中枢及外周自主神经系统对内脏功能的调控机制
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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。
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.
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。
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.
引文
1. Abrahamsson H. Studies on the inhibitory nervous control of gastric motility. Acta Physiol Scand 390 Suppl: 1973, 1-3
2. Akashi M, Nishida E. Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock. Genes Dev. 2000 Mar 15; 14(6): 645-9.
3. Balsaobre A, Damiola F, Schibler U. A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell. 1998 Jun 12; 93(6): 929-37.
4. Balsalobre A, Marcacci L, Schibler U. Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts. Curr Biol. 2000 Oct 19; 10(20): 1291-4.
5. Bos NP, Mirmiran M. Circadian rhythms in spontaneous neuronal discharges of the cultured suprachiasmatic nucleus.Brain Res. 1990 Mar 12; 511(1):158-62.
6. Broussard DL, Li H, Altschuler SM. Colocalization of GABA (A) and NMDA receptors within the dorsal motor nucleus of the vagus nerve (DMV) of the rat.Brain Res. 1997 Jul 18; 763(1):123-6.
7. Buijs RM, Hermes MH, Kalsbeek A. The suprachiasmatic nucleus-paraventricular nucleus interactions: a bridge to the neuroendocrine and autonomic nervous system. Prog Brain Res. 1998; 119: 365-82.
8. Buijs RM, Kalsbeek A, van der Woude TP, van Heerikhuize JJ; Shinn S. Suprachiasmatic nucleus lesion increases corticosterone secretion. Am J Physiol. 1993 Jun; 264(6 Pt 2):R1186-92.
9. Buijs RM, Wortel J, Van Heerikhuize JJ, Feenstra MG, Ter Horst GJ, Romijn HJ, Kalsbeek A. Anatomical and functional demonstration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway. Eur J Neurosci. 1999 May; 11(5): 1535-1544.
10. Bult H. Boeckxstaens GE. Pelckmans PA. et al. Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurolransmitter. Nature, 1990, 345:346～349
11. Chai NL, Dong L, Li ZF,et al. Effects of neurotrophins on gastrointestinalmyoelectric activities of rats. World J Gastroenterol, 2003 Aug;9(8):1874～1877.
12. Chansard M, Iwahana E, Liang J, Fukuhara C. Regulation of cAMP-induced arylalkylamine N-acetyltransferase, Period1, and MKP-1 gene expression by mitogen-activated protein kinases in the rat pineal gland. Brain Res Mol Brain Res. 2005 Oct 3; 139(2): 333-40.
13. Damiola F, Le Minh N, Preitner N, Kornmann B, Fleury-Olela F, Schibler U. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev. 2000 Dec 1; 14(23): 2950-61.
14. David M.A., Lesie M., John W.H., et al. Co-localization of choline acetyltransferase and tyrosine hydroxylase within neurons of the dorsal motor nuclers of the vagus. J.Chemixal Neuroanatomy, 3: 133-140
15. Dawson TW, Bredt DS, Fotuhi M, et al.Nitric oxide synthase and neuronal NADPH-disphorase are indentical in brain and peripheral tissuse, Proc Natl Acad Sci USA, 1991,88(13):7797～7801.
16. Emmanuel K.T., Williams R.G. Catecholaminergic parasympathetic efferents within the dorsal motor nucleus of the vagus in the rat: a quantitative analysis. Neuroscience Letter, 1988(90): 1-5
17. Ewart WR, Jones MV, King BF. Central origin of vagal nerve fibers innervating the fundus and corpus of the stomach in rat. J Auton Nerv Syst. 1988 Dec; 25(2-3): 219-31.
18. Ferreira M Jr, Browning KN, Sahibzada N, et al. Glucose effects on gastric motility and tone evoked from the rat dorsal vagal complex. J Physiol. 2001 Oct 1; 536(Pt 1): 141-52.
19. Fukuhara C, Yamazaki S, Liang J. Pineal circadian clocks gate arylalkylamine N-acetyltransferase gene expression in the mouse pineal gland. J Neurochem. 2005 Apr; 93(1): 156-62.
20. Gererdai I, Halasz B. Central nervous system structures connected with theendocrine glands. Findings obtained with the viral transneuronal tracing technique. Exp Clin Endocrinol Diabetes. 2000; 108(6): 389-95.
21. Glavin GB, Murison R, Overmier JB, Pare WP, Bakke HK, Henke PG, and Hernandez DE. The neurobilolgy of stress ulcers. Brain Res Rev 1991, 16: 301-343,
22. Goncharuk VD, van Heerikhuize J, Dai JP, Swaab DF, Buijs RM. Neuropeptide changes in the suprachiasmatic nucleus in primary hypertension indicates functional impairment of the biological clock. J Comp Neurol. 2001 Mar 12; 431(3): 320-30.
23. Grabauskas G, Moises HC. Gastrointestinal-projecting neurones in the dorsal motor nucleus of the vagus exhibit direct and viscerotopically organized sensitivity to orexin.J Physiol. 2003 May 15; 549(Pt 1):37-56.
24. Groos G, Hendriks J. Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro. Neurosci Lett. 1982 Dec 31; 34(3): 283-288.
25. 郭海军,林洁,李国成.功能性消化不良的动物模型研究.中国中西医结合杂志,2001,9(3):1411～1412
26. Guo J.J, Browning KN, Rogers RC, et al. Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus. Am J Physiol Gastrointest Liver Physiol 2001, 280: G361-G367
27. Hayakawa T, Takanaga A, Tanaka K, et al. Cells of origin of vagal motor neurons projecting to different parts of the stomach in the rat: confocal laser scanning and electron miroscopic study. Anat Embryol. 2003 Dec; 207(4-5): 289-297
28. Hirano T, Niijima A. Effects of 2-deoxy-D-glucose, glucose and insulin on efferent activity in gastric vagus nerve. Experientia. 1980 Oct 15; 36(10): 1197-8
29. Hirota T, Fukada Y. Resetting mechanism of central and peripheral circadian clocks in mammals. Zoolog Sci. 2004 Apr; 21(4): 359-368.
30. Hirota T, Okano T, Kokame K, Shirotani-Ikejima H, Miyata T, Fukada Y. Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expressionin cultured Rat-1 fibroblasts. J Biol Chem. 2002 Nov 15; 277(46): 44244-51.
31. Izumo M, Johnson CH, Yamazaki S. Circadian gene expression in mammalian fibroblasts revealed by real-time luminescence reporting: temperature compensation and damping. Proc Natl Acad Sci U S A. 2003 Dec 23; 100(26): 16089-16094.
