本文選題：動(dòng)脈粥樣硬化 + 地高辛　； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：第一部分地高辛對(duì)動(dòng)脈粥樣硬化形成的影響目的：體外實(shí)驗(yàn)證實(shí)地高辛可以顯著抑制炎癥反應(yīng),同時(shí)影響脂質(zhì)代謝。然而,地高辛對(duì)動(dòng)脈粥樣硬化是否有影響,一直以來(lái)缺少相關(guān)的研究報(bào)道。本研究的目的是通過(guò)ApoE-/-動(dòng)脈粥樣硬化小鼠來(lái)研究地高辛對(duì)動(dòng)脈粥樣硬化斑塊形成的影響。方法：隨機(jī)分組,每組8只ApoE-/-小鼠,分為對(duì)照組、低劑量地高辛組、高劑量地高辛組,給予高脂飼料Western-type diet (WD,含有0.15%膽固醇和21%脂肪)。每天監(jiān)測(cè)小鼠體重,根據(jù)小鼠的體重給予相應(yīng)劑量的地高辛干預(yù)。其中對(duì)照組腹腔注射相應(yīng)體積的PBS；第二組為低劑量組,腹腔注射注射(i.p.)地高辛接近20μg(1mg/kg perday);第三組為高劑量組,腹腔注射(i.p.)地高辛接近40μg (2 mg/kg per day),高脂喂養(yǎng)12周。在12周結(jié)束時(shí),同一天將所有小鼠戊巴比妥鈉(50 mg/kg)腹腔注射麻醉、眶靜脈采血后,快速頸椎脫臼法執(zhí)行安樂(lè)死,分離脾臟用于流式實(shí)驗(yàn),主動(dòng)脈、肝臟、血樣本保存在-80℃的冰箱中進(jìn)一步分析。通過(guò)地高辛干預(yù)12周后,通過(guò)主動(dòng)脈大體油紅O染色以及主動(dòng)脈瓣油紅O染色斑塊定量分析,評(píng)價(jià)地高辛對(duì)動(dòng)脈粥樣硬化斑塊形成的影響。檢測(cè)血漿中總膽固醇、低密度脂蛋白膽固醇、甘油三脂、高密度脂蛋白膽固醇的水平以及炎癥因子的表達(dá),為相關(guān)機(jī)制研究提供理論依據(jù)。結(jié)果：地高辛劑量依賴性的降低了動(dòng)脈粥樣化斑塊和血漿中脂質(zhì)水平(總膽固醇減少41%,甘油三酯降低了54%,低密度膽固醇降低了20%,對(duì)照組與高劑量地高辛組相比)。另外,地高辛顯著降低了白介素(IL)-17A的水平以及1L-17A相關(guān)的炎癥反應(yīng),同時(shí)增加調(diào)節(jié)性T細(xì)胞(Tregs)的比例。結(jié)論：我們的實(shí)驗(yàn)數(shù)據(jù)證實(shí),地高辛作為維甲酸相關(guān)核孤兒受體γ的阻斷劑,通過(guò)降低血脂和IL-17A相關(guān)的炎癥反應(yīng),顯著抑制了動(dòng)脈粥樣硬化斑塊的形成。第二部分地高辛對(duì)動(dòng)脈粥樣硬化斑塊穩(wěn)定性的影響目的：動(dòng)脈粥樣硬化斑塊的的穩(wěn)定性在冠心病事件的發(fā)生中起著重要作用,其穩(wěn)定性易受免疫反應(yīng)和炎癥反應(yīng)的影響,前期研究證實(shí)地高辛影響著白介素(IL)-17A的水平以及IL-17A相關(guān)炎癥反應(yīng),因此本研究的目的,評(píng)價(jià)地高辛對(duì)動(dòng)脈粥樣斑塊的穩(wěn)定性的影響。方法：隨機(jī)分組每組8只ApoE-/-小鼠，分為對(duì)照組、低劑量地高辛組、高劑量地高辛組,給予Western-type diet (WD,含有0.15%膽固醇和21%脂肪),三組分別進(jìn)行不同的干預(yù),其中對(duì)照組腹腔注射相應(yīng)體積的PBS,低劑量組每天腹腔注射1 mg/kg地高辛,高劑量組每天腹腔注射2mg/kg地高辛,12周后通過(guò)免疫組化對(duì)斑塊內(nèi)巨噬細(xì)胞、平滑肌細(xì)胞、膠原以及CD4+T細(xì)胞的變化檢測(cè),對(duì)動(dòng)脈粥樣硬化斑塊穩(wěn)定性做出評(píng)估。采用RT-PCR檢測(cè)主動(dòng)脈中IL-17A相關(guān)因子TNF-α,IL-1β, MCP-1, IFN-γ,MMP-2和MMP-9的mRNA水平變化。結(jié)果：地高辛劑量依賴性的加強(qiáng)了動(dòng)脈粥樣硬化斑塊的穩(wěn)定性,顯著降低CD68+陽(yáng)性的巨噬細(xì)胞,斑塊纖維帽中α-SMA+陽(yáng)性的平滑肌細(xì)胞和Collagen+顯著增加與對(duì)照組相比,同時(shí)在地高辛治療組斑塊中CD4+T細(xì)胞顯著降低。采用RT-PCR檢測(cè)IL-17A相關(guān)因子mRNA的表達(dá),發(fā)現(xiàn)在主動(dòng)脈中地高辛治療組劑量依賴性的降低了TNF-α,IL-1β,MCP-1,IFN-γ,MMP-2和MMP-9的mRNA水平。結(jié)論：與對(duì)照組相比,地高辛治療組通過(guò)抑制斑塊中IL-17A相關(guān)的炎癥反應(yīng)以及增加了斑塊纖維帽中膠原、平滑肌細(xì)胞,增強(qiáng)動(dòng)脈粥樣硬化斑塊的穩(wěn)定性。
[Abstract]:Part 1 the effect of digoxin on the formation of atherosclerosis: in vitro experiments have demonstrated that digoxin can significantly inhibit inflammatory response and affect lipid metabolism. However, the effect of digoxin on atherosclerosis has been lacking related research reports. The aim of this study was to pass ApoE-/- atherosclerosis. The effects of digoxin on the formation of atherosclerotic plaque. Methods: randomly divided into groups of 8 ApoE-/- mice in each group, divided into control group, low dose digoxin group, high dose digoxin group, high fat diet Western-type diet (WD, containing 0.15% cholesterol and 21% fat). The control group was treated with digoxin. The control group was intraperitoneally injected with the corresponding volume of PBS; the second group was the low dose group, the intraperitoneal injection (i.p.) of digoxin was close to 20 g (1mg/kg perday); the third group was the high dose group, the peritoneal injection (i.p.) of digoxin was close to 40 mu (2 mg/kg per day), and the high fat was fed for 12 weeks. At the end of 12 weeks, all of them would be on the same day. The mice were intraperitoneally injected with pentobarbital sodium (50 mg/kg). After the orbital vein was