T淋巴细胞凋亡在脓毒症患者免疫抑制和预后中的作用

Role of T lymphocytes apoptosis in immunosuppression and prognosis of sepsis

目的：研究脓毒症患者外周血T淋巴细胞亚群和T淋巴细胞凋亡的变化,探讨其在免疫抑制和预后中的作用。方法：收集2014年10月至2015年12月重庆医科大学附属第一医院ICU收治的脓毒症患者55例,随访患者28 d生存率,根据预后不同,分为脓毒症患者存活组和脓毒症患者死亡组;同时收集健康志愿者30例为对照组。应用流式细胞仪检测患者外周血T细胞亚群计数,分析其与APACHEⅡ评分及预测死亡危险率（R）的相关性;用免疫磁珠分选外周血CD3＋T淋巴细胞,通过流式细胞技术和Tunel法检测细胞凋亡。用脓毒症患者和健康志愿者血清体外刺激Jurkat细胞24 h,采用流式细胞技术和Tunel染色法检测细胞凋亡,Western blot法检测激活型天冬氨酸特异性半胱氨酸蛋白酶（cleaved-caspase 3）,B细胞淋巴瘤-2（B-cell lymphoma-2,Bcl-2）和髓样细胞白血病-1（myeloid cell leukemia 1,Mcl-1）蛋白表达水平。结果：脓毒症患者存活组、死亡组T淋巴细胞各亚群绝对值计数均明显低于对照组,分别为：CD3＋374.0（274.5～514.0）、154.0（87.5～256.2）、1534.5（1255.0～1756.7）,CD3＋CD4＋198.0（148.5～299.0）、68.0（37.7～108.5）、766.5（629.3～923.5）,CD3＋CD8＋142.0（89.0～184.0）、76.0（48.7～134.5）、600.5（400.2～717.0）（全部P〈0.05）;脓毒症患者死亡组CD3＋、CD3＋CD4＋细胞计数减少均较脓毒症患者存活组更显著,差异具有统计学意义（P=0.011,P=0.005）。脓毒症患者死亡组外周血T淋巴细胞中CD3＋CD4＋细胞占（42.40±5.71）%,较对照组（53.46±9.28）%明显减少（P=0.003）;CD3＋CD8＋细胞占（54.40±4.76）%,较对照组（39.33±7.26）%增加（P=0.003）;CD4＋/CD8＋比值为（0.79±0.17）,较对照组（1.44±0.51）明显下降（P=0.023）。CD3＋、CD3＋CD4＋、CD3＋CD8＋、CD4＋/CD8＋比值与患者APACHEⅡ评分和R均呈负相关关系（全部P〈0.05）。脓毒症患者存活组、死亡组CD3＋T淋巴细胞凋亡率分别为（15.59±0.54）%、（39.57±4.49）%,较对照组（9.53±1.91）%明显增加（P=0.040,P=0.000）,脓毒症患者死亡组较脓毒症患者存活组增加更明显（P=0.000）。脓毒症患者存活组、死亡组血清诱导Jurkat细胞凋亡率分别为（16.60±3.55）%、（32.93±5.89）%,较对照组（8.56±0.89）%明显增加（P=0.049、P=0.000）;Cleaved-caspase3蛋白水平（2.01±0.21,2.68±0.28）较对照组（1.00±0.00）上调（P=0.001、P=0.000）;抗凋亡蛋白Mcl-1（0.77±0.03,0.61±0.01）、Bcl-2（0.68±0.07,0.48±0.03）较对照组（1.00±0.00）明显下调（P=0.000、P=0.000）（P=0.000、P=0.000）;与脓毒症患者存活组比较,脓毒症患者死亡组中Jurkat细胞凋亡、Cleaved-caspase3、Mcl-1、Bcl-2变化更加为明显（P=0.002、P=0.007、P=0.000、P=0.001）。结论：脓毒症患者免疫失衡,T淋巴细胞凋亡增加,T淋巴细胞数明显减少,发生免疫抑制,且与病情严重程度和预后密切相关。

