【作者】 雷君；

【导师】 茹立强； Ruud M. Buijs；

【作者基本信息】 华中科技大学 ， 中西医结合基础， 2006， 博士

【摘要】 胃肠动力障碍病是临床常见消化系统的病症,致病原因和临床症状表现形式多种多样,主要分为器质性和功能性两种。消化间期移行性复合运动(MMC)作为胃肠运动的一种重要形式已成为研究重点。同时,针刺对胃肠运动功能的调整作用一直是人们关心的热点。越来越多的临床观察和实验研究证实,针刺“足三里”穴能明显缓解消化系统的急慢性炎症,调节胃肠功能,治疗胃肠动力障碍病。但针刺胃经穴位是否对MMC有调整作用及其作用的神经化学机制是什么,目前还不完全清楚。胃由自主神经系统所支配,并以肠神经系统做为转换神经元。副交感神经系统主要作用是促进胃肠运动,起源于脑干的迷走神经运动背核(DMV)。DMV是副交感神经节前纤维的主要启始位点,其神经元上有接受来自不同核团投射神经元所发出的神经递质的多种受体,对DMV神经元的活动产生调控作用。DMV神经元自身也包含多种神经递质,除了乙酰胆碱经典神经递质外,还有垂体腺苷酸环化酶激活肽(PACAP)、多巴胺、一氧化氮等,对外周的生理活动进行调节。目前有关自主神经系统对胃肠道的调控机制进行了卓有成效的研究,并已证实摄食是一个非常重要的影响因素,但对DMV投射至胃的神经元中神经化学物质的分子水平变化至今未有报道。下丘脑是调控自主神经系统的高级中枢,它不仅调控胃的生理功能,也对如肾上腺、肝脏、心脏等其它内脏器官的生理功能进行调控,对生物钟的调控是其中一项重要内容。调控生物钟节律的重要核团位于下丘脑的视交叉上核(SCN)。SCN是哺乳动物内源性昼夜节律的一个主要的起搏器,被称为人体的“生物钟”,负责调控哺乳动物生理及行为的节律性活动。通过跨节示踪已显示了SCN和许多外周器官如肝脏、胰腺、肾上腺、松果体等存在的多突触解剖学联系。其中,室旁核(PVN)被认为是一个重要的中继站,包含有前自主交感和前自主副交感神经元。近十年来,研究发现存在于SCN的钟基因(clock gene)参与了SCN生物钟的分子调节机制。对于哺乳动物而言,钟基因主要包括CLOCK、BMAL1、PERIOD(Per1/2/3)和CRY(CRY1/2)。研究人员发现钟基因不仅表达于SCN,在脑SCN以外其他结构和外周组织中也广泛表达。损毁SCN和体外培养研究则提示外周组织的钟基因依赖于SCN的调控。综合以上研究结果,我们假设SCN活动可以影响外周钟基因的表达,而外周钟基因的变化可能与组织器官特异性功能有关。研究目的与内容:1.电针调节胃肠功能时胃、十二指肠运动功能(MMC)的变化及胃、十二指肠肌间神经丛AChE、NOS阳性神经成分的变化。2.DMV-胃投射神经元调控胃生理功能过程中神经递质及受体在分子水平的变化。3.下丘脑SCN活动变化对外周组织器官中钟基因的影响及这些变化是否与某些生理过程相偶联。研究方法:1.采用夹尾刺激激怒法制成大鼠胃肠动力障碍模型,用应力传感器记录大鼠胃和十二指肠消化间期移行性复合运动(MMC),采用组织化学技术观测胃、十二指肠肌间神经丛AChE、NOS阳性神经成分的变化。2.应用Aglient 2100生物分析仪和RT-PCR比较应用于激光显微切割技术(LMPC)的大鼠非固定、不同固定时程脑组织总RNA质量以及神经肽mRNA水平表达,建立采用激光显微切割技术的最佳动物材料的实验研究方法学。3. LMPC结合CTB追踪技术,检测DMV-胃投射神经元中PACAP、NO、ChAT、GABA-AR等神经递质和受体分子水平在禁食和重新喂食时的变化。4.分别采用白天PVN微量渗析GABA受体拮抗剂荷包牡丹碱(BIC)及夜间光照大鼠,观察外周组织器官:松果体、肾上腺、肝脏、心脏及肌肉组织中钟基因的mRNA变化。研究结果:1.(1)MMC的改变:与对照组比较,胃肠动力障碍组胃-肠MMC周期、Ⅰ、Ⅱ相延长、Ⅲ相缩短(P<0.01),Ⅲ相频率减慢(P<0.01)、幅度下降(P<0.05),胃Ⅲ相发生率降低(P<0.01),胃-肠平均协调收缩率显著下降(P<0.01);西药治疗组、电针治疗组与胃肠动力障碍组比较,胃-肠MMC周期、Ⅰ、Ⅱ相缩短、Ⅲ相延长(P<0.01),Ⅲ相频率加快(P<0.01)、幅度上升(P<0.05),胃Ⅲ相发生率升高(P<0.01),胃-肠平均协调收缩率显著升高(P<0.01)。(2)胃窦、十二指肠肌间神经丛AChE阳性神经元表达:与对照组比较,胃肠动力障碍组显著减少(P<0.01);与胃肠动力障碍组比较,西药治疗组、电针治疗组显著增加(P<0.01);(3)胃窦、十二指肠肌间神经丛NOS阳性神经元表达:与对照组比较,胃肠动力障碍组显著增加(P<0.01);与胃肠动力障碍组比较,西药治疗组、电针治疗组显著减少(P<0.01)。2.(1)非固定下丘脑脑片总RNA浓度、完整性均高于固定脑片,随着固定时间延长,总RNA浓度下降、降解增加;(2)短片段目的基因GAPDH、NSE、MCH、MC4R均能从非固定和固定脑片扩增得到,长片段目的基因GR只能从非固定脑片扩增得到;(3) CRH、TRH在PVN小细胞部高表达,在SON及PVN大细胞部不表达或低表达;AVP、OT在PVN大细胞部和SON高表达,在PVN小细胞部低表达; (4) DMV标记神经元和非标记神经元看家基因表达含量无显著差异。3.与对照组比较,禁食48小时后,DMV投射至胃神经元神经递质PACAP和NOS的mRNA水平下降(P<0.05),ChAT及TH无显著性变化;重新喂食60分钟后, PACAP恢复至对照组水平, NOS仍低于正常对照组(P<0.05)。与对照组比较,禁食组GABA-AR的mRNA水平下降(P<0.05),恢复进食组GABA-AR无显著性差异。其他受体GR、TRHR、orexin-R、oxytocin-R、NK1R无显著性差异。4.在2小时BIC渗析作用下外周钟基因发生不同程度的改变。松果体钟基因在30分钟及60分钟夜间光照下发生改变,其他组织外周钟基因30分钟光照无变化,延长光照时间至60分钟后,肾上腺、肝脏、心脏钟基因均有不同程度变化,而肌肉组织钟基因仍无变化。结论:1.(1)胃、十二指肠协调运动障碍可能是胃肠动力障碍大鼠发病机制之一;(2)大鼠胃、十二指肠肌间神经丛胆碱能神经、氮能神经紊乱可能是胃肠动力障碍胃、十二指肠协调运动障碍的神经化学基础;(3)针刺能改善胃肠动力障碍胃、十二指肠运动协调性,其作用机理可能与调节ACh、NO神经递质的释放有关。2.激光显微切割技术适用于不同来源大脑神经元定性及定量研究,取材尽量采用非固定或短暂固定的组织;其优越性在于特异地从各种脑核团细胞、各种细胞结构的组织中分离出单细胞类型或组织结构,将形态和分子生物学结合,对该单细胞种类或组织结构进行形态和分子生物学分析。3. PACAP、NO和GABA-AR可能参与了副交感神经系统调控胃生理功能的不同神经化学机制。4.SCN对外周钟基因具有调控作用,但外周钟基因可能不与外周生理活动直接偶联,而主要与调节纪录外周时间系统有关。更多还原

