本文選題：睡眠呼吸暫停 + 間歇缺氧��； 參考：《華中科技大學》2016年博士論文

【摘要】：目的：睡眠呼吸暫停綜合征可引起患者認知障礙,但其具體機制尚不清楚。慢性間歇缺氧是睡眠呼吸暫停最重要的特征,此缺氧模型常用于對睡眠呼吸暫停綜合癥的研究。P2X7受體是一種常見于多種神經系統(tǒng)疾病的嘌呤受體,在腦組織中廣泛表達。本實驗擬研究P2X7受體在慢性間歇缺氧導致的神經損傷中的作用及其可能的通路。方法：實驗使用8周齡C57BL/6小鼠。為探究P2X7受體表達的變化,每個時間點(第3,7,14,21天)采用8只小鼠,隨機分為缺氧組(氧濃度21%～6%,40循環(huán)/小時,一天8小時)和常氧組(氧濃度21%,其余條件一致)2組。使用蛋白質印跡法和實時定量熒光PCR檢測P2X7受體。之后在為期21天的BBG (P2X7受體拮抗劑)實驗中,采用48只小鼠,隨機分為慢性間歇缺氧+生理鹽水組,慢性缺氧+BBG組,常氧+生理鹽水組,常氧+BBG組4組。使用Morris水迷宮實驗檢測小鼠的認知功能。使用蛋白質印跡法檢測NFκB和NOX2水平。使用實時定量熒光PCR測量腫瘤壞死因子α (TNF-α),白介素-1β(IL-1β), IL-18, IL-6的水平。使用相應試劑盒檢測丙二醛和超氧化物歧化酶水平。用蘇木精-伊紅染色法和TUNEL染色法檢測神經細胞損傷和凋亡。結果：實驗中發(fā)現P2X7受體mRNA于間歇缺氧3天后開始增高,持續(xù)到21天,P2X7受體蛋白水平于間歇缺氧第7天開始增高持續(xù)到21天。在BBG實驗中,慢性間歇缺氧小鼠在Morris水迷宮的表現差于其他組小鼠,表現為潛伏期更長,在平臺的穿越次數更少,目標象限停留時間更少。BBG處理后認知功能改善。慢性間歇小鼠中NFκB和NOX2升高,使用BBG后其水平下降。慢性間歇小鼠中TNF-α, IL-1β, IL-18, IL-6的水平均高于正常組。BBG處理CIH組炎癥因子的水平下降。慢性間歇小鼠中氧化應激水平均高于正常組。BBG可使MDA下降,SOD增高。慢性間歇缺氧小鼠的海馬神經細胞出現細胞腫脹,核聚縮等細胞退化,細胞凋亡的改變,BBG使細胞受損改善。結論：拮抗P2X7受體可以改善間歇缺氧導致的神經炎癥、氧化應激、神經損害和空間認知能力損傷,提示P2X7受體可能為睡眠呼吸暫停綜合征所致認知缺損的新靶點,為今后藥物的研究提供更多的選擇。目的：海馬神經損傷為睡眠呼吸暫停導致的認知損害的重要原因。阿托伐他汀是一種廣泛使用的降脂藥物,有文獻報導其能改善認知損傷。TLR4受體是一種參與天然免疫的Toll受體,近期發(fā)現其在阿爾茨海默病等多種神經疾病中發(fā)揮作用。本實驗擬研究阿托伐他汀是否能改善慢性間歇缺氧所致小鼠海馬神經細胞損傷,以及TLR4及其下游通路是否參與其中。方法：實驗使用40只8周齡C57BL/6小鼠。小鼠隨機分為慢性間歇缺氧(氧濃度21%-6%,40循環(huán)/小時,一天8小時)組,慢性間歇缺氧+阿托伐他汀組,常氧組,常氧+阿托伐他汀組4組。使用蛋白質印跡法和實時定量熒光PCR檢測TLR4受體及其下游通路蛋白MYD88和TRIF。使用酶聯免疫吸附測定法(ELISA)檢測腫瘤壞死因子a (TNF-a),白介素-1β(IL-β)。使用相應試劑盒檢測丙二醛和超氧化物歧化酶水平。使用用蘇木精-伊紅染色法和TUNEL染色法檢測神經細胞損傷和凋亡。結果：慢性間歇小鼠中TLR4受體,MYD88和TRIF水平升高,阿托伐他汀的使用降低TLR4受體及下游通路蛋白的表達。慢性間歇缺氧海馬和血清中TNF-α, IL-1β水平均高于正常組,阿托伐他汀處理使炎癥因子水平下降。慢性間歇小鼠中氧化應激水平高于正常組。阿托伐他汀可使SOD增高,MDA下降。慢性間歇缺氧小鼠的海馬神經細胞出現細胞腫脹,核聚縮等細胞退化的改變,神經細胞凋亡增加,阿托伐他汀使細胞受損改善。結論：阿托伐他汀可以改善慢性間歇缺氧所致海馬神經損傷,其作用可能涉及TLR4/MYD88和TLR4/TRIF通路。目的：海馬神經損傷為睡眠呼吸暫停導致認知損害的主要原因。TLR2受體是-種在腦缺血等多種神經疾病中發(fā)揮重要作用的I型受體,在OSAS患者中表達亦有增高。鄰香草醛為新近發(fā)現的TLR2通路抑制劑,且具有抗氧化等作用。本實驗擬研究鄰香草醛是否能通過抑制TLR2通路改善慢性間歇缺氧所致小鼠海馬神經細胞損傷。方法：實驗使用40只8周齡C57BL/6小鼠。小鼠隨機分為慢性間歇缺氧(氧濃度21%-6%,40循環(huán)/小時,一天8小時)組,慢性間歇缺氧+鄰香草醛組,常氧組,常氧+鄰香草醛組4組。使用免疫共沉淀和蛋白質印跡法檢測TLR2受體與其下游通路蛋白MYD88相互作用,使用蛋白質印跡法檢測NFκB激活狀態(tài)。使用酶聯免疫吸附測定法檢(ELISA)測腫瘤壞死因子α (TNF-α),白介素-1β(IL-β)。使用相應試劑盒檢測丙二醛和超氧化物歧化酶水平。使用用免疫組織化學方法檢測小膠質細胞的表達,使用TUNEL染色法檢測細胞凋亡。結果：慢性間歇小鼠中TLR2/MYD88通路被激活,鄰香草醛的使用抑制TLR2通路激活及下游NFkB的表達。慢性間歇缺氧海馬和血清中TNF-α, IL-1β水平均高于正常組,鄰香草醛的處理使炎癥因子水平下降。慢性間歇小鼠中氧化應激水平高于正常組。鄰香草醛可使MDA下降,SOD增高。慢性間歇缺氧小鼠的海馬小膠質細胞聚集增加,活化程度較高,神經細胞出現細胞腫脹,核聚縮等細胞退化的改變,神經細胞凋亡增加,鄰香草醛可以減少小膠質細胞的聚集,減輕神經細胞凋亡。結論：鄰香草醛可以改善慢性間歇缺氧所致海馬神經損傷,其作用涉及TLR2/MYD88通路。
[Abstract]:Objective: sleep apnea syndrome can cause cognitive impairment, but its specific mechanism is not clear. Chronic intermittent hypoxia is the most important feature of sleep apnea. This anoxia model is commonly used in the study of sleep apnea syndrome. The.P2X7 receptor is a purinine receptor common in a variety of nervous system diseases and in the brain tissue. This experiment aims to study the role and possible pathway of P2X7 receptor in the nerve injury caused by chronic intermittent hypoxia. Methods: the experiment used 8 weeks old C57BL/6 mice. To explore the changes of the expression of P2X7 receptor, 8 mice were used at each time point (day 3,7,14,21) and randomly divided into hypoxia group (oxygen concentration 21% ~ 6%, 40 circulation / small) In the 2 groups, 8 hours a day) and the normal oxygen group (the oxygen concentration 21%, the rest of the conditions). The P2X7 receptor was detected by Western blot and real-time quantitative fluorescence PCR. In the 21 day