心脏骤停(CA)的SD大鼠复苏后CD4^+T细胞功能的改变

Changes of CD4^+T cell functions after restoration of spontaneous circulation from cardiac arrest (CA) in SD rats

目的观察心脏骤停大鼠心肺复苏后CD4^+ T细胞功能的改变。方法将SD大鼠随机分为心脏骤停(cardiac arrest,CA)组和假手术(sham-operated,SO)组,每组24只,每组按3、24、72 h 时间点各分为3个亚组,每组8只。CA组建立窒息6 min致心脏骤停模型;SO组为假手术对照组。并分别于建模或操作后相应时间点行心脏采血。运用流式细胞仪检测T细胞及CD4^+、CD8^+亚群比例,CD4^+ T细胞表面分子CD28、细胞毒性T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)和程序性死亡受体1(programmed cell death-1,PD-1)的表达;采用酶联免疫吸附法检测血清中肿瘤坏死因子α(tumor necrosis factor,TNF-α)、白介素6(interleukin 6,IL-6)、IL-10、干扰素γ(interferon γ,IFN-γ)、IL-4的浓度。结果 CA组IL-4浓度在3个时间点均显著高于SO组( P <0.05),IL-6、IL-10在3 h、24 h显著高于SO组( P <0.05),IFN-γ在24 h、72 h较SO组显著降低( P <0.05)。CA组CD4^+ T细胞表面CD28、PD-1和CTLA-4的表达在24 h时显著高于SO组( P < 0.05 )。结论心脏骤停最初3 h、24 h抗炎反应和促炎反应同时存在,72 h后抗炎反应为主;CD4^+ T细胞在心脏骤停24 h时共抑制因子表达升高,处于受抑制调控状态。

Objective To investigate the changes of CD4^+ T cell functions in return of spontaneous circulation (ROSC) from asphyxial cardiac arrest rat model. Methods Sprague-Dawley rats were randomly divided into cardiac arrest (CA) group and sham-operated (SO) group (24 rats for each group).CA group were induced cardiac arrest by 6 min of asphyxia,while SO group were just done the same procedures with that of CA group only without asphyxia.The 24 surviving rats in CA group after successful ROSC were randomly assigned to three subgroups (eight rats per subgroup) at 3 h,24 h and 72 h.Likewise,OA group was divided into three subgroups too.The proportion of T cells (CD4^+,CD8^+ subsets) and the expression of surface molecules (CTLA-4,PD-1,CD28) on T cells were identified by flow cytometry.The protein concentrations of cytokines (TNF-α,IL-6,IL-10,IL-4,IFN-γ) in serum were measured by enzyme-linked immunosorbent assay (ELISA). Results It was observed that IL-4 was significantly increased in the CA subgroups compared with the SO subgroups at three different post-ROSC times ( P <0.05).IL-6 and IL-10 at 3 h and 24 h were significantly higher than those in SO group ( P <0.05).While the concentrations of IFN-γ were significantly decreased in the CA groups compared with the SO groups at 24 h and 72 h post-ROSC ( P <0.05).Besides,the expression levels of CD28,PD-1,and CTLA-4 on CD4 ^+ T cells at 24 h were markedly increased in CA group at 24 h post-ROSC,significantly higher than those in SO group ( P <0.05). Conclusions At the early stage of CA (3 h and 24 h after ROSC),proinflammatory and anti-inflammatory exists in the same time.However,at the late stage (72 h after ROSC),anti-inflammatory greatly dominate proinflammatory in the immunity action.The co-inhibitory molecule on CD4 ^+ T cells increases at 24 h after ROSC,which was at the inhibitory stage.