正丁酸钠对糖尿病T细胞亚群的影响

发布时间：2018-07-18 16:41

【摘要】：目的:观察1型糖尿病(T1DM)模型小鼠体内T细胞亚群的变化及HMGB1蛋白的表达,正丁酸钠作为晚期炎症因子HMGB1的拮抗剂,通过对HMGB1的抑制作用,探讨其延缓1型糖尿病发展的可能机制与对辅助性T淋巴(Th)细胞的影响,阐明HMGB1及Th细胞在1型糖尿病发生和发展中的作用,为探索干预1型糖尿病的免疫策略提供新思路。方法:SPF级5-6周龄雄性C57BL/6品系小鼠30只,用常规饲料喂养1周,使小鼠适应新的环境,一周后采用随机分组方法将30只小鼠分为正常对照(NC)组,糖尿病(DM)组,正丁酸钠(SB)组,每组小鼠各10只。称取体重,按照40mg/kg的体重剂量,DM组、SB组腹腔注射由柠檬酸盐缓冲液溶解的STZ(配置及注射过程于冰上操作),1次/天,连续5天;同时SB组给予生理盐水溶解的正丁酸钠500mg/kg,1次/天,持续到实验结束;DM组及NC组则同时腹腔注射相同量的0.9%的生理盐水。小鼠造模前,测取一次基础血糖;造模后,给予实验小鼠充足的食物和水,于第7天开始监测随机血糖,第7次血糖监测后处死取材。观察小鼠成模前后及正丁酸钠干预后血糖水平,流式细胞术检测脾脏Th1、Th2、Th17及胰腺引流淋巴结(PLNs)中Th17细胞的分化比例,Western Blotting方法检测HMGB1蛋白在小鼠胰腺组织中的表达水平,Elisa方法检测小鼠血清中IL-1β细胞因子水平。结果:DM组小鼠在第2周末时血糖水平显著高于NC组(P0.01),且多饮、多食、多尿,显示DM组小鼠已存在糖尿病症状;与NC组相比,DM组小鼠脾脏Th1细胞比例显著升高(P0.05),Th2细胞比例降低(P0.05),Th1/Th2比值明显升高(P0.01),Th17细胞比例轻度下降,差异无统计学意义(P0.05);PLNs中Th17细胞比例明显升高(P0.01);DM组小鼠外周血清IL-1β细胞因子水平显著高于NC组(P0.01);胰腺组织HMGB1蛋白表达水平显著高于NC组(P0.01)。与DM组相比,SB治疗组小鼠发病时间延缓,发病率显著降低(P0.05),血糖水平显著降低(P0.05),脾脏组织中Th1细胞显著减少(P0.05),Th2细胞轻度增加,但差异无统计学意义(P0.05),Th1/Th2比值显著降低(P0.01),Th17细胞比例降低,差异无统计学意义(P0.05),PLNs中Th17细胞明显减少(P0.01);胰腺组织中HMGB1蛋白水平显著低于DM组(P0.01),外周血清IL-1β细胞因子水平相比DM组降低(P0.01)。结论:(1)在T1DM动物模型中,应用正丁酸钠干预可以延缓T1DM发病并减少T1DM发病率。(2)T1DM小鼠脾脏内Th1细胞比例上调,Th2细胞比例下降,PLNs中Th17细胞上调,血清中IL-1β促炎细胞因子增多,以及胰腺组织中晚期炎症介质HMGB1蛋白表达增多,均有可能导致β细胞的持续破坏,进而促进T1DM的发生和发展。(3)正丁酸钠可能通过调节小鼠体内Th1、Th2及Th17细胞比例和血清中IL-1β促炎细胞因子水平来改善小鼠发病情况和血糖水平;在胰腺组织中正丁酸钠可能通过抑制HMGB1晚期炎症介质的产生,减轻胰腺局部的炎症反应,保护胰岛β细胞免受破坏,从而延缓T1DM的发病进程。
[Abstract]:Aim: to observe the changes of T cell subsets and the expression of HMGB1 protein in type 1 diabetes mellitus (T1DM) mice. Sodium butyrate was used as an antagonist of late inflammatory factor HMGB1. To explore the possible mechanism of delaying the development of type 1 diabetes and its effect on helper T lymphoid (Th) cells, to elucidate the role of HMGB1 and Th cells in the development and development of type 1 diabetes, and to provide a new way of thinking on the immunological intervention of type 1 diabetes. Methods Thirty C57BL / 6 male C57BL / 6 strain mice with SPF grade 5-6 weeks of age were fed with conventional diet for one week to adapt to the new environment. After one week, 30 mice were randomly divided into normal control (NC) group, diabetes mellitus (DM) group and sodium butyrate (SB) group. There were 10 mice in each group. According to the body weight dose of 40mg/kg, group SB was injected intraperitoneally with sodium butyrate 500 mg / kg / day, which was dissolved in citrate buffer solution once a day for 5 days, while the group SB was given sodium butyrate 500 mg / kg / day, and the control group was given sodium butyrate 500 mg / kg / day. At the end of the experiment, the DM group and NC group were intraperitoneally injected with 0.9% saline at the same time. The mice were given sufficient food and water to monitor