【摘要】：目的隨著肥胖和代謝綜合癥的發(fā)病率不斷升高,近年來阻塞性睡眠呼吸暫停低通氣綜合癥(obstructive sleep apnea hypopnea syndrome, OSAHS)的發(fā)病率也逐年增加。該疾病以睡眠過程中反復出現(xiàn)的上氣道阻塞為主要特征。長期的低氧、高碳酸血癥和影響睡眠質量可誘發(fā)高血壓,心肌梗塞,中風等疾病。目前OSAHS發(fā)病的原因和機制還未完全闡明,哪些因素在OSAHS的發(fā)生和發(fā)展過程中影響了呼吸中樞的調節(jié)尚不明了。近來的研究發(fā)現(xiàn),下丘腦合成的一種具有重要作用的神經(jīng)肽--Orexin (OX,食欲素),參與攝食、能量代謝、內分泌、心血管活動、睡眠-覺醒等多種生理功能,最新的研究還發(fā)現(xiàn)其可能參與了中樞對呼吸活動的調節(jié),臨床研究報告指出OSAHS患者血漿中Orexin的含量顯著下降,給予患者正壓通氣治療后,不僅臨床癥狀有明顯改善,患者血漿中的Orexin水平也顯著上升了。另有動物實驗證明：Orexin基因敲除小鼠會出現(xiàn)反復的睡眠呼吸暫停的現(xiàn)象。因此本實驗利用低壓氧艙處理誘導大鼠模擬OSAHS病程,觀察慢性低壓低氧(chronic hypobaric hypoxia, CHH)模型大鼠動脈血氣、肺組織病理學、肺功能和舌下神經(jīng)放電活動的改變,以及中樞OXA的表達。并進一步觀察在正常大鼠下丘腦外側區(qū)特異性損毀Orexin神經(jīng)元后,肺功能和舌下神經(jīng)放電活動的改變。本文研究OX對慢性低壓低氧模型大鼠呼吸活動的調節(jié)作用。為進一步探討OX在OSAHS發(fā)生和發(fā)展過程中對呼吸活動的調節(jié)機制提供實驗依據(jù)。 方法1.慢性低壓低氧模型大鼠制備：本研究選用正常成年雄性Sprague-Dawley (SD)大鼠制備慢性低壓低氧模型。利用低壓氧艙對大鼠進行為期28天,每天6小時的間歇性低壓低氧處理(低壓氧艙模擬海拔高度5000米,氧分壓53.9KPa,氧濃度10%-11.2%)。并觀察大鼠在艙內和出艙后的表現(xiàn)。2.28天造模后：(1)對CHH模型大鼠進行血氣分析、肺組織病理學和肺功能測定；(2)下丘腦Orexin A神經(jīng)元的免疫組織化學染色,并進行相對光密度(relative optical density, ROD)值分析；(3)舌下神經(jīng)放電活動的記錄；(4)上氣道(喉+主支氣管)的CT斷層掃描,并對圖像進行三維立體重建,測量上氣道容積。3.特異性損毀正常大鼠下丘腦Orexin神經(jīng)元,飼養(yǎng)兩周后：(1)下丘腦尼氏染色和OrexinA免疫組織化學觀察；(2)肺功能測定；(3)舌下神經(jīng)放電活動的記錄。 結果 1.觀察到大鼠在入艙開始減壓低氧后,即出現(xiàn)呼吸加快、變深、腹式呼吸明顯,即早期的過度通氣的生理反應；六小時出艙后觀察到大鼠耳廓、足爪色澤變淺、口唇輕度紫紺等現(xiàn)象,表明其血氧飽和度可能降低。低壓低氧28天后模型組大鼠與對照組相比,血氣分析顯示：pH明顯下降,PCO2增加,PO2下降；肺組織病理學顯示：模型組大鼠的肺組織表現(xiàn)為彌漫性的充血、出血、水腫；氣道阻力明顯增大(P0.05,n=6),肺動態(tài)順應性減小(P0.05,n=6),潮氣量減小,但無統(tǒng)計學差異,表明肺的通氣功能下降。 2.與對照組相比,模型組下丘腦Orexin A神經(jīng)元的免疫組織化學染色相對光密度(ROD)值顯著升高(P0.01,n=5)。 3.低壓低氧28天后模型組大鼠與對照組相比,舌下神經(jīng)放電的頻率降低(P0.05,n=6)和幅度變小(P0.01,n=6),提示舌下神經(jīng)放電活動減弱。 4.利用重建后的三維立體圖像,測量大鼠上氣道容積,對照組與模型組相比無統(tǒng)計學差異。 5.正常SD大鼠雙側LH區(qū)注射Orexin-SAP (0.043mg/ml, 100nl/側)兩周后,尼氏染色結果顯示與生理鹽水對照組比較LH區(qū)的神經(jīng)元數(shù)量明顯減少,通過免疫組織化學實驗也觀察到LH的Orexin A神經(jīng)元數(shù)量明顯減少,僅有少量殘存。應用電生理的實驗技術,觀察到損毀組大鼠與生理鹽水對照組相比：氣道阻力明顯增大(P0.001,n=6),肺動態(tài)順應性減小(P0.001,n=6)。舌下神經(jīng)放電的頻率降低(P0.05,n=6)和幅度變小(P0.01,n=6)。 結論 1.低壓氧艙能成功制作慢性低壓低氧模型大鼠。 2.28天的慢性低壓低氧導致大鼠pH和PO2下降,CO2潴留；并引起大鼠肺的通氣功能明顯下降；舌下神經(jīng)放電的幅度減小、呼吸頻率減慢,提示舌下神經(jīng)放電活動明顯減弱；但還未造成大鼠上氣道容積的器質性改變。 3.慢性低壓低氧模型大鼠下丘腦OXA神經(jīng)元的合成加速,提示OXA可能參與慢性低壓低氧的病理過程,其在中樞的顯著增高為其參與中樞對呼吸活動的調節(jié)提供了實驗證據(jù)。 4.正常SD大鼠特異性損毀LH區(qū)Orexin神經(jīng)元后,Orexin A神經(jīng)元表達明顯減少,肺的通氣功能下降,舌下神經(jīng)放電活動減弱。提示,Orexin神經(jīng)元分泌內源性Orexin可以興奮舌下神經(jīng)、促進對上氣道的控制。 5.本文的結果提示Orexin神經(jīng)元參與了慢性低壓低氧模型大鼠OSAHS的病理生理學過程。Orexin神經(jīng)元的分泌增加,可能是為了對抗慢性低壓低氧引起的大鼠呼吸活動的減弱。
[Abstract]:Objective With the increasing incidence of obesity and metabolic syndrome, the incidence of obstructive sleep apnea-hypopnea syndrome (OSAHS) has increased year by year. The disease is mainly characterized by the repeated upper airway obstruction in the sleep process. Long-term hypoxia, hypercapnia and effects on sleep quality can induce hypertension, myocardial infarction, stroke and other diseases. The causes and mechanisms of OSAHS are not fully set out, and the factors that affect the adjustment of the respiratory center in the development and development of OSAHS are unknown. Recent studies have found that a neuropeptide--Orexin (OX, orexin), which has an important role in the synthesis of the hypothalamus, is involved in various physiological functions such as food consumption, energy