高迁移率族蛋白B1对小鼠调节性树突状细胞免疫功能影响的实验研究

发布时间：2018-02-04 06:44

本文关键词： 高迁移率族蛋白B1 调节性树突状细胞脓毒症脾T淋巴细胞白介素-2 白介素-10 白介素-12 Toll样受体4 增殖反应功能性极化　出处：《中国人民解放军军医进修学院》2012年博士论文　论文类型：学位论文

【摘要】：目的：高迁移率族蛋白B1（HMGB1）作为一种晚期炎症介质，介导了脓毒症和其他全身炎症的致死性效应，研究其病理生理作用与意义将有助于深化对脓毒症发病机制的认识，并为免疫反应的调控提供潜在的干预目标。本实验拟采用HMGB1体外刺激小鼠脾脏调节性树突状细胞（DCreg），重点围绕HMGB1对DCreg免疫功能的影响及其受体机制进行初步探讨，旨在为脓毒症及多器官功能障碍综合征的预防和治疗提供新思路。方法：1.分离培养正常Balb/c小鼠脾脏DCreg（CD11c~(low)CD45RB~(high)DCs），采用不同剂量的HMGB1（10、100、1000ng/ml）刺激12小时，并以100ng/ml HMGB1于不同时间点（2、12、24小时）刺激，观察HMGB1刺激与DCreg分泌白细胞介素（IL）-10和IL-12水平、表面共刺激分子CD40、CD80、CD86和主要组织相容性复合物（MHC）-II表达的剂量-效应关系及时间-效应关系。采用ELISA法检测上清中IL-10和IL-12水平，DCreg共刺激分子表达采用流式细胞仪分析。2. DCreg经100ng/ml的HMGB1刺激12小时后，与脾T淋巴细胞以1:100的细胞比例混合培养，于共同作用后3天观察T淋巴细胞增殖反应、功能性极化（Th1/Th2）、IL-2表达情况。3. HMGB1刺激后，分别采用流式细胞术和PCR技术检测DCreg表面Toll样受体（TLR）4的表达，进一步观察封闭TLR4受体对HMGB1刺激后DCreg分泌IL-10和IL-12水平、以及对T淋巴细胞增殖反应、功能性极化、IL-2表达的影响。结果：1. HMGB1对小鼠脾脏DCreg功能的影响：（1）HMGB1刺激后，DCreg分泌IL-10、IL-12水平于12~24小时分别明显升高和下降（P0.01），，其中以作用12小时后达稳态；10ng/ml、100ng/ml、1000ng/ml的HMGB1刺激均可诱导DCregIL-10、IL-12分泌水平分别明显上升和下降（P0.01），其中HMGB1的浓度为100ng/ml时，DCreg分泌IL-10、IL-12趋于稳定。（2）与正常对照组相比，100ng/ml的HMGB1刺激DCreg12小时后，表面共刺激分子CD40、CD80、CD86和MHC-II表达均明显下调（P0.01）。2. HMGB1诱导的DCreg对小鼠脾T淋巴细胞功能的影响：HMGB1诱导的DCreg与T淋巴细胞以1:100的比例相互作用3天，与对照组比较，T淋巴细胞对丝裂原刺激的增殖反应明显减弱，上清液中干扰素（IFN）-γ/IL-4比值显著降低，IL-2水平明显降低（P0.01）。3. HMGB1介导小鼠脾脏DCreg作用的受体机制：（1）100ng/ml HMGB1刺激12小时后，DCreg表面TLR4表达显著上调（P0.01）；（2）拮抗TLR4后，HMGB1刺激的DCreg分泌IL-10、IL-12水平与对照组比较变化不明显，T淋巴细胞增殖反应、功能性极化、IL-2水平亦未见显著改变（P0.05）。结论：1. HMGB1能促进IL-10的合成、释放增加，抑制IL-12的合成、释放，诱导DCreg表面共刺激分子表达减弱，HMGB1可能是诱导DCreg功能分化的重要免疫刺激信号。2. HMGB1诱导的DCreg使脾T淋巴细胞对丝裂原刺激的增殖反应明显抑制，并促进T淋巴细胞向Th2功能性极化，抑制IL-2的合成、释放。3. HMGB1能上调DCreg受体TLR4表达，TLR4可能是参与HMGB1诱导DCreg功能分化的主要受体之一。
[Abstract]:Objective: as a late inflammatory mediator, high mobility group protein B1HMGB1 mediates the lethal effect of sepsis and other systemic inflammation. The study of its pathophysiological role and significance will help to deepen the understanding of the pathogenesis of sepsis. In this experiment, HMGB1 was used to stimulate mouse splenic regulatory dendritic cells in vitro. The effect of HMGB1 on the immune function of DCreg and its receptor mechanism were discussed in order to provide new ideas for the prevention and treatment of sepsis and multiple organ dysfunction syndrome. Methods 1. The spleen of normal Balb/c mice was isolated and cultured. Different doses of HMGB1 / 100ng / ml were used to stimulate for 12 hours, and 100ng / ml HMGB1 was used at different time points. The levels of IL-10 and IL-12 secreted by HMGB1 and DCreg were observed, and the surface costimulatory molecules CD40 and CD80 were observed. The dose-effect relationship and time-effect relationship of the expression of CD86 and MHC-II. The levels of IL-10 and IL-12 in the supernatant were detected by ELISA method. The expression of DCreg costimulatory molecules was analyzed by flow cytometry. DCreg