32. Jin L, Oliveira A, Inwards C, et al. Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and RT-PCR. Diagn Mol Pathol, 2003, 12(4): 224-230
33. Kalsbeek A, Garidou ML, Palm IF, Van Der Vliet J, Simonneaux V, Pevet P, Buijs RM. Melatonin sees the light: blocking GABA-ergic transmission in the paraventricular nucleus induces daytime secretion of melatonin. Eur J Neurosci. 2000 Sep; 12(9):3146-54.
34. Kalsbeek A, La Fleur S, Van Heijningen C, Buijs RM. Suprachiasmatic GABAergic inputs to the paraventricular nucleus control plasma glucose concentrations in the rat via sympathetic innervation of the liver. J Neurosci. 2004 Sep 1; 24(35):7604-7613.
35. Kalsbeek A, Ruiter M, La Fleur SE, Van Heijningen C, Buijs RM. The diurnal modulation of hormonal responses in the rat varies with different stimuli.J Neuroendocrinol. 2003 Dec; 15(12):1144-55.
36. Kalsbeek A, Van Heerikhuize JJ, Wortel J, Buijs RM. A diurnal rhythm of stimulatory input to the hypothalamo-pituitary-adrenal system as revealed by timed intrahypothalamic administration of the vasopressin V1 antagonist.J Neurosci. 1996 Sep 1; 16(17): 5555-65.
37. Kubek M.J., Rea M.A., Hodes Z.I., et al. Quantitation and characterization of thyrotropin-releasing hormone in vagal nuclei and other regions of the medulla oblongata of the rat. J. Neurohemistry. 1983, 40(5): 13071313
38. Ladic LA, Buchan AM. Association of substance P and its receptor with efferent neurons projecting to the greater curvature of the rat stomach. J Auton Nerv Syst.1996 Apr 20; 58(1-2): 25-34.
39. Leahy A , Besberdas K , Clayman C , et al. Abnormalities of the electrogastrogram in functional gastrointestinal disorder. Am J Gastroenterol,1999,94(4):1023～1027
40. La Fleur SE, Kalsbeek A, Wortel J, Buijs RM. A suprachiasmatic nucleus generated rhythm in basal glucose concentrations. J Neuroendocrinol. 1999 Aug; 11(8): 643-52.
41. La Fleur SE, Kalsbeek A, Wortel J, Buijs RM. Polysynaptic neural pathways between the hypothalamus, includes the suprachiasmatic nucleus, and the liver. Brain Res. 2000 Jul 14; 871(1): 50-6.
42. Larsen PJ, Enquist LW, Card JP. Characterization of the multisynaptic neuronal control of the rat pineal gland using viral transneuronal tracing. Eur J Neurosci. 1998 Jan; 10(1):128-45.
43. Liu Y, Heintzen C, Loros J, Dunlap JC. Regulation of clock genes. Cell Mol Life Sci. 1999 Aug 15; 55(10): 1195-205.
44. Lefebvre RA, De Vriese A, and Smits GJM. Influence of vasoactive intestinal polypeptide and NG-nito-L-arginine methylester on cholinergic neurotransmission in the rat gastric fundus. Eur J Pharmacol1992, 221: 235-242,
45. Lewis MW, Travagli RA. Effects of substance P on identified neurons of the rat dorsal motor nucleus of the vagus. Am J Physiol Gastrointest Liver Physiol. 2001 Jul; 281(1): G164-G172
46. Manier M., Mouchet P, Feuerstein C., et al. Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurons of the vagal motor nuclers in the adult rat, Neurosci. Lett.80 (1987): 141-146
47. Masuda N, Ohnishi T, Kawamoto S, et al. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Moore RY. Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus. FedProc. 1983 Aug; 42(11): 2783-9.
48. Nakamura TJ, Shinohara K, Funabashi T, Mitsushima D, Kimura F. Circadian and photic regulation of cryptochrome mRNAs in the rat pineal gland. Neurosci Res. 2001 Sep; 41(1): 25-32.
49. Mutsuga N, Shahar T, Verbalis J, et al. Selective gene expression in magnocelllular neurons in rat supraoptic nucleus. Journal of Neuroscience. 2004, 24 (32): 7174-7185
50. Nicole L, Robert F, and John W, et al. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Review, 1998, 14(7): 272-276.
51. Obiakor H, Sehgal D, Dasso JF, et al. A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ. Analytical Biochemistry, 2002, 306(1): 55-62
52. Ozawa M, Aono M, Moriga M. Central effects of pituitary adenylate cyclase activating polypeptide (PACAP) on gastric motility and emptying in rats. Dig Dis Sci. 1999 Apr; 44(4): 735-743
53. Parsons RL, Neel DS, McKeon TW, et al.Organization of a vertebrate ganglion: a correlated biochemical and histochemical study. J Neurosci 7: 837-846
54. Perreau-Lenz S, Kalsbeek A, Garidou ML, et al. Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J Biol Chem. 1998 Oct 16; 273(42): 27039-42.
55. Randall WC, Wurster RD. Peripheral innervation of the heart. In: Levy MN, Schwartz PJ, and editors. Vagal control of the heart: experimental basis and clinical implications. New York: Futura Puvlishing. 1994: 21-32
56. Rogers RC, Hermann GE, and Travagli RA. Brainstem pathways responsible for oesophageal control of gastric motility and tone in the rat. J Physiol(Lond) 1999, 514:369-383,
57. 茹立强.胆碱脂酶的组织化学及超微定位。神经介质及有关酶类组织化学(艾民康主编)。北京:人民卫生出版社,1987,110～113
58. 茹立强.消化管壁内神经丛及其神经元,现代科技综述大辞典(主编:朱永和)北京:科学出版社,1998, 1301～1302.
59. Rwppert SM, Weaver DR. Molecular analysis of mammalian circadian rhythms.Annu Rev Physiol. 2001; 63:647-76. Sakaguchi T, Yamaguchi K. Changes in efferent activities of the gastric vagus nerve by administration of glucose in the portal vein. Experientia. 1979 Jul 15; 35(7): 875-6.
60. Scheer FA, Ter Horst GJ, van Der Vliet J, Buijs RM. Physiological and anatomic evidence for regulation of the heart by suprachiasmatic nucleus in rats. Am J Physiol Heart Circ Physiol. 2001 Mar; 280(3): H1391-9.
