肝移植术后合并脓毒症患者外周血调节性T细胞比例及功能变化的分析

Analysis on percentage and function changes of peripheral regulatory T cell in patients with sepsis after liver transplantation

目的探讨肝移植患者在脓毒症不同阶段外周血调节性T细胞(Treg)比例和功能的变化。方法选取自2009年1月至2010年12月期间在中山大学附属第三医院行外科手术,术后合并脓毒症的47例患者作为研究对象,根据手术方式和美国胸科和危重症医师协会制订的脓毒症诊断和分期标准指南分为4组:肝移植脓毒症组(sepsis after liver transplantation group,TS组;11例)、肝移植严重脓毒症组(severe sepsis after liver transplantation group,TSS组;10例)、非肝移植脓毒症组(sepsis without liver transplantation group,NTS组;15例)、非肝移植严重脓毒症组(severe sepsis without liver transplantation group,NTSS组;11例),另外选取20名健康正常人作为健康对照组。4组脓毒症患者通过急性生理和慢性健康评估(acute physiology and chronic health evaluation,APACHE)Ⅱ和感染相关的序贯器官衰竭评估(sequential organ failure assessment,SOFA)来评价与比较脓毒症严重程度。分别采集各组研究对象的外周血,采用流式细胞术检测CD4+CD25+Foxp3+调节性T细胞比例(Treg%),采用荧光定量逆转录聚合酶链反应检测Foxp3信使核糖核酸(messenger RNA,mRNA)。结果 TSS和NTSS组的APACHEⅡ评分、SOFA评分均高于TS组和NTS组(均为P<0.01),且TS组的APACHEⅡ评分高于NTS组,TSS组的APACHEⅡ评分和SOFA评分均高于NTSS组(均为P<0.01)。与健康对照组比较,NTS组Treg%明显降低(P<0.001),NTSS组明显升高(P=0.003);而TS组与健康对照组相比差异无统计学意义(P=0.398),TSS组也高于健康对照组(P=0.006),但变化幅度不如NTSS组显著。4组脓毒症组的组间比较发现,NTSS组患者Treg%显著高于NTS组(P<0.01),而TSS组与TS组两组比较差异无统计学意义(P=0.099),NTS组患者Treg%低于TS组(P=0.05),而NTSS组则显著高于TSS组(P=0.002)。与健康对照组比较,NTSS组的Foxp3 mRNA表达显著升高,差异有统计学意义(P<0.05)。4组脓毒症组的组间比较发现,TSS组和NTSS组Foxp3 mRNA表达值均高于TS组和TSS组,但移植组内(TS组与TSS组)的差异没有非移植组(NTS组与NTSS组)显著(分别为P=0.038、P<0.001);另外NTSS组的Foxp3 mRNA表达显著高于TSS组(P=0.012)。结论免疫抑制剂的应用使移植患者在发生脓毒症时Treg的比例和功能的变化有别于普通人群,评估机体免疫状态时需要综合多个免疫指标。

Objective To investigate the change of percentage and function of peripheral regulatory T cell （Treg） in patients at different stage of sepsis after liver transplantation. Methods Forty-seven patients complicated with sepsis after abdominal surgery in the Third Affiliated Hospital of Sun Yat-sen University from January 2009 to December 2010 were selected as study objects. According to type of surgery and definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis recommended by American College of Chest Physicians/Society of Critical Care Medicine, 47 patients were divided into 4 groups ： sepsis after liver transplantation （TS, n = 11 ）, severe sepsis after liver transplantation （TSS, n = 10）, sepsis without liver transplantation （NTS, n = 15） and severe sepsis without liver transplantation （ NTSS, n = 11 ）. And 20 healthy volunteers were selected as healthy control group. The severity of sepsis was evaluated and compared by acute physiology and chronic health evaluation （APACHE） II and sequential organ failure assessment （SOFA） in 4 groups. Peripheral blood samples were collected from patients. The percentage of CD4 ＋ CD25 ＋ Foxp3 ＋ Treg was measured by flow cytometry. And the expression of Foxp3 messenger ribonucleic acid （mRNA） was measured by reverse transcription polymerase chain reaction （RT-PCR）. Results The scores of APACHE 11 and SOFA of TSS and NTSS group were significantly higher than those of TS and NTS group （ all in P 〈 0. 01 ）. The scores of APACHE II of TS group were higher than those of NTS group and The scores of APACHE 11 and SOFA of TSS group were higher than those of NTSS group （all in P 〈 0. 01 ）. Compared with healthy control group, Treg% was significantly lower in NTS group （ P 〈 0. 001 ） and higher in NTSS group （P = 0. 003）. There was no significant difference of Treg% between TS group and healthy control group （ P = 0. 398）. The Treg% in TSS group was significantly higher than healthy control group （P = 0. 006）, but the change was not as significant as that in NTSS group. The result of comparison among 4 sepsis groups showed that Treg% in NTSS group was significantly higher than that in NTS group （P 〈 0. 01 ） and there was no significant difference between TSS group and TS group （ P = 0. 099）. The Treg% in NTS group was lower than that in TS group （P = 0. 05）, but Treg% in NTSS group was significantly higher than that in TSS group （P = 0. 002）. Compared with healthy group, the expression of Foxp3 mRNA in NTSS group was significantly higher （P 〈 0. 05）. The result of comparison among 4 sepsis groups showed that the expression of Foxp3 mRNA in TSS and NTSS group was significantly higher than that in TS and TSS group, but the difference between transplant groups （TS and TSS group） was not as significant as that between non-transplant groups （NTS and NTSS group） （P = 0. 038, P 〈0. 001, respectively）. And the expression of Foxp3 mRNA in NTSS group was significantly higher than that in TSS group （P = 0. 012）. Conclusions The percentage and function of Treg in liver transplant patients are different from those in non-transplant ones for the usage of immunosuppressant. Multiple immune variables should be taken to evaluate immune status.