程序性死亡受体1缺乏对动脉粥样硬化性免疫反应的影响

Effect of PD-1 deficiency on atherogenic immune responses

目的 探讨程序性死亡受体1（PD-1）缺乏小鼠中T淋巴细胞功能的改变对动脉粥样硬化的影响.方法 将PD^-1/-小鼠与ApoE^-/-小鼠进行交配,获得双基因敲除PD-1-/-ApoE^-/-小鼠为实验组,以同龄野生型PD-1＋/-＋ApoE^-/-小鼠为对照组,2组小鼠高脂喂养12周后,取主动脉根部的标本,油红染色观察动脉粥样硬化情况,免疫组化观察斑块内T淋巴细胞浸润;取脾脏和血清标本,经流式细胞仪（FACS）进行脾脏淋巴细胞计数和检测淋巴细胞增殖、酶联免疫吸附试验（ELISA）检测淋巴细胞内和血清中细胞因子的分泌.结果 PD-1-/-ApoE^-/-小鼠主动脉根部切片示动脉粥样硬化斑块面积为（0.51 ±0.08） mm2,对照组为（0.29±0.06）mm2,差异有统计学意义（t=2.36,P＜0.0l）;动脉斑块中CD3＋T淋巴细胞含量为（154.88±36.64）个/mm2,较对照组的（59.38±25.28）个/mm2（t=2.14,P＜0.01）显著增多;脾脏所含细胞总数为（8.59±0.55）×107个,CD4＋、CD8＋T淋巴细胞数分别为（1.53±0.18） ×10 7、（1.41±0.15）×107个,均较对照组的（6.29±0.39） ×107、（0.94±0.10）×10 7、（0.70±0.09）×107个显著增多（t=3.43、2.88、4.03,均P＜0.05）,其中活性部分CD3＋ CD62L-、CD3＋ CD25＋的表达也显著增高;缺乏PD-1的CD4＋和CD8＋T淋巴细胞,无论是在巨噬细胞还是在树突状细胞的递呈抗原条件下,均具有更强的增殖能力;来源于实验组的T淋巴细胞经抗CD3抗体激活后,培养上清液中的白细胞介素-2（IL-2）、干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）浓度分别为（53.38±5.94）、（114.50±9.69）、（326.00±22.25） pg/ml,均高于对照组的（15.63±2.23）、（33.25±4.16）、（64.50±8.17） pg/ml,差异有统计学意义（=5.95、7.70、11.03,均P ＜0.01）,而血清中只有TNF-α在实验组中浓度显著增高.结论 PD-1的缺失导致ApoE^-/-小鼠体内T淋巴细胞免疫反应显著增强,是促动脉粥样硬化作用的重要机制之一.

Objective To explore the functional changes of T lymphocyte from PD^-1-/-mice and the effects on atherogenic immune responses.Methods PD-1-/-mice were mated with ApoE^-/-mice to obtain PD^-1-/-ApoE^-/-mice.PD-1 ^＋/＋ ApoE^-/-mice were used as control.Both groups had a high-lipid diet for 12 weeks.Then the samples of aortic sinuses were harvested for oil-red and immunohistochemical stains.Samples of spleen and sera were obtained.Numbers of lymphocytes in spleen were evaluated by flow cytometry （FACS）.And the cells of proliferation were also measured.The intracellular and serum cytokines were detected by enzyme-linked immunosorbent assay （ELISA）.Results After a 12-week high-lipid diet,aortic root revealed more lesions in PD-1-/-ApoE^-/-mice than in PD-1 ＋/＋ ApoE^-/-controls （（0.51 ± 0.08） vs （0.29 ±0.06） mm2,t =2.36,P 〈0.01））.There was a significant increase in CD3 T cells in the lesions of PD-1-/-ApoE^-/-mice （（154.88 ± 36.64）/mm2 vs （59.38 ± 25.28）/mm2,t =2.14,P 〈 0.01）.In PD-1-/-ApoE^-/-mice,the numbers of total cells （（8.59 ± 0.55） × 107） and cells in CD4 ＋ and CD8 ＋ T lymphocyte subsets （（1.53 ±0.18） × 107 and （1.41 ±0.15） × 107）,increased significantly than those in control mice （（6.29 ±0.39） × 107,（0.94 ±0.10） × 107 and （0.70 ±0.09） × 107 respectively,t =3.43,2.88,4.03,all P〈0.05）.Same changes were detected in both CD3 ＋ CD62L-and CD3＋ CD25＋ cells.PD-1 deficiency in both CD4 ＋ and CD8 ＋ T cells resulted in enhanced proliferation by either macrophages or dendritic cells as antigen presenting cells （APCs）.The supernatant levels of interleukin-2 （IL-2） （（53.38 ± 5.94） pg/ml）,interferon-γ （IFN-γ） （（114.50 ± 9.69） pg/ml） and tumor necrosis factor-α （TNF-o） （（326.00 ±22.25） pg/ml） from PD-1-/-T cells co-cultured with CD3 were significantly higher than those of control T cells （（15.63 ±2.23）,（33.25 ±4.16） and （64.50 ± 8.17） pg/ml respectively,t =5.95,7.70,11.03,all P 〈 0.01）.However,only the serum levels of TNF-α increased in PD-1-/-ApoE^-/-mice compared with control.Conclusions PD-1 deficiency in T cells increase markedly immune responses.And it is one of the mechanisms in atherogenesis.