61. Scott SM. Manometric techniques for the evaluation of colonic motor activity:current status, Neurogastroenterol Motil.2003 Oct;15(5):483～513
62. Simonneaux V, Pevet P, Buijs RM. Suprachiasmatic control of melatonin synthesis in rats: inhibitory and stimulatory mechanisms. Eur J Neurosci. 2003 Jan; 17(2): 221-8.
63. Simonneaux V, Piorel VJ, Garidou ML, Nguyen D, Diaz-Rodriguez E, Pevet P. Daily rhythm and regulation of clock gene expression in the rat pineal gland. Brain Res Mol Brain Res. 2004 Jan 5; 120(2): 164-72.
64. Steffens AB. A method for frequent sampling blood and continuous infusion of fluids in the rat without disturbing the animal. Physiol. Behav. 4: 833-836.
65. Stoll S., Konig S., Thanos S. Isolation of total-RNA from formalin-fixed rat retina. Journal of Neuroscience Methods, 2002, 120(1): 55-63.
66. Susan M, Stockton S, Carol S, et al. Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Molecular Carcinogenesis, 1999, 25:86-91
67. Swanson LW, Sawchenko PE. Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology. 1980, 31(6): 410-417.
68. Swanson LW, Sawchenko PE, Wiegand SJ, et al. Separate neurons in the paraventricular nucleus project to the median eminence and to the medulla or spinal cord. Brain Res. 1980, 198(1): 1950-1955.
69. Takahashi T and Owyang C. Characterization of vagal pathways mediating gastric accommodation reflex in rats. J Physiol(Lond) 1997, 504: 479-488,
70. Takanaga A, Hayakawa T, Tanaka K et al. Immunohistochemical characterization of cardiac vagal preganglionic neurons in the rat. Autonomic Neuroscience: Basic and Clinical. 2003, 106: 132-137
71. Tatewaki M, Harris M, Uemura K, et al. Dual effects of acupuncture on gastric motility in conscious rats. Am J Physiol Regul Integr Comp Physiol, 2003, 285(4): 862～867
72. Teclemariam-Mesbah R, Ter Horst GJ, Postema F, Wortel J, Buijs RM. Anatomical demonstration of the suprachiasmatic nucleus-pineal pathway. J Comp Neurol. 1999 Apr 5; 406(2): 171-82.
73. Tony E, Sun-Hun Kim, Marielena Chavira, et al. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5’ nuclease quantitative reverse transcription-polymerase chain reaction. Journal of Molecular Diagnostics, 2000, 2(2): 84-91.
74. Twstoni PA, Fanti L, Bagnolo F, et al. Manometric evalution of the interdigestive antroduodenal motility subjects with fasting bile reflux with and without antigastritis, Gut 1989; 30: 443-448
75. Vivianna M, Lisa H, Peter T. Optimizing gene expression analysis in archival brain tissue. Neurochemical Research, 27(10): 993-1003.
76. Wang ZS, Elsenbruch S, Orr WC, et al. Detection of gastric slow wave uncoupling from multi-channel electrogastrogram: validations and applications. Neurogastroenterol Motil. 2003 Oct; 15(5): 457-465.
77. Weaver DR. The suprachiasmatic nucleus: a 25-year retrospective. J Biol Rhythms. 1998 Apr; 13(2): 100-12.
78. Welsh DK, Yoo SH, Liu AC, Takahashi JS, Kay SA. Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.Curr Biol. 2004 Dec 29;14(24):2289-95.
79. 徐三平,易粹琼.西沙必利对部分胃肠神经递质释放的影响.临床消化病学杂志,1999;11(2):71～72
80. 张经济,许冠荪.消化道生理与病理生理学.第一版. 广东:广东科技出版社,1997,10:42
81. Yamamoto T, Nakahata Y, Soma H, Akashi M, Mamine T, Takumi T. Transcriptional oscillation of canonical clock genes in mouse peripheral tissues. BMC Mol Biol. 2004 Oct 9; 5: 18.
82. Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M, Tei H. Resetting central and peripheral circadian oscillators in transgenic rats. Science. 2000 Apr 28; 288(5466): 682-685.
83. Young MW, Kay SA. Time zones: a comparative genetics of circadian clocks. Nat Rev Genet. 2001 Sep; 2(9): 702-715.
84. Zhou L,Xie YK. A strain gauge for measuring gastrointestinal motility in animals. Chin J Appl Physiol, 1988,4:346-349
85. Zylka MJ, Shearnan LP, Levine JD, Jin X, Weaver DR, Reppert SM. Molecular analysis of mammalian timeless. Neuron. 1998 Nov; 21(5): 1115-1122.
86. Zylka MJ, Shearman LP, Weaver DR, Reppert SM. Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron. 1998 Jun; 20(6): 1103-1110.
1. Albrecht U, Sun Z S, Eichele G, et al. Adifferentialresponse of two putative mammalian circadian regulators, mPer1 and mPer2, to light. Cell, 1997, 91: 1055-1064
2. Bae K, Jin X W, Maywood E S, et al. Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron, 2001, 30(2): 525-536
3. Balsalobre A, Brown S A, Marcacci L, et al. Resetting of circadian time in peripheral tissues by glucocorticoidsignaling. Science, 2000, 2 8 9(5488): 2344-2347
4. Balsalobre A, Damiola F, Schibler U. A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell, 1998, 93(6): 929-937
5. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells that set the circadian clock. Science, 2002, 295:1070-1073
6. Bouskila Y, Dudek FE. Can a population of suprachiasmatic nucleus neurons with different period lengths produce a stable circadian rhythm? Brain Res, 1995, 670∶333-336.
7. Bunger M K, Wilsbacher L D, Moran S M, et al. Mop3 is an essential component of the master circadian pacemaker in mammals. Cell, 2000, 103:1009-1017
8. Chaurasia SS, Pozdeyev N, Haque R, Visser A, Ivanova TN, Iuvone PM. Circadian clockwork machinery in neural retina: evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures.Mol Vis. 2006 Mar 30;12: 215-23.
