CD38基因干扰对H9c2心肌细胞缺氧复氧损伤的保护作用及机制研究

发布时间：2018-05-14 09:31

本文选题：CD38 + 缺氧复氧　；参考：《南昌大学》2016年硕士论文

【摘要】：背景与目的:CD38是一种多功能酶,具有ADPR环化酶和cADPR水解酶活性,它可以分别催化NAD+合成cADPR或水解cADPR为ADPR。CD38广泛分布于各种组织,具有多种生物学功能。有研究表明,CD38基因缺失可导致小鼠组织内NAD+浓度显著升高,SIRT1活性增加。SIRT1作为一个重要的NAD+依赖性的去乙酰酶可以去乙酰化激活FOXO1信号通路而发挥抗氧化应激作用。本实验室的前期工作显示,CD38基因敲除小鼠的胚胎成纤维细胞(MEFs)可显著的耐受H2O2和缺氧复氧(Hypoxia Reoxygeneation,H/R)诱导的损伤,提示CD38基因缺失可能在心肌缺血再灌注损伤中发挥重要作用,但其在心肌缺血再灌注损伤中的作用尚无报道。本文采用体外缺氧复氧模型观察CD38基因干扰对H/R诱导心肌细胞损伤的影响并探讨其分子机制,从而为临床心脏缺血性疾病的防治提供实验依据。实验方法:1.细胞缺氧复氧模型的制备:以95%N2,5%CO2混合气体通气模拟体外缺氧环境处理H9c2细胞。缺氧4小时,复氧不同时间后,通过CCK8实验检测细胞活性以明确模型制备的条件。2.细胞缺氧复氧损伤表型的检测:缺氧4h复氧3h后,流式细胞仪检测线粒体膜电位及细胞凋亡,多功能酶标仪检测H9c2细胞上清中的LDH释放量。3.氧自由基(ROS)检测:荧光探针DCFH-DA染色,流式细胞仪和荧光显微镜检测细胞缺氧复氧后ROS生成。4.通过细胞免疫荧光检测CD38基因干扰对FOXO1核转移的影响。5.利用实时荧光定量PCR检测CD38基因干扰细胞系中CD38基因转录水平干扰效率及抗氧化蛋白Catalase和SOD2在H/R处理后mRNA表达水平。6.免疫印迹Western Blot检测H/R处理后抗氧化蛋白Catalase和SOD2的蛋白表达水平。实验结果:1.心肌细胞缺氧4h,复氧不同时间后,H9c2细胞活性均表现出明显的降低,并呈现出一定的时间依赖性。但缺氧4h复氧3h即足够模拟体外的急性心肌缺血再灌注损伤。2.H/R处理后,对照组H9c2细胞出现明显的细胞活性降低,上清LDH释放增加,线粒体膜电位降低以及细胞凋亡,而CD38基因干扰可显著减轻以上细胞损伤的表型。3.CD38基因干扰可以明显减轻H/R诱导的氧自由基生成。4.CD38基因干扰可以促进FOXO1蛋白的核定位。5.CD38基因干扰促进抗氧化蛋白Catalase和SOD2的表达。结论:CD38基因干扰对缺氧复氧诱导的心肌细胞损伤具有保护作用,其可能的作用机制是:敲减CD38基因可激活心肌细胞的SIRT1/FOXO1抗氧化信号通路,促进其下游抗氧化蛋白Catalase和SOD2转录表达,从而抑制氧化应激诱导的缺氧复氧损伤。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R54

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