予LPS 100μg/L+PDTC 25 mg/L)、LPS+磷酸盐缓冲液(PBS)组(给予LPS 100μg/L+PBS 10μL)、LPS+PDTC+重组Sema3A(rSema3A)组(给予LPS 100μg/L+PDTC 25 mg/L+rSema3A 300μg/L)和LPS+PBS+rSema3A组(给予LPS 100μg/L+PBS 10μL+rSema3A 300μg/L)。各组培养24 h后,采用反转录-聚合酶链反应(RT-PCR)和免疫荧光法检测CD4^(+)CD25^(+)Tregs特异性标志物叉头翼状转录因子-3(Foxp-3)、细胞毒性T淋巴细胞相关抗原-4(CTLA-4)和膜相关转化型生长因子-β1(TGF-β1^(m+))的基因及蛋白表达,采用酶联免疫吸附试验(ELISA)检测细胞上清液中白细胞介素-10(IL-10)和分泌型转化生长因子-β1(sTGF-β1)的水平,采用免疫荧光法检测细胞凋亡,采用甲基化特异性聚合酶链反应(MSP)检测Foxp-3-Tregs特异性去甲基化区(Foxp-3-TSDR)的去甲基化程度,以反映CD4^(+)CD25^(+)Tregs的稳定性,采用电泳迁移率分析(EMSA)检测NF-κB信号通路的DNA结合活性,采用蛋白质免疫印迹试验(Western blotting)检测NF-κB信号通路活性。结果与对照组比较,LPS能够增加细胞稳定性,表现为Foxp-3、CTLA-4和TGF-β1^(m+)的基因及蛋白表达上调,IL-10和sTGF-β1分泌增加,细胞凋亡减少,Foxp-3-TSDR去甲基化程度增加;同时LPS可增加细胞内NF-κB信号通路的DNA结合活性,以及主要分子NF-κB抑制蛋白激酶β(IKKβ)和p65的磷酸化水平,说明LPS增加细胞稳定性的机制与NF-κB信号通路有关。与LPS组比较,PBS并未对细胞稳定性和NF-κB信号通路产生影响;但添加rSema3A后能进一步�Objective To investigate the effect and mechanism of semaphorin-3A(Sema3A)in maintaining the cellular stability of CD4^(+)CD25^(+)regulatory T cells(Tregs)induced by lipopolysaccharide(LPS).Methods In vitro,using immunomagnetic beads,splenic CD4^(+)CD25^(+)Tregs of C57BL/6J mice were isolated and cultured.According to the random number table,the isolated cells were divided into control group(treated with anti-CD3e and anti-CD28 for polyclonal activation),LPS group(on the basis of control group,treated with LPS at the dose of 100μg/L),LPS+nuclear factor kappa B(NF-κB)inhibitor pyrrolidine dithiocarbamate(PDTC)group(treated with LPS at the dose of 100μg/L and PDTC at the dose of 25 mg/L),LPS+phosphate buffer solution(PBS)group(treated with LPS at the dose of 100μg/L and PBS at the volume of 10μL),LPS+PDTC+recombinant Sema3A(rSema3A)group(treated with LPS at the dose of 100μg/L,PDTC at the dose of 25 mg/L and rSema3A at the dose of 300μg/L),and LPS+PBS+rSema3A group(treated with LPS at the dose of 100μg/L,PBS at the volume of 10μL and rSema3A at the dose of 300μg/L).mRNA and protein expressions of the specific markers of CD4^(+)CD25^(+)Tregs,including forkhead box protein P-3(Foxp-3),cytotoxic T lymphocyte-associated antigen-4(CTLA-4)and membrane-associated transforming growth factor-β1(TGF-β1^(m+))were detected by reverse transcription-polymerase chain reaction(RT-PCR)and immunofluorescence methods after 24 hours.The supernatant interleukin-10(IL-10)and secretory type TGF-β1(sTGF-β1)were detected by enzyme-linked immunosorbent assay(ELISA).The apoptotic level was detected by immunofluorescence.The demethylation of Foxp3-Tregs-specific demethylated region(Foxp-3-TSDR)was detected by methylation specific PCR(MSP)in order to reflect the cellular stability of CD4^(+)CD25^(+)Tregs.DNA binding activity of NF-κB signaling pathway was determined by electrophoretic mobility shift assay(EMSA),and activity of NF-κB signaling pathway was determined by Western blotting.Results Compared with control group,LPS cou

关 键 词：脓毒症 调节性T细胞 细胞稳定性 轴突导向因子-3A 核转录因子-ΚB 

分 类 号：R392.12[医药卫生—免疫学]