Tim-3和Th17细胞在免疫性肝损伤中作用及相关机制的研究

发布时间：2018-10-29 20:00

【摘要】：目的用刀豆蛋白A(Con A)建立免疫性肝损伤模型,探讨Tim-3和Th17细胞相关因子在发病过程中的动态变化及意义;靶向干预Tim-3通路后探讨其对Th17细胞的影响。方法60只Wistar雄性大鼠随机分为两组,即模型组(50只)和对照组(10只)。模型组大鼠尾静脉注射12.5mg/kg Con A,每周1次连续8周。对照组大鼠尾静脉注射无菌PBS,每周1次连续8周。随机从模型组中取10只大鼠,在第0周(0w)、第2周(2w)、第4周(4w)、第6周(6w)、第8周(8w)时对大鼠实施体外心尖取血1.5 ml,分离血清后于-20℃备用。8周后随机从模型组中取出20只大鼠,酒精浸泡20分钟后无菌取脾制备脾淋巴细胞。在脾淋巴细胞中加入Tim-3的单克隆抗体来阻断Tim-3通路;用Tim-3的重组配体来激活Tim-3通路,37℃、5%CO2培养箱中培养72h,收集细胞上清液于-20℃保存。生化分析法测血清中ALT、AST和ALB的表达;HE染色观察肝脏组织的病理变化;ELISA法测血清中Tim-3、IL-17A、IL-6、TGF-β和IL-23的表达及上清液中IL-17A和IL-6的表达;免疫组化法测肝脏组织中Tim-3、IL-17A和ROR-γt蛋白的表达;实时定量PCR测各组脾淋巴细胞ROR-γt m RNA的表达。结果与对照组相比,模型组大鼠的ALT及AST水平均显著升高(P0.05),ALB水平显著下降(P0.05)。在模型组的不同阶段中,HE染色检测肝组织可见4w时开始偶见假小叶,6w时量明显增多,而8w时发现有明显的炎性细胞和肝脏损伤,镜下假小叶较多见;ELISA法检测相应的细胞因子可见IL-17A、IL-6、IL-23的动态趋势是先升高,4w后开始降低(P0.05);Tim-3的动态趋势则是先降低,4w以后开始升高(P0.05);免疫组化检测相应的蛋白可见4w时IL-17A和ROR-γt蛋白的水平明显升高,而Tim-3蛋白的水平明显降低。靶向干预后,与阻断对照组相比,阻断实验组中IL-17A和IL-6的水平升高(P0.05);与激活对照组相比,激活实验组中IL-17A和IL-6的水平降低;实时定量PCR显示与阻断对照组相比,阻断实验组中ROR-γt m RNA的水平明显增加(P0.05)。结论Tim-3及Th17细胞参与了免疫性肝损伤的发生发展,其水平的变化可以在一定程度上反应病情的严重程度,可望为免疫性肝损伤发生机制提供新的线索;免疫调控Tim-3通路可通过影响Th17细胞效应,进而影响肝损伤的程度,以期为临床治疗提供新的依据。
[Abstract]:Objective to establish an immunological liver injury model with concanavalin A (Con A), to investigate the dynamic changes and significance of Tim-3 and Th17 cytokines in the pathogenesis of liver injury, and to explore the effect of targeted intervention of Tim-3 pathway on Th17 cells. Methods 60 male Wistar rats were randomly divided into two groups: model group (50 rats) and control group (10 rats). Rats in the model group were injected with 12.5mg/kg Con A once a week for 8 weeks. Rats in control group were injected with sterile PBS, once a week for 8 weeks. Ten rats were randomly selected from the model group and were given extracorporeal apical blood for 1.5 ml, at week 0 (0w), week 2 (2w), week 4 (4w), week 6 (6w) and week 8 (8w). 20 rats were randomly removed from the model group at -20 鈩，

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