葡聚糖硫酸钠诱导的结肠炎小鼠中TRAIL-R基因敲除对Th17细胞凋亡的影响  

Effect of TRAIL-R deficiency on apoptosis of Th17 cells in experimental colitis mice induced by dextran sodium sulfate

作　　者：金颖莉 夏宣平[1] 曹曙光[1] 林道泼 胡定元[1] 夏盛隆[1] 蒋益[1] JIN Yingli;XIA Xuanping;CAO Shuguang;LIN Daopo;HU Dingyuan;XIA Shenglong;JIANG Yi(Department of Gastroenterology,the Second Affiliated Hospital&Yuying Children’s Hospital of Wenzhou Medical University,Wenzhou 325027,China)

机构地区：[1]温州医科大学附属第二医院育英儿童医院消化内科,浙江温州325027

出　　处：《温州医科大学学报》2020年第12期947-954,共8页Journal of Wenzhou Medical University

基　　金：浙江省自然科学基金资助项目(LY15H030018);浙江省中医药科学研究基金资助项目(2019ZB075);温州市科技局科研基金资助项目(Y20170062,Y20180637)。

摘　　要：目的:采用葡聚糖硫酸钠(DSS)构建实验性结肠炎小鼠模型,探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)的受体基因敲除(TRAIL-R-/-)对结肠炎症及Th17细胞凋亡的影响。方法:将C57BL/6品系小鼠分为4组:TRAIL-R-/-结肠炎组、TRAIL-R-/-对照组、野生型(WT)结肠炎组、WT对照组,每组各9只。结肠炎组饮用3.5%的DSS,对照组饮用清水。7 d后,从临床表现和组织病理学上评估小鼠结肠炎的严重程度。取外周血单个核细胞(PBMCs)和结肠黏膜固有层单个核细胞(LPMCs),采用流式细胞术检测Th17细胞占CD4+T细胞的比例,采用实时荧光定量聚合酶链反应(RT-q PCR)、Western blot和酶联免疫吸附测定(ELISA)检测视黄酸相关孤儿受体(ROR-γt)和IL-17A的m RNA和蛋白表达水平。采用TUNEL荧光染色技术及激光共聚焦显微镜观察结肠组织中Th17细胞的凋亡率。通过比色法检测结肠组织中凋亡蛋白酶Caspase 3、Caspase 8和Caspase 9的活性。结果:DSS诱导后,TRAIL-R-/-小鼠较WT小鼠发生了更为严重的结肠炎。TRAIL-R-/-结肠炎小鼠PBMCs中Th17细胞的比例(P<0.01),以及ROR-γt和IL-17A的m RNA和蛋白表达水平均显著高于WT结肠炎小鼠(均P<0.05)。TRAIL-R-/-结肠炎小鼠LPMCs中Th17细胞的比例(P<0.01),以及ROR-γt和IL-17A的m RNA和蛋白表达水平均显著高于WT结肠炎小鼠(均P<0.05)。与WT结肠炎小鼠相比,TRAIL-R-/-结肠炎小鼠的结肠组织中Th17细胞的凋亡率(P<0.01)以及Caspase 3和Caspase 8的酶活性均显著降低(均P<0.01)。结论:DSS诱导后,TRAIL-R-/-小鼠的结肠炎症较WT小鼠更为严重,原因可能是Th17细胞凋亡减少,其数目和活性增高所致。Objective:To investigate the effect of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)receptor gene knockout(TRAIL-R-/-)on the severity of colitis and the apoptosis of Th17 cells in mice induced by 3.5%dextran sodium sulfate(DSS).Methods:C57BL/6 mice were assigned into 4 groups:TRAIL-R-/-colitis group,TRAIL-R-/-control group,wild type(WT)colitis group and WT control group,with 9 mice in each group.Colitis groups were orally administrated by 3.5%DSS.Control groups were fed with the same volume of water.After 7 days,the severity of colitis in mice was evaluated clinically and histopathologically.In peripheral blood mononuclear cells(PBMCs)and colonic lamina propria mononuclear cells(LPMCs),the proportion of Th17 cells in CD4+T cells was detected by means of flow cytometry.The m RNA and protein expressions of retinoic acid-related orphan receptor-γt(ROR-γt)and IL-17A were measured using real-time quantitative polymerase chain reaction(RT-q PCR),western blot and enzyme linked immunosorbent assay(ELISA).The apoptosis rate of Th17 cells in colonic tissue was examined by TUNEL fluorescence staining technique and observed by laser confocal microscope.The activity of Caspase 3,Caspase 8,Caspase 9 in colonic tissue was assayed by colorimetric method.Results:After administration of 3.5%DSS,TRAIL-R-/-colitis mice exhibited more severe colitis than WT colitis group did.The proportion of Th17 cells in PBMCs of TRAIL-R-/-colitis group was shown to be significantly higher in TRAIL-R-/-colitis group than in WT colitis group(P<0.01).The same conclusions were also drawn for the m RNA and protein levels of ROR-γt and IL-17A in PBMCs when compared WT colitis group with TRAIL-R-/-colitis group(both P<0.05).In comparison with WT colitis group,the proportion of Th17 cells in colonic LPMCs of TRAIL-R-/-colitis group was significantly enhanced in TRAIL-R-/-colitis group(P<0.01).The m RNA and protein levels of ROR-γt and IL-17A were also found to be higher in TRAIL-R-/-colitis group than in WT colitis group(both P<0.05).

关 键 词：受体 TNF相关凋亡诱导配体 结肠炎 Th17细胞 细胞凋亡 

分 类 号：R574.621[医药卫生—消化系统]