collected, the method of rapid cervical dislocation was performed to perform euthanasia and the spleens were separated for flow test. The aorta, liver and blood samples were further analyzed in the refrigerator of -80 C. After 12 weeks of digoxin intervention, the aorta was stained with gross oil red O and the aortic valve oil red O staining. Quantitative analysis of color plaques to evaluate the effect of digoxin on the formation of atherosclerotic plaque. The detection of plasma total cholesterol, low density lipoprotein cholesterol, glycerol three fat, high density lipoprotein cholesterol level and the expression of inflammatory factors provide the rationale for the study of related mechanisms. Results: the dose dependence of digoxin is reduced. Lipid levels in atherosclerotic plaque and plasma (total cholesterol decreased by 41%, triglycerides decreased by 54%, low density cholesterol decreased by 20%, compared with the high dose digoxin group). In addition, digoxin significantly decreased the level of IL -17A and the inflammatory response in 1L-17A phase, and increased the ratio of regulatory T cells (Tregs). Conclusion: our experimental data confirm that digoxin, as a blocker of retinoic acid related nuclear orphan receptor gamma, significantly inhibits the formation of atherosclerotic plaque by lowering blood lipid and IL-17A related inflammatory responses. Second the effects of partial digoxin on atherosclerotic plaque stability: atherosclerotic plaques Stability plays an important role in the occurrence of coronary heart disease. Its stability is susceptible to the effects of immune response and inflammatory response. Previous studies have confirmed that digoxin affects the level of IL -17A and the IL-17A related inflammatory response. Method: 8 ApoE-/- mice in each group were divided into two groups: control group, low dose digoxin group, high dose digoxin group, Western-type diet (WD, 0.15% cholesterol and 21% fat). The control group was injected with the corresponding volume of PBS, and the low dose group was injected with 1 mg/kg digoxin every day, high dose. The dose group was intraperitoneally injected with 2mg/kg digoxin every day. After 12 weeks, the changes of macrophages, smooth muscle cells, collagen and CD4+T cells were detected by immunohistochemistry, and the stability of atherosclerotic plaque was evaluated. RT-PCR was used to detect the mRNA levels of IL-17A related factors TNF- alpha, IL-1 beta, MCP-1, IFN- gamma, MMP-2, and MMP-9. Results: the dose-dependent manner of digoxin enhanced the stability of atherosclerotic plaques, significantly reduced CD68+ positive macrophages, and a significant increase in the -SMA+ positive smooth muscle cells and Collagen+ in the plaque fibrous cap compared with the control group, while the CD4+T cells in the plaque of the digoxin treatment group were significantly reduced. RT-PCR was used to detect I. The expression of L-17A related factor mRNA showed that the dose-dependent reduction of TNF- alpha, IL-1 beta, MCP-1, IFN- gamma, MMP-2 and MMP-9 in the aortic digoxin treatment group was in a dose-dependent manner. Conclusion: compared with the control group, the digoxin treatment group could increase the collagen and smooth muscle of the plaque in the plaque of the plaque by inhibiting the IL-17A related inflammation in the plaque. Cells enhance the stability of atherosclerotic plaque.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R543.5

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