本文選題：SIRT1 + 急性呼吸窘迫綜合征��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：背景與目的:ARDS是以嚴(yán)重呼吸窘迫,進(jìn)行性呼吸衰竭為主要表現(xiàn)的臨床急、危重癥[1]。其病理特征為肺泡上皮屏障和肺毛細(xì)血管內(nèi)皮屏障的彌漫性損害,導(dǎo)致蛋白質(zhì)滲漏、肺水腫、炎性細(xì)胞因子浸潤(rùn)及透明膜形成等[1、6]。直接或間接的肺損傷均引起炎癥細(xì)胞的活化及炎癥介質(zhì)的大量釋放,繼而觸發(fā)炎癥級(jí)聯(lián)反應(yīng),導(dǎo)致全身炎癥失控[4]。臨床上ARDS患者即使有相似的發(fā)病因素、治療方案,其發(fā)展為ARDS的概率,炎癥程度,進(jìn)展情況及預(yù)后也存在差異。因此深入研究與ARDS的炎癥發(fā)生密切相關(guān)的基因?qū)?duì)ARDS的防治提供新的方向。SIRT1是Ⅲ類(lèi)組蛋白去乙�；�(histone deacetylase,HDAC),近來(lái)研究證實(shí),SIRT1可通過(guò)對(duì)NF-κB、FOXO3、p53等組蛋白和非組蛋白的脫乙酰基作用,啟動(dòng)炎癥的發(fā)生發(fā)展[7、11]。但SIRT1對(duì)炎癥的作用是雙向的,SIRT1可以抑制炎癥因子的活化,持續(xù)增高的水平又會(huì)出現(xiàn)免疫抑制并造成死亡率增加,SIRT1對(duì)炎癥的作用可因環(huán)境的不同而不同[15-17]。疾病階段、作用靶點(diǎn)的差異等,SIRT1對(duì)機(jī)體的炎癥調(diào)控及疾病轉(zhuǎn)歸可能都存在差異。而SIRT1對(duì)ARDS的炎癥反應(yīng)究竟有著何種作用尚不清楚,而p38 MAPK信號(hào)通路作為ARDS的主要炎癥調(diào)控途徑,因此推測(cè)p38 MAPK信號(hào)通路可能參與SIRT1對(duì)ARDS的炎癥作用。因此本課題擬運(yùn)用SIRT1+/-基因敲減小鼠和野生型小鼠,在不同SIRT1基因背景下,用LPS建立ARDS模型,評(píng)估SIRT1基因減弱對(duì)肺損傷炎癥程度的影響,以及觀察肺組織中p38 MAPK通路信號(hào)分子表達(dá)的變化,從而初步探討SIRT1對(duì)LPS致ARDS炎癥發(fā)生發(fā)展的具體作用及其可能機(jī)制。方法:1.本研究采用SIRT1+/-基因敲減小鼠作為實(shí)驗(yàn)動(dòng)物,進(jìn)行大量配對(duì)繁殖,用PCR法進(jìn)行基因型鑒定,篩選出足夠數(shù)量的SIRT1+/-小鼠,用于后續(xù)實(shí)驗(yàn)研究。2.運(yùn)用SIRT1+/-基因敲減和野生型小鼠,使用q RT-PCR和Western Blot檢測(cè)兩種小鼠肺組織中SIRT1表達(dá)的差異。3.用LPS腹腔注射法建立ARDS模型,觀察兩種不同SIRT1基因背景小鼠的肺組織病理形態(tài)學(xué)變化,測(cè)定肺濕干比(W/D比),進(jìn)行支氣管肺泡灌洗液(BALF)的白細(xì)胞計(jì)數(shù),BCA法測(cè)定BALF中總蛋白濃度,ELISA法檢測(cè)BALF及血漿中炎癥因子TNF-ɑ、IL-6水平,Western Blot及免疫組化法檢測(cè)兩種小鼠肺組織中p-p38MAPK、p-ATF2的表達(dá)變化。結(jié)果:1.SIRT1+/-基因敲減雜合子小鼠(HET)與野生型小鼠(WT)相比,其肺組織SIRT1在m RNA和蛋白表達(dá)均顯著降低(P0.01)。2.HET+LPS組與WT+LPS組分別與各自生理鹽水對(duì)照組相比,病理形態(tài)學(xué)觀察均表現(xiàn)為肺組織的明顯損傷,肺損傷病理評(píng)分顯著增高(P0.05),而HET+LPS組中肺組織結(jié)構(gòu)的破壞更嚴(yán)重,肺損傷病理評(píng)分進(jìn)一步增加(P0.05);HET+LPS組與WT+LPS組相比于對(duì)照組,肺W/D比,BALF中白細(xì)胞總數(shù)、總蛋白濃度,BALF及血漿中炎癥因子TNF-ɑ、IL-6水平均顯著增加(P0.05),而HET+LPS組與WT+LPS組相比,上述指標(biāo)的增加更為顯著(P0.05)。3.HET+LPS組與WT+LPS組分別和各自對(duì)照組相比,肺組織中p-p38MAPK、p-ATF2的表達(dá)增加(P0.05),而HET+LPS組與WT+LPS組相比,上述蛋白的增加更明顯(P0.05)。結(jié)論:1.SIRT1+/-小鼠肺組織中的SIRT1表達(dá)顯著降低,SIRT1表達(dá)的差異為后續(xù)實(shí)驗(yàn)提供了必要的前提和有效的保障。2.LPS致ARDS的疾病模型中,SIRT1+/-小鼠與野生型小鼠相比,表現(xiàn)為更嚴(yán)重的炎癥反應(yīng),提示SIRT1敲減對(duì)LPS致ARDS小鼠有著促炎作用,SIRT1在LPS致ARDS的炎癥反應(yīng)過(guò)程中起著保護(hù)作用。3.LPS致ARDS的疾病模型中,SIRT1+/-小鼠肺組織中p-p38MAPK、p-ATF2的表達(dá)與野生型小鼠相比明顯增加,提示p38 MAPK-p-ATF2信號(hào)通路可能參與了SIRT1對(duì)LPS致ARDS的炎癥調(diào)控作用。
[Abstract]:Background and purpose: ARDS is an acute clinical manifestation of severe respiratory distress and progressive respiratory failure. The pathological features of critical [1]. are diffuse impairment of the alveolar epithelial barrier and the barrier of pulmonary capillary endothelium, resulting in protein leakage, pulmonary edema, inflammatory cytokines infiltration, and the formation of transparent membrane and other direct or indirect lung lesions, such as [1,6].. The injury causes the activation of inflammatory cells and the massive release of the inflammatory mediators, and then triggers the inflammatory cascade reaction, which leads to the [4]. patients with systemic inflammation, even if there are similar pathogenesis, treatment schemes, the probability of developing ARDS, the degree of inflammation, progress and preconditioning. Therefore, the study and the inflammation of the ARDS are deeply studied. Closely related genes will provide a new direction for the prevention and control of ARDS..SIRT1 is class III histone deacetylase (histone deacetylase, HDAC). Recent studies have confirmed that SIRT1 can initiate the occurrence and development of inflammation by the deacetylation of NF- kappa B, FOXO3, p53 and other histones, but the effect of SIRT1 on inflammation is bidirectional. SIRT1 can inhibit the activation of inflammatory factors, the level of sustained increased levels of immunosuppression and the increase in mortality, the effect of SIRT1 on inflammation can be different in the [15-17]. disease stage, the difference of target target, and the difference in