was stimulated by 100ng / ml HMGB1 for 12 hours. After co-culture with spleen T lymphocytes at 1: 100, T lymphocyte proliferation and functional polarization Th1 / Th2 were observed 3 days after co-treatment. After HMGB1 stimulation, the expression of Toll like receptor (TLR4) on the surface of DCreg was detected by flow cytometry and PCR. The levels of IL-10 and IL-12 secreted by DCreg stimulated by HMGB1, the proliferation response of T lymphocytes and the functional polarization of T lymphocytes were further observed by blocking TLR4 receptor. The effect of IL-2 expression. Results 1. The effect of HMGB1 on the function of DCreg in the spleen of mice. IL-10 was secreted by HMGB1 stimulated by HMGB1. The level of IL-12 increased and decreased significantly at 12h after 24 hours, and reached steady state after 12 hours of exposure. 10ng / ml 100ng / ml HMGB1 stimulation could induce DCregIL-10. The level of IL-12 secretion was increased and decreased significantly (P 0.01), and IL-10 was secreted when the concentration of HMGB1 was 100ng / ml. Compared with the control group, the surface costimulatory molecule CD40 and CD80 were stimulated by 100ng / ml HMGB1 for DCreg12 hours. The expression of CD86 and MHC-II were significantly down-regulated (P0.01). Effect of DCreg induced by HMGB1 on the function of spleen T lymphocytes in mice; the ratio of 1: 100 of DCreg to T lymphocytes induced by 1: HMGB1 interacted for 3 days. Compared with the control group, the proliferative response of T lymphocytes to mitogen stimulation was significantly decreased, and the ratio of IFN- 纬 / IL-4 in the supernatant was significantly decreased. The level of IL-2 decreased significantly. The receptor mechanism of HMGB1 mediated the effect of DCreg in spleen of mice was induced by 100 ng / ml HMGB1 for 12 hours. The expression of TLR4 on the surface of DCreg was significantly upregulated (P 0.01). (2) the level of IL-10 IL-12 secreted by DCreg stimulated by HMGB1 after TLR4 was not significantly changed compared with the control group. There was no significant change in IL-2 level. Conclusion 1. HMGB1 can promote the synthesis of IL-10, increase its release, inhibit the synthesis of IL-12, and induce the decrease of the expression of costimulatory molecules on the surface of DCreg. HMGB1 may be an important immunostimulatory signal to induce the differentiation of DCreg. 2. DCreg induced by HMGB1 can significantly inhibit the proliferation of splenic T lymphocytes to mitogen stimulation. It also promoted T lymphocytes to functional polarization of Th2, inhibited the synthesis of IL-2, and released .3.The expression of DCreg receptor TLR4 was up-regulated by HMGB1. TLR4 may be one of the major receptors involved in DCreg differentiation induced by HMGB1.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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