metabolism, endocrine, cardiovascular activity, sleep-wakefulness, and the like, The most recent study also found that it may be involved in the regulation of respiratory activity in the central nervous system. The clinical study report indicates that the content of Orexin in the plasma of the patients with OSAHS is significantly reduced, and after the positive-pressure ventilation treatment of the patient, not only the clinical symptoms are obviously improved, and the Orexin level in the plasma of the patient also increases significantly. A further animal experiment demonstrated that repeated sleep apnea was observed in the Orexin knockout mice. In this experiment, the course of OSAHS induced by low-pressure oxygen chamber was used to study the changes of arterial blood gas, lung tissue pathology, pulmonary function and hypoglossal nerve discharge activity in rats with chronic low-pressure hypoxia (CHH) model, and the expression of central OXA. The changes of the pulmonary function and the hypoglossal nerve discharge activity were further observed following the specific destruction of the Orexin neurons in the outside of the hypothalamic area of the normal rat. In this paper, the effect of OX on respiratory activity in rats with chronic low-pressure hypoxia was studied. In order to further explore the mechanism of OX in the development and development of OSAHS, the experimental basis is provided. The method 1. The preparation of chronic low-pressure low-pressure model rats: the normal adult male Sprague-Dawley (SD) rats were selected to prepare the chronic low-pressure hypoxia model. Type. The rats were subjected to an intermittent low-pressure low-pressure treatment (low-pressure oxygen chamber at a simulated altitude of 5000 meters, an oxygen partial pressure of 53.9 KPa, and an oxygen concentration of 10-11.2%) for a period of 28 days with a low-pressure oxygen chamber. (1) The blood gas analysis, lung tissue pathology and lung function test were performed on the CHH model rats. (2) The immunohistochemical staining of the hypothalamic Orexin A neurons was performed and the relative optical density (ROD) value was measured. (3) the recording of the hypoglossal nerve discharge activity; (4) the CT scan of the upper airway (larynx + main bronchus) and the three-dimensional reconstruction of the image to measure the upper airway volume. 3. Specific damage to the hypothalamic Orexin neurons in the normal rat, after two weeks of feeding: (1) the hypothalamic Neshlet staining and the OrexinA immunohistochemical study; (2) the lung function determination; and (3) the recording of the hypoglossal nerve discharge activity. Record. Results 1. After the initial decompression of the rat into the cabin, the rats were observed to have a rapid, deep and abdominal breathing, that is, the physiological response of the early over-ventilation. After six hours of the capsule, the auricle of the rat was observed, the color of the foot claw was light, and the lips were mild. the phenomenon of purpuria and the like, indicating the saturation of the blood oxygen The degree of lung tissue in the model group was significantly lower than that in the control group. The lung tissue pathology showed that the lung tissue of the model group was diffuse congestion, hemorrhage, and edema, and the airway resistance was significantly increased (P0.05). (n = 6), the pulmonary dynamic compliance decreased (P0.05, n = 6), and the tidal volume decreased, but there was no statistical difference, indicating that the lung 2. Compared with the control group, the immunohistochemical staining of the hypothalamic Orexin A neurons in the model group was significantly higher than that of the control group (P0. 