氧化應激在支氣管哮喘中的作用機制研究
發(fā)布時間：2019-06-22 13:34
【摘要】：目的：研究哮喘外周血單個核細胞氧化應激的情況以及核因子轉錄蛋白κB（NF-κB）和8-羥基鳥嘌呤DNA糖苷酶（OGG1）表達，探討人B淋巴母細胞在氧化應激下的細胞增殖，研究氧化應激在哮喘發(fā)病中發(fā)揮的可能作用機制。內(nèi)容與方法：第一部分：收集2011.2-2011.12瀘州醫(yī)學院附屬醫(yī)院呼吸內(nèi)科就診的哮喘患者16例，，按照支氣管哮喘GINA（全球哮喘防治創(chuàng)議）標準，按其嚴重程度分為輕中度哮喘組，重癥哮喘組（所有研究患者均處于急性發(fā)作期），正常健康體檢者作為對照。淋巴細胞分離液分離外周血單個核細胞（PBMC）備用。分別以2’，7’-二氯熒光素二乙酸酯（2’，7’-dichlorofluorescin-diacetate，DCFH-DA）法和二氫乙啶（DHE）法檢測胞內(nèi)活性氧（ROS）的產(chǎn)生情況；WST法檢測血清中SOD的表達。單細胞凝膠電泳法檢測胞內(nèi)DNA損傷的情況。采用間接熒光免疫法測定胞內(nèi)OGG1蛋白和核因子轉錄蛋白κB（NF-κB）的表達。用酶聯(lián)免疫吸附試驗（ELISA）檢測血清中腫瘤壞死因子a（TNF-a）含量。第二部分：用脂多糖（LPS）和維生素C分別刺激人B淋巴母細胞建立氧化和抗氧化細胞模型，2’，7’-二氯熒光素二乙酸酯（2’，7’-dichlorofluorescin-diacetate，DCFH-DA）法檢測細胞內(nèi)ROS的產(chǎn)生情況；總SOD活性檢測試劑盒（Total SuperoxideDismutase Assay Kit with WST-1）酶標儀檢測顯示患者血清中超氧化物歧化酶（SOD）單細胞凝膠電泳法檢測去氧核糖核酸（DNA）損傷情況；用酶聯(lián)免疫吸附試驗（ELISA）測定上清液中腫瘤壞死因子a（TNF-a）變化。用細胞增殖示蹤熒光探針(CFDA SE)法間接檢測細胞增殖的情況。結果：1.臨床試驗：1.通過熒光探針DCFH-DA和DHE探針檢測發(fā)現(xiàn)哮喘患者外周血單個核細胞內(nèi)平均熒光強度明顯高于正常對照組，重癥哮喘中熒光強度升高最為明顯。總SOD活性檢測試劑盒（Total SuperoxideDismutase Assay Kit with WST-1）酶標儀檢測顯示患者體內(nèi)超氧化物歧化酶（SOD）的表達明顯下降。重癥組中SOD表達最低；單細胞凝膠電泳激光共聚焦下觀察DNA損傷情況：哮喘患者存在DNA損傷，重癥哮喘患者DNA損傷最為明顯；間接熒光免疫法測定OGG1陽性表達結果顯示重癥哮喘組和輕中度哮喘組陽性表達率較對照組表達率明顯增高，重癥哮喘組陽性表達率最高；間接熒光免疫法測定外周血單個核細胞NF-κB表達，輕中哮喘組和重癥哮喘組較對照組明顯增高；2.細胞（人B淋巴母細胞）試驗：通過熒光探針DCFH-DA法測定，顯示LPS+維生素C刺激組較LPS刺激組ROS的產(chǎn)生減少，與正常對照組比較無明顯差別；單細胞凝膠電泳結果顯示，LPS刺激組細胞DNA損傷最為明顯，而LPS+維生素C刺激組DNA損傷程度減輕，但較正常對照組仍存在一定程度的損傷;上清液中TNF-a表達LPS組最為明顯; CFDASE法檢測細胞增殖，顯示LPS細胞增殖成劑量相關性，而LPS+維生素C可以抑制細胞增殖。結論：1.氧化應激參與了哮喘的發(fā)生發(fā)展，在哮喘疾病發(fā)病機理中占有重要的作用，且哮喘疾病嚴重度與氧化應激密切關系；氧化應激參與DNA的損傷，介導外周血單個核細胞OGG1和NF-κB的表達增加。2.LPS能夠成功建立細胞氧化應激模型，引起DNA的損傷，抗氧化能夠減輕DNA氧化損傷。氧化應激促進細胞增殖，參與上調(diào)細胞因子TNF-a表達。
[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.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R562.25
本文編號：2504628
[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.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R562.25
【共引文獻】
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