9. Damiola F, LeM N , Preitner N , et al. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev, 2000, 14: 2950- 2961.
10. Delaunay F, Laudet V. Circadian clock and microarrays: mammalian genome gets rhythm. Trends Genet, 2002, 18 (12): 595-597
11. Dunlap J.C. Molecular bases for circadian clocks.Cell. 1999 Jan 22; 96(2):271-90.
12. Edery I. Circadian rhythms in a nutshell. PhysiolGenomics, 2000, 3: 59-74
13. 范镇海.依时辰辨治内科杂病三法.湖南中医学院学报 1995;15(2):402
14. Foster RG, Lucas RJ. Clocks, criteria and critical genes.Nat Genet. 1999 Jul; 22(3):217-219.
15. Fukada Y, Okano T. Circadian clock system in the pineal gland. Mol Neurobiol, 2002,25(1):19-30
16. Gekakis N, Staknis D, Nguyen H B, et al. Role of the CLOCK protein in the mammalian circadian mechanism. Science, 1998, 280: 1564-1569
17. Grosse J, Davis FC. Melatonin entrains the restored circadian activity rhythms of syrian hamsters bearing fetal suprachiasmatic nucleus grafts. J Neurosis, 1998, 18∶8032-8037.
18. Hamada T, L esauter J, V enuti JM, et al. Exp ression of Period genes: rhythmic and nonrhythmic compartments of the suprachiasmatic nucleus pacemaker. J N euro sci, 2001, 21: 7742- 7750.
19. Hao H P, Allen D L, Hardin P E. A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila melanogaster. Mol Cell Biol, 1997, 17 : 3687-3693
20. Hogenesch J B, Gu Y Z, Jain S, et al. The basic-helix-loophelix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci USA, 1998, 95 (10): 5474-5479
21. Horst TJ, Muijtjens M, Konayashi K, et al. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature, 1999, 398: 627-630
22. 侯恒太,等.乘时用药医案三则.河南中医杂志 1996;16(2):1172
23. Hsu D S, Zhao X D, Zhao Y, et al. A putative human bluelight photoreceptors hCRY1 and hCRY2 are flavoproteins. Biochemistry, 1996, 35(14): 13871-13877
24. 黄娟,杜怀荣.运用时间医学方法治愈外感发热1例.北京中医药大学学报 1997;20(3):653
25. Inouye S2IT and Kawamura H. Characteristics of a circadian pacemaker in the suprachiasmatic nucleus. J Comp Physiol, 1982, 146∶153-160.
26. Jones C R, Campbell S S, Zone S E, et al. Familial advanced sleep-phasesyndrome: ashort-period circadian rhythm variant in humans. Nat Med, 1999, 5: 1062-1065
27. King D P, Zhao Y L, Sangoram A M, et al. Positional cloning ofthemousecircadian clock gene. Cell, 1997, 89(4): 641-653
28. Kondo Y, Kanai Y, Sakamoto M, Mizokami M, Ueda R, Hirohashi S. Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpGislands in microdissected specimens from patients with hepatocellular carcinoma. Hepatology. 2000 Nov; 32(5):970-979.
29. 孔凡涵,赵传莲,张锐.中医时间医学在临床应用.光明中医 2000;17(5):182
30. Konopka R J, Benzer S. Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci USA, 1971, 68: 2112-2116
31. Kovacikova Z, Sladek M, Bendova Z, Illnerova H, Sumova A. Expression of Clock and Clock-Driven Genes in the Rat Suprachiasmatic Nucleus during Late Fetal and Early Postnatal Development.J Biol Rhythms. 2006 Apr; 21(2):140-148.
32. Liu C, Peppert SM. GABA synch ronizes clock celles w ith in the sup rach iasmatic circadian clock. Neuron, 2000, 25: 123- 128.
33. 刘世兴,杜淑云.简析中风发病与死亡时间.中医杂志 1998;39(8):5082
34. 陆保年.应用中医时间医学理论探讨对发热的辨证论治.四川中医 1994;12(5):123
35. McNamara P, Seo S P, Rudic R D, et al. Regulation of CLOCK and MOP4 by nuclear hormone receptors in the vasculature: a humoral mechanism to reset a peripheral clock. Cell, 2001, 105(7): 877-889
36. Mueller RL. Evolutionary rates, divergence dates, and the performance of mitochondrial genes in bayesian phylogenetic analysis.Syst Biol. 2006 Apr;55(2):289-300
37. Okamura H, Miyake S, Sumi Y, et al. Photic induction of mPer1 and mPer2 in Cry-deficient mice lacking a biological clock. Science, 1999, 286: 2531-2534
38. Panda S, Antoch M P, Miller B H, et al. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell, 2002, 109(3): 307-320
39. Pruitt K, Pestell RG, Der CJ. Ras inactivation of the retinoblastoma pathway by distinct mechanisms in NIH 3T3 fibroblast and RIE-1 epithelial cells. J Biol Chem. 2000 Dec 29;275(52):40916-40924
40. 秦庆云,秦昕昕.中医治疗高热的时间医学探讨.中西医结合实用临床急救杂志 1997;4(8):3372
41. Reppert SM, Weaver DR. Forward genetic approach strikes gold: cloning of a mammalianclock gene.Cell. 1997 May 16; 89(4):487-490.
42. Shearman LP, Zylka MJ, Weaver DR, Kolakowski LF Jr, Reppert SM. Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei. Neuron. 1997 Dec;19(6):1261-1269
43. Shearman L P, Jin X W, Lee C, et al. Targeted disruption of the mPer3 gene: subtle effects on circadian clock function. Mol Cell Biol, 2000, 20: 6269-6275
44. Shgeyoshi Y, Taguchi K, Yamamoto S, et al. Light induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript. Cell, 1999, 9: 1043-1053
45. Stokkan K. A, Yamazaki S, Tei H, et al. Entrainment of the circadian clock in the liver by feeding. Science, 2001, 291 (5503): 490-493
46. Sun Z S, Albrecht U, Zhuchenko O, et al. RIGUI, a putative mammalian ortholog of the Drosophila period gene. Cell, 1997, 90 : 1003-1011
47. Tei H, Okamura H, Shigeyoshi Y, et al. Circadianoscillation of a mammalian homologue of the Drosophila period gene. Nature, 1997, 389: 512-516
48. Veleri S, Brandes C, Helfrich-Forster C et al. A self-sustaining, Light-entrainable circadian oscillator in the drosophila brain. Current Biology, 2003, 13: 1758–1767.
49. Vitaterna M H, King D P, Chang A M, et al. Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior. Science, 1994, 264: 719-725
50. Whitmore D, Foulkes NS, Sassone-Corsi P. Light acts directly on organs and cells in culture to set the vertebrate circadian clock. Nature. 2000 Mar 2; 404(6773):87-91.
51. Weinstein IB. Disorders in cell circuitry during multistage carcinogenesis: the role of homeostasis.Carcinogenesis. 2000 May; 21(5):857-64.
