高迁移率族蛋白B1对大鼠脾脏树突状细胞免疫功能影响及机制研究

发布时间：2018-04-30 08:01

本文选题：高迁移率族蛋白B1 + 树突状细胞　；参考：《中国人民解放军军医进修学院》2007年硕士论文

【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系统性炎症的致死性效应，研究其病理生理作用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。本试验拟应用HMGB1体外刺激大鼠脾脏树突状细胞(DC)，，阐明HMGB1对DC免疫功能的影响并对其机制进行初步探讨，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：分离正常Wistar大鼠脾脏DC，在含10％小牛血清的1640培养液中调整细胞浓度1×10~6/孔后置于24孔培养板。实验一：采用HMGB1刺激，观察HMGB1刺激与DC表面共刺激分子CD80、CD86和主要组织相容性复合物(MHC)Ⅱ表达、细胞因子白介素(IL)-12、肿瘤坏死因子(TNF)-α基因表达和蛋白合成的时间-效应关系及剂量-效应关系。实验二：HMGB1刺激后，采用激光扫描共聚焦显微镜和流式细胞术检测DC表面晚期糖基化终末产物受体(RAGE)的表达强度。同时观察抗RAGE抗体对HMGB1刺激后DC表面共刺激分子CD80、CD86和MHCⅡ表达的影响。实验三：采用1μg/ml的HMGB1刺激48小时(h)，DC与脾T淋巴细胞的细胞比例分别为1：50、1：100、1：150、1：200混合，于共同作用后3天、5天观察T淋巴细胞增殖反应、功能性极化、IL-2、白介素-2受体(IL-2R)基因/蛋白表达及核因子(NF)-κB活性的情况。结果：1．HMGB1对大鼠脾脏DC成熟分化的影响及机制：(1)与正常对照组相比，HMGB1作用后DC表面共刺激分子CD80、CD86和MHCⅡ表达分别于24～72h明显上调(P＜0.05或P＜0.01)，其中以作用48h后DC表面共刺激分子表达上调尤为显著(P＜0.01)；0.1μg/ml、1μg/ml、10μg/ml的HMGB1刺激均可诱导DC表面共刺激分子CD80、CD86和MHCⅡ表达增强(P＜0.05或P＜0.01)，其中HMGB1的浓度在1μg/ml时大鼠DC表面共刺激分子表达增强最明显(P＜0.01)。(2)HMGB1刺激后，DC IL-12、TNF-α基因表达和蛋白合成分别于24～72h明显上调(P＜0.05或P＜0.01)，其中以作用48h后其表达上调尤为显著(P＜0.01)；0.1μg/ml、1μg/ml、10μg/ml的HMGB1刺激均可诱导DC IL-12、TNF-α基因表达和蛋白合成增强(P＜0.01)，其中HMGB1的浓度在1μg/ml时，DC IL-12、TNF-α基因/蛋白表达水平最高(P＜0.01)。2．HMGB1介导大鼠脾脏DC作用的受体机制：1μg/ml HMGB1刺激48h后，DC表面受体RAGE表达显著上调(P＜0.01)；1：50、1：100、1：200稀释度的抗RAGE多克隆抗体处理均可使HMGB1诱导DC表面共刺激分子CD80、CD86和MHCⅡ表达减弱(P＜0.01)，其中抗RAGE多克隆抗体的稀释度在1：100时，大鼠DC表面共刺激分子表达减弱最明显(P＜0.01)。3．HMGB1诱导的DC对大鼠脾T淋巴细胞功能的影响：(1)DC与T淋巴细胞以1：50、1：100、1：150、1：200的比例相互作用，脾T淋巴细胞对丝裂原刺激的增殖反应均明显增强、上清液中干扰素(IFN)-γ含量显著升高和IL-4含量明显降低(P＜0.01)，其中细胞比例1：150组最为明显。脾T淋巴细胞对丝裂原刺激的增殖反应增强、IFN-γ含量增加和IL-4含量降低均以第3天最为显著。(2)实验研究显示，DC与T淋巴细胞以1：50、1：100、1：150、1：200的比例相互作用，脾T淋巴细胞IL-2、IL-2R基因/蛋白表达及NF-κB活性明显增强(P＜0.01)，其中细胞比例1：150组尤为明显。脾T淋巴细胞IL-2、IL-2R基因/蛋白表达及NF-κB活性均以第3天最为显著。结论：1．HMGB1能诱导DC表面共刺激分子表达增强和IL-12、TNF-α的合成、释放，HMGB1可能是诱导DC成熟的重要免疫刺激信号。2．HMGB1能上调DC受体RAGE表达，RAGE可能是参与HMGB1诱导DC成熟分化的主要受体之一。3．HMGB1诱导的DC使脾T淋巴细胞对丝裂原刺激的增殖反应明显增强，并促进T淋巴细胞向Th1功能性极化。4．初步明确了HMGB1诱导的DC影响脾T淋巴细胞IL-2生成和IL-2R表达的相关信号转导通路。
[Abstract]:Objective: high mobility group protein B1 (HMGB1), as a late inflammatory mediator, mediates the lethal effects of sepsis and other systemic inflammation. The study of its pathophysiological role will help to deepen the understanding of the pathogenesis of sepsis and provide potential intervention targets for the regulation of immune responses. This test is intended to be applied to HMGB1 in vitro spines. The rat splenic dendritic cells (DC) were stimulated to elucidate the effect of HMGB1 on the immune function of DC and to explore its mechanism. The aim is to provide new ideas for the prevention and treatment of sepsis and multiple organ dysfunction syndrome.
Methods: the spleen DC of normal Wistar rats was isolated, and the cell concentration was adjusted in the 1640 culture solution containing 10% calf serum. The cell concentration was adjusted to the 24 hole culture plate. Experiment 1: HMGB1 stimulation was used to observe the expression of CD80, CD86 and the major histocompatibility complex (MHC) II (MHC) II of the HMGB1 stimulation and the DC surface, and the cytokine interleukins (IL) -12, swelling. The time effect relationship and dose effect relationship of tumor necrosis factor (TNF) - alpha gene expression and protein synthesis. Experiment two: HMGB1 stimulation, using laser scanning confocal microscope and flow cytometry to detect the expression intensity of advanced glycosylated end product receptor (RAGE) on the surface of DC. Meanwhile, the anti RAGE antibody was observed on the DC surface after HMGB1 stimulation. The influence of the expression of CD80, CD86 and MHC II. Experiment three: the HMGB1 stimulation of 1 g/ml was 48 hours (H), the proportion of DC to spleen T lymphocyte was 1:50,1:100,1:150,1:200 mixed, 3 days after the joint action, and 5 days to observe the proliferation reaction of T lymphocyte, functional polarization, IL-2, interleukin--2 receptor gene / protein table And the activity of nuclear factor (NF) - kappa B.
Results: the effect and mechanism of 1.HMGB1 on the maturation and differentiation of DC in spleen of rats: (1) compared with the normal control group, the expression of CD80, CD86 and MHC II on the DC surface was up significantly up (P < 0.05 or P < 0.01) after HMGB1 action, especially after the action 48h, the expression of the expression of CO stimulants on the DC surface was particularly significant (0.01); 0.1 mu, The HMGB1 stimulation of 1 mu g/ml and 10 mu g/ml could induce the expression of CD80, CD86 and MHC II (P < 0.05 or P < 0.01) on the surface of DC (P < 0.05 or P < 0.01). The expression of CO stimulatory molecules on the DC surface was the most obvious (2) when the concentration of HMGB1 was 1 mu g/ml. The regulation (P < 0.05 or P < 0.01) was especially significant after the action of 48h (P < 0.01); HMGB1 stimulation of 0.1 mu g/ml, 1 mu g/ml and 10 mu g/ml could induce DC IL-12, TNF- alpha gene expression and protein synthesis enhanced (P < 0.01). The receptor mechanism of spleen DC action in rats was mediated: after 1 mu g/ml HMGB1 stimulated 48h, the expression of DC surface receptor RAGE was significantly up-regulated (P < 0.01), and the anti RAGE polyclonal antibody treatment of 1:50,1:100,1:200 dilution can induce HMGB1 to induce DC surface CO stimulation of molecular CD80. At 1:100, the expression of CO stimulatory molecules on DC surface weakened the most obvious (P < 0.01).3.HMGB1 induced DC on the function of T lymphocyte in the spleen of rats: (1) the interaction between DC and T lymphocytes was proportional to the proportion of 1:50,1:100,1:150,1:200, and the proliferation of mitogen stimulated by the spleen T lymphocyte was obviously enhanced, and the supernatant was in the supernatant. The content of interferon (IFN) - gamma was significantly increased and the content of IL-4 decreased significantly (P < 0.01). The proportion of cell ratio in 1:150 group was the most obvious. The proliferation response of splenic T lymphocytes to mitogen stimulated proliferation, the increase of IFN- gamma content and the decrease of IL-4 content were the most significant. (2) the experimental study showed that DC and T lymphocyte were 1:50,1:100,1:150,1. 200 ratio interaction, the IL-2 of spleen T lymphocyte, IL-2R gene / protein expression and NF- kappa B activity were obviously enhanced (P < 0.01), in which the proportion of cells in 1:150 group was particularly obvious. The IL-2 of spleen T lymphocyte, IL-2R gene / protein expression and NF- kappa B activity were the most significant in third days.
Conclusion: 1.HMGB1 can induce the enhancement of the expression of CO stimulatory molecules on the surface of DC and the synthesis and release of IL-12, TNF- alpha, and HMGB1 may be an important stimulant signal to induce the maturation of DC..2.HMGB1 can up regulate the RAGE expression of DC receptor. RAGE may be a major receptor involved in HMGB1 inducible DC mature differentiation. The proliferative response of the spur stimulation was obviously enhanced, and the T lymphocyte was promoted to Th1 functional polarization.4. to clarify that HMGB1 induced DC affects the related signal transduction pathway of IL-2 generation and IL-2R expression in spleen T lymphocyte.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R631.2

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