CAJ | 학술논문

目的探讨共刺激信号OX40对体外诱导的小鼠CD4+ CD25+适应性调节性T淋巴细胞(iTreg)的Foxp3表达的影响.方法制备C57BL/6小鼠淋巴细胞悬液,经免疫磁珠法分选,获得CD4+ CD25-静息T淋巴细胞,与抗CD3单克隆抗体、抗CD28单克隆抗体、转化生长因子β1、白细胞介素2共孵育,诱导产生Foxp3+ iTreg.在此基础上,于培养体系中加入OX40激动型抗体及其对照抗体,利用流式细胞仪分析研究OX40信号刺激对iTreg Foxp3表达的影响.结果 C57BL/6小鼠淋巴结中CD4+ CD25+天然调节性T淋巴细胞(Treg)比例为(5.0±0.4)％,体外诱导培养的CD4+CD25+ Treg比例为(71.8±13.4)％,其中Foxp3阳性表达占(74.9±1.9)％.OX40激动型抗体组CD4+ CD25+ Treg细胞比例为(80.0±1.6)％,其中Foxp3表达水平为(59.2±0.7)％；OX40激动型抗体对照抗体组CD4+ CD25+ Treg细胞比例为(86.0±1.4)％,其中Foxp3表达水平为(70.0±0.8)％,两组间差异有统计学意义(P＜0.05).结论静息T淋巴细胞可以在体外诱导培养获得高纯度iTreg；OX40信号刺激可以显著抑制CD25+ iTreg细胞Foxp3的表达.
목적 탐토공자격신호OX40대체외유도적소서CD4+ CD25+괄응성조절성T림파세포(iTreg)적Foxp3표체적영향.방법 제비C57BL/6소서림파세포현액,경면역자주법분선,획득CD4+ CD25-정식T림파세포,여항CD3단극륭항체、항CD28단극륭항체、전화생장인자β1、백세포개소2공부육,유도산생Foxp3+ iTreg.재차기출상,우배양체계중가입OX40격동형항체급기대조항체,이용류식세포의분석연구OX40신호자격대iTreg Foxp3표체적영향.결과 C57BL/6소서림파결중CD4+ CD25+천연조절성T림파세포(Treg)비례위(5.0±0.4)％,체외유도배양적CD4+CD25+ Treg비례위(71.8±13.4)％,기중Foxp3양성표체점(74.9±1.9)％.OX40격동형항체조CD4+ CD25+ Treg세포비례위(80.0±1.6)％,기중Foxp3표체수평위(59.2±0.7)％；OX40격동형항체대조항체조CD4+ CD25+ Treg세포비례위(86.0±1.4)％,기중Foxp3표체수평위(70.0±0.8)％,량조간차이유통계학의의(P＜0.05).결론 정식T림파세포가이재체외유도배양획득고순도iTreg；OX40신호자격가이현저억제CD25+ iTreg세포Foxp3적표체.
Objective To evaluate the regulatory effect of OX40 co-stimulatory signal on the expression of Foxp3 in inductive regulatory T cells (iTreg) in vitro.Method CD4+ CD25+ naive T cells were isolated from C57BL/6 mouse lymphocyte suspension by MASC CD4+ CD25+ regulatory T cell isolation kit.Inductive Tregs were generated by stimulation of naive T cells in the presence of transforming growth factor beta (TGFβ1),anti-CD3,anti-CD28 and IL-2.The regulatory effect on iTregs was shown by use of OX40 stimulation monoclonal antibody (OX86) or control antibody.Using flow cytometric analysis (FACS),we examined the antibody-based identification of Tregs surface markers CD4 and CD25,along with the intracellular activation marker FoxP3.Results The ratio of CD4+ CD25+ nTregs isolated from mouse lymphatic node was (5.0 ± 0.4)％ vs.(71.8 ± 13.4)％ of TGFβ1-driven iTregs.The ratio of CD4+ CD25+ Tregs was (80.0 ± 1.6) ％ in OX40 stimulation McAb group vs.(86.0 ± 1.4)％ in control antibody group.Furthermore,the expression of Foxp3 was (59.2 ± 0.7) ％ in OX40 stimulation McAb group vs.(70.0 ± 0.8) ％ in control antibody group (P＜0.05).Conclusion TGFβ1-dependent protocol may induce the conversion of naive CD4+ T cells into CD25+ Foxp3+ iTregs.OX40 stimulation can down-regulate the expression of Foxp3 in CD4+ CD25 + iTreg significantly.Thus OX40 molecular may become an attractive target in Tregs-induced transplant tolerance.Further study should be performed to increase the suppressive activity of iTregs through blockade of OX40 signal.