52. 吴晓红.疼痛按“时间”论治.新疆中医药杂志 1995;(3):82
53. Yamazaki S, Numano R, Abe M, et al. Resetting central and periphera lcircadian oscillators intransgenic rats. Science, 2000, 288(5466): 682-685
54. Yan L, Takekida S, Shigeyosi Y et al. Per1 and Per2 gene expression in the rat suprachiasmatic nucleus: circadian profile and the compartment-specific response to light. Neuroscience, 1999, 94: 141-150
55. 杨普选,李长荣.运用中医时间医学治疗慢性气管炎486例.陕西中医 1998;19(4):1482
56. 杨一民.中医时间医学临床应用举隅.河北中医学院学报 1998;13(4):132
57. 张莉莎,刘燕华.因时制宜临证举隅.中国医药学报 1999;27(6):552
58. Zheng B H, Albrecht U, Kaasik K, et al. Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell, 2001, 105(5): 683-694
59. 周汉清.运用《内经》时间医学治疗五更泻临床观察.新中医 1994;26(11):202
60. ZylkaM J, Shearman L P,W eaver D R, et al. Three period homo logs in mammals: differential ligh t responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron, 1998, 20: 1103- 1110.
2. Akashi M, Nishida E. Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock. Genes Dev. 2000 Mar 15; 14(6): 645-9.
3. Balsaobre A, Damiola F, Schibler U. A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell. 1998 Jun 12; 93(6): 929-37.
4. Balsalobre A, Marcacci L, Schibler U. Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts. Curr Biol. 2000 Oct 19; 10(20): 1291-4.
5. Bos NP, Mirmiran M. Circadian rhythms in spontaneous neuronal discharges of the cultured suprachiasmatic nucleus.Brain Res. 1990 Mar 12; 511(1):158-62.
6. Broussard DL, Li H, Altschuler SM. Colocalization of GABA (A) and NMDA receptors within the dorsal motor nucleus of the vagus nerve (DMV) of the rat.Brain Res. 1997 Jul 18; 763(1):123-6.
7. Buijs RM, Hermes MH, Kalsbeek A. The suprachiasmatic nucleus-paraventricular nucleus interactions: a bridge to the neuroendocrine and autonomic nervous system. Prog Brain Res. 1998; 119: 365-82.
8. Buijs RM, Kalsbeek A, van der Woude TP, van Heerikhuize JJ; Shinn S. Suprachiasmatic nucleus lesion increases corticosterone secretion. Am J Physiol. 1993 Jun; 264(6 Pt 2):R1186-92.
9. Buijs RM, Wortel J, Van Heerikhuize JJ, Feenstra MG, Ter Horst GJ, Romijn HJ, Kalsbeek A. Anatomical and functional demonstration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway. Eur J Neurosci. 1999 May; 11(5): 1535-1544.
10. Bult H. Boeckxstaens GE. Pelckmans PA. et al. Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurolransmitter. Nature, 1990, 345:346～349
11. Chai NL, Dong L, Li ZF,et al. Effects of neurotrophins on gastrointestinalmyoelectric activities of rats. World J Gastroenterol, 2003 Aug;9(8):1874～1877.
12. Chansard M, Iwahana E, Liang J, Fukuhara C. Regulation of cAMP-induced arylalkylamine N-acetyltransferase, Period1, and MKP-1 gene expression by mitogen-activated protein kinases in the rat pineal gland. Brain Res Mol Brain Res. 2005 Oct 3; 139(2): 333-40.
13. Damiola F, Le Minh N, Preitner N, Kornmann B, Fleury-Olela F, Schibler U. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev. 2000 Dec 1; 14(23): 2950-61.
14. David M.A., Lesie M., John W.H., et al. Co-localization of choline acetyltransferase and tyrosine hydroxylase within neurons of the dorsal motor nuclers of the vagus. J.Chemixal Neuroanatomy, 3: 133-140
15. Dawson TW, Bredt DS, Fotuhi M, et al.Nitric oxide synthase and neuronal NADPH-disphorase are indentical in brain and peripheral tissuse, Proc Natl Acad Sci USA, 1991,88(13):7797～7801.
16. Emmanuel K.T., Williams R.G. Catecholaminergic parasympathetic efferents within the dorsal motor nucleus of the vagus in the rat: a quantitative analysis. Neuroscience Letter, 1988(90): 1-5
17. Ewart WR, Jones MV, King BF. Central origin of vagal nerve fibers innervating the fundus and corpus of the stomach in rat. J Auton Nerv Syst. 1988 Dec; 25(2-3): 219-31.
18. Ferreira M Jr, Browning KN, Sahibzada N, et al. Glucose effects on gastric motility and tone evoked from the rat dorsal vagal complex. J Physiol. 2001 Oct 1; 536(Pt 1): 141-52.
19. Fukuhara C, Yamazaki S, Liang J. Pineal circadian clocks gate arylalkylamine N-acetyltransferase gene expression in the mouse pineal gland. J Neurochem. 2005 Apr; 93(1): 156-62.
20. Gererdai I, Halasz B. Central nervous system structures connected with theendocrine glands. Findings obtained with the viral transneuronal tracing technique. Exp Clin Endocrinol Diabetes. 2000; 108(6): 389-95.
21. Glavin GB, Murison R, Overmier JB, Pare WP, Bakke HK, Henke PG, and Hernandez DE. The neurobilolgy of stress ulcers. Brain Res Rev 1991, 16: 301-343,
22. Goncharuk VD, van Heerikhuize J, Dai JP, Swaab DF, Buijs RM. Neuropeptide changes in the suprachiasmatic nucleus in primary hypertension indicates functional impairment of the biological clock. J Comp Neurol. 2001 Mar 12; 431(3): 320-30.
23. Grabauskas G, Moises HC. Gastrointestinal-projecting neurones in the dorsal motor nucleus of the vagus exhibit direct and viscerotopically organized sensitivity to orexin.J Physiol. 2003 May 15; 549(Pt 1):37-56.
24. Groos G, Hendriks J. Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro. Neurosci Lett. 1982 Dec 31; 34(3): 283-288.
25. 郭海军,林洁,李国成.功能性消化不良的动物模型研究.中国中西医结合杂志,2001,9(3):1411～1412
26. Guo J.J, Browning KN, Rogers RC, et al. Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus. Am J Physiol Gastrointest Liver Physiol 2001, 280: G361-G367
27. Hayakawa T, Takanaga A, Tanaka K, et al. Cells of origin of vagal motor neurons projecting to different parts of the stomach in the rat: confocal laser scanning and electron miroscopic study. Anat Embryol. 2003 Dec; 207(4-5): 289-297
28. Hirano T, Niijima A. Effects of 2-deoxy-D-glucose, glucose and insulin on efferent activity in gastric vagus nerve. Experientia. 1980 Oct 15; 36(10): 1197-8
29. Hirota T, Fukada Y. Resetting mechanism of central and peripheral circadian clocks in mammals. Zoolog Sci. 2004 Apr; 21(4): 359-368.
