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Orexin对慢性低压低氧模型大鼠呼吸活动的调节及机制研究

发布时间:2019-06-29 19:43
【摘要】:目的随着肥胖和代谢综合症的发病率不断升高,近年来阻塞性睡眠呼吸暂停低通气综合症(obstructive sleep apnea hypopnea syndrome, OSAHS)的发病率也逐年增加。该疾病以睡眠过程中反复出现的上气道阻塞为主要特征。长期的低氧、高碳酸血症和影响睡眠质量可诱发高血压,心肌梗塞,中风等疾病。目前OSAHS发病的原因和机制还未完全阐明,哪些因素在OSAHS的发生和发展过程中影响了呼吸中枢的调节尚不明了。近来的研究发现,下丘脑合成的一种具有重要作用的神经肽--Orexin (OX,食欲素),参与摄食、能量代谢、内分泌、心血管活动、睡眠-觉醒等多种生理功能,最新的研究还发现其可能参与了中枢对呼吸活动的调节,临床研究报告指出OSAHS患者血浆中Orexin的含量显著下降,给予患者正压通气治疗后,不仅临床症状有明显改善,患者血浆中的Orexin水平也显著上升了。另有动物实验证明:Orexin基因敲除小鼠会出现反复的睡眠呼吸暂停的现象。因此本实验利用低压氧舱处理诱导大鼠模拟OSAHS病程,观察慢性低压低氧(chronic hypobaric hypoxia, CHH)模型大鼠动脉血气、肺组织病理学、肺功能和舌下神经放电活动的改变,以及中枢OXA的表达。并进一步观察在正常大鼠下丘脑外侧区特异性损毁Orexin神经元后,肺功能和舌下神经放电活动的改变。本文研究OX对慢性低压低氧模型大鼠呼吸活动的调节作用。为进一步探讨OX在OSAHS发生和发展过程中对呼吸活动的调节机制提供实验依据。 方法1.慢性低压低氧模型大鼠制备:本研究选用正常成年雄性Sprague-Dawley (SD)大鼠制备慢性低压低氧模型。利用低压氧舱对大鼠进行为期28天,每天6小时的间歇性低压低氧处理(低压氧舱模拟海拔高度5000米,氧分压53.9KPa,氧浓度10%-11.2%)。并观察大鼠在舱内和出舱后的表现。2.28天造模后:(1)对CHH模型大鼠进行血气分析、肺组织病理学和肺功能测定;(2)下丘脑Orexin A神经元的免疫组织化学染色,并进行相对光密度(relative optical density, ROD)值分析;(3)舌下神经放电活动的记录;(4)上气道(喉+主支气管)的CT断层扫描,并对图像进行三维立体重建,测量上气道容积。3.特异性损毁正常大鼠下丘脑Orexin神经元,饲养两周后:(1)下丘脑尼氏染色和OrexinA免疫组织化学观察;(2)肺功能测定;(3)舌下神经放电活动的记录。 结果 1.观察到大鼠在入舱开始减压低氧后,即出现呼吸加快、变深、腹式呼吸明显,即早期的过度通气的生理反应;六小时出舱后观察到大鼠耳廓、足爪色泽变浅、口唇轻度紫绀等现象,表明其血氧饱和度可能降低。低压低氧28天后模型组大鼠与对照组相比,血气分析显示:pH明显下降,PCO2增加,PO2下降;肺组织病理学显示:模型组大鼠的肺组织表现为弥漫性的充血、出血、水肿;气道阻力明显增大(P0.05,n=6),肺动态顺应性减小(P0.05,n=6),潮气量减小,但无统计学差异,表明肺的通气功能下降。 2.与对照组相比,模型组下丘脑Orexin A神经元的免疫组织化学染色相对光密度(ROD)值显著升高(P0.01,n=5)。 3.低压低氧28天后模型组大鼠与对照组相比,舌下神经放电的频率降低(P0.05,n=6)和幅度变小(P0.01,n=6),提示舌下神经放电活动减弱。 4.利用重建后的三维立体图像,测量大鼠上气道容积,对照组与模型组相比无统计学差异。 5.正常SD大鼠双侧LH区注射Orexin-SAP (0.043mg/ml, 100nl/侧)两周后,尼氏染色结果显示与生理盐水对照组比较LH区的神经元数量明显减少,通过免疫组织化学实验也观察到LH的Orexin A神经元数量明显减少,仅有少量残存。应用电生理的实验技术,观察到损毁组大鼠与生理盐水对照组相比:气道阻力明显增大(P0.001,n=6),肺动态顺应性减小(P0.001,n=6)。舌下神经放电的频率降低(P0.05,n=6)和幅度变小(P0.01,n=6)。 结论 1.低压氧舱能成功制作慢性低压低氧模型大鼠。 2.28天的慢性低压低氧导致大鼠pH和PO2下降,CO2潴留;并引起大鼠肺的通气功能明显下降;舌下神经放电的幅度减小、呼吸频率减慢,提示舌下神经放电活动明显减弱;但还未造成大鼠上气道容积的器质性改变。 3.慢性低压低氧模型大鼠下丘脑OXA神经元的合成加速,提示OXA可能参与慢性低压低氧的病理过程,其在中枢的显著增高为其参与中枢对呼吸活动的调节提供了实验证据。 4.正常SD大鼠特异性损毁LH区Orexin神经元后,Orexin A神经元表达明显减少,肺的通气功能下降,舌下神经放电活动减弱。提示,Orexin神经元分泌内源性Orexin可以兴奋舌下神经、促进对上气道的控制。 5.本文的结果提示Orexin神经元参与了慢性低压低氧模型大鼠OSAHS的病理生理学过程。Orexin神经元的分泌增加,可能是为了对抗慢性低压低氧引起的大鼠呼吸活动的减弱。
[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,
【学位授予单位】:复旦大学
【学位级别】:硕士
【学位授予年份】:2011
【分类号】:R766

【引证文献】

相关硕士学位论文 前1条

1 金春艳;OREXIN在非洲鸵鸟小肠内的分布及发育性变化[D];华中农业大学;2013年



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