CTLA4Ig诱导T细胞对氧化修饰低密度脂蛋白无能的体外研究

目的 在体外诱导T细胞对氧化修饰低密度脂蛋白(ox-LDL)的免疫无能,以期预防免疫损伤在动脉粥样硬化(AS)发病中的作用,为防治AS提供新的思路.方法 分离外周血单个核细胞诱导树突状细胞(DC).分别加入LPS、LDL、ox-LDL等刺激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MTY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(DC:T-1:5,1.6717±0.3152 vs 1.4250±0.2874,P<0.05;DC:T=1:10,1.5458±0.2748 vs 1.3352士0.2991,P<0.05);应用CTLA4Ig后,SI较未应用时明显降低(CTLA4Ig 1.25 Ixg/ml,0.96±0.30 vs 1.64±0.33,P<0.01;CTLA4Ig0.62μg/ml,1.12±0.33 vs 1.64±0.33,P<0.05;CTLA4Ig 0.31μg/ml,1.29±0.28vs 1.64±0.33,P<0.05);CTLA4Ig可明显减少T细胞CD25的表达(CTLA4Ig 1.25μg/ml,11.26士0.58 vs 14.25±1.02,P<0.05;CTLA4Ig 10μg/ml,8.42±0.45,P<0.01),增加T细胞的凋亡(CTLA4Ig 1.25μg/ml,12.54±3.69 vs 6.09±2.24,P<0.05;CTLA4Ig 10μg/ml,26.87±5.06 vs 6.09±2.24,P<0.01).ELISPOT表明,CTLA4Ig可减少IL-2(CTLA4Ig 1.25μg/ml,386±42 vs 534±54,P<0.05;CTLA4Ig 10μg/ml,230±27 vs 534±54,P<0.01)和IFN-γ(CTLA4Ig 1.25μg/ml,445±48 v8672±46,P<0.05;CTLA4Ig 10μg/ml,193±39 vs 672±46,P<0.01)的ELISPOT计数,增加IL-4的ELISPOT计数(CTLA4Ig 1.25μg/ml,401±32 vs 332±41,P<0.05;CTLA4Ig 10μg/ml,453±57 vs332±41,P<0.05).结论 CTLA4Ig可在体外诱导T细胞对ox-LDL的免疫无能;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T细胞免疫无能.

Duction of immunoincompetence of T cells to oxidized-low density lipoprotein in vitro by CTLA4Ig

Objective To induce the immunoincompetence of T cells to oxidized-low density lipoprotein(ox-LDL)in vitro,in order to prevent immune injuries in atherosclerosis(AS)and to find new strategies to prevent AS.Methods Monocytes were separated from peripheral blood to induce dendritic cells (DC).DCs were treated with LPS(30 ng/m1),ox-LDL(10μg/m1)and LDL(10μg/m1)for 48 h,respectively.Then DCs were mixed with allogeneic T lymphocytes to earry out mixed lymphoeytes reaction (MLR).CTLA4Ig in different concentrations was added into the MLR of ox-LDL group.MTr method was used to assay the proliferation of T cells.The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.And the excretion of IL-2,IFN-γ and IL-4 were assayed by ELISPOT method.Results SI of the ox-LDL group was higher than that of the LDL group significantly(DC:T=1:5,1.6717±0.3152vs 1.4250±0.2874.P<0.05;DC:T=1:10,1.5458±0.2748 vs 1.3352±0.2991,P<0.05),and CTLA4Ig inhibited the SI of the ox-LDL group in dose-dependence(CTLA4Ig 1.25μg/ml,0.96±0.30 vs μg/ml 1.29±0.28 vs 1.64±0.33 P<0.05).CTLA4Ig caused the decrease of CD25 expression(CTLA4Ig 1.25 μg/ml,11.26±0.58 vs 14.25±1.02,P<0.05;CTLA4Ig 10μg/rnl 8.42±0.45,P<0.01)and induced apoptosis of T cells in MLR(CTLA4Ig 1.25μg/ml,12.54±3.69 vs 6.09 4-2.24,P<0.05;CTLA4Ig 10μg/ml,26.87±5.06 VS 6.09±2.24,P<0.01).CTLA4Ig caused the decrease of ELISPOT counts of IL-2(CTLA4Ig 1.25μg/ml,386±42 VS 534±54,P<0.05;CTLA4Ig 10μg/rnl,230±27 VS 534±54,P<0.01)and IFN-γ(CTLA4Ig 1.25μg/ml,445±48 VS 672±46,P<0.05;CTLA4Ig 10μg/ml,193±39 VS 672±46,P<0.01),while that of IL-4 increased(CTLA4Ig 1.25μg/ml,401±32 VS 332±41,P<0.05;CTLA4Ig 10μg/ml,453±57 VS 332±41,P<0.05).Conclusion CTLA4Ig can induce T cens immunoin competence to ox-LDL in vitro by inhibiting T cells activation,inducing T cells apoptosis and TH 1/TH2 immune deviation.