Orai1蛋白介导钙池操纵的钙内流在脓毒症小鼠CD4^(+)T细胞免疫功能损害中的作用  

Role of Orai 1-mediated store-operated calcium entry in the immune function of CD4^(+)T cells in septic mice

作　　者：连洁[1] 陈晨松 方俊杰 陈隆望[1] 蔡文超 赵光举[1] 洪广亮[1] 卢中秋[1] Lian Jie;Chen Chensong;Fang Junjie;Chen Longwang;Cai Wenchao;Zhao Guangju;Hong Guangliang;Lu Zhongqiu(Emergency Department,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China;Xiangshan Hospital Affiliated to Wenzhou Medical University,Ningbo 315700,China)

机构地区：[1]温州医科大学附属第一医院急诊医学中心,温州325000 [2]温州医科大学附属象山医院,宁波315700

出　　处：《中华医学杂志》2021年第7期504-510,共7页National Medical Journal of China

基　　金：国家自然科学基金(81772112,81871583);浙江省“十三五”中医药重点专科(急诊科);温州市科技计划项目(Y20170185,Y20170177)。

摘　　要：目的探讨Orai1介导钙池操纵的钙内流(SOCE)在脓毒症小鼠CD4^(+)T细胞免疫功能损害中的作用。方法采用清洁级雄性Balb/c小鼠建立盲肠结扎穿孔(CLP)脓毒症模型,分别于术后1、3、5 d处死小鼠,无菌留取脾脏,免疫磁珠法分选CD4^(+)T细胞,Western印迹法检测各组脾脏CD4^(+)T细胞Orai1蛋白表达,流式细胞术检测SOCE,流式细胞术检测CD4^(+)T细胞凋亡,CCK-8细胞计数试剂盒检测CD4^(+)T细胞增殖,酶联免疫吸附法(ELISA)检测干扰素(IFN)-γ和白细胞介素(IL)-4水平。然后调节Orai1蛋白的表达,实验分为4组,sham组、CLP3组、Orai1下调组(Orai1-down组)、Orai1上调组(Orai1-up组),进一步检测小鼠脾脏CD4^(+)T细胞SOCE和免疫功能。结果sham组脾脏CD4^(+)T细胞Orai1蛋白的相对表达量为1.03±0.16,与sham组相比,CLP组的Orai1蛋白水平明显降低(F=19.64,P=0.0005)。与sham组相比,CLP组SOCE水平明显降低(F=30.01,P=0.001)。sham组的CD4^(+)T细胞早期和晚期凋亡比率为8.7%±1.5%,与sham组相比,CLP组明显升高(F=32.29,P=0.0001)。与sham组相比,CLP组小鼠脾脏CD4^(+)T细胞增殖能力明显降低,差异有统计学意义(F=7.26,P=0.0018)。与sham组相比,CLP组小鼠脾脏CD4^(+)T细胞分泌IFN-γ和IL-4的能力均明显降低,差异均有统计学意义(F=19.690、6.183,均P<0.05)。与sham组相比,CLP组IFN-γ/IL-4比值变小(F=11.23,P=0.0031)。与CLP3组相比,Orai1-down组CD4^(+)T细胞SOCE的水平明显下降,早期和晚期凋亡比率明显升高,脾脏CD4^(+)T细胞增殖能力显著下降,分泌IFN-γ和IL-4下降,IFN-γ/IL-4值下降,差异均有统计学意义(t=4.819、7.952、2.988、28.760、3.140、7.670,均P<0.05);而Orai1-up组则均获得相反的结果,差异亦均有统计学意义(t=2.983、6.796、9.390、19.670、3.692、15.870,均P<0.05)。结论Orai1介导SOCE减轻脓毒症小鼠CD4^(+)T细胞免疫功能障碍。Objective To investigate the role of Orai1-mediated store-operated calcium entry in the immune damage of CD4^(+)T cells in septic mice.Methods Sepsis mouse model was established by cecal ligation and puncture(CLP).Balb/c mice of clean grade were sacrificed 1,3,and 5 days after operation.Spleen samples were harvested at given intervals.Splenic CD4^(+)T cells were selected by immunomagnetic beads and the expression of Orai1 protein was detected by western blotting,the storage operated calcium entry(SOCE)was detected by flow cytometry,the apoptosis of CD4^(+)T cells was detected by flow cytometry,the proliferation of CD4^(+)T cells was detected by CCK-8,and the IFN-γ and IL-4 were detected by enzyme-linked immunosorbent assay(ELISA).Then the expression of Orai1 protein was regulated to further detect the SOCE and immune function of splenic CD4^(+)T cells in mice.The experiment was divided into 4 groups,sham group,CLP3 group,Orai1 down group(Orai1-down group)and Orai1 up regulation group(Orai1-up group).Results The relative expression of Orai1 protein in splenic CD4^(+)T cells in sham group was 1.03±0.16.Compared with sham group,Orai1 protein levels in CLP Group were all significantly lower(F=19.64,P=0.0005).The increased value of splenic CD4^(+)T cells fluorescence intensity in sham group was 494±41.Compared with sham group,the levels of SOCE in CLP Group were all lower(F=30.01,P=0.001).The ratio of early and late apoptosis of CD4^(+)T cells in sham group was 8.7%±1.5%.Compared with sham group,the early and late apoptosis rates of CLP Group were significantly higher(F=32.29,P=0.0001).The OD of sham group was 0.81±0.10 at 450 nm.Compared with sham group,the proliferation ability of splenic CD4^(+)T cells in CLP Group were significantly decreased(F=7.26,P=0.0018).Compared with sham group,the secretion of IFN-γ and IL-4 by CD4^(+)T cells and the ratio of IFN-γ/IL-4 in CLP Group were all significantly decreased(F=19.690,6.183,11.230,all P<0.05).Compared with CLP3 group,the increased value of fluorescence intensit

关 键 词：脓毒症 Orai1 钙池操纵的钙内流 淋巴细胞 免疫功能 

分 类 号：R459.7[医药卫生—急诊医学]