孤束核促食欲素系统参与呼吸运动的调节

发布时间：2018-03-25 11:34

本文选题：孤束核　切入点：促食欲素　出处：《河北医科大学》2017年硕士论文

【摘要】：呼吸化学感受器反射通过排出CO_2参与调节内环境酸碱平衡,是一种重要的稳态调节机制。呼吸频率和幅度的改变主要由脑干复杂的神经环路调控实现。控制节律呼吸的神经环路包括从脑桥到低位脑干的一些呈串联排列、功能上相互影响的神经核团,其中Botzinger complex和pre-Botzinger complex等是产生呼吸节律的关键脑区;中枢呼吸化学感受器和脑桥则为节律中枢和中枢模式发生器提供兴奋性驱动。孤束核(nucleus tractus solitarii,NTS)是心血管和呼吸系统传入信息的初级整合中枢,存在中枢呼吸化学感受器神经元。虽然NTS神经元可直接投射到延髓腹侧呼吸组,通过突触活动影响节律中枢的活动,但是,参与投射的神经环路基础还不明确,投射神经元的生物化学表型也有待揭示。促食欲素(Orexin)是由下丘脑Orexin能神经元合成和分泌,参与体内许多生理功能的调节,包括能量代谢、摄食行为、睡眠-觉醒的交替、奖赏等。Orexin是中枢性调控呼吸的重要内源性物质。研究表明,Pre-Botzinger complex、延髓腹外侧头端区、Kolliker-Fuse核、舌下神经核、延髓中缝核和斜方体后核的Orexin系统均参与呼吸的频率和/或幅度的调节,最终的呼吸改变形式取决于局部神经环路的构成、实验动物的状态(清醒和睡眠)和生物节律等。NTS富集Orexin受体(OXR),然而,NTS的Orexin系统是否以及如何参与呼吸运动的调节还未见报道。目的:观察NTS的Orexin系统在呼吸调控中的作用。方法:实验动物分为两组:OX1R敲低组(注射AAV-U6-OX1R-sh RNA-EGFP)和对照组(注射AAV-U6-Scrambled-shRNA EGFP)。利用RNA干扰技术,敲低SD大鼠NTS的Ⅰ型Orexin受体(OX1R)。免疫荧光技术用于检测病毒载体的感染效率。qRT-PCR技术用于测定OX1R的敲低效率。应用无创体积描计系统观察敲低OX1R受体对清醒大鼠高碳酸性肺通气反应的影响,观察指标为潮气量(TV)、呼吸频率(BF)和每分通气量(MV)等。最后,观察在NTS注射Orexin对麻醉大鼠膈神经放电、血压和心率的影响。结果:1免疫荧光结果显示NTS富集OX1R;OX1R-shRNA-EGFP在NTS成功表达。2 qRT-PCR结果表明OX1R敲低效率约为15%。3基础状态下(100%O_2,0%CO_2),两组大鼠的TV和MV无显著差异,但敲低组大鼠的BF低于对照组(P0.0001)。4阶梯式增加吸入气中CO_2浓度(2~8%),两组大鼠的TV、BF和MV均升高,但OX1R敲低组的TV升高的幅度大于对照组(P0.05~0.0001),而OX1R敲低组的BF和MV升高的幅度均小于对照组(P0.05~0.0001)。5 NTS微量注射OrexinA可使麻醉大鼠膈神经放电频率减少(P0.0001),幅度不变,但放电积分值减少(呼吸输出,P0.0001);同时,收缩压、舒张压和平均动脉压降低(P0.001),心率减慢(P0.01)。结论:清醒状态下抑制NTS的Orexin系统可减弱高碳酸性通气反应;麻醉状态下激活NTS的Orexin系统可抑制中枢性呼吸驱动。因此,孤束核Orexin系统对呼吸的调控作用可能呈状态依赖性。
[Abstract]:Respiratory chemical receptor reflex is involved in regulating the acid-base balance in the internal environment by excreting CO_2. The changes in respiratory frequency and amplitude are mainly controlled by the complex neural loop of the brain stem. The neural loops that control rhythmic respiration are arranged in series from the pons to the lower brain stem. Botzinger complex and pre-Botzinger complex are the key areas to produce respiratory rhythm. Central respiratory chemosensors and pontine provide excitatory drive for rhythm center and central pattern generator. Nucleus solitarii (NTSs) is the primary integration center of cardiovascular and respiratory afferent information. Although NTS neurons can be directly projected to ventral medulla oblongata and affect the activity of rhythmic center through synaptic activity, the basis of the neural loop involved in the projection is not clear. The biochemical phenotype of projected neurons is also to be revealed. Orexin is synthesized and secreted by Orexin neurons in the hypothalamus, involved in the regulation of many physiological functions in the body, including energy metabolism, feeding behavior, sleep and arousal. Orexin is an important endogenous substance in central respiratory regulation. Studies have shown that Pre-Botzinger complex, Kolliker-Fuse nucleus of ventrolateral medulla oblongata, hypoglossal nucleus, raphe nucleus of medulla oblongata and posterior nucleus obliquus participate in the regulation of respiratory frequency and / or amplitude. The final form of respiratory change depends on the composition of the local neural loop, The state (awake and sleep) and biological rhythm of experimental animals. NTS enriched the Orexin receptor OXRN. However, whether and how the Orexin system of NTS is involved in the regulation of respiratory movement has not been reported. Objective: to observe the Orexin system of NTS in respiratory regulation. Methods: experimental animals were divided into two groups: AAV-U6-OX1R-sh RNA-EGFP group (injected with AAV-U6-OX1R-sh RNA-EGFP) and control group (control group). RNA interference technique was used. Immunofluorescence technique was used to detect the infection efficiency of virus vector. QRT-PCR was used to detect the knockout efficiency of OX1R. The noninvasive volumetric system was used to observe the response of low OX1R receptor to high carbon content in awake rats. Effects of acidic lung ventilation, The indexes of observation were tidal volume, respiratory frequency and ventilation volume, etc. Finally, the phrenic nerve discharges in anesthetized rats were observed by NTS injection of Orexin. Results NTS enriched OX1RX OX1R-shRNA-EGFP was successfully expressed in NTS. The results showed that the OX1R knockdown rate was about 15.3 basic state. The results showed that there was no significant difference in TV and MV between the two groups. However, the BF of the rats in the knockout group was lower than that of the control group (P 0.0001n.4), and the CO_2 concentration in the inhaled gas was increased in step by step. The TV-BF and MV of the rats in the two groups were both increased. However, the amplitude of the increase of TV in the OX1R knockout group was greater than that in the control group (P 0.05 0. 0001), while the increase in BF and MV in the OX1R knockdown group was lower than that in the control group (P 0.05) 0.0001.5 NTS injection of OrexinA could decrease the phrenic nerve discharge frequency of anesthetized rats (P 0. 0001). At the same time, systolic blood pressure, diastolic blood pressure and mean arterial pressure decreased by P 0.001, and the heart rate decreased by P0.010.Conclusion: the Orexin system which suppresses NTS in awake state can weaken the high carbon acid ventilation response. The central respiratory drive can be inhibited by the Orexin system that activates NTS under anesthesia. Therefore, the regulation of respiration by the Orexin system of the nucleus tractus solitarii may be state dependent.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R332.1

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