【Abstract】 The disorder of motility of gastrointestinal tract is common in clinical digestive diseases. Its pathogeny and clinical pattern are varied, which are divided into functional and organic. The interdigestive migrating motor complex is the study point as an important motility formation of gastrointestinal tract. In the meantime, the regulation of the acupuncture on the motility of gastrointestinal tract is also interesting. More and more clinical and experimental investigation have shown that electroacupuncture at T Zusanli can relieve the inflammation of gastrointestinal tract; regulate the function of gastrointestinal tract. However, the role of electroacupuncture on MMC and its neurochemical mechanism are still not clear.Mucosal lining and smooth muscle tissue of the stomach are innervated by the autonomic nervous system (ANS) through enteric nervous system (ENS) indirectly. Activation of the parasympathetic innervation increases motility and secretion. The parasympathetic innervation mainly originates in the middle and caudal parts of the dorsal motor nucleus of the nervus vagus (DMV). The neurons in the DMV contain several transmitter types. Besides acetylcholine (Ach), the classical transmitter of the parasympathetic nervous system, the presence of pituitary adenylyl cyclase-activating polypeptide (PACAP), dopamine, and NO in the DMV have been described as well. Many different afferent projection systems have been described to be present in the DMV and influence DMV neuronal activity. Among them are metabolically important peptides like, GABA, TRH, orexin and Substance P. Although the exact regulatory mechanism of the autonomic control of the gastro-intestinal tract is still subject of ongoing investigation, it is well established that the feeding condition is an important factor. Despite the extensive knowledge of the DMV it is at present not known which type of DMV neurons is involved in the gastric response to metabolic challenge.The hypothalamus is the superior central of controlling the autonomous nervous system. It does not only regulate the physiology of stomach, but also regulate other visceral organs as adrenal, liver, heart etc. The regulation of rhythms is involved in importantly. The suprachiasmatic nucleus (SCN), the mammalian central circadian pacemaker, located in the hypothalamus, is responsible for circadian rhythms in physiology and behavior. SCN neurons endogenously display spontaneous circadian rhythms of metabolic and electrical activity in intact animals as well as in vitro, these rhythms have a period of～24h and are reset by light daily. Anatomical studies using transneuronal tracing, revealed polysynaptic pathways between SCN and many organs, such as liver, pancreas, adrenal, pineal and others. Herein the paraventricular nucleus (PVN) was recognized as a critical relay, which contains both preautonomic-sympathetic and