氧化应激在支气管哮喘中的作用机制研究

发布时间：2019-06-22 13:34

【摘要】：目的：研究哮喘外周血单个核细胞氧化应激的情况以及核因子转录蛋白κB（NF-κB）和8-羟基鸟嘌呤DNA糖苷酶（OGG1）表达，探讨人B淋巴母细胞在氧化应激下的细胞增殖，研究氧化应激在哮喘发病中发挥的可能作用机制。内容与方法：第一部分：收集2011.2-2011.12泸州医学院附属医院呼吸内科就诊的哮喘患者16例，，按照支气管哮喘GINA（全球哮喘防治创议）标准，按其严重程度分为轻中度哮喘组，重症哮喘组（所有研究患者均处于急性发作期），正常健康体检者作为对照。淋巴细胞分离液分离外周血单个核细胞（PBMC）备用。分别以2’，7’-二氯荧光素二乙酸酯（2’，7’-dichlorofluorescin-diacetate，DCFH-DA）法和二氢乙啶（DHE）法检测胞内活性氧（ROS）的产生情况；WST法检测血清中SOD的表达。单细胞凝胶电泳法检测胞内DNA损伤的情况。采用间接荧光免疫法测定胞内OGG1蛋白和核因子转录蛋白κB（NF-κB）的表达。用酶联免疫吸附试验（ELISA）检测血清中肿瘤坏死因子a（TNF-a）含量。第二部分：用脂多糖（LPS）和维生素C分别刺激人B淋巴母细胞建立氧化和抗氧化细胞模型，2’，7’-二氯荧光素二乙酸酯（2’，7’-dichlorofluorescin-diacetate，DCFH-DA）法检测细胞内ROS的产生情况；总SOD活性检测试剂盒（Total SuperoxideDismutase Assay Kit with WST-1）酶标仪检测显示患者血清中超氧化物歧化酶（SOD）单细胞凝胶电泳法检测去氧核糖核酸（DNA）损伤情况；用酶联免疫吸附试验（ELISA）测定上清液中肿瘤坏死因子a（TNF-a）变化。用细胞增殖示踪荧光探针(CFDA SE)法间接检测细胞增殖的情况。结果：1.临床试验：1.通过荧光探针DCFH-DA和DHE探针检测发现哮喘患者外周血单个核细胞内平均荧光强度明显高于正常对照组，重症哮喘中荧光强度升高最为明显。总SOD活性检测试剂盒（Total SuperoxideDismutase Assay Kit with WST-1）酶标仪检测显示患者体内超氧化物歧化酶（SOD）的表达明显下降。重症组中SOD表达最低；单细胞凝胶电泳激光共聚焦下观察DNA损伤情况：哮喘患者存在DNA损伤，重症哮喘患者DNA损伤最为明显；间接荧光免疫法测定OGG1阳性表达结果显示重症哮喘组和轻中度哮喘组阳性表达率较对照组表达率明显增高，重症哮喘组阳性表达率最高；间接荧光免疫法测定外周血单个核细胞NF-κB表达，轻中哮喘组和重症哮喘组较对照组明显增高；2.细胞（人B淋巴母细胞）试验：通过荧光探针DCFH-DA法测定，显示LPS+维生素C刺激组较LPS刺激组ROS的产生减少，与正常对照组比较无明显差别；单细胞凝胶电泳结果显示，LPS刺激组细胞DNA损伤最为明显，而LPS+维生素C刺激组DNA损伤程度减轻，但较正常对照组仍存在一定程度的损伤;上清液中TNF-a表达LPS组最为明显; CFDASE法检测细胞增殖，显示LPS细胞增殖成剂量相关性，而LPS+维生素C可以抑制细胞增殖。结论：1.氧化应激参与了哮喘的发生发展，在哮喘疾病发病机理中占有重要的作用，且哮喘疾病严重度与氧化应激密切关系；氧化应激参与DNA的损伤，介导外周血单个核细胞OGG1和NF-κB的表达增加。2.LPS能够成功建立细胞氧化应激模型，引起DNA的损伤，抗氧化能够减轻DNA氧化损伤。氧化应激促进细胞增殖，参与上调细胞因子TNF-a表达。
[Abstract]:Objective: To study the oxidative stress of mononuclear cells in peripheral blood of asthma and the expression of nuclear factor transcription factor B (NF-B) and 8-hydroxytryptin DNA glycosidase (OGG1), and to study the cell proliferation of human B lymphoblast cells under oxidative stress. To study the possible mechanism of oxidative stress in the pathogenesis of asthma. Content and method: The first part: collect 16 cases of asthma in the respiratory department of the Affiliated Hospital of Luzhou Medical College, 2011.2-2011.12. According to the standard of the GINA (Global Asthma Control), it is divided into mild and moderate asthma group according to the severity. The severe asthma group (all the study patients were in the acute attack period), and the normal health check-up was used as the control. The peripheral blood mononuclear cells (PBMC) were separated from the peripheral blood mononuclear cells (PBMC). The production of reactive oxygen (ROS) was detected in 2 ',7'-dichlorofluorescein diacetate, DCFH-DA and DHE respectively. The expression of SOD in the serum was detected by the WST method. The intracellular DNA damage was detected by single cell gel electrophoresis. The expression of the intracellular OGG1 protein and the nuclear factor transcript B (NF-EMAB) was determined by indirect fluorescence immunoassay. The tumor necrosis factor a (TNF-a) in the serum was detected by an enzyme-linked immunosorbent assay (ELISA). 