高迁移率族蛋白B1诱导急性肺损伤小鼠调节性T细胞免疫反应的受体机制

发布时间：2018-08-12 17:18

【摘要】：目的:探讨高迁移率族蛋白B1(HMGB1)通过Toll样受体4(TLR4)介导小鼠急性肺损伤时CD4+CD25+调节性T细胞(CD4+CD25+Treg)免疫功能及免疫抑制活性的变化;研究CD4+CD25+Treg与CD4+CD25-T细胞相互作用的影响。方法:分别给健康清洁级C57BL/10J和C57BL/10Sc[分别为TLR4野生型(TLR4+/+)和敲除型(TLR4-/-)]小鼠气管内注射HMGB1(20μg/只)为实验组,同时设立气管内注射生理盐水野生型小鼠为对照组;(1)于造模后饲养48小时眼球取血法法处死小鼠收集血清及肺泡灌洗液(BALF),光镜下观察肺脏病理改变,称重测定肺脏干湿比;采用免疫磁珠分离脾脏CD4+CD25+Treg和CD4+CD25-T细胞。(2)将各组部分CD4+CD25+Treg体外培养24小时后,采用流式细胞仪检测CD4+CD25+Treg表达叉头翼状螺旋转录因子(FOXP3)和淋巴细胞相关抗原4(CTLA-4)的表达强度,并且用酶联免疫吸附试验(ELISA)法检测BALF中白细胞介素-1(IL-1),肿瘤坏死因子-α(TNF-α)及CD4+CD25+Treg分泌的白细胞介素-10(IL-10)和转化生长因子-β(TGF-β)的含量。(3)将各组CD4+CD25+Treg与对照组CD4+CD25-T细胞按1:1比例共培养,包被CD3单抗和可溶性CD28单抗辅助活化;CCK-8法检测CD4+CD25+Treg对CD4+CD25-T细胞增殖的抑制作用。并采用ELISA法检测共培养体系中分泌的白细胞介素-4(IL-4)、IL-2和干扰素-γ(IFN-γ)的含量。结果:1、与对照组相比,两实验组小鼠气管内注射HMGB1后,肺组织损伤程度明显严重,肺脏干湿比值及BALF灌洗液中TNF-α,IL-1含量显著增高,且TLR4野生型小鼠较TLR4敲除型小鼠病变程度更加严重;2、流式细胞学检测免疫磁珠分选的CD4+CD25+Treg纯度在90%以上;TLR4野生型急性肺损伤小鼠FOXP3和CTLA-4表达强度低于对照组和TLR4敲除型(P0.05);野生型实验组小鼠Treg分泌的IL-10及TGF-β含量较对照组含量降低(P0.05),而TLR4敲除型较对照组IL-10及TGF-β含量升高(P0.05);3、经CD3和CD28辅助活化后,TLR4野生型急性肺损伤小鼠CD4+CD25+Treg与效应T细胞共培养上清中,IL-2,IFN-γ含量高于对照组(P0.05),IL-4含量低于对照组(P0.05),提示CD4+CD25+Treg可使T细胞极化Th2细胞能力减弱,向Th1细胞方向极化;而TLR4敲除型急性肺损伤小鼠CD4+CD25+Treg与效应T细胞共培养上清中,IL-2,IFN-γ含量低于对照组(P0.05),IL-4含量高于对照组(P0.05),CD4+CD25+Treg介导的免疫抑制效应促使T细胞极化为Th2细胞,引起Th1/Th2的漂移。结论:1、气管内注射HMGB1可以诱导小鼠急性肺损伤模型;2、免疫磁珠分选的CD4+CD25+Treg细胞纯度较高,可以满足后续实验的要求;3、在急性肺损伤时HMGB1可通过TLR4下调CD4+CD25+Treg的免疫功能,使CD4+CD25+Treg中Foxp3和CTLA-4分子的表达下调,从而减弱其免疫抑制功能的发挥;4、在急性肺损伤时HMGB1可通过TLR4使CD4+CD25+Treg极化Th2细胞能力减弱,向Th1细胞方向极化,弱化了CD4+CD25+Treg的功能,影响炎症反应的结局;为ARDS开辟新的治疗靶点。
[Abstract]:Aim: to investigate the effects of high mobility group protein B1 (HMGB1) on the immune function and immunosuppressive activity of CD4 CD25 regulatory T cells (CD4 CD25 Treg) in mice with acute lung injury mediated by Toll like receptor 4 (TLR4), and to investigate the interaction between CD4 CD25 Treg and CD4 CD25-T cells. Methods: healthy clean grade C57BL/10J and C57BL/10Sc [TLR4 wild-type (TLR4 /) and knockout type (TLR4-r-)] mice were given intratracheal injection of HMGB1 (20 渭 g / mouse) respectively as experimental group. At the same time, the wild-type mice with intratracheal injection of normal saline were set up as the control group. (1) after 48 hours of feeding, the mice were killed to collect serum and alveolar lavage fluid (BALF),) to observe the pathological changes of lung, and the lung dry-wet ratio was measured. Splenic CD4 CD25 Treg and CD4 CD25-T cells were isolated by immunomagnetic beads. (2) after cultured in vitro for 24 hours, the expression of CD4 CD25 Treg, FOXP3 and CTLA-4 were detected by flow cytometry. The levels of interleukin-1 (IL-1), tumor necrosis factor- 伪 (TNF- 伪), interleukin-10 (IL-10) and transforming growth factor- 尾 (TGF- 尾) secreted by CD4 CD25 Treg in BALF were detected