preautonomic-parasympathetic neurons.The molecular mechanism of the SCN’s clock function is proposed to be composed of several rhythmically expressed genes, consisting of interacting positive and negative transcription/translation feedback loops. However, besides in the SCN, local sets of the clock genes are also rhythmically expressed in peripheral tissues. SCN-lesion and culture experiments on peripheral clock genes have implied that the SCN seems to synchronize these peripheral oscillators. This raises the possibility that SCN efferent may modulate peripheral clock gene expression directly, and subsequently lead to changs in rhythms in both physiology and behavior. Taking all data together, we hypothesize that with change of SCN activity, the synchronized output of the SCN can regulate peripheral circadian oscillations including clock genes, which may be involved in the tissue-specific function.Objectives:1. To investigate the regulatory effects and possible neurochemical mechanism of electroacupuncture on MMC and the changed of AChE and NOS of ENS.2. To determine the expression of several genes, which are known to be present in the DMV projecting to the stomach in response to fasting and refeeding?3. To examine the effect by PVN administration of BIC in the daytime and light exposure in the nighttime on the expression of clock genes (Per1, Per2, Per3, Cry1, Cry2 and DBP) and some tissue-specific physiological related genes in peripheral organs.Methods:1. By provoking rats with pinching their tails continuously the animal model was made. The gastroduodenal motor was recorded by stress gauge. The expression of acetylcholinesterase(AChE) and nitric oxide synthase(NOS) in gastroduodenal myenteric plexus were demonstrated histochemistrically and measured by image analysis system.2. (1) Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12h hypothalamus sections; (2)Different neurons of PVN and SON were collected by LMPC, CRH、TRH、AVP、OT mRNA level were measured by RT-PCR; (3)labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR.3. CTB retrograde tracing was used to get labeled neurons in DMV, by isolating labeled neurons and RT-PCR, the levels of transmitters and receptors were measured.4. Under the stimulations of light exposure (day-signal from SCN) and BIC infusion into the PVN (night-signal from SCN), respectively, the mRNA level of clock genes in pineal, adrenal, liver, heart and muscle were measured by RT-PCR.Results:1. (1) The changes of MMC: compared with the control group the cycle, phaseⅠ, phaseⅡelongated (P<0.01), phaseⅢshortened (P<0.01), the frequency and sewing reduced (P<0.01, P<0.05), the incidence rate of phaseⅢdecreased (P<0.01) and the average coordinated contract rate decreased (P<0.01) in the model group. In the gastrointestinal motility disorder groups plus western medicine and electroacupuncture respectively, compared with the model group, phaseⅠ, phaseⅡshortened (P<0.01), phaseⅢelongated (P<0.01), the frequency and sewing elevated (P<0.01, P<0.05), the incidence rate of phaseⅢelevated (P<0.01), and the average coordinated contract rate increased (P<0.01). (2) The expression of AChE: compared with the control group, the model group decreased (P<0.01); compared with the model group, the gastrointestinal motility disorder groups plus western medicine and electroacupuncture respectively increased (P<0.01); (3)The expression of NOS: compared with the control group, the model group increased (P<0.01); compared with the model group, the gastrointestinal motility disorder groups plus western medicine and electroacupuncture respectively decreased (P<0.01).2. (1) unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC; relative short amplicons such as GAPDH, NSE, MCH and MC4R were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; (2) In magnocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two; (3) the expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons.3. By fasting, for transmitter the mRNA expressions of PACAP, NOS decreased (P<0.05) significantly compared with control. For receptor, the gene expressions of GABA-AR decreased compared with control. By refeeding, PACAP and GABA-AR recovered as the control level (P>0.05), while the level of NOS still decreased (P <0.05).4. 2hr of bicuculin infusion changed the peripheral clock genes differentially. The expression of clock genes altered in the pineal under both 30 and 60min of light exposure, however only with 60min of light exposure clock genes changed differentially in adrenal, liver and heart. No significant changes can be observed in muscle by 60min of light exposure.Conclusions:1. (1) The dyscoorordination of gastroduoenal motility may play an important role in the pathogenesis of gastrointestinal motility disorder. (2) The decrease of the expression of AChE and the increase of the expression of NOS suggested that the disorders of cholinergic and nitronergic neurons might partake into the neurochemical mechanism of the dyscoorordination of gastroduoenal motility. (3) the act of electroacupuncture on the improvement of coordination of gastroduoenal motility maybe through the cholinergic and nitronergic neurons. 2. The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC; LMPC is a novel technique making isolation of homogeneous cells more rapidly in high quality and purity. This could be very useful in the brain because it is divided into numerous small nuclei or irregularly shaped even single neurons to be accurately dissected by hand.3. PACAP, NO and GABA-AR might partake the machanism of regulation of DMV neurons projecting to stomach during fasting and refeeding.4. SCN activity can regulate peripheral clock genes; however, the changes of clock genes were not coupled with the peripheral functions, suggesting the peripheral physiology can happen without the direct involvement of peripheral clocks.更多还原