国际中医中药杂志
國際中醫中藥雜誌
국제중의중약잡지
INTERNATIONAL JOURNAL OF TRIDITIONAL CHINESE MEDICINE
2014年
10期
889-893
,共5页
黄立锋%李金凤%姚咏明%张淑文%李文雄
黃立鋒%李金鳳%姚詠明%張淑文%李文雄
황립봉%리금봉%요영명%장숙문%리문웅
高迁移率族蛋白B1%调节性T细胞%黄芪甲苷%免疫抑制%脓毒症
高遷移率族蛋白B1%調節性T細胞%黃芪甲苷%免疫抑製%膿毒癥
고천이솔족단백B1%조절성T세포%황기갑감%면역억제%농독증
High mobility group box-1 protein%Regulatory T cell%AstragalosideⅣ%Immune suppression%Sepsis
目的：观察高迁移率族蛋白B1（HMGB1）刺激的小鼠调节性T细胞（CD4＋CD25＋Treg）对CD4＋CD25-T细胞免疫功能的影响及黄芪甲苷（AST Ⅳ）对HMGB1介导Treg免疫功能的拮抗作用。方法采用清洁级BALB/c小鼠，活杀后分离脾脏的CD4＋CD25＋Treg、CD4＋CD25-T细胞，将CD4＋CD25-T细胞在48孔板上随机分为4组（每组12孔）培养，对照组：单纯CD4＋CD25- T细胞；Treg实验组：CD25＋∶CD25-按照1∶10比例加入100μl CD4＋CD25＋Treg与CD4＋CD25-T细胞共同培养；HMGB1＋Treg组：按CD25＋∶CD25-比例为1∶10加入100μl经HMGB1（1μg/ml）刺激72 h的Treg与CD4＋CD25-T细胞进行共培养；HMGB1＋AST IV＋Treg组： CD25＋∶CD25-按照1∶10比例加入100μl经HMGB1（1μg/ml）、AST IV（100μg/ml）刺激72 h的CD4＋CD25＋Treg与CD4＋CD25-T细胞共同培养。以上4组均于培养后72 h重新分离收集CD4＋CD25-T细胞及其上清液，采用噻唑蓝（MTT）法检测脾脏CD4＋CD25-T细胞增殖活性；ELISA法检测CD4＋CD25-T细胞活化核因子（NFAT）活性、孵育上清液IL-2表达。结果将CD4＋CD25＋Treg加入CD4＋CD25-T细胞培养后，CD4＋CD25-T细胞增殖活性受到抑制（0.166±0.039），P＜0.01，NFAT活性（0.156±0.035）、IL-2水平（2.38±0.58）pg/ml均较对照组下降（P＜0.01）。加入经HMGB1刺激的Treg后，CD4＋CD25-T细胞增殖活性受抑制程度逆转（0.477±0.097），P＜0.01。与CD4＋CD25＋Treg组比较，HMGB1＋Treg组NFAT活性（0.409±0.094）、IL-2（4.55±0.96）pg/ml均升高（P＜0.01）。与HMGB1＋Treg组比较，HMGB1＋AST Ⅳ＋Treg组CD4＋CD25-T细胞增殖受抑制程度加重（0.287±0.052）、NFAT活性（0.261±0.046）、IL-2水平（2.73±0.62）pg/ml降低（P＜0.01）。结论 HMGB1在体外可抑制小鼠Treg免疫功能发挥，而ASTⅣ可拮抗HMGB1对Treg免疫功能的抑制作用，表明其对HMGB1介导的促炎效应具有治疗作用。
目的：觀察高遷移率族蛋白B1（HMGB1）刺激的小鼠調節性T細胞（CD4＋CD25＋Treg）對CD4＋CD25-T細胞免疫功能的影響及黃芪甲苷（AST Ⅳ）對HMGB1介導Treg免疫功能的拮抗作用。方法採用清潔級BALB/c小鼠，活殺後分離脾髒的CD4＋CD25＋Treg、CD4＋CD25-T細胞，將CD4＋CD25-T細胞在48孔闆上隨機分為4組（每組12孔）培養，對照組：單純CD4＋CD25- T細胞；Treg實驗組：CD25＋∶CD25-按照1∶10比例加入100μl CD4＋CD25＋Treg與CD4＋CD25-T細胞共同培養；HMGB1＋Treg組：按CD25＋∶CD25-比例為1∶10加入100μl經HMGB1（1μg/ml）刺激72 h的Treg與CD4＋CD25-T細胞進行共培養；HMGB1＋AST IV＋Treg組： CD25＋∶CD25-按照1∶10比例加入100μl經HMGB1（1μg/ml）、AST IV（100μg/ml）刺激72 h的CD4＋CD25＋Treg與CD4＋CD25-T細胞共同培養。以上4組均于培養後72 h重新分離收集CD4＋CD25-T細胞及其上清液，採用噻唑藍（MTT）法檢測脾髒CD4＋CD25-T細胞增殖活性；ELISA法檢測CD4＋CD25-T細胞活化覈因子（NFAT）活性、孵育上清液IL-2錶達。結果將CD4＋CD25＋Treg加入CD4＋CD25-T細胞培養後，CD4＋CD25-T細胞增殖活性受到抑製（0.166±0.039），P＜0.01，NFAT活性（0.156±0.035）、IL-2水平（2.38±0.58）pg/ml均較對照組下降（P＜0.01）。加入經HMGB1刺激的Treg後，CD4＋CD25-T細胞增殖活性受抑製程度逆轉（0.477±0.097），P＜0.01。與CD4＋CD25＋Treg組比較，HMGB1＋Treg組NFAT活性（0.409±0.094）、IL-2（4.55±0.96）pg/ml均升高（P＜0.01）。與HMGB1＋Treg組比較，HMGB1＋AST Ⅳ＋Treg組CD4＋CD25-T細胞增殖受抑製程度加重（0.287±0.052）、NFAT活性（0.261±0.046）、IL-2水平（2.73±0.62）pg/ml降低（P＜0.01）。結論 HMGB1在體外可抑製小鼠Treg免疫功能髮揮，而ASTⅣ可拮抗HMGB1對Treg免疫功能的抑製作用，錶明其對HMGB1介導的促炎效應具有治療作用。
