LIPUS对LPS诱导的巨噬细胞系U937炎症反应的抑制作用及其机制研究

发布时间：2018-05-21 07:59

本文选题：LIPUS + LPS　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:本实验用脂多糖(Lipopolysaccharide,LPS)诱导U937细胞,构建炎症模型。应用低强度脉冲超声(Low intensity pulsed ultrasound,LIPUS)处理,探讨LIPUS是否对炎症存在抑制作用,并探讨其主要作用机制。方法:1.体外应用佛波酯(Phorbol 12-myristate 13-acetate,PMA)诱导人巨噬细胞系U937细胞成熟。筛选LPS浓度。分组:U937细胞;不同浓度的LPS(0.05-10μg/ml)诱导U937细胞组。24h后收集上清液,使用酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测炎症因子肿瘤坏死因子-α(Tumour necrosis factor-α,TNF-α)的含量。筛选LIPUS强度。分组:U937细胞组;LPS诱导U937细胞组;不同参数LIPUS(10,30,60,90m W/cm2)刺激LPS介导下的U937细胞组。收集上清液及细胞团块,采用ELISA检测各组TNF-α及白介素-8(Interleukin-8,IL-8)的表达情况,实时聚合酶链反应检测(Real-time polymerase chain reaction,RT-PCR)各组中IL-8的基因变化情况。2.U937细胞增殖活力检测。采用96孔板构建模型,分组:单独U937细胞组;LPS诱导U937细胞组;LIPUS刺激LPS介导的U937细胞组;LIPUS刺激U937细胞组;Bay 11-7082刺激LPS介导的U937细胞组。应用细胞技术试剂盒(Cell counting kit-8,CCK-8)检测U937细胞增殖活力。洗去CCK-8检测液,换成普通培养基继续培养,于第3,5,7天再次检测。U937细胞凋亡检测。采用10cm培养皿构建模型,分组同上,处理后胰酶消化,流式细胞检测仪检测细胞凋亡情况。3.探讨LIPUS刺激对LPS介导的U937细胞炎症反应的作用。分组:U937细胞组;LPS诱导U937细胞组;LIPUS刺激LPS介导的U937细胞组;LIPUS刺激U937细胞组。ELISA及RT-PCR检测炎症因子(TNF-α、IL-1β、IL-6及IL-8)表达情况。4.探讨LIPUS刺激对LPS介导的U937细胞炎症反应的机制。分组:U937细胞组;LPS诱导U937细胞组;LIPUS刺激LPS介导的U937细胞组;Bay 11-7082刺激LPS介导的U937细胞组。RT-PCR检测炎症因子及TLR4基因表达;聚丙烯酰氨凝胶电泳技术(Western blot,WB)检测TLR4、p65、p-IκBα和IκBα蛋白含量;激光共聚焦扫描显微镜观察p65入核情况。结果:1.成功获得成熟U937细胞并构建炎症模型,筛选LPS浓度为1μg/ml、LIPUS强度为60m W/cm2。2.CCK-8检测及流式细胞仪检测发现,LPS明显抑制U937细胞增殖、促进U937细胞凋亡,而LIPUS对此反应有一定抑制作用。3.ELISA和RT-PCR检测结果显示:LPS促进炎症因子的表达,而LIPUS起明显的抑制作用。4.RT-PCR检测示:LIPUS抑制炎症因子以及TLR4的表达;WB检测结果示:LPS加速了IκBα的磷酸化、p65的跨膜转位入核以及TLR4的蛋白表达,而LIPUS阻断了IκBα的磷酸化和p65入核的过程,但对TLR4蛋白表达的抑制作用不明显;激光共聚焦扫描显微镜观察可见:LPS促进p65入核,LIPUS抑制p65入核的效果明显。结论:LIPUS能增强细胞活性,抑制细胞凋亡,并有效地抑制了IκBα蛋白的磷酸化和p65入核,从而抑制炎症因子的分泌,TLR4-核转录因子-κB(Nuclear factor-kappa B,NF-κB)信号通路参与了该反应。
[Abstract]:Aim: to establish an inflammatory model of U937 cells induced by lipopolysaccharide (LPS). Low intensity pulsed ultrasound (LIPUS) was used to investigate the inhibitory effect of LIPUS on inflammation and its main mechanism. Method 1: 1. Human macrophage cell line U937 was matured by Phorbol 12-myristate 13-acetate PMA in vitro. LPS concentration was screened. The supernatants of U937 cells were induced by LPS(0.05-10 渭 g / ml at different concentrations. The supernatants of U937 cells were collected after 24 hours. The levels of tumor necrosis factor- 伪 Tumour necrosis factor- 伪 (TNF- 伪) were measured by enzyme linked immunosorbent assay (Elisa). LIPUS strength was screened. The U937 cells were divided into two groups: one group was induced by LPS-induced U937 cells, and the other was U937 cell group stimulated by LPS with different parameters (LIPUS1030) and 6090m / cm ~ (2). The supernatant and cell mass were collected and the expression of TNF- 伪 and Interleukin-8 IL-8 in each group were detected by ELISA, and the gene changes of IL-8 in real-time polymerase chain reactionation RT-PCR.2.U937 cell proliferation activity were detected by real-time polymerase chain reaction. The model was constructed with 96-well plate and divided