CTLA4Ig诱导T细胞对氧化修饰低密度脂蛋白无能的体外研究

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

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48h，与同种异体淋巴细胞行混合淋巴细胞反应（met）。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MLRl中T细胞分泌IL-2、IFN-γ和IL4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±0．3152vs1．4250±0．2874，P〈0．05；DC：T=1：10，1．5458±0．2748vs1．3352±0．2991，P〈0．05）；应用CTIA4Ig后，sI较未应用时明显降低（CTIA4Ig1．25μg/ml，0．96±0．30vs1．64±0．33，P〈0．01；CTIA4Ig0．62μg/ml，1．12±0．33vs1．64±0．33，P〈0．05；CTIA4Ig0．31μg/ml，1．29±0．28vs1．64±0．33，P〈0．05）；CTIA4Ig可明显减少T细胞CD25的表达（CTIA4Ig1．25μg/ml，11．26±0．58vs14．25±1．02，P〈0．05；CTIA4Ig10μg/ml，8．42±0．45，P〈0．01），增加T细胞的凋亡（CT-IA4Ig1．25μg/ml，12．54±3．69vs6．09±2．24，P〈0．05；CTIA4Ig10μg/ml，26．87±5．06vs6．09±2．24，P〈0．01）。ELISPOT表明，CTIA4Ig可减少IL-2（CTIA4Ig1．25μg/ml，386±42vs534±54，P〈0．05；CTIA4Ig10μg/ml，230±27vs534±54，P〈0．01）和IFN-γ（CTIA4Ig1．25μg/ml，445±48vs672±46，P〈0．05；CTIA4Ig10μg/ml，193±39vs672±46，P〈0．01）的ELISPOT计数，增加IL4的ELISPOT计数（CTIA4Ig1．25μg/ml，401±32vs332±41，P〈0．05；CTIA4Ig10μg/ml，453±57vs332±41，P〈0．05）。结论CTIA4Ig可在体外诱导T细胞对ox-LDL的免疫无能；CTIA4Ig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。

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/ml）, ox-LDL （ 10 μg/ml） and LDL（ 10 μg/ml） for 48 h, respectively. Then DCs were mixed with allogeneic T lymphocytes to carry out mixed lymphocytes reaction （MLR）. CTLA4Ig in different concentrations was added into the MLR of ox-LDL group. MTT 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-T 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 ± 0.3152 vs 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 1.25 μg/ml, 0.96 ± 0.30 vs 1.64 ± 0.33, P 〈 0.01 ; CTLA4Ig 0.62 μg/ml, 1.12 ± 0.33 vs 1.64 ± 0.33, P 〈 0.05 ; CTLA4Ig 0.31 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 10 μg/ml 8.42 ± 0.45, P 〈 0.01 ） and induced apoptosis of T cells in MLR（ CTLA4Ig 1.25 μg/ml, 12.54 ± 3.69 vs 6.09 ± 2.24, P 〈 0.05 ; CTLA4Ig 10 μg/ml 26.87 ±5.06 vs 6.09 ±2.24, P〈0.01）. CTLA4Ig caused the decrease of ELISPOT counts of IL-2（CTLA4Ig 1.25 iμg/ml 386 ±42 vs 534 ±54, P 〈0.05; CTLA4Ig 10 μg/ml, 230 ±27 vs 534 ±54, P〈0.01） and IFN-±/（CTLA4Ig 1.25 Iμg/ml 445 ±48 vs 672 ±46, P 〈0.05; CTLA4Ig 10 pee/ml, 193 ± 39 vs 672 ± 46, P 〈 0.01 ）, while that of IL-4 increased （ CTLA4Ig 1.25 μg/ml 401 ± 32 vs 332 ±41, P〈0.05; CTLA4Ig 10 Iμg/ml 453 ±57 vs 332 ±41, P 〈0.05）. Conclusion CTLA4Ig can induce T cells immunoin competence to ox-LDL in vitro by inhibiting T cells activation, inducing T cells apoptosis and TH1/TH2 immune deviation.