BBG (P2X7 receptor antagonist) experiment, 48 mice were randomly divided into chronic intermittent oxygen deficiency + physiological saline group, chronic hypoxia +BBG group, and normal oxygen + physiological saline group. The 4 groups of the normal oxygen +BBG group. The cognitive function of mice was detected by the Morris water maze test. The level of NF kappa B and NOX2 was detected by Western blot. The level of tumor necrosis factor alpha (TNF- a), interleukins -1 beta (IL-1 beta), IL-18, IL-6 were measured by real-time quantitative fluorescent PCR. The level of malondialdehyde and superoxide dismutase was detected by the corresponding kit. Results: the hematoxylin eosin staining and TUNEL staining were used to detect nerve cell damage and apoptosis. Results: in the experiment, the P2X7 receptor mRNA began to increase after 3 days of intermittent hypoxia, and the P2X7 receptor protein level began to increase to 21 days at seventh days of intermittent hypoxia. In the BBG experiment, the chronically intermittent anoxic mice were in the Morris water maze table. It is worse than the other groups, showing longer latency, fewer crossing times on the platform, less time for the target quadrant and less.BBG treatment. The level of NF kappa B and NOX2 in chronic intermittent mice and the decrease in BBG level. The level of TNF- alpha, IL-1 beta, IL-18 and IL-6 in chronic intermittent mice is higher than that of.BBG CIH in the normal group. The level of inflammatory factors decreased in the group of chronic intermittent mice. The average oxidative stress in the chronic intermittent mice was higher than that of the normal group.BBG, and the MDA decreased and the SOD increased. The hippocampal neurons in the chronic intermittent hypoxia mice showed cell swelling, the degeneration of nuclear polycondensation, the change of cell apoptosis, and the improvement of the cell damage by BBG. Conclusion: the antagonistic P2X7 receptor can improve the interval. Anoxia induced neuroinflammation, oxidative stress, nerve damage, and spatial cognitive impairment suggest that P2X7 receptor may be a new target for cognitive impairment induced by sleep apnea syndrome, and provides more options for future research on drugs. Atorvastatin is a widely used lipid lowering drug. It has been reported that it can improve the cognitive impairment.TLR4 receptor is a kind of Toll receptor involved in natural immunity. It has recently been found to play a role in a variety of neurodiseases such as Alzheimer's disease. Nerve cell injury, and whether TLR4 and its downstream pathways were involved. Methods: 40 8 weeks old C57BL/6 mice were used in the experiment. The mice were randomly divided into groups of chronic intermittent hypoxia (oxygen concentration 21%-6%, 40 circulation / hour, 8 hours a day), chronic intermittent hypoxia + atorvastatin group, normooxy group, and oxygen + atorvastatin group 4 groups. Detection of TLR4 receptor and its downstream pathway protein MYD88 and TRIF. using enzyme linked immunosorbent assay (ELISA) for detection of tumor necrosis factor A (TNF-a) and interleukin -1 beta (IL- beta) by real-time quantitative fluorescence PCR. Detection of malondialdehyde and superoxide dismutase by corresponding kits. The use of hematoxylin eosin staining and TUNEL staining Results: the levels of TLR4 receptor, MYD88 and TRIF increased in chronic intermittent mice. The