into two groups: single U937 cell group was induced by LPS-induced LPS-mediated LPS stimulation of U937 cell group and LIPUS stimulated U937 cell group and Bay 11-7082 stimulated LPS mediated U937 cell group. Cell counting kit-8 CCK-8 was used to detect the proliferative activity of U937 cells. The CCK-8 assay solution was washed out and then cultured on the normal culture medium. The apoptosis of. U937 cells was detected again on the 7th day of the 3rd day. The model was constructed with 10cm dish and divided into two groups. After treatment, trypsin digestion and flow cytometry were used to detect apoptosis. To investigate the effect of LIPUS stimulation on the inflammatory response of U937 cells mediated by LPS. The expression of TNF- 伪, IL-1 尾, IL-6 and IL-8 was detected by RT-PCR and LPS mediated by LIPUS in the U937 cell group induced by LPS-induced LPS-induced U937 cell group (U937 cell group). The expression of TNF- 伪, IL-1 尾, IL-6 and IL-8 was detected by Elisa. The expression of the inflammatory factor TNF- 伪, IL-1 尾, IL-6 and IL-8 was detected by RT-PCR. To investigate the mechanism of LIPUS stimulation on LPS mediated inflammation of U937 cells. The expression of inflammatory factor and TLR4 gene was detected by RT-PCR in U937 cell group induced by LPS-induced LPS and LPS mediated by LPS, and the protein levels of TLR4p65p-I 魏 B 伪 and I 魏 B 伪 were detected by polyacrylamide gel electrophoresis. The nucleation of p65 was observed by confocal laser scanning microscope. The result is 1: 1. Mature U937 cells were successfully obtained and inflammatory models were established. The LPS concentration of 1 渭 g / ml LIPUS was 60 m W/cm2.2.CCK-8 and flow cytometry showed that LPs significantly inhibited the proliferation of U937 cells and promoted the apoptosis of U937 cells. LIPUS inhibited the reaction. 3. The results of Elisa and RT-PCR showed that LIPUS promoted the expression of inflammatory factors. RT-PCR showed that LIPUS inhibited the expression of inflammatory factors and TLR4. The results showed that LIPUS accelerated the transmembrane translocation of p65 of I 魏 B 伪 into the nucleus and the expression of TLR4 protein, while LIPUS blocked the phosphorylation of I 魏 B 伪 and the entry of p65 into the nucleus. However, the inhibitory effect of TLR4 on the expression of p65 protein was not obvious, and it was observed by confocal laser scanning microscopy that the effect of LIPUS on the inhibition of p65 entry was obvious. Conclusion: WLIPUS can enhance cell activity, inhibit cell apoptosis, and inhibit the phosphorylation of I 魏 B 伪 protein and p65 entry into the nucleus effectively, thus inhibiting the secretion of inflammatory factor TLR4- 魏 B(Nuclear factor-kappa BNF- 魏 B signal pathway involved in this reaction. [WT5HZ] [WT5HZ] [WT5 "BZ] [WT5BZ] LIPUS can inhibit the phosphorylation of I 魏 B 伪 protein and p65 entry into the nucleus.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

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