【摘要】：目的: 本研究構(gòu)建了磨損微粒(wear particles,WP)刺激巨噬細(xì)胞的炎癥反應(yīng)模型,觀察超高分子聚乙烯磨損顆粒(Ultra-high molecular weight polyethylene wear particles,UHMWPE-WP)誘導(dǎo)小鼠單核巨噬細(xì)胞產(chǎn)生炎癥反應(yīng)中炎癥因子,尤其是晚期炎癥蛋白高遷移率族蛋白 B1(high mobility group protein,HMGB1)表達(dá)的影響及可能的機(jī)制,為臨床上人工關(guān)節(jié)無菌性松動(dòng)的機(jī)制研究提供的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。 方法: 第一部分:磨損微粒誘導(dǎo)巨噬細(xì)胞炎癥反應(yīng)模型的構(gòu)建 實(shí)驗(yàn)分為三組:①空白組;②模型組(WP 組);③陽性對照組(LPS 組)。 1. MTT 法檢測各分組條件下的細(xì)胞活力。 2. ELISA 法檢測細(xì)胞外周上清液中炎癥因子的含量以分析炎癥反應(yīng)程度。 第二部分:磨損微粒對巨噬細(xì)胞炎癥因子的影響及可能機(jī)制 實(shí)驗(yàn)分為五組:①空白組;②模型組(WP 組);③模型加 HMGB1 蛋白拮抗劑組(WP+A 組);④HMGB1 蛋白拮抗劑對照組(A 組);⑤陽性對照組(LPS組)。 1. ELISA 法檢測細(xì)胞外周上清液中炎癥因子的含量以分析炎癥反應(yīng)程度。 2. ELISA 法檢測各組外周上清液中 HMGB1 蛋白含量,Western Blot 法檢測細(xì)胞內(nèi) HMGB1 蛋白含量。 3. Western Blot 法檢測細(xì)胞在各分組條件下 NF-κB p65 磷酸化影響。結(jié)果第一部分:磨損微粒誘導(dǎo)巨噬細(xì)胞炎癥反應(yīng)模型的構(gòu)建1.MTT法分別測定LPS和WP對小鼠單核巨噬細(xì)胞細(xì)胞活力的影響,確定實(shí)驗(yàn)中LPS的適宜濃度為1μg/ml,WP的適宜濃度為250ng/ml。2.ELISA法測定各組細(xì)胞炎癥因子的濃度,模型組與空白組相比,炎癥因子TNF-α、IL-1和IL-6的濃度具有顯著差異(P0.05),說明模型構(gòu)建成功,WP誘發(fā)炎癥反應(yīng)。模型組與模型加蛋白拮抗劑組的炎癥因子濃度差異具有顯著性(P0.05),說明WP誘發(fā)的炎癥反應(yīng)與HMGB1的表達(dá)有一定的聯(lián)系。第二部分:磨損微粒對巨噬細(xì)胞炎癥因子的影響及可能機(jī)制1.ELISA法測定各組細(xì)胞外周上清液炎癥因子濃度。模型組與空白組相比,炎癥因子的表達(dá)具有顯著差異(P0.05),當(dāng)加入拮抗劑HMGB1 A-box后,炎癥因子含量降低。結(jié)果說明WP誘發(fā)的炎癥反應(yīng)與HMGB1的表達(dá)增多具有相關(guān)性。2.ELISA法測定各組細(xì)胞外周上清液HMGB1濃度,Western Blot法檢測細(xì)胞胞內(nèi)HMGB1表達(dá)量。模型組與空白組相比,HMGB1的表達(dá)具有顯著差異(P0.05),當(dāng)加入拮抗劑HMGB1 A-box后,炎癥反應(yīng)程度降低同時(shí)HMGB1蛋白表達(dá)下調(diào)。上述結(jié)果說明WP誘發(fā)的炎癥反應(yīng)與HMGB1的表達(dá)增多具有相關(guān)性。3.Western Blot法檢測WP對NF-κB p65磷酸化程度的影響,通過模型組與空白組對比發(fā)現(xiàn),在WP刺激作用下,NF-κB的p-65的活化程度顯著上升(P0.05),HMGB1的表達(dá)上調(diào)與NF-κB活化具有相關(guān)性。結(jié)論磨損微�？梢哉T導(dǎo)小鼠單核巨噬細(xì)胞產(chǎn)生炎癥反應(yīng),上調(diào)小鼠單核巨噬細(xì)胞HMGB1蛋白的表達(dá),其機(jī)制可能與NF-κB信號通路有關(guān)。
[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- 偽, 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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R687.4

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