목적：관찰고천이솔족단백B1（HMGB1）자격적소서조절성T세포（CD4＋CD25＋Treg）대CD4＋CD25-T세포면역공능적영향급황기갑감（AST Ⅳ）대HMGB1개도Treg면역공능적길항작용。방법채용청길급BALB/c소서，활살후분리비장적CD4＋CD25＋Treg、CD4＋CD25-T세포，장CD4＋CD25-T세포재48공판상수궤분위4조（매조12공）배양，대조조：단순CD4＋CD25- T세포；Treg실험조：CD25＋∶CD25-안조1∶10비례가입100μl CD4＋CD25＋Treg여CD4＋CD25-T세포공동배양；HMGB1＋Treg조：안CD25＋∶CD25-비례위1∶10가입100μl경HMGB1（1μg/ml）자격72 h적Treg여CD4＋CD25-T세포진행공배양；HMGB1＋AST IV＋Treg조： CD25＋∶CD25-안조1∶10비례가입100μl경HMGB1（1μg/ml）、AST IV（100μg/ml）자격72 h적CD4＋CD25＋Treg여CD4＋CD25-T세포공동배양。이상4조균우배양후72 h중신분리수집CD4＋CD25-T세포급기상청액，채용새서람（MTT）법검측비장CD4＋CD25-T세포증식활성；ELISA법검측CD4＋CD25-T세포활화핵인자（NFAT）활성、부육상청액IL-2표체。결과장CD4＋CD25＋Treg가입CD4＋CD25-T세포배양후，CD4＋CD25-T세포증식활성수도억제（0.166±0.039），P＜0.01，NFAT활성（0.156±0.035）、IL-2수평（2.38±0.58）pg/ml균교대조조하강（P＜0.01）。가입경HMGB1자격적Treg후，CD4＋CD25-T세포증식활성수억제정도역전（0.477±0.097），P＜0.01。여CD4＋CD25＋Treg조비교，HMGB1＋Treg조NFAT활성（0.409±0.094）、IL-2（4.55±0.96）pg/ml균승고（P＜0.01）。여HMGB1＋Treg조비교，HMGB1＋AST Ⅳ＋Treg조CD4＋CD25-T세포증식수억제정도가중（0.287±0.052）、NFAT활성（0.261±0.046）、IL-2수평（2.73±0.62）pg/ml강저（P＜0.01）。결론 HMGB1재체외가억제소서Treg면역공능발휘，이ASTⅣ가길항HMGB1대Treg면역공능적억제작용，표명기대HMGB1개도적촉염효응구유치료작용。
Objective Based the previous studies, the present study was performed to investigate the antagonistic effects of different doses of Astragaloside IV on the immune function of Treg mediated by HMGB1 in vitro and its potential mechanism.Methods CD4+CD25-T cells isolated from the spleens of male BABL/c mice by magnetic beads were seeded on 48-well cell culture plates and were randomly divided into four groups as follows(12 holes per group). Normal control group: CD4+CD25-T cells were cultured merely. Treg group: Tregs(100μl) and CD4+CD25-T cells were co-cultured in ratio of 1:10. HMGB1+Treg group: Tregs(100μl) stimulated by HMGB1(1μg/ml) for 72 h and CD4+CD25-T cells were co-cultured in ratio of 1∶10. HMGB1+AST IV+Treg group: Tregs(100μl) stimulated by HMGB1(1μg/ml) and AST IV(100μg/ml)for 72 h were co-cultured with CD4+CD25-T cells in ratio of 1:10. CD4+CD25-T cells and supernatants were again collected on post-culture 72 hour. The proliferation of CD4+CD25- T cells was analyzed by MTT test, the activity of NFAT and the contents of cytokines of IL-2 released into supernatants were also determined by means of ELISA. Results When CD4+CD25-T cells were co-cultured with Tregs, the cell proliferation(0.166±0.039) and the levels of NFAT(0.156±0.035) and IL-2(2.38±0.58) in supernatant were markedly decreased as compared with those in the control group(P<0.01). However, the contrary results were found when CD4+CD25-T cells were co-cultured with Treg stimulated by HMGB1. Compared with those in the(HMGB1+Treg) group, the contrary results were showed with a dose-dependent in the(HMGB1+ASTⅣ+Treg) group.Conclusion ASTⅣcan rivalry the effects of HMGB1 on immune function of Treg in vitro, this result indicate that ASTⅣhas the therapeutic action on inflammation promoted by HMGB1.