2 ',7'-dichlorofluorescein diacetate (DCFH-DA) method was used to detect the production of ROS in cells. The total Superoxide Dismutase Assay Kit with WST-1 was used to detect the damage of deoxyribonucleic acid (DNA) in the serum of the patient. Tumor necrosis factor a (TNF-a) in the supernatant was determined by an enzyme-linked immunosorbent assay (ELISA). The cell proliferation was indirectly detected by a cell proliferation-tracing fluorescent probe (CFDA SE) method. Results:1. Clinical trial:1. The average fluorescence intensity of the peripheral blood mononuclear cells of the patients with asthma was significantly higher than that of the normal control group by the fluorescence probe DCFH-DA and the DHE probe, and the increase of the fluorescence intensity in the severe asthma was the most obvious. Total Superoxide Dismutase Assay Kit with WST-1 showed a significant decrease in the expression of superoxide dismutase (SOD) in the patient. The expression of SOD in severe group was the lowest. DNA damage was observed by single-cell gel electrophoresis. DNA damage in the patients with asthma and the most obvious DNA damage in the patients with severe asthma were observed. The positive expression of the positive expression of OGG1 in the severe asthma group and the mild-to-moderate asthma group was significantly higher than that of the control group, and the positive expression rate of the severe asthma group was the highest, and the indirect fluorescence immunoassay method was used to determine the expression of NF-EMAB in the peripheral blood mononuclear cells. The asthmatic group and the severe asthma group were significantly higher than those in the control group. The cells (human B lymphoblasts) were tested by means of the fluorescence probe DCFH-DA method. The results showed that the production of ROS in the LPS + vitamin C-stimulated group was less than that of the LPS-stimulated group, and there was no significant difference with the normal control group, and the single-cell gel electrophoresis showed that the DNA damage of the LPS-stimulated group was the most obvious. The degree of DNA damage in the LPS + vitamin C-stimulated group was reduced, but a certain degree of injury was still in the normal control group; the expression of TNF-a in the supernatant was the most obvious; the proliferation of the cells was detected by the CFDASE method, and the proliferation of the LPS cells was shown to be a dose-related, and the LPS + vitamin C inhibited cell proliferation. Conclusion:1. Oxidative stress is involved in the development of the asthma, plays an important role in the pathogenesis of the asthma, and the severity of the asthma disease is closely related to the oxidative stress; and the oxidative stress is involved in the DNA damage, 2. The expression of OGG1 and NF-EMAB in peripheral blood mononuclear cells was increased. Oxidative stress promotes cell proliferation and participates in up-regulation of the expression of cytokine TNF-a.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25

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