脾脏源性髓源性抑制细胞在脓毒症小鼠肾上腺损伤中的作用及机制  

Role and mechanism of splenic myeloid-derived suppressor cells in sepsis-induced adrenal injury in mice

作　　者：徐前程 王涛 曹迎亚 祁羽鹏 曹玉涵 傅聪 陶秀彬 于涛 鲁卫华 姜小敢 Xu Qiancheng;Wang Tao;Cao Yingya;Qi Yupeng;Cao Yuhan;Fu Cong;Tao Xiubin;Yu Tao;Lu Weihua;Jiang Xiaogan(Department of Critical Care Medicine,the First Affiliated Hospital of Wannan Medical College,Yijishan Hospital,Wuhu 241000,Anhui,China;Department of Nephrology,the First Affiliated Hospital of Wannan Medical College,Yijishan Hospital,Wuhu 241000,Anhui,China;Department of Cardiovascular,the First Affiliated Hospital of Wannan Medical College,Yijishan Hospital,Wuhu 241000,Anhui,China;Department of Neurosurgery Intensive Care Unit,the First Affiliated Hospital of Wannan Medical College,Yijishan Hospital,Wuhu 241000,Anhui,China)

机构地区：[1]皖南医学院第一附属医院(弋矶山医院)重症医学科,安徽芜湖241000 [2]皖南医学院第一附属医院(弋矶山医院)肾脏内科,安徽芜湖241000 [3]皖南医学院第一附属医院(弋矶山医院)心血管内科,安徽芜湖241000 [4]皖南医学院第一附属医院(弋矶山医院)神经外科ICU,安徽芜湖241000

出　　处：《中华危重病急救医学》2020年第1期33-38,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金青年基金(81700265,81702092);安徽省重点研究和开发计划项目(201904a07020034);安徽省高校自然科学研究重点项目(KJ2016A719);安徽省自然科学基金青年基金(1908085QH360)。

摘　　要：目的探讨脾脏源性髓源性抑制细胞(MDSCs)在脓毒症诱导肾上腺损伤(SAI)中的作用及其机制。方法采用随机数字表法将30只6~8周龄C57雄性小鼠分为正常对照组(n=5)、假手术组(Sham组,n=5)、脓毒症模型组〔盲肠结扎穿孔术(CLP)组,n=10〕和脓毒症+脾切除组(CLPS组,n=10)。采用CLP法制备小鼠脓毒症模型;Sham组仅开腹分离盲肠后关腹,不给予盲肠结扎穿孔;CLPS组于CLP前切除脾脏;正常对照组不给予任何处理。各组于制模后24 h处死小鼠,收集外周血、脾脏、骨髓及双侧肾上腺;采用苏木素-伊红(HE)染色后光镜下观察肾上腺组织病理学改变,采用流式细胞仪测定外周血、脾脏和骨髓中MDSCs细胞比例,采用实时定量反转录-聚合酶链反应(PCR)检测肾上腺组织中MDSCs表面抗原CD11b、Gr-1和白细胞介素(IL-6、IL-1β)的mRNA表达,采用蛋白质免疫印迹试验(Western Blot)检测肾上腺组织中哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白总mTOR(T-mTOR)、磷酸化mTOR(p-mTOR)和天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)的蛋白表达。结果光镜下显示,正常对照组和Sham组肾上腺皮质、髓质完整,结构清晰;CLP组肾上腺皮质肿胀、出血,可见细胞水肿;CLPS组上述肾上腺组织损伤较CLP组明显减轻。与正常对照组和Sham组相比,CLP组外周血中MDSCs细胞比例明显增加,脾脏中MDSCs细胞比例明显降低,骨髓中MDSCs细胞比例差异无统计学意义,肾上腺组织中CD11b、Gr-1、IL-6、IL-1β的mRNA和caspase-3蛋白表达均明显增加,p-mTOR蛋白表达明显降低,T-mTOR蛋白表达差异无统计学意义;与CLP组相比,CLPS组外周血中MDSCs细胞比例明显降低(0.143±0.011比0.324±0.023,P<0.01),肾上腺组织中CD11b、Gr-1、IL-6和IL-1β的mRNA以及caspase-3的蛋白表达均明显降低〔CD11b mRNA(2^-ΔΔCt):2.90±0.56比5.74±0.13,Gr-1 mRNA(2^-ΔΔCt):2.71±0.14比4.59±0.46,IL-6 mRNA(2^-ΔΔCt):2.44±0.64比5.17±1.04,IL-1βmRNA(2^-ΔΔCObjective To investigate the role and mechanism of splenic myeloid-derived suppressor cells(MDSCs)in sepsis-induced adrenal injury(SAI).Methods Thirty male C57 mice aged 6-8 weeks were randomly divided into normal control group(n=5),sham operation group(Sham group,n=5),sepsis model group[cecal ligation and perforation(CLP)group,n=10]and sepsis+splenectomy group(CLPS group,n=10).The sepsis model of mice was reproduced by CLP method.In Sham group,only the cecum was opened and separated,then closed,without CLP.In CLPS group,the spleen was removed before CLP.In normal control group,no challenge was given.After 24 hours,the rats were sacrificed by anesthesia,and peripheral blood,spleen,bone marrow,and bilateral adrenal glands were harvested.The pathological of adrenal gland was assessed by hematoxylin-eosin(HE)staining under optical microscope.The ratio of MDSCs in peripheral blood,spleen and bone marrow was determined by flow cytometry.The expressions of MDSCs surface antigen CD11b,Gr-1 and interleukins(IL-6,IL-1β)mRNA in adrenal tissue were measured by real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR).Western Blot was used to detect the expressions of mammalian rapamycin target protein(mTOR)pathway related proteins including total mTOR(T-mTOR),phosphorylation of mTOR(p-mTOR)and caspase-3.Results The adrenal cortex and medulla of the normal control group and Sham group were intact and the structure was clear under optical microscope,while in the CLP group,the adrenal gland showed edema,cortical hemorrhage and cell edema.Compared with the CLP group,the adrenal tissue injury was significantly reduced in the CLPS group.Compared with the normal control group and Sham group,MDSCs ratio in the peripheral blood was significantly increased and significantly reduced in the spleen in the CLP group,but there was no significant difference in bone marrow,the expression levels of CD11b,Gr-1,IL-6,IL-1βmRNA and caspase-3 protein were increased significantly and p-mTOR protein expression was significant

关 键 词：脓毒症 肾上腺损伤 髓源性抑制细胞 炎症 哺乳动物雷帕霉素靶蛋白 

分 类 号：R459.7[医药卫生—急诊医学]