用MyD88 siRNA制备半成熟树突状细胞致免疫耐受的实验研究

Experimental Study on Immune Tolerance Induced by Semi-mature Dendritic Cells with MyD88 siRNA Transfection

目的探讨利用Toll样受体关键蛋白转录水平抑制剂髓细胞样分化标记物(myeloid differentiation marker88,MyD88)siRNA,制备半成熟树突状细胞(dendritic cell,DC)的表型和致耐受功能。方法取BALB/c小鼠骨髓接种、培养,加入浓度为10ng/ml的重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM-CSF)诱导,培养至第8d将DC分为3组,空白对照组:于培养过程中不加其它任何物质;LPS组:加入终浓度为1μg/ml的LPS;实验组:加入MyD88 siRNA 4h后,再加入1μg/ml的LPS。采用流式细胞术检测3组DC的表型,用酶联免疫吸附测定(ELISA)检测培养上清的白细胞介素10(IL-10)、白细胞介素12(IL-12)的含量,初次和再次混合淋巴细胞实验检测DC刺激T细胞增殖能力,并观察术后鼠移植心存活时间。结果空白对照组DC表型为CD11c+、CD25-、CD40low、CD80low、CD86low和MHC-Ⅱlow未成熟表型DC,再经LPS刺激后成为成熟表型DC;而实验组DC表型为CD11c+、CD25-、CD40mid、CD80low、CD86low和MHC-Ⅱmid半成熟表型,其IL-10/IL-12比率明显增高;可诱导同种异型T细胞无反应性,并能延长移植心存活时间。结论MyD88 siRNA转染结合LPS刺激可使未成熟DC进一步分化为一种半成熟DC,较未成熟DC拥有更稳定有效的致耐受特性。

Objective To investigate the phonetype and tolerogenic function of semi-mature dendritic cells （DC） transfected by myeloid differentiation marker 88（MyD88） small interfering ribonucleic acid（siRNA）. Methods Bone marrow of BALB/c mice was inoculated and cultured in vitro,and induced into DC by 10ng/ml recombinated granulocyte macrophage colony stimulating factor （rmGM-CSF） ,then DC was divided into three groups at the 8th day： blank control group： added nothing; lipopolysaccharide （LPS）group： added with 1μg/ml LPS; and experimental group： added with 1μg/ml LPS after transfected by MyD88 siRNA for 4 hours. The phonetype of three groups was analysed by fluorescence-activated cell sorting （FACS）. The concentration of interleukin 10（IL-10）and interleukin 12 （IL-12） in culture supernatant was detected by enzyme linked immunosorbent assay （ELISA）. The function of stimulating alloreactive T cell proliferation were evaluated by primary and secondary mixed lymphocyte reaction （MLR）. The cardiac allograft survival time was compared after DC of three groups injected into recipient mice. Results The phonetype of blank control group DC was CD11c^＋ , CD25^-, CD40^low, CD80^low, CD86^low, MHC- Ⅱ^low, which could be induced to mature DC by LPS. Experimental group DC was phonetypically semi-mature DC （CD11c^＋, CD25^-,CD40^mid,CD80^low,CD86^low, MHC-Ⅱ^mid） and the IL-10/IL-12 ratio of semi-mature DC increases significantly. Hyporesponsiveness of alloactive T cells can be induced by experimental group DC and the survive time of heart allograft was prolonged. Conclusion Immature DC could become semi-mature DC after transfected by MyD88 siRNA and stimulation by LPS. These semi-mature DC are more tolerogenic than immature DC.