Changes in the expression of three markers in T lymphocytes of peripheral blood and immunoregulatory mechanisms of burned mice with sepsis at early stage
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摘要: 目的 观察烧伤脓毒症小鼠伤后早期外周血T淋巴细胞中膜联蛋白A1(ANXA1)、GATA-3、T-bet的表达情况,分析3种标志物的免疫调控机制。 方法 将780只清洁级雄性小鼠按随机数字表法分为假伤组60只、烧伤组240只、脓毒症组240只及烧伤脓毒症组240只。假伤组小鼠背部于37 ℃温水中浸浴10 s模拟致伤;烧伤组小鼠背部于100 ℃沸水中浸浴10 s造成20%TBSA深Ⅱ度烧伤;脓毒症组小鼠腹腔注射LPS(6 mg/kg);烧伤脓毒症组小鼠同烧伤组处理后,立即同脓毒症组腹腔注射等剂量LPS。(1)伤后即刻,假伤组按照随机数字表法选取6只小鼠,每只小鼠收集1管外周血淋巴细胞悬液。其余3组按照随机数字表法,于伤后12、24、48及72 h各选取6只小鼠,每只小鼠收集1管外周血淋巴细胞悬液。每管细胞悬液等分为2份,一份加入异硫氰酸荧光素(FITC)标记的人抗小鼠CD4单克隆抗体、藻红蛋白(PE)标记的人抗小鼠γ干扰素单克隆抗体,以标记Th1;另一份加入FITC标记的人抗小鼠CD4单克隆抗体、PE标记的人抗小鼠IL-4单克隆抗体,以标记Th2。采用流式细胞仪检测Th1、Th2百分比,并计算Th1/Th2比值。(2)伤后即刻,假伤组按照随机数字表法取18只小鼠,每只小鼠收集1管外周血淋巴细胞悬液。其余3组按照随机数字表法,于伤后12、24、48及72 h各选取18只小鼠,每只小鼠收集1管外周血淋巴细胞悬液。采用实时荧光定量RT-PCR法检测淋巴细胞中ANXA1、GATA-3、T-bet mRNA的表达量。(3)伤后即刻,假伤组按照随机数字表法取36只小鼠,每只小鼠收集1管外周血淋巴细胞悬液,分成6次进行实验。一次取6管细胞悬液分别加入6种抗体组合:标记Th1和Th2的抗体分别与PE-花青素7标记的人抗小鼠ANXA1单克隆抗体、PE-花青素7标记的人抗小鼠GATA-3单克隆抗体、PE-花青素7标记的人抗小鼠T-bet单克隆抗体联用,以分别标记Th1、Th2的3种标志物。其余3组按照随机数字表法,于伤后12、24、48及72 h各取36只小鼠,每只小鼠收集1管外周血淋巴细胞悬液,各时相点分成6次进行实验。一次取6管细胞悬液分别加入上述6种抗体组合进行标记。采用流式细胞仪检测Th1、Th2中ANXA1、GATA-3、T-bet 3种标志物的阳性细胞百分比。对数据行单因素方差分析、析因设计方差分析、SNK检验。对淋巴细胞中的ANXA1 mRNA与GATA-3 mRNA表达量及Th1、Th2中ANXA1阳性细胞百分比与GATA-3阳性细胞百分比均行直线相关分析。 结果 (1)与假伤组伤后即刻比较,烧伤组小鼠淋巴细胞中Th1、Th2百分比及Th1/Th2比值从伤后24 h起明显降低(
P 值均小于0.05),脓毒症组及烧伤脓毒症组小鼠淋巴细胞中Th1、Th2百分比及Th1/Th2比值从伤后12 h起明显降低(P 值均小于0.05)。(2)与假伤组伤后即刻比较,烧伤组小鼠淋巴细胞中ANXA1和GATA-3 mRNA表达量从伤后24 h起明显下降(P 值均小于0.05);T-bet mRNA表达量于伤后24 h明显下降,但伤后48、72 h明显升高,P 值均小于0.05。与假伤组伤后即刻比较,脓毒症组小鼠淋巴细胞中ANXA1和GATA-3 mRNA表达量从伤后12 h起明显降低,T-bet mRNA表达量从伤后12 h起明显增加,P 值均小于0.05;烧伤脓毒症组小鼠淋巴细胞中ANXA1、GATA-3、T-bet mRNA表达量从伤后12 h起明显降低(P 值均小于0.05),于伤后72 h达最低值(分别为0.50±0.04、0.45±0.03、0.21±0.05)。(3)淋巴细胞中ANXA1 mRNA表达量与GATA-3 mRNA表达量呈明显正相关(r =0.862,P <0.05)。(4)与假伤组伤后即刻比较,烧伤组小鼠Th1、Th2中ANXA1、GATA-3阳性细胞百分比从伤后24 h起均明显降低,T-bet阳性细胞百分比于伤后24 h明显降低,但从伤后48 h起明显升高,P 值均小于0.05。脓毒症组小鼠Th1和Th2中ANXA1、GATA-3阳性细胞百分比从伤后12 h起持续下调,大部分时相点显著低于假伤组伤后即刻(P 值均小于0.05);T-bet阳性细胞百分比从伤后12 h起明显高于假伤组伤后即刻(P 值均小于0.05)。烧伤脓毒症组小鼠Th1和Th2中ANXA1、GATA-3、T-bet 3种标志物的阳性细胞百分比从伤后12 h起持续下调,大部分时相点显著低于假伤组伤后即刻(P 值均小于0.05)。(5)在Th1、Th2中,GATA-3阳性细胞百分比与ANXA1阳性细胞百分比均呈明显正相关(r 值分别为0.747、0.787,P 值均小于0.05)。 结论 在烧伤脓毒症小鼠中,ANXA1、GATA-3及T-bet表达持续下调,在Th1/Th2平衡向Th2方向漂移及免疫抑制过程中发挥重要作用。Abstract: Objective To study the expression levels of annexin A1 (ANXA1), GATA-3, and T-bet in T lymphocytes of peripheral blood in burned mice with sepsis at early stage, and to analyze their immune regulatory mechanisms. Methods Seven-hundred and eighty male mice of clean grade were divided into sham injury group (n =60, sham injured on the back by immersing in 37 ℃ warm water for 10 s), burn group (n =240, inflicted with 20% TBSA deep partial- thickness burn on the back by immersing in 100 ℃ hot water for 10 s), sepsis group (n =240, intraperitoneally injected with 6 mg/kg lipopolysaccharide), and burn+ sepsis group (n =240) according to the