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10s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Th 1/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
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
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2016年第2期89-96,共8页
Chinese Journal of Burns
基金
国家自然科学基金(81272091)National Natural Science Foundation of China