the regulation of inflammation and the outcome of the disease by SIRT1, and SIRT1 to ARDS What is the role of the disease response is not clear, and the p38 MAPK signaling pathway is the main regulation of inflammation in ARDS, so it is presumed that the p38 MAPK signaling pathway may be involved in the inflammatory effect of SIRT1 on ARDS. Therefore, we should use SIRT1+/- knockout mice and wild mice to establish ARDS mode with LPS in different SIRT1 background. To assess the effect of SIRT1 gene weakening on the degree of inflammation in lung injury, and to observe the changes in the expression of signal molecules in the p38 MAPK pathway in lung tissue, and to explore the specific role and possible mechanism of SIRT1 on the development of LPS induced ARDS inflammation. Methods: 1. studies were conducted with SIRT1+/- gene knockout mice as experimental animals. For reproduction, PCR method was used to identify the genotypes and to screen out a sufficient number of SIRT1+/- mice. For the follow-up experiment,.2. was used to use SIRT1+/- gene knockout and wild type mice. Q RT-PCR and Western Blot were used to detect the difference of SIRT1 expression in the lung tissues of two mice..3. LPS abdominal injection was used to establish ARDS model, and two different kinds of bases were observed. The pulmonary wetness and dry ratio (W/D ratio), the white cell count of bronchoalveolar lavage fluid (BALF), the total protein concentration in BALF were measured by the BCA method. The ELISA method was used to detect the BALF and the inflammatory factor TNF- in the plasma, the IL-6 level, Western Blot and immunohistochemistry were used to detect the p-p38MAPK in the lung tissues of two mice. P-A Results: the expression of TF2 in the 1.SIRT1+/- gene knockout heterozygote mice (HET) and the wild type mice (WT), the expression of SIRT1 in the lung tissue was significantly decreased (P0.01) in the m RNA and protein (P0.01) in the.2.HET+LPS group and the WT+LPS group, compared with the normal saline control group, the pathological morphologic observation all showed the obvious injury of the lung tissue and the lung injury disease. The lung tissue structure in HET+LPS group was significantly increased (P0.05), but the damage of lung tissue structure was more serious and the pathological score of lung injury was further increased (P0.05). Compared with the control group, the HET+LPS group was compared with the control group, the W/D ratio in the lung, the total white blood cells in the BALF, the total protein concentration, the BALF and the IL-6 water increased significantly (P0.05), while the HET+LPS group was with the WT+LPS. Compared with the +LPS group, the above index increased significantly (P0.05) the expression of p-p38MAPK and p-ATF2 increased in the lung tissue compared with the WT+LPS group and the control group (P0.05), while the increase of the above protein was more obvious in the group HET+LPS than in the WT+LPS group (P0.05). Conclusion: the SIRT1 expression in the lung tissue of the 1.SIRT1+/- mice decreased significantly. The difference in expression provides the necessary premise for the follow-up experiment and the effective guarantee of the disease model of.2.LPS induced ARDS. The SIRT1+/- mice are more severe inflammatory response compared with the wild type mice, suggesting that the SIRT1 knockout has the proinflammatory effect on the ARDS mice induced by LPS, and the SIRT1 plays a protective role in the process of ARDS's inflammation induced by LPS.3.LPS. In the ARDS disease model, the expression of p-p38MAPK and p-ATF2 in the lung tissue of SIRT1+/- mice increased significantly compared with the wild type mice, suggesting that the p38 MAPK-p-ATF2 signaling pathway may be involved in the regulation of SIRT1 to LPS induced ARDS.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R563.8

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相關(guān)期刊論文 前1條

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本文編號(hào)：2000692