use of atorvastatin reduced the expression of TLR4 receptor and downstream pathway protein. The level of TNF- alpha and IL-1 beta in the hippocampus and serum of chronic intermittent hypoxia was higher than that in the normal group. The level of inflammatory factors decreased in the treatment of atorvastatin. The level of oxidative stress in the mice was higher than that in the normal group. Atorvastatin increased the SOD and MDA. The hippocampal neurons in the chronic intermittent hypoxia mice showed cell swelling, the degeneration of nuclear polycondensation, the increased apoptosis of the nerve cells, and the improvement of the atorvastatin. Conclusion: atorvastatin could improve the chronic intermittent deficiency. The effects of oxygen induced hippocampal nerve damage may involve TLR4/MYD88 and TLR4/TRIF pathways. Objective: hippocampal nerve injury is the main cause of cognitive impairment caused by sleep apnea, the.TLR2 receptor is a type of I receptor that plays an important role in a variety of neurological diseases such as cerebral ischemia. The expression is also elevated in OSAS patients. The recent discovery of TLR2 pathway inhibitor and antioxidant activity. This experiment is to investigate whether ortho vanillin can improve the damage of hippocampal neurons in mice induced by chronic intermittent hypoxia by inhibiting the TLR2 pathway. Methods: 40 8 weeks old C57BL/6 mice were used. The mice were randomly divided into chronic intermittent hypoxia (oxygen concentration 21%-6%, 40 cycle / hour). 8 hours a day) group, chronic intermittent hypoxia + ortho vanillin group, normooxy group, normoxic Oxal group 4 groups. The interaction between TLR2 receptor and downstream pathway protein MYD88 was detected by immunoprecipitation and Western blot, and the activation state of NF kappa B was detected by Western blot. The cause of cancer death was measured by enzyme linked immunosorbent assay (ELISA). Zi (TNF- alpha), interleukin -1 beta (IL- beta). Use the corresponding kits to detect the level of malondialdehyde and superoxide dismutase. The use of immunohistochemistry to detect the expression of microglia and the detection of apoptosis by TUNEL staining. Results: the TLR2/ MYD88 pathway was activated in chronic intermittent mice and the use of ortho vanillin inhibited the TLR2 pathway. The level of TNF- A and IL-1 beta in the hippocampus and serum of chronic intermittent hypoxia was higher than that in the normal group. The treatment of ortho vanillin decreased the level of inflammatory factors in chronic intermittent hypoxia. The level of oxidative stress in chronic intermittent mice was higher than that in the normal group. The MDA decreased and the SOD increased. The microglia in the hippocampus of mice with slow intermittent hypoxia could accumulate microglia. The increase of the set, the higher activation degree, the cell swelling of the nerve cells, the degeneration of the nuclear polycondensation, the increase of the apoptosis of the nerve cells, the reduction of the aggregation of microglia and the reduction of the apoptosis of the nerve cells. Conclusion: ortho vanillin can improve the damage of the hippocampal nerve caused by chronic intermittent oxygen deficiency, and its effect involves the TLR2/MYD88 pass. Road.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R766

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