by enzyme-linked immunosorbent assay (ELISA). (3) CD4 CD25 Treg and CD4 CD25-T cells in control group were co-cultured at 1:1. The inhibitory effect of CD4 CD25 Treg on the proliferation of CD4 CD25-T cells was detected by CCK-8 method coated with CD3 McAb and soluble CD28 McAb. The levels of interleukin-4 (IL-4) IL-2 and interferon- 纬 (IFN- 纬) secreted in co-culture system were detected by ELISA method. Results compared with the control group, the lung tissue injury of the mice in the two experimental groups was obviously serious after intratracheal injection of HMGB1, and the ratio of lung dry-wet and the content of TNF- 伪 IL-1 in the BALF lavage fluid were significantly higher than those in the control group. The pathological changes of TLR4 wild-type mice were more serious than those of TLR4 knockout mice. The CD4 CD25 Treg purity of immunomagnetic beads was more than 90% in TLR4 wild-type mice with acute lung injury, and the expression of FOXP3 and CTLA-4 in mice with wild type lung injury was lower than that in control group. The levels of IL-10 and TGF- 尾 secreted by Treg in wild type mice were lower than those in control group (P0.05), while those of TLR4 knockout type were higher (P0.05) than those of control group (P0.05). After CD3 and CD28 activation, the contents of TGF- 尾 in wild type of lung injury mice were significantly lower than those in control group (P0.05). The content of IL-2IFN- 纬 in the supernatant of Treg and effector T cell culture was higher than that in the control group (P0.05), which suggested that CD4 CD25 Treg could weaken the ability of polarized Th2 cells. The level of IL-2tIFN- 纬 in the supernatant of CD4 CD25 Treg and effector T cells in mice with acute lung injury induced by TLR4 knockout type was lower than that in the control group (P0.05) and the immunosuppressive effect mediated by CD4 CD25 Treg in the control group was higher than that in the control group (P0.05). Cause the drift of Th1/Th2. Conclusion HMGB1 injected into trachea can induce acute lung injury model in mice. The purity of CD4 CD25 Treg cells separated by immunomagnetic beads is high, which can meet the requirements of subsequent experiments. HMGB1 can down-regulate the immune function of CD4 CD25 Treg through TLR4 during acute lung injury. The expression of Foxp3 and CTLA-4 molecules in CD4 CD25 Treg was down-regulated and the immunosuppressive function was weakened. In acute lung injury, HMGB1 could weaken the ability of CD4 CD25 Treg polarized Th2 cells through TLR4, polarize CD4 CD25 Treg cells in the direction of Th1 cells, and weaken the function of CD4 CD25 Treg. To influence the outcome of inflammatory reaction; to open up a new therapeutic target for ARDS.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563

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