30. Hirota T, Okano T, Kokame K, Shirotani-Ikejima H, Miyata T, Fukada Y. Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expressionin cultured Rat-1 fibroblasts. J Biol Chem. 2002 Nov 15; 277(46): 44244-51.
31. Izumo M, Johnson CH, Yamazaki S. Circadian gene expression in mammalian fibroblasts revealed by real-time luminescence reporting: temperature compensation and damping. Proc Natl Acad Sci U S A. 2003 Dec 23; 100(26): 16089-16094.
32. Jin L, Oliveira A, Inwards C, et al. Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and RT-PCR. Diagn Mol Pathol, 2003, 12(4): 224-230
33. Kalsbeek A, Garidou ML, Palm IF, Van Der Vliet J, Simonneaux V, Pevet P, Buijs RM. Melatonin sees the light: blocking GABA-ergic transmission in the paraventricular nucleus induces daytime secretion of melatonin. Eur J Neurosci. 2000 Sep; 12(9):3146-54.
34. Kalsbeek A, La Fleur S, Van Heijningen C, Buijs RM. Suprachiasmatic GABAergic inputs to the paraventricular nucleus control plasma glucose concentrations in the rat via sympathetic innervation of the liver. J Neurosci. 2004 Sep 1; 24(35):7604-7613.
35. Kalsbeek A, Ruiter M, La Fleur SE, Van Heijningen C, Buijs RM. The diurnal modulation of hormonal responses in the rat varies with different stimuli.J Neuroendocrinol. 2003 Dec; 15(12):1144-55.
36. Kalsbeek A, Van Heerikhuize JJ, Wortel J, Buijs RM. A diurnal rhythm of stimulatory input to the hypothalamo-pituitary-adrenal system as revealed by timed intrahypothalamic administration of the vasopressin V1 antagonist.J Neurosci. 1996 Sep 1; 16(17): 5555-65.
37. Kubek M.J., Rea M.A., Hodes Z.I., et al. Quantitation and characterization of thyrotropin-releasing hormone in vagal nuclei and other regions of the medulla oblongata of the rat. J. Neurohemistry. 1983, 40(5): 13071313
38. Ladic LA, Buchan AM. Association of substance P and its receptor with efferent neurons projecting to the greater curvature of the rat stomach. J Auton Nerv Syst.1996 Apr 20; 58(1-2): 25-34.
39. Leahy A , Besberdas K , Clayman C , et al. Abnormalities of the electrogastrogram in functional gastrointestinal disorder. Am J Gastroenterol,1999,94(4):1023～1027
40. La Fleur SE, Kalsbeek A, Wortel J, Buijs RM. A suprachiasmatic nucleus generated rhythm in basal glucose concentrations. J Neuroendocrinol. 1999 Aug; 11(8): 643-52.
41. La Fleur SE, Kalsbeek A, Wortel J, Buijs RM. Polysynaptic neural pathways between the hypothalamus, includes the suprachiasmatic nucleus, and the liver. Brain Res. 2000 Jul 14; 871(1): 50-6.
42. Larsen PJ, Enquist LW, Card JP. Characterization of the multisynaptic neuronal control of the rat pineal gland using viral transneuronal tracing. Eur J Neurosci. 1998 Jan; 10(1):128-45.
43. Liu Y, Heintzen C, Loros J, Dunlap JC. Regulation of clock genes. Cell Mol Life Sci. 1999 Aug 15; 55(10): 1195-205.
44. Lefebvre RA, De Vriese A, and Smits GJM. Influence of vasoactive intestinal polypeptide and NG-nito-L-arginine methylester on cholinergic neurotransmission in the rat gastric fundus. Eur J Pharmacol1992, 221: 235-242,
45. Lewis MW, Travagli RA. Effects of substance P on identified neurons of the rat dorsal motor nucleus of the vagus. Am J Physiol Gastrointest Liver Physiol. 2001 Jul; 281(1): G164-G172
46. Manier M., Mouchet P, Feuerstein C., et al. Immunohistochemical evidence for the coexistence of cholinergic and catecholaminergic phenotypes in neurons of the vagal motor nuclers in the adult rat, Neurosci. Lett.80 (1987): 141-146
47. Masuda N, Ohnishi T, Kawamoto S, et al. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Moore RY. Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus. FedProc. 1983 Aug; 42(11): 2783-9.
48. Nakamura TJ, Shinohara K, Funabashi T, Mitsushima D, Kimura F. Circadian and photic regulation of cryptochrome mRNAs in the rat pineal gland. Neurosci Res. 2001 Sep; 41(1): 25-32.
49. Mutsuga N, Shahar T, Verbalis J, et al. Selective gene expression in magnocelllular neurons in rat supraoptic nucleus. Journal of Neuroscience. 2004, 24 (32): 7174-7185
50. Nicole L, Robert F, and John W, et al. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Review, 1998, 14(7): 272-276.
51. Obiakor H, Sehgal D, Dasso JF, et al. A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ. Analytical Biochemistry, 2002, 306(1): 55-62
52. Ozawa M, Aono M, Moriga M. Central effects of pituitary adenylate cyclase activating polypeptide (PACAP) on gastric motility and emptying in rats. Dig Dis Sci. 1999 Apr; 44(4): 735-743
53. Parsons RL, Neel DS, McKeon TW, et al.Organization of a vertebrate ganglion: a correlated biochemical and histochemical study. J Neurosci 7: 837-846
54. Perreau-Lenz S, Kalsbeek A, Garidou ML, et al. Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J Biol Chem. 1998 Oct 16; 273(42): 27039-42.
55. Randall WC, Wurster RD. Peripheral innervation of the heart. In: Levy MN, Schwartz PJ, and editors. Vagal control of the heart: experimental basis and clinical implications. New York: Futura Puvlishing. 1994: 21-32
56. Rogers RC, Hermann GE, and Travagli RA. Brainstem pathways responsible for oesophageal control of gastric motility and tone in the rat. J Physiol(Lond) 1999, 514:369-383,
57. 茹立强.胆碱脂酶的组织化学及超微定位。神经介质及有关酶类组织化学(艾民康主编)。北京:人民卫生出版社,1987,110～113
58. 茹立强.消化管壁内神经丛及其神经元,现代科技综述大辞典(主编:朱永和)北京:科学出版社,1998, 1301～1302.