Objective：To investigate changes of T lymphocyte subsets and T lymphocytes apoptosis in sepsis and to discuss its role in immunosuppression and prognosis. Methods：Totally 55 sepsis patients from Intensive Care Unit of The First Affiliated Hospital of Chongqing Medical University from October 1,2014 to December 31,2015 ,were enrolled. Sepsis patients were divided into survival group and death group according to their prognosis. The APACHE Ⅱ score was calculated and the risk of death（R） was predicat- ed according to APACHE Ⅱ score,and the survival rate of 28 days were followed up. The absolute counts of peripheral blood T-lymphocyte subsets were measured by flow cytometry;T-lym- phocyte apoptosis were detected by flow cytometry and TUNEL assay. Immortalized line of human T lymphocyte ceIls（Jurkat） was cultured and cells were treated with serum from sepsis patients or healthy control subjects,then apoptosis was measured by flow cytometry and TUNEL assay. Cleaved-caspase 3, B-cell lymphoma-2 （Bcl-2） and myeloid cell leukemia 1 （Mcl-1） expressions were detected by Western blot analysis. Results：Absolute counts of pe- ripheral blood T lymphocyte subsets in survival group and death group were decreased as compared with those in control group：CD3＋ cell counts were respectively 374.0（274.5-514.0）, 154.0（87.5-256.2）, 1534.5 （1255.0-1756.7） ;CD3＋CD4＋ cell counts were respec- tively 198.0（148.5-299.0） ,68.0（37.7-108.5） ,766.5 （629.3-923.5） ;CD3＋CD8＋ cell counts were respectively 142.0（89.0-184.0） ,76.0 （48.7-134.5）, 600.5 （400.2-717.0）（P〈0.05）. Compared with those in survival group, C D3＋ and CD3＋CD4＋ cell counts decreased more obviously in death group （P=0.011, P=0.005）. CD3＋CD4＋ proportion was lower in death group （42.40 ± 5.71 ） % than in control group （53.46 ± 9.28）% ; CD3＋CD8＋ proportion was obviously higher in death group（54.40 ±4.76）% than in control group（39.33 ± 7.26）% ; CD4＋/CD8＋ ratio was significantly lower in death group （0.79 ± 0.17） than in control group （ 1.44 ± 0.51 ） （ P=-0.003, P=0.003, P=0.023）. CD3＋,CD3＋CD4＋,CD3＋CD8＋ and CD4＋/CD8＋ ratio exhibited a negative relationship with APACHE I1 score and predicted risk of death score（R）（P〈0.05）. CD3＋ T-lymphocyte apoptosis ratio in survival group（15.59 ±0.54）% and death group（39.57 ±4.49）% both increased as compared with that in control group （9.53 ± 1.91 ）% （P=0.040, P=0.000）. Apoptosis ratio of CD3＋ T-lymphocyte in death group increased more obviously than that in survival group（P=0.000）. Apoptosis ratio of Jurkat cell induced by serum from survival group（16.60 ± 3.55）% and death group （32.93 ± 5.89）% both increased obviously as compared with that in control group （8.56 ± 0.89）%（P=-0.049,P=0.000）. Protein expressions of Cleaved-caspase 3 in survival group（2.01 ± 0.21） and death group（2.68 ± 0.28） were obviously higher than those in control group（P=0.001 ,P=0.000）. Mcl-1 expressions in survival group（0.77 ± 0.03） and death group（0.61 ± 0.01） were lower than those in control group（P=0.000,P=0.000）. Bcl-2 expressions in survival group（0.68 ± 0.07） and death group（0.48 ± 0.03） were lower than those in control group （P=0.000,P=0.000）. Compared with those in survival group,the changes of apoptosis rate,Cleaved-caspase3,Mcl-1 and Bcl-2 in Jurkat cell were more obvious in death group（P=0.002, P=0.007,P=-0.000,P=0.001 ）. Conclusion：Declined T-lymphocyte cell counts and increased T lymphocyte apoptosis induce im- munosuppression in sepsis,which are related to the severity of illness and prognosis.