3. The frequency of hypoglossal nerve discharge decreased (P0.05, n = 6) and the amplitude (P0.01, n = 6) in the model group rats after 28 days of low-pressure and low-pressure hypoxia (P0.01, n = 6). 4. Using the reconstructed three-dimensional image, the volume of the upper airway of the rat and the control group were measured. Compared with the model group, there was no statistical difference in the model group.5. After two weeks of injection of Orexin-SAP (0.043 mg/ ml,100 nl/ side) in the bilateral LH region of the normal SD rats, the number of neurons in the LH region was significantly reduced compared with the normal saline control group, and the Ore of the LH was also observed by immunohistochemistry. xin A neuron The experimental technique of electrophysiology showed that the airway resistance increased significantly (P 0.001, n = 6) and the pulmonary dynamic compliance decreased in the damaged group compared with the normal saline control group. Small (P0.001, n = 6). The frequency of hypoglossal nerve discharge (P0.05, n = 6) and amplitude to become smaller (P0.01, n = 6). Conclusion 1. Low voltage The rats with chronic low-pressure and low-pressure hypoxia model were successfully produced by the oxygen cabin. The chronic low-pressure hypoxia of 2.28 days resulted in the decrease of pH and PO2 in the rats and the retention of CO2, and the ventilation function of the lung of the rats decreased significantly; the amplitude of the hypoglossal nerve discharge was reduced, and the respiration The frequency of the hypoglossal nerve was decreased and the hypoglossal nerve discharge activity was significantly reduced. 3. There was no organic change in the volume of the upper airway of the rat.3. The synthesis and acceleration of the OXA neurons in the hypothalamus of the rats with chronic low-pressure and low-oxygen model suggested that the OXA could be involved in the pathological process of chronic low-pressure hypoxia, which was significantly higher in the central nervous system. In order to provide experimental evidence for the adjustment of the respiratory activity in the central nervous system, the expression of the Orexin A neurons in the normal SD rats after the specific destruction of the Orexin neurons in the LH region The decrease of the ventilation function of the lung and the decrease of the hypoglossal nerve discharge activity. Rexin can stimulate the hypoglossal nerve and promote the control of the upper airway. Compared with the pathophysiological process of OSAHS in rats with chronic low-pressure hypoxia, the secretion of Orexin neurons increased,
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R766

【引證文獻】

相關碩士學位論文 前1條

1 金春艷;OREXIN在非洲鴕鳥小腸內的分布及發(fā)育性變化[D];華中農業(yè)大學;2013年

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本文編號：2508058