脓毒症小鼠脾脏树突状细胞焦亡及其对炎症反应和免疫功能的影响  

Pyroptosis of splenic dendritic cells in septic mice and its effect on inflammatory response and immune function

作　　者：贺鹏翼 董宁[2,3] 吴瑶 郝济伟 祝筱梅[2,3] 姚咏明[2,3] He Peng-Yi;Dong Ning;Wu Yao;Hao Ji-Wei;Zhu Xiao-Mei;Yao Yong-Ming(Medical College of PLA,Beijing 100853,China;Medical Innovation Research Division of Chinese PLA General Hospital,Beijing 100853,China;Fourth Medical Center of Chinese PLA General Hospital,Beijing 100048,China)

机构地区：[1]解放军医学院,北京100853 [2]解放军总医院医学创新研究部,北京100853 [3]解放军总医院第四医学中心,北京100048

出　　处：《解放军医学杂志》2023年第5期537-544,共8页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81871557,82130062,82241062)。

摘　　要：目的 初步观察脓毒症小鼠脾脏树突状细胞(DC)焦亡情况及其与炎性因子水平、DC免疫功能的相关性。方法 70只BALB/c小鼠随机分为假手术组(n=20)、脓毒症模型组(CLP组,n=30)与胱天蛋白酶(CASP)-1抑制剂干预组(CLP+YVAD组,n=20),CLP组和CLP+YVAD组按术后不同时间点分为CLP 12 h组、CLP 24 h组、CLP 48 h组、CLP72 h组及CLP+YVAD24 h组、CLP+YVAD72 h组。所有小鼠于术后预定时间眼眶取血、处死并取脾脏组织。流式细胞仪测定脾脏DC焦亡率及表面标志物(CD80、CD86、MHC-Ⅱ)表达水平,激光共聚焦显微镜观察CASP-1在小鼠脾脏DC中的活化情况,Western blotting检测DC中CASP-1的表达情况,ELISA法检测血清中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-12、IL-1β、IL-6浓度;观察CLP术后小鼠死亡情况。提取BALB/c小鼠T细胞与各组小鼠脾脏DC共培养,采用流式细胞仪测定T细胞增殖率,ELISA法检测共培养上清中γ干扰素(IFN-γ)、IL-4、IL-10浓度。结果 与假手术组比较,DC焦亡率在CLP术后12 h升高(P<0.05),于24 h达高峰(P<0.01),随后呈下降趋势,至72 h仍高于假手术组(P<0.01)。激光共聚焦显微镜观察显示,CLP术后小鼠脾脏DC CASP-1活化明显。Western blotting检测结果显示,DC中焦亡蛋白CASP-1表达在脓毒症早期(12 h和24 h)较假手术组明显上调(P<0.01)。而给予CASP-1特异性抑制剂Ac-YVAD-cmk后,CLP+YVAD 24 h组DC焦亡率较CLP 24 h组下降(P<0.01),CLP+YVAD 72 h组DC表面标志物CD80、MHC-Ⅱ表达水平较CLP 72 h组上调(P<0.01),术后7 d小鼠生存率提高(P<0.01)。ELISA法检测结果显示,CLP小鼠血清TNF-α、IL-12、IL-1β和IL-6浓度在脓毒症早期(24 h)和后期(72 h)均较假手术组明显升高(P<0.01),而CLP+YVAD组血清内上述因子浓度明显下降(P<0.01)。共培养实验结果显示,与DC-CLP 24 h组比较,DC-CLP+YVAD 24 h组T细胞增殖率明显增高(P<0.01),共培养上清中IFN-γ水平下降,而IL-4、IL-10水平上升(P<0.01)。结论 DC焦亡在�Objective To preliminarily observe the pyroptosis of splenic dendritic cells(DC)in septic mice and its correlation with the levels of inflammatory factors and DC immune function.Methods Seventy BALB/c mice were randomly divided into sham group(n=20),sepsis model group(CLP group,n=30)and caspase(CASP)-1 inhibit group(CLP+YVAD group,n=20).The CLP group and CLP+YVAD group were then divided into CLP 12 h group,CLP 24 h group,CLP 48 h group and CLP 72 h group,and CLP+YVAD 24 h group and CLP+YVAD 72 h group at different time points after operation.Orbital blood was collected from all mice at a predetermined time after operation,and the mice were sacrificed and spleen tissues were extracted.The pyroptosis rate of DC and the expression levels of DC surface markers(CD80,CD86,MHC-Ⅱ)were determined by flow cytometry.Activation of CASP-1 in mouse spleen DC was observed using confocal laser microscope.Western blotting was used to determine the expression of CASP-1 in DC.ELISA was performed to measure the levels of tumor necrosis factor(TNF)-α,interleukin(IL)-12,IL-1β,and IL-6 in serum.Observe the death of mice after CLP operation.BALB/c mouse T cells were extracted and co-cultured with the mouse spleen DC of each group,the T cell proliferation rate was measured by flow cytometry,and the concentrations of interferon(IFN)-γ,IL-4 and IL-10 in the co-cultured supernatants were detected by ELISA.Results The pyroptosis rate of DC was increased 12 h after CLP compared to sham group(P<0.05),peaked at 24 h(P<0.01),and then progressively declined,but remained higher than sham group at 72 h after CLP(P<0.01).Confocal laser microscopy revealed that the activation of DC CASP-1 in spleen of mice after CLP was obvious.Western blotting showed the expression of CASP-1 in the early stage of sepsis(12 h and 24 h)was significantly higher than that in sham group(P<0.01).After administration of CASP-1 specific inhibitor Ac-YVAD-cmk,the DC pyroptosis rate at CLP+YVAD 24 h group was lower than that in CLP 24 h group(P<0.01).The expression level

关 键 词：脓毒症 炎症反应 免疫抑制 树突状细胞 焦亡 

分 类 号：R392[医药卫生—免疫学]