CAJ | 학술논문

目的揭示人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制.方法从猪的主动脉分离血管内皮细胞(PEC)并培养扩增;从人单个核细胞(PBMC)中纯化CD4+T淋巴细胞和单核细胞.建立PEC和人PBMC混合培养体系,培养后收集细胞,然后加入荧光标记的单克隆抗体,通过流式细胞术检测CD14+单核细胞表面共刺激分子表达情况.为了检测淋巴细胞增殖反应以及阻断共刺激分子对PEC免疫反应的作用,在PEC和人PBMC混合培养体系中分别加入抗CD154、CD80和CD86单克隆抗体.在培养的最后24 h加入同位素,于培养结束后收集细胞并经同位素计数仪进行检测.纯化的单核细胞经PEC刺激后与CD4+T淋巴细胞共培养来研究这些单核细胞诱导CD4+T淋巴细胞的增殖以及阻断共刺激分子的作用.结果 PEC和人PBMC混合培养后可检测到PBMC对异种PEC的高度免疫增殖反应;流式细胞术检测到PBMC中的CD14+单核细胞表面无CD40和CD80的表达,但表达CD86,经PEC刺激后,CD14+单核细胞膜表面显著上调CD40和CD80蛋白分子的表达,CD86表达上调.与未经刺激的单核细胞相比较,经PEC刺激后的单核细胞和CD4+T淋巴细胞共培养后可诱导CD4+T淋巴细胞明显增殖,抗人CD154、CD80、CD86单克隆抗体可以阻断CD4+T淋巴细胞对PEC的增殖反应.结论人CD14+单核细胞在异种免疫反应过程的间接抗原提呈和共刺激信号传导中发挥重要作用,通过上调其共刺激分子的表达与CD4+T淋巴细胞共刺激分子CD154和CD28相互作用形成第二信号,并诱导CD4+T淋巴细胞对PEC的增殖反应;阻断共刺激分子可抑制异种细胞免疫反应.
목적 게시인단핵세포공자격분자재이충면역반응중적표체급기작용궤제.방법 종저적주동맥분리혈관내피세포(PEC)병배양확증;종인단개핵세포(PBMC)중순화CD4+T림파세포화단핵세포.건립PEC화인PBMC혼합배양체계,배양후수집세포,연후가입형광표기적단극륭항체,통과류식세포술검측CD14+단핵세포표면공자격분자표체정황.위료검측림파세포증식반응이급조단공자격분자대PEC면역반응적작용,재PEC화인PBMC혼합배양체계중분별가입항CD154、CD80화CD86단극륭항체.재배양적최후24 h가입동위소,우배양결속후수집세포병경동위소계수의진행검측.순화적단핵세포경PEC자격후여CD4+T림파세포공배양래연구저사단핵세포유도CD4+T림파세포적증식이급조단공자격분자적작용.결과 PEC화인PBMC혼합배양후가검측도PBMC대이충PEC적고도면역증식반응;류식세포술검측도PBMC중적CD14+단핵세포표면무CD40화CD80적표체,단표체CD86,경PEC자격후,CD14+단핵세포막표면현저상조CD40화CD80단백분자적표체,CD86표체상조.여미경자격적단핵세포상비교,경PEC자격후적단핵세포화CD4+T림파세포공배양후가유도CD4+T림파세포명현증식,항인CD154、CD80、CD86단극륭항체가이조단CD4+T림파세포대PEC적증식반응.결론 인CD14+단핵세포재이충면역반응과정적간접항원제정화공자격신호전도중발휘중요작용,통과상조기공자격분자적표체여CD4+T림파세포공자격분자CD154화CD28상호작용형성제이신호,병유도CD4+T림파세포대PEC적증식반응;조단공자격분자가억제이충세포면역반응.
Objective To explore the expression and the role of monocyte-derived costimulatory molecuels during xenogeneic immune responses. Methods Porcine endothelial cells (PEC) were isolated from aorta, and subcultures were performed. CD4+ cells and monocytes were purified from human peripheral blood mononuclear cells (PBMC). PBMC-PEC co-cultures were established, and the cells were collected followed by staining with florescent-labeled monoclonal antibodies and analyzing by FACS. In selected experiments, monoclonal antibodies specific for CD154, CD80 and CD86 were added into PBMC-PEC co-cultures, and the effects of co-stimulatory molecule blockade in inhibiting lymphocyte proliferation in response to PEC were determined by 3H-thymidine up-take. The proliferation of CD4+ cells induced by PEC-conditioned monocytes with or without co-stimulation blockade was evaluated. Results PBMC-PEC co-incubation demonstrated dramatic lymphocyte proliferation as determined by 3H-thymidine up-take. FACS found that resting monocytes expressed only CD86 but not CD40 and CD80. CD14+ monocytes from PEC-stimulated PBMC demonstrated up-regulation of CD80 and CD40 expression. The up-regulation of CD86 was revealed. PEC-activated monocytes induced CD4+ cell proliferation while resting monocytes did not and this proliferation was inhibited by anti-CD154, anti-CD80 or anti-CD86 antibodies. Conclusions CD14+ monocytes play an important role during xenogeneie immune responses in indirect antigen presentation and co-stimulation- The interaction between monocyte-derived co-stimulatory molecules and CD4+ cell-derived CD154 and CD28 delivers secondary signal and induces CD4+ proliferation, and the co-stimulation blockade inhibits xe-nogeneic cell-mediated immune responses.