CTLA4Ig诱导对氧化修饰低密度脂蛋白免疫耐受的机制研究

CTLA4Ig induces immune tolerance of T cells to oxidized-low density lipoprotein in vitro

目的:动脉粥样硬化(AS)是一种炎症过程,获得性免疫应答参与AS发生和发展。氧化修饰的低密度脂蛋白(ox-LDL)是目前认为最重要的AS相关自身抗原。本研究拟应用融合蛋白CTLA4Ig,在体外建立对ox-LDL的免疫耐受模型,从而有可能预防免疫应答导致的炎症损伤在AS发病中的作用,为防治AS提供新的策略。方法:分离人外周血单个核细胞诱导树突状细胞(DC)。分别加入LPS、LDL、ox-LDL刺激48 h,与同种异体淋巴细胞行混合淋巴细胞反应(MLR)。ox-LDL组的MLR中,分别加入不同浓度的CTLA4Ig。以MTT法检测T细胞的增殖。流式细胞仪检测MLR中T细胞活化和T细胞凋亡。ELISpot检测MLR中T细胞分泌IL-2、IFN-γ和IL-4的情况。结果:ox-LDL组MTT中的刺激指数(SI)明显高于LDL组(P<0.05);应用CTLA4Ig后,SI较未应用时明显降低(P<0.05,P<0.01);CTLA4Ig可明显减少T细胞CD25的表达(P<0.05,P<0.01),增加T细胞的凋亡(P<0.05,P<0.01)。CTLA4Ig可减少T细胞分泌IL-2和IFN-γ的ELISpot计数(P<0.01),增加IL-4的ELISpot计数(P<0.05)。结论:CTLA4Ig可在体外诱导对ox-LDL的免疫耐受;CTLA4Ig通过抑制T细胞活化、诱导T细胞凋亡和促进Th1/Th2免疫偏移等机制,诱导免疫耐受。

AIM： Recently, it is widely accepted that atherosclerosis （AS） is an auto -immune related disease and the oxidized - low density lipoprotein （ ox - LDL） is the most important AS - related antigen. In order to prevent immune injuries in AS and find new strategies to prevent AS, the immune tolerance of T cells to ox - LDL in vitro was induced in this study. METHODS： Human monocytes were separated from peripheral blood to induce dendritic cells （DCs）. DCs were treated with LPS （30 μg/L）, ox -LDL （10 mg/L） and LDL （10 mg/L） for48 h. Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction （MLR）. CTLA4Ig in different concentrations was added in the MLR of ox - LDL group. MTT method was used to assay the proliferation of T cells and expressed in stimulation index （IS）. The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry. The excretion of IL - 2, IFN - γ and IL - 4 was assayed by ELISpot method. RESULTS ： SI in ox - LDL group was higher than that in LDL group significantly （ P 〈 0.05） and CTLA4Ig inhibited the SI in ox - LDL group with dose - dependent effect （P 〈 0. 05, P 〈 0.01 ）. CTLA4Ig decreased the CD25 expression （P 〈0. 05, P 〈 0. 01 ） and induced apoptosis of T cells in MLR （P 〈 0.05, P 〈 0.01 ）. CTLA4Ig decreased the ELISpot counts of IL - 2 and IFN - γ （ P 〈 0. 01 ）, while increased that of IL-4 （P 〈0. 05）. CONCLUSION： CTLA4Ig induces T cells tolerance to ox- LDL in vitro. CTLA4Ig inhibits T cells activation, promotes T cells apoptosis and Th1/Th2 immune deviation, which is the important mechanism in it＇s induction of tolerance.