59. Rwppert SM, Weaver DR. Molecular analysis of mammalian circadian rhythms.Annu Rev Physiol. 2001; 63:647-76. Sakaguchi T, Yamaguchi K. Changes in efferent activities of the gastric vagus nerve by administration of glucose in the portal vein. Experientia. 1979 Jul 15; 35(7): 875-6.
60. Scheer FA, Ter Horst GJ, van Der Vliet J, Buijs RM. Physiological and anatomic evidence for regulation of the heart by suprachiasmatic nucleus in rats. Am J Physiol Heart Circ Physiol. 2001 Mar; 280(3): H1391-9.
61. Scott SM. Manometric techniques for the evaluation of colonic motor activity:current status, Neurogastroenterol Motil.2003 Oct;15(5):483～513
62. Simonneaux V, Pevet P, Buijs RM. Suprachiasmatic control of melatonin synthesis in rats: inhibitory and stimulatory mechanisms. Eur J Neurosci. 2003 Jan; 17(2): 221-8.
63. Simonneaux V, Piorel VJ, Garidou ML, Nguyen D, Diaz-Rodriguez E, Pevet P. Daily rhythm and regulation of clock gene expression in the rat pineal gland. Brain Res Mol Brain Res. 2004 Jan 5; 120(2): 164-72.
64. Steffens AB. A method for frequent sampling blood and continuous infusion of fluids in the rat without disturbing the animal. Physiol. Behav. 4: 833-836.
65. Stoll S., Konig S., Thanos S. Isolation of total-RNA from formalin-fixed rat retina. Journal of Neuroscience Methods, 2002, 120(1): 55-63.
66. Susan M, Stockton S, Carol S, et al. Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Molecular Carcinogenesis, 1999, 25:86-91
67. Swanson LW, Sawchenko PE. Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology. 1980, 31(6): 410-417.
68. Swanson LW, Sawchenko PE, Wiegand SJ, et al. Separate neurons in the paraventricular nucleus project to the median eminence and to the medulla or spinal cord. Brain Res. 1980, 198(1): 1950-1955.
69. Takahashi T and Owyang C. Characterization of vagal pathways mediating gastric accommodation reflex in rats. J Physiol(Lond) 1997, 504: 479-488,
70. Takanaga A, Hayakawa T, Tanaka K et al. Immunohistochemical characterization of cardiac vagal preganglionic neurons in the rat. Autonomic Neuroscience: Basic and Clinical. 2003, 106: 132-137
71. Tatewaki M, Harris M, Uemura K, et al. Dual effects of acupuncture on gastric motility in conscious rats. Am J Physiol Regul Integr Comp Physiol, 2003, 285(4): 862～867
72. Teclemariam-Mesbah R, Ter Horst GJ, Postema F, Wortel J, Buijs RM. Anatomical demonstration of the suprachiasmatic nucleus-pineal pathway. J Comp Neurol. 1999 Apr 5; 406(2): 171-82.
73. Tony E, Sun-Hun Kim, Marielena Chavira, et al. Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5’ nuclease quantitative reverse transcription-polymerase chain reaction. Journal of Molecular Diagnostics, 2000, 2(2): 84-91.
74. Twstoni PA, Fanti L, Bagnolo F, et al. Manometric evalution of the interdigestive antroduodenal motility subjects with fasting bile reflux with and without antigastritis, Gut 1989; 30: 443-448
75. Vivianna M, Lisa H, Peter T. Optimizing gene expression analysis in archival brain tissue. Neurochemical Research, 27(10): 993-1003.
76. Wang ZS, Elsenbruch S, Orr WC, et al. Detection of gastric slow wave uncoupling from multi-channel electrogastrogram: validations and applications. Neurogastroenterol Motil. 2003 Oct; 15(5): 457-465.
77. Weaver DR. The suprachiasmatic nucleus: a 25-year retrospective. J Biol Rhythms. 1998 Apr; 13(2): 100-12.
78. Welsh DK, Yoo SH, Liu AC, Takahashi JS, Kay SA. Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression.Curr Biol. 2004 Dec 29;14(24):2289-95.
79. 徐三平,易粹琼.西沙必利对部分胃肠神经递质释放的影响.临床消化病学杂志,1999;11(2):71～72
80. 张经济,许冠荪.消化道生理与病理生理学.第一版. 广东:广东科技出版社,1997,10:42
81. Yamamoto T, Nakahata Y, Soma H, Akashi M, Mamine T, Takumi T. Transcriptional oscillation of canonical clock genes in mouse peripheral tissues. BMC Mol Biol. 2004 Oct 9; 5: 18.
82. Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M, Tei H. Resetting central and peripheral circadian oscillators in transgenic rats. Science. 2000 Apr 28; 288(5466): 682-685.
83. Young MW, Kay SA. Time zones: a comparative genetics of circadian clocks. Nat Rev Genet. 2001 Sep; 2(9): 702-715.
84. Zhou L,Xie YK. A strain gauge for measuring gastrointestinal motility in animals. Chin J Appl Physiol, 1988,4:346-349
85. Zylka MJ, Shearnan LP, Levine JD, Jin X, Weaver DR, Reppert SM. Molecular analysis of mammalian timeless. Neuron. 1998 Nov; 21(5): 1115-1122.
86. Zylka MJ, Shearman LP, Weaver DR, Reppert SM. Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron. 1998 Jun; 20(6): 1103-1110.
1. Albrecht U, Sun Z S, Eichele G, et al. Adifferentialresponse of two putative mammalian circadian regulators, mPer1 and mPer2, to light. Cell, 1997, 91: 1055-1064
2. Bae K, Jin X W, Maywood E S, et al. Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron, 2001, 30(2): 525-536
3. Balsalobre A, Brown S A, Marcacci L, et al. Resetting of circadian time in peripheral tissues by glucocorticoidsignaling. Science, 2000, 2 8 9(5488): 2344-2347
4. Balsalobre A, Damiola F, Schibler U. A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell, 1998, 93(6): 929-937
5. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells that set the circadian clock. Science, 2002, 295:1070-1073
6. Bouskila Y, Dudek FE. Can a population of suprachiasmatic nucleus neurons with different period lengths produce a stable circadian rhythm? Brain Res, 1995, 670∶333-336.
