本文選題：LIPUS + LPS��； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：目的:本實驗用脂多糖(Lipopolysaccharide,LPS)誘導(dǎo)U937細胞,構(gòu)建炎癥模型。應(yīng)用低強度脈沖超聲(Low intensity pulsed ultrasound,LIPUS)處理,探討LIPUS是否對炎癥存在抑制作用,并探討其主要作用機制。方法:1.體外應(yīng)用佛波酯(Phorbol 12-myristate 13-acetate,PMA)誘導(dǎo)人巨噬細胞系U937細胞成熟。篩選LPS濃度。分組:U937細胞;不同濃度的LPS(0.05-10μg/ml)誘導(dǎo)U937細胞組。24h后收集上清液,使用酶聯(lián)免疫吸附試驗(Enzyme-linked immunosorbent assay,ELISA)檢測炎癥因子腫瘤壞死因子-α(Tumour necrosis factor-α,TNF-α)的含量。篩選LIPUS強度。分組:U937細胞組;LPS誘導(dǎo)U937細胞組;不同參數(shù)LIPUS(10,30,60,90m W/cm2)刺激LPS介導(dǎo)下的U937細胞組。收集上清液及細胞團塊,采用ELISA檢測各組TNF-α及白介素-8(Interleukin-8,IL-8)的表達情況,實時聚合酶鏈反應(yīng)檢測(Real-time polymerase chain reaction,RT-PCR)各組中IL-8的基因變化情況。2.U937細胞增殖活力檢測。采用96孔板構(gòu)建模型,分組:單獨U937細胞組;LPS誘導(dǎo)U937細胞組;LIPUS刺激LPS介導(dǎo)的U937細胞組;LIPUS刺激U937細胞組;Bay 11-7082刺激LPS介導(dǎo)的U937細胞組。應(yīng)用細胞技術(shù)試劑盒(Cell counting kit-8,CCK-8)檢測U937細胞增殖活力。洗去CCK-8檢測液,換成普通培養(yǎng)基繼續(xù)培養(yǎng),于第3,5,7天再次檢測。U937細胞凋亡檢測。采用10cm培養(yǎng)皿構(gòu)建模型,分組同上,處理后胰酶消化,流式細胞檢測儀檢測細胞凋亡情況。3.探討LIPUS刺激對LPS介導(dǎo)的U937細胞炎癥反應(yīng)的作用。分組:U937細胞組;LPS誘導(dǎo)U937細胞組;LIPUS刺激LPS介導(dǎo)的U937細胞組;LIPUS刺激U937細胞組。ELISA及RT-PCR檢測炎癥因子(TNF-α、IL-1β、IL-6及IL-8)表達情況。4.探討LIPUS刺激對LPS介導(dǎo)的U937細胞炎癥反應(yīng)的機制。分組:U937細胞組;LPS誘導(dǎo)U937細胞組;LIPUS刺激LPS介導(dǎo)的U937細胞組;Bay 11-7082刺激LPS介導(dǎo)的U937細胞組。RT-PCR檢測炎癥因子及TLR4基因表達;聚丙烯酰氨凝膠電泳技術(shù)(Western blot,WB)檢測TLR4、p65、p-IκBα和IκBα蛋白含量;激光共聚焦掃描顯微鏡觀察p65入核情況。結(jié)果:1.成功獲得成熟U937細胞并構(gòu)建炎癥模型,篩選LPS濃度為1μg/ml、LIPUS強度為60m W/cm2。2.CCK-8檢測及流式細胞儀檢測發(fā)現(xiàn),LPS明顯抑制U937細胞增殖、促進U937細胞凋亡,而LIPUS對此反應(yīng)有一定抑制作用。3.ELISA和RT-PCR檢測結(jié)果顯示:LPS促進炎癥因子的表達,而LIPUS起明顯的抑制作用。4.RT-PCR檢測示:LIPUS抑制炎癥因子以及TLR4的表達;WB檢測結(jié)果示:LPS加速了IκBα的磷酸化、p65的跨膜轉(zhuǎn)位入核以及TLR4的蛋白表達,而LIPUS阻斷了IκBα的磷酸化和p65入核的過程,但對TLR4蛋白表達的抑制作用不明顯;激光共聚焦掃描顯微鏡觀察可見:LPS促進p65入核,LIPUS抑制p65入核的效果明顯。結(jié)論:LIPUS能增強細胞活性,抑制細胞凋亡,并有效地抑制了IκBα蛋白的磷酸化和p65入核,從而抑制炎癥因子的分泌,TLR4-核轉(zhuǎn)錄因子-κB(Nuclear factor-kappa B,NF-κB)信號通路參與了該反應(yīng)。
[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- 偽 Tumour necrosis factor- 偽 (TNF- 偽) 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- 偽 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- 偽, 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- 偽, IL-1 尾, IL-6 and IL-8 was detected by Elisa. The expression of the inflammatory factor TNF- 偽, 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 偽 and I 魏 B 偽 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 偽 into the nucleus and the expression of TLR4 protein, while LIPUS blocked the phosphorylation of I 魏 B 偽 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 偽 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 偽 protein and p65 entry into the nucleus.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.4

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