random blood glucose on the 7th day, and then killed after the seventh blood glucose monitoring. The blood glucose levels were observed before and after the establishment of the model and the intervention of sodium butyrate in mice. The differentiation ratio of Th17 cells in spleen Th2T and pancreatic drainage lymph nodes (PLNs) was detected by flow cytometry. Western blotting was used to detect the expression of HMGB1 protein in mouse pancreas. Elisa was used to detect IL-1 尾 cytokines in serum of mice. Results at the end of the second week, the blood glucose level of the mice in the weight DM group was significantly higher than that in the NC group (P0.01). Compared with NC group, the proportion of Th1 cells in spleen of DM group increased significantly (P0.05) the proportion of Th2 cells decreased (P0.05) the ratio of Th1 / Th2 increased significantly (P0.01) the proportion of Th17 cells decreased slightly. There was no significant difference (P0.05) in the proportion of Th17 cells in PLNs (P0.01). The level of IL-1 尾 cytokines in peripheral blood of DM group was significantly higher than that of NC group (P0.01), and the expression level of HMGB1 protein in pancreatic tissue was significantly higher than that in NC group (P0.01). Compared with DM group, the onset time was delayed, the incidence rate was significantly decreased (P0.05), blood glucose level was significantly decreased (P0.05), Th1 cells in spleen tissue were significantly decreased (P0.05) and Th2 cells in spleen tissue were slightly increased. But the ratio of Th1 / Th2 decreased significantly (P0.01), the ratio of Th17 cells in PLNs decreased significantly (P0.01), the level of HMGB1 protein in pancreatic tissue was significantly lower than that in DM group (P0.01), and the level of IL-1 尾 cytokines in peripheral blood was lower than that in DM group (P0.01). Conclusion: (1) in the animal model of T1DM, sodium butyrate can delay the onset of T1DM and reduce the incidence of T1DM. (2) the proportion of Th1 cells in spleen of T1DM mice is up-regulated and Th17 cells in PLNs are up-regulated, and IL-1 尾 pro-inflammatory cytokines are increased in serum of T1DM mice. The increase of HMGB1 protein expression in pancreatic tissue may lead to the sustained destruction of 尾 cells. (3) Sodium butyrate may improve the incidence and blood sugar level of mice by regulating the proportion of Th1 Th2 and Th17 cells and the level of IL-1 尾 inflammatory cytokines in serum. Sodium butyrate may slow down the pathogenesis of T1DM by inhibiting the production of late HMGB1 inflammatory mediators, reducing the local inflammatory response of pancreas and protecting islet 尾 cells from destruction.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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