7. Bunger M K, Wilsbacher L D, Moran S M, et al. Mop3 is an essential component of the master circadian pacemaker in mammals. Cell, 2000, 103:1009-1017
8. Chaurasia SS, Pozdeyev N, Haque R, Visser A, Ivanova TN, Iuvone PM. Circadian clockwork machinery in neural retina: evidence for the presence of functional clock components in photoreceptor-enriched chick retinal cell cultures.Mol Vis. 2006 Mar 30;12: 215-23.
9. Damiola F, LeM N , Preitner N , et al. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev, 2000, 14: 2950- 2961.
10. Delaunay F, Laudet V. Circadian clock and microarrays: mammalian genome gets rhythm. Trends Genet, 2002, 18 (12): 595-597
11. Dunlap J.C. Molecular bases for circadian clocks.Cell. 1999 Jan 22; 96(2):271-90.
12. Edery I. Circadian rhythms in a nutshell. PhysiolGenomics, 2000, 3: 59-74
13. 范镇海.依时辰辨治内科杂病三法.湖南中医学院学报 1995;15(2):402
14. Foster RG, Lucas RJ. Clocks, criteria and critical genes.Nat Genet. 1999 Jul; 22(3):217-219.
15. Fukada Y, Okano T. Circadian clock system in the pineal gland. Mol Neurobiol, 2002,25(1):19-30
16. Gekakis N, Staknis D, Nguyen H B, et al. Role of the CLOCK protein in the mammalian circadian mechanism. Science, 1998, 280: 1564-1569
17. Grosse J, Davis FC. Melatonin entrains the restored circadian activity rhythms of syrian hamsters bearing fetal suprachiasmatic nucleus grafts. J Neurosis, 1998, 18∶8032-8037.
18. Hamada T, L esauter J, V enuti JM, et al. Exp ression of Period genes: rhythmic and nonrhythmic compartments of the suprachiasmatic nucleus pacemaker. J N euro sci, 2001, 21: 7742- 7750.
19. Hao H P, Allen D L, Hardin P E. A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila melanogaster. Mol Cell Biol, 1997, 17 : 3687-3693
20. Hogenesch J B, Gu Y Z, Jain S, et al. The basic-helix-loophelix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci USA, 1998, 95 (10): 5474-5479
21. Horst TJ, Muijtjens M, Konayashi K, et al. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature, 1999, 398: 627-630
22. 侯恒太,等.乘时用药医案三则.河南中医杂志 1996;16(2):1172
23. Hsu D S, Zhao X D, Zhao Y, et al. A putative human bluelight photoreceptors hCRY1 and hCRY2 are flavoproteins. Biochemistry, 1996, 35(14): 13871-13877
24. 黄娟,杜怀荣.运用时间医学方法治愈外感发热1例.北京中医药大学学报 1997;20(3):653
25. Inouye S2IT and Kawamura H. Characteristics of a circadian pacemaker in the suprachiasmatic nucleus. J Comp Physiol, 1982, 146∶153-160.
26. Jones C R, Campbell S S, Zone S E, et al. Familial advanced sleep-phasesyndrome: ashort-period circadian rhythm variant in humans. Nat Med, 1999, 5: 1062-1065
27. King D P, Zhao Y L, Sangoram A M, et al. Positional cloning ofthemousecircadian clock gene. Cell, 1997, 89(4): 641-653
28. Kondo Y, Kanai Y, Sakamoto M, Mizokami M, Ueda R, Hirohashi S. Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpGislands in microdissected specimens from patients with hepatocellular carcinoma. Hepatology. 2000 Nov; 32(5):970-979.
29. 孔凡涵,赵传莲,张锐.中医时间医学在临床应用.光明中医 2000;17(5):182
30. Konopka R J, Benzer S. Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci USA, 1971, 68: 2112-2116
31. Kovacikova Z, Sladek M, Bendova Z, Illnerova H, Sumova A. Expression of Clock and Clock-Driven Genes in the Rat Suprachiasmatic Nucleus during Late Fetal and Early Postnatal Development.J Biol Rhythms. 2006 Apr; 21(2):140-148.
32. Liu C, Peppert SM. GABA synch ronizes clock celles w ith in the sup rach iasmatic circadian clock. Neuron, 2000, 25: 123- 128.
33. 刘世兴,杜淑云.简析中风发病与死亡时间.中医杂志 1998;39(8):5082
34. 陆保年.应用中医时间医学理论探讨对发热的辨证论治.四川中医 1994;12(5):123
35. McNamara P, Seo S P, Rudic R D, et al. Regulation of CLOCK and MOP4 by nuclear hormone receptors in the vasculature: a humoral mechanism to reset a peripheral clock. Cell, 2001, 105(7): 877-889
36. Mueller RL. Evolutionary rates, divergence dates, and the performance of mitochondrial genes in bayesian phylogenetic analysis.Syst Biol. 2006 Apr;55(2):289-300
37. Okamura H, Miyake S, Sumi Y, et al. Photic induction of mPer1 and mPer2 in Cry-deficient mice lacking a biological clock. Science, 1999, 286: 2531-2534
38. Panda S, Antoch M P, Miller B H, et al. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell, 2002, 109(3): 307-320
39. Pruitt K, Pestell RG, Der CJ. Ras inactivation of the retinoblastoma pathway by distinct mechanisms in NIH 3T3 fibroblast and RIE-1 epithelial cells. J Biol Chem. 2000 Dec 29;275(52):40916-40924
40. 秦庆云,秦昕昕.中医治疗高热的时间医学探讨.中西医结合实用临床急救杂志 1997;4(8):3372
41. Reppert SM, Weaver DR. Forward genetic approach strikes gold: cloning of a mammalianclock gene.Cell. 1997 May 16; 89(4):487-490.
42. Shearman LP, Zylka MJ, Weaver DR, Kolakowski LF Jr, Reppert SM. Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei. Neuron. 1997 Dec;19(6):1261-1269
43. Shearman L P, Jin X W, Lee C, et al. Targeted disruption of the mPer3 gene: subtle effects on circadian clock function. Mol Cell Biol, 2000, 20: 6269-6275
44. Shgeyoshi Y, Taguchi K, Yamamoto S, et al. Light induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript. Cell, 1999, 9: 1043-1053
45. Stokkan K. A, Yamazaki S, Tei H, et al. Entrainment of the circadian clock in the liver by feeding. Science, 2001, 291 (5503): 490-493
46. Sun Z S, Albrecht U, Zhuchenko O, et al. RIGUI, a putative mammalian ortholog of the Drosