超高分子聚乙烯微粒对小鼠单核巨噬细胞HMGB1蛋白表达的影响

发布时间：2018-12-28 14:52

【摘要】：目的: 本研究构建了磨损微粒(wear particles,WP)刺激巨噬细胞的炎症反应模型,观察超高分子聚乙烯磨损颗粒(Ultra-high molecular weight polyethylene wear particles,UHMWPE-WP)诱导小鼠单核巨噬细胞产生炎症反应中炎症因子,尤其是晚期炎症蛋白高迁移率族蛋白 B1(high mobility group protein,HMGB1)表达的影响及可能的机制,为临床上人工关节无菌性松动的机制研究提供的理论基础和实验依据。方法: 第一部分:磨损微粒诱导巨噬细胞炎症反应模型的构建实验分为三组:①空白组;②模型组(WP 组);③阳性对照组(LPS 组)。 1. MTT 法检测各分组条件下的细胞活力。 2. ELISA 法检测细胞外周上清液中炎症因子的含量以分析炎症反应程度。第二部分:磨损微粒对巨噬细胞炎症因子的影响及可能机制实验分为五组:①空白组;②模型组(WP 组);③模型加 HMGB1 蛋白拮抗剂组(WP+A 组);④HMGB1 蛋白拮抗剂对照组(A 组);⑤阳性对照组(LPS组)。 1. ELISA 法检测细胞外周上清液中炎症因子的含量以分析炎症反应程度。 2. ELISA 法检测各组外周上清液中 HMGB1 蛋白含量,Western Blot 法检测细胞内 HMGB1 蛋白含量。 3. Western Blot 法检测细胞在各分组条件下 NF-κB p65 磷酸化影响。结果第一部分:磨损微粒诱导巨噬细胞炎症反应模型的构建1.MTT法分别测定LPS和WP对小鼠单核巨噬细胞细胞活力的影响,确定实验中LPS的适宜浓度为1μg/ml,WP的适宜浓度为250ng/ml。2.ELISA法测定各组细胞炎症因子的浓度,模型组与空白组相比,炎症因子TNF-α、IL-1和IL-6的浓度具有显著差异(P0.05),说明模型构建成功,WP诱发炎症反应。模型组与模型加蛋白拮抗剂组的炎症因子浓度差异具有显著性(P0.05),说明WP诱发的炎症反应与HMGB1的表达有一定的联系。第二部分:磨损微粒对巨噬细胞炎症因子的影响及可能机制1.ELISA法测定各组细胞外周上清液炎症因子浓度。模型组与空白组相比,炎症因子的表达具有显著差异(P0.05),当加入拮抗剂HMGB1 A-box后,炎症因子含量降低。结果说明WP诱发的炎症反应与HMGB1的表达增多具有相关性。2.ELISA法测定各组细胞外周上清液HMGB1浓度,Western Blot法检测细胞胞内HMGB1表达量。模型组与空白组相比,HMGB1的表达具有显著差异(P0.05),当加入拮抗剂HMGB1 A-box后,炎症反应程度降低同时HMGB1蛋白表达下调。上述结果说明WP诱发的炎症反应与HMGB1的表达增多具有相关性。3.Western Blot法检测WP对NF-κB p65磷酸化程度的影响,通过模型组与空白组对比发现,在WP刺激作用下,NF-κB的p-65的活化程度显著上升(P0.05),HMGB1的表达上调与NF-κB活化具有相关性。结论磨损微粒可以诱导小鼠单核巨噬细胞产生炎症反应,上调小鼠单核巨噬细胞HMGB1蛋白的表达,其机制可能与NF-κB信号通路有关。
[Abstract]:Objective: to establish an inflammatory response model of macrophages stimulated by wear particles (wear particles,WP) and observe the ultrahigh molecular weight polyethylene (Ultra-high molecular weight polyethylene wear particles,) wear particles. UHMWPE-WP) induces the expression of inflammatory factors, especially advanced inflammatory protein high mobility group B1 (high mobility group protein,HMGB1, in mouse mononuclear macrophages and its possible mechanism. To provide a theoretical and experimental basis for the clinical study of aseptic loosening of artificial joints. Methods: the first part: the model of macrophage inflammation induced by wear particles was divided into three groups: 1 blank group, 2 model group (WP group), 3 positive control group (LPS group). 1. MTT assay was used to detect the viability of the cells in each group. 2. The level of inflammatory factors in peripheral supernatant was measured by ELISA method to analyze the degree of inflammatory reaction. The second part: the effect of wear particles on macrophage inflammatory factors and its possible mechanism were divided into five groups: 1 blank group, 2 model group (WP group), 3 model plus HMGB1 protein antagonist group (WP A group); 4HMGB1 protein antagonist control group (group A) and positive control group (LPS group). 1. The level of inflammatory factors in peripheral supernatant was measured by ELISA method to analyze the degree of inflammatory reaction. 2. The content of HMGB1 protein in peripheral supernatant of each group was detected by ELISA method. The content of HMGB1 protein in cells was detected by, Western Blot method. 3. The phosphorylation of NF- 魏 B p65 was detected by Western Blot. Results the first part: the construction of the model of macrophage inflammation induced by wear particles. The effects of LPS and WP on the activity of mouse mononuclear macrophages were determined by 1.MTT method. The optimum concentration of LPS was 1 渭 g / ml in the experiment. The suitable concentration of WP was determined by 250ng/ml.2.ELISA method. The concentration of TNF- 伪, IL-1 and IL-6 in the model group was significantly different from that in the blank group (P0.05). The results showed that the model was successfully constructed and WP induced inflammatory response. There was significant difference in the concentration of inflammatory factors between the model group and the model plus protein antagonist group (P0.05), which indicated that the inflammatory response induced by WP was related to the expression of HMGB1. Part two: the effect of wear particles on macrophage inflammatory factors and its possible mechanism 1.ELISA assay was used to determine the concentration of inflammatory factors in the supernatant of each group. The expression of inflammatory factors in the model group was significantly different from that in the blank group (P0.05). When the antagonist HMGB1 A-box was added, the content of inflammatory factor decreased. The results showed that the inflammatory response induced by WP was correlated with the increase of HMGB1 expression, and the expression of HMGB1 in the cells was detected by 2.ELISA assay, the HMGB1 concentration in the supernatant of the cells was measured by, Western Blot method. The expression of HMGB1 in the model group was significantly different from that in the blank group (P0.05). When the antagonist HMGB1 A-box was added, the degree of inflammatory reaction was decreased and the expression of HMGB1 protein was down-regulated. The results indicated that the inflammatory response induced by WP was correlated with the increase of HMGB1 expression. The effect of WP on the phosphorylation of NF- 魏 B p65 was detected by 3.Western Blot assay. The activation of p-65 of NF- 魏 B increased significantly (P0.05), and the up-regulation of HMGB1 expression was correlated with the activation of NF- 魏 B. Conclusion Wear particles can induce inflammatory reaction in murine mononuclear macrophages and up-regulate the expression of HMGB1 protein in murine mononuclear macrophages. The mechanism may be related to the NF- 魏 B signaling pathway.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R687.4

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