CAJ | 학술논문

目的研究新一代兔抗人免疫细胞多克隆抗体(newRALG)对异种细胞免疫反应的抑制作用.方法应用活化的淋巴细胞和单核细胞致敏新西兰兔后获得newRALG.将PKH-26标记的猪血管内皮细胞(PEC)和正常人单个核细胞(PBMC)建立混合培养体系,培养液中分别加入newRALG、正常兔IgG、Thymoglobulin和Scavenger受体(SR)阻断剂Poly G,培养后收集PBMC,然后分别加入异硫氰基荧光素(FITC)标记的鼠抗人CD14、CD40、CD80、CD86和HLA-DR单克隆抗体.通过流式细胞术检测单核细胞对PEC膜的吞噬作用;通过淋巴细胞与PEC的混合培养体系,加入newRALG,以观察淋巴细胞的增殖反应和newRALG对增殖反应的阻断作用.结果 PBMC与PKH-26标记的PEC共培养后,PBMC中的CD86+单核细胞表达PKH-26,进一步研究发现PKH-26阳性的CD86+单核细胞不但表达CD14、CD86和HLA-DR,同时上调共刺激分子CD40和CD80的表达水平.正常兔IgG(作为阴性对照)对单核细胞吞噬PEC膜无阻断作用,Thymoglobulin具有较低的阻断效果,newRALG与Poly G相似,均具有较高的阻断作用.正常未经刺激的人淋巴细胞不具备增殖能力,经灭活的PEC刺激后,人淋巴细胞具有高水平的免疫增殖反应.正常兔IgG不能阻断PEC对人淋巴细胞的免疫增殖反应;Thymoglobulin的抑制作用随浓度的降低而增强;高浓度的newRALG不能抑制人淋巴细胞的免疫增殖反应,但低浓度的newRALG则显示出强大地抑制人淋巴细胞免疫增殖反应的作用.结论单核细胞在异种免疫反应过程中发挥重要作用;newRALG可有效抑制单核细胞的吞噬功能和淋巴细胞的免疫增殖反应,从而有效的抑制异种移植排斥反应.
목적 연구신일대토항인면역세포다극륭항체(newRALG)대이충세포면역반응적억제작용.방법 응용활화적림파세포화단핵세포치민신서란토후획득newRALG.장PKH-26표기적저혈관내피세포(PEC)화정상인단개핵세포(PBMC)건립혼합배양체계,배양액중분별가입newRALG、정상토IgG、Thymoglobulin화Scavenger수체(SR)조단제Poly G,배양후수집PBMC,연후분별가입이류청기형광소(FITC)표기적서항인CD14、CD40、CD80、CD86화HLA-DR단극륭항체.통과류식세포술검측단핵세포대PEC막적탄서작용;통과림파세포여PEC적혼합배양체계,가입newRALG,이관찰림파세포적증식반응화newRALG대증식반응적조단작용.결과 PBMC여PKH-26표기적PEC공배양후,PBMC중적CD86+단핵세포표체PKH-26,진일보연구발현PKH-26양성적CD86+단핵세포불단표체CD14、CD86화HLA-DR,동시상조공자격분자CD40화CD80적표체수평.정상토IgG(작위음성대조)대단핵세포탄서PEC막무조단작용,Thymoglobulin구유교저적조단효과,newRALG여Poly G상사,균구유교고적조단작용.정상미경자격적인림파세포불구비증식능력,경멸활적PEC자격후,인림파세포구유고수평적면역증식반응.정상토IgG불능조단PEC대인림파세포적면역증식반응;Thymoglobulin적억제작용수농도적강저이증강;고농도적newRALG불능억제인림파세포적면역증식반응,단저농도적newRALG칙현시출강대지억제인림파세포면역증식반응적작용.결론 단핵세포재이충면역반응과정중발휘중요작용;newRALG가유효억제단핵세포적탄서공능화림파세포적면역증식반응,종이유효적억제이충이식배척반응.
Objective Delayed xenograft rejection (DXR) is a major barrier to the long-term xenograft survial.This study evaluated the interaction between human peripheral blood mononuelear cells (PBMC) and porcine endothelial cells (PEC),and the effects of new generation of rabbit antihuman leukocyte polyclonal antibody (newRALG) inhibiting xenogeneic cell-mediated immune responses.Methods newRALG was obtained from rabbits after immunization with activated lymphocytes and monoeytes.PEC were isolated from aorta,and human PBMC were isolated from peripheral blood.Co-cultures of PKH-26 labeled PEC with PBMC were established,newRALG,thymoglobulin,isotype Ig and scavenger receptor (SR) ligand poly G were added into the co-cultures.Cells were collected,then FACS analysis was carried out to detect the up-take of PEC membrane by monocytes and the expression of costimulatory molecules.Lymphocyte proliferative responses to PEC with or without antibody were evaluated by a xenogeneie mixed lymphocyte-endothelial cell reaction (xMLER).Results FACS analysis revealed that monocytes from PBMC-PEC co-cultures became positive for PKH-26 following their interaction with PKH-26 labeled PEC,indicating that they engulfed PEC membranes during activation.PKH-26 positive monocytes up-regulated the CD40 and CD80 expression.Furthermore,SR blockade with poly-G prevented PEC membrane up-take by monocytes,newRALG greatly reduced SR-mediated PEC membrane up-take.The effects of thymoglobulin in inhibiting PEC membrane uptake were limited.xMLER demonstrated strong lymphocyte proliferation in response to PEC,and lymphocyte proliferation was dramatically inhibited by newRALG but not isotype Ig at a dosmdependent manner.Conclusions Monocytes play an important role in xenogeneic immune responses.SR ligand poly G inhibits PEC membrane up-take.newRALG inhibits PEC membrane up-take by monocytes,suggesting that newRALG blocks SR.Additionally,newRALG inhibits lymphocyte proliferation in response to PEC.These results suggest that this new polyclonal preparation may thus impair the initiation of xeno-specific immune responses and prevent xenograft rejection.