random number table. Mice of burn+ sepsis group were treated as that in burn group at first, and then they were treated as that in sepsis group. (1) Immediately after injury, six mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table. According to the random number table, 6 mice of each of the other three groups were respectively selected at post injury hour (PIH) 12, 24, 48, and 72 for the collection of lymphocyte suspension from peripheral blood (1 tube each mouse). Each tube of cell suspension was equally divided into two parts. Fluorescein isothiocyanate (FITC)-labeled human anti-mouse CD4 monoclonal antibody and phycoerythrin (PE)-labeled human anti-mouse interferon-γ monoclonal antibody were added to one part of cell suspension to mark helper T lymphocyte 1 (Th1). FITC-labeled human anti-mouse CD4 monoclonal antibody and PE-labeled human anti-mouse interleukin-4 (IL-4) monoclonal antibody were added to the other part of cell suspension to mark Th2. The percentages of Th1 and Th2 were determined with flow cytometer, and the ratio of Th1 to Th2 was calculated. (2) According to the random number table, 18 mice in sham injury group were selected immediately after injury for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and 18 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 to collect the lymphocyte suspension of peripheral blood (1 tube each mouse). The mRNA expression levels of ANXA1, GATA-3, and T-bet were determined by real-time fluorescent quantitative reverse transcription-PCR. (3) Immediately after injury, 36 mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table, and then 36 tubes of cell suspension were divided into 6 batches (6 tubes each batch). Each one of 6 kinds of antibody combinations: antibodies for labeling Th1 and Th2 in combination with PE-anthocyanin 7 labeled human anti-mouse ANXA1 monoclonal antibody, PE-anthocyanin 7 labeled human anti-mouse GATA-3 monoclonal antibody, and PE-anthocyanin 7 labeled human anti-mouse T-bet monoclonal antibody was added to 1 tube of cell suspension at each batch. According to the random number table, 36 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and then 36 tubes of cell suspension at each time point were divided into 6 batches for marking with 3 kinds of surface markers of Th1 and Th2 (6 tubes each batch). Each one of above-mentioned 6 kinds of antibodies was added to 1 tube of cell suspension at each time point for each batch. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 were determined with flow cytometer. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and SNK test. The relationship between the percentages of ANXA1 positive cell and the percentages of GATA-3 positive cell in Th1 and Th2, and mRNA expression level of ANXA1 and mRNA expression level of GATA-3 in lymphocytes were assessed by linear correlation analysis. Results (1) Compared with those in sham injury group immediately after injury, the percentages of Th1 and Th2 and the ratio of Th1 to Th2 of mice in burn group were significantly decreased from PIH 24 on, withP values below 0.05; the percentages of Th1 and Th2 and the ratios of Th1 to Th2 of mice in sepsis group and burn+ sepsis group were significantly decreased from PIH 12 on, withP values below 0.05. (2) Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocyte of mice in burn group were significantly decreased from PIH 24 on, withP values below 0.05; the mRNA expression level of T-bet was significantly decreased at PIH 24 but significantly increased at PIH 48 and 72, withP values below 0.05. Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocytes of mice in sepsis group were significantly decreased from PIH 12 on, and the mRNA expression level of T-bet was increased significantly from PIH 12 on, withP values below 0.05; the mRNA expression levels of ANXA1, GATA-3, and T-bet in lymphocytes of mice in burn+ sepsis group were significantly decreased from PIH 12 on, withP values below 0.05, reaching the nadir at PIH 72 (0.50±0.04, 0.45±0.03, 0.21±0.05, respectively). (3) A significant positive correlation was observed between ANXA1 mRNA expression level and GATA-3 mRNA expression level in lymphocytes of peripheral blood (r =0.862,P <0.05). (4) Compared with those in sham injury group immediately after injury, the percentages of ANXA1 and GATA-3 positive cellsin Th1 and Th2 of mice in burn group were significantly lowered from PIH 24 on, and the percentage of T-bet positive cells was significantly decreased at PIH 24, but it was increased from PIH 48 on, withP values below 0.05. The percentages of ANXA1 and GATA-3 positive cells in Th1 and Th2 of mice in sepsis group were continuously decreased from PIH 12 on, which were lower at most time points than those in sham injury group immediately after injury, withP values below 0.05. The percentages of T-bet positive cells in Th1 and Th2 of mice in sepsis group were significantly increased since PIH 12 as compared with those in sham injury group immediately after injury, withP values below 0.05. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 of mice in burn+ sepsis group were continuously lowered from PIH 12, with significantly statistical differences at most time points as compared with those in sham injury group immediately after injury, withP values below 0.05. (5) The percentages of GATA-3 positive cells in Th1 and Th2 were significantly positively correlated with those of ANXA1 (withr values respectively 0.747 and 0.787,P values below 0.05). Conclusions The expression levels of ANXA1, GATA-3, and T-bet were continuously lowered in burned mice with sepsis, and it may play an important role in Th1/Th2 balance switching to Th2 bias and immunosuppressive process.-
Key words:
- Burns /
- Sepsis /
- Lymphocytes /
- Annexin A1 /
- GATA3 transcription factor /
- T-bet
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