本文選題：CD38 + 缺氧復(fù)氧　； 參考：《南昌大學(xué)》2016年碩士論文

【摘要】：背景與目的:CD38是一種多功能酶,具有ADPR環(huán)化酶和cADPR水解酶活性,它可以分別催化NAD+合成cADPR或水解cADPR為ADPR。CD38廣泛分布于各種組織,具有多種生物學(xué)功能。有研究表明,CD38基因缺失可導(dǎo)致小鼠組織內(nèi)NAD+濃度顯著升高,SIRT1活性增加。SIRT1作為一個重要的NAD+依賴性的去乙酰酶可以去乙�；せ頕OXO1信號通路而發(fā)揮抗氧化應(yīng)激作用。本實(shí)驗(yàn)室的前期工作顯示,CD38基因敲除小鼠的胚胎成纖維細(xì)胞(MEFs)可顯著的耐受H2O2和缺氧復(fù)氧(Hypoxia Reoxygeneation,H/R)誘導(dǎo)的損傷,提示CD38基因缺失可能在心肌缺血再灌注損傷中發(fā)揮重要作用,但其在心肌缺血再灌注損傷中的作用尚無報道。本文采用體外缺氧復(fù)氧模型觀察CD38基因干擾對H/R誘導(dǎo)心肌細(xì)胞損傷的影響并探討其分子機(jī)制,從而為臨床心臟缺血性疾病的防治提供實(shí)驗(yàn)依據(jù)。實(shí)驗(yàn)方法:1.細(xì)胞缺氧復(fù)氧模型的制備:以95%N2,5%CO2混合氣體通氣模擬體外缺氧環(huán)境處理H9c2細(xì)胞。缺氧4小時,復(fù)氧不同時間后,通過CCK8實(shí)驗(yàn)檢測細(xì)胞活性以明確模型制備的條件。2.細(xì)胞缺氧復(fù)氧損傷表型的檢測:缺氧4h復(fù)氧3h后,流式細(xì)胞儀檢測線粒體膜電位及細(xì)胞凋亡,多功能酶標(biāo)儀檢測H9c2細(xì)胞上清中的LDH釋放量。3.氧自由基(ROS)檢測:熒光探針DCFH-DA染色,流式細(xì)胞儀和熒光顯微鏡檢測細(xì)胞缺氧復(fù)氧后ROS生成。4.通過細(xì)胞免疫熒光檢測CD38基因干擾對FOXO1核轉(zhuǎn)移的影響。5.利用實(shí)時熒光定量PCR檢測CD38基因干擾細(xì)胞系中CD38基因轉(zhuǎn)錄水平干擾效率及抗氧化蛋白Catalase和SOD2在H/R處理后mRNA表達(dá)水平。6.免疫印跡Western Blot檢測H/R處理后抗氧化蛋白Catalase和SOD2的蛋白表達(dá)水平。實(shí)驗(yàn)結(jié)果:1.心肌細(xì)胞缺氧4h,復(fù)氧不同時間后,H9c2細(xì)胞活性均表現(xiàn)出明顯的降低,并呈現(xiàn)出一定的時間依賴性。但缺氧4h復(fù)氧3h即足夠模擬體外的急性心肌缺血再灌注損傷。2.H/R處理后,對照組H9c2細(xì)胞出現(xiàn)明顯的細(xì)胞活性降低,上清LDH釋放增加,線粒體膜電位降低以及細(xì)胞凋亡,而CD38基因干擾可顯著減輕以上細(xì)胞損傷的表型。3.CD38基因干擾可以明顯減輕H/R誘導(dǎo)的氧自由基生成。4.CD38基因干擾可以促進(jìn)FOXO1蛋白的核定位。5.CD38基因干擾促進(jìn)抗氧化蛋白Catalase和SOD2的表達(dá)。結(jié)論:CD38基因干擾對缺氧復(fù)氧誘導(dǎo)的心肌細(xì)胞損傷具有保護(hù)作用,其可能的作用機(jī)制是:敲減CD38基因可激活心肌細(xì)胞的SIRT1/FOXO1抗氧化信號通路,促進(jìn)其下游抗氧化蛋白Catalase和SOD2轉(zhuǎn)錄表達(dá),從而抑制氧化應(yīng)激誘導(dǎo)的缺氧復(fù)氧損傷。
[Abstract]:Background and objective CD38 is a multifunctional enzyme with the activities of ADPR cyclase and cADPR hydrolase. It can catalyze the synthesis of cADPR by NAD or hydrolyze cADPR as ADPR.CD38 widely distributed in various tissues and has a variety of biological functions. It has been shown that the deletion of CD38 gene can significantly increase the activity of SIRT1 in mouse tissues. As an important NAD dependent deacetylase, it can deacetylation and activate the FOXO1 signaling pathway and play the role of antioxidant stress. Our previous work has shown that the embryonic fibroblasts of CD38 knockout mice can significantly tolerate H2O2 and hypoxia reoxygenation induced by H / R, suggesting that CD38 gene deletion may play an important role in myocardial ischemia-reperfusion injury. However, its role in myocardial ischemia reperfusion injury has not been reported. In this paper, the effects of CD38 gene interference on H / R induced cardiomyocyte injury were observed in vitro hypoxia reoxygenation model, and its molecular mechanism was discussed, thus providing experimental basis for the prevention and treatment of clinical heart ischemic diseases. Experimental method: 1. Preparation of anoxic reoxygenation model: H9c2 cells were treated with 95% N _ 2 and 5 ~ 5 CO _ 2 mixed gas ventilation under anoxic environment in vitro. After hypoxia for 4 hours and reoxygenation for different time, the cell activity was detected by CCK8 experiment to determine the conditions of model preparation. The phenotype of anoxia and reoxygenation injury: after 4 h reoxygenation for 3 h, the mitochondrial membrane potential and apoptosis were detected by flow cytometry, and the LDH release from supernatant of H9c2 cells was detected by multifunctional enzyme marker. Oxygen free radical detection: fluorescence probe DCFH-DA staining, flow cytometry and fluorescence microscopy to detect ROS production after anoxia and reoxygenation. The effect of CD38 gene interference on the nuclear metastasis of FOXO1 was detected by immunofluorescence. Real-time fluorescence quantitative PCR was used to detect the interference efficiency of CD38 gene transcription level and the mRNA expression level of Catalase and SOD2 after H / R treatment in CD38 gene interference cell lines. Western blot Western Blot was used to detect the protein expression of antioxidant protein Catalase and SOD2 after H / R treatment. The result of the experiment was 1: 1. After hypoxia for 4 h and reoxygenation for different time, the activity of H9c2 cells decreased significantly and showed a time-dependent manner. However, after hypoxia for 4 h and reoxygenation for 3 h, which was enough to simulate acute myocardial ischemia-reperfusion injury in vitro, the H9c2 cells in the control group showed a significant decrease in cell activity, increased LDH release in the supernatant, decreased mitochondrial membrane potential and apoptosis. The interference of CD38 gene can significantly attenuate the phenotype of above cell damage. 3. CD38 gene interference can significantly reduce the oxygen free radical production induced by H / R. 4. CD38 gene interference can promote the nuclear localization of FOXO1 protein .5.CD38 gene interference can promote the expression of antioxidant protein Catalase and SOD2. Conclusion the interference of CD38 gene can protect cardiomyocytes from hypoxia and reoxygenation. The possible mechanism is that knockout of CD38 gene can activate the SIRT1/FOXO1 antioxidant signaling pathway of cardiomyocytes. Promote the expression of Catalase and SOD2, and inhibit the hypoxia and reoxygenation injury induced by oxidative stress.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R54

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Maria-Giulia Perrelli;Pasquale Pagliaro;Claudia Penna;;Ischemia/reperfusion injury and cardioprotective mechanisms:Role of mitochondria and reactive oxygen species[J];World Journal of Cardiology;2011年06期

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本文編號：1887310