CAJ | 학술논문

目的探讨他克莫司诱导的人源耐受性树突细胞(tDC)的生物学特性及免疫抑制功能.方法分离人外周血单核细胞,在含GM-CSF和IL-4的药品生产质量管理规范级树突细胞(DC)培养液中诱导培养DC,于第3、第5天加入0.1μmol/L他克莫司进一步培养诱导tDC,流式细胞术检测细胞分子标志及存活率.用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)活细胞染色检测诱导的tDC与效应T细胞共培养后的异源CD4+效应T细胞增殖情况及tDC对成熟DC引起的混合淋巴细胞反应的抑制能力.检测他克莫司诱导的tDC经冻存复苏后的分子标志及免疫抑制功能.结果他克莫司诱导的tDC显示出典型的耐受性细胞表型:共刺激分子CD80、CD83、CD86及HLA-DR表达水平分别为(2.95±1.32)％、(2.33±1.60)％、(90.02±7.42)％、(91.80±6.18)％,其存活率为(85.24±4.72)％.他克莫司诱导的tDC与效应T细胞共孵育5d,效应T细胞的增殖率为0.42％,且它能有效抑制成熟DC引起的混合淋巴细胞反应,抑制率达(67.01±19.73)％.其在冻存复苏后仍具有表型和功能的稳定性.结论他克莫司诱导培养的tDC具有典型的免疫耐受细胞表型,不能诱导异源CD4+T细胞增殖,并可有效抑制成熟DC引起的混合淋巴细胞反应.
목적 탐토타극막사유도적인원내수성수돌세포(tDC)적생물학특성급면역억제공능.방법 분리인외주혈단핵세포,재함GM-CSF화IL-4적약품생산질량관리규범급수돌세포(DC)배양액중유도배양DC,우제3、제5천가입0.1μmol/L타극막사진일보배양유도tDC,류식세포술검측세포분자표지급존활솔.용간기형광소이작산염호박선아알지(CFSE)활세포염색검측유도적tDC여효응T세포공배양후적이원CD4+효응T세포증식정황급tDC대성숙DC인기적혼합림파세포반응적억제능력.검측타극막사유도적tDC경동존복소후적분자표지급면역억제공능.결과 타극막사유도적tDC현시출전형적내수성세포표형:공자격분자CD80、CD83、CD86급HLA-DR표체수평분별위(2.95±1.32)％、(2.33±1.60)％、(90.02±7.42)％、(91.80±6.18)％,기존활솔위(85.24±4.72)％.타극막사유도적tDC여효응T세포공부육5d,효응T세포적증식솔위0.42％,차타능유효억제성숙DC인기적혼합림파세포반응,억제솔체(67.01±19.73)％.기재동존복소후잉구유표형화공능적은정성.결론 타극막사유도배양적tDC구유전형적면역내수세포표형,불능유도이원CD4+T세포증식,병가유효억제성숙DC인기적혼합림파세포반응.
Objective To explore the biological characteristics and the immuno-suppression function of tolerogenic dendritic cells (tDC) induced by tacrolimus.Methods Human monocytes derived from peripheral blood were cultured in the cGMP-compliant CellGro DC medium supplemented with GM-CSF and IL-4 to obtain dendritic cells (DCs),and 0.1 μmol/L immunosuppressive drug tacrolimus was added to the culture medium at the third and fifth day to obtain tDCs.The molecular markers of them and the livability were assayed by flow cytometry.Then the tolerance functionality of tDCs induced by many agents and these tDCs modulated allogeneic CD4 T cells was determined via CFSE proliferation assay.And the research also analyzed the biological characters and immunosuppression function of tDCs induced by tacrolimus after storing.Results tDCs induced by tacrolimus exhibit a typical tolerogenic phenotype,whose level of costimulatory molecules CD80,CD83,CD86 and HLA-DR is (2.95± 1.32)％,(2.33±1.60)％,(90.02±7.42)％ and (91.80±6.18)％,respectively.It' s survival rate was (85.24±4.72) ％.And immunosuppressive drugs didn' t influence the differentiation of tDCs from monocytes.tDCs induced by immunosuppressive drugs dexamethasone,cyclosporin A and tacrolimus had lower immunogenic than control DCs as CD4+ T proliferation rate of tDCs induced by tacrolimus is 0.42％ and could not primed allogeneic CD4T cells proliferation.Functional analyses showed that tDCs induced by tacrolimus can more effectively suppressed mature DC-induced T cell proliferation than other tDCs,whose inhibition rate can reach (67.01 ± 19.73)％.Importantly,tDCs induced by tacrolimus had phenotypical and functional stability after storing.Conclusion tDCs induced by tacrolimus with tolerance functionality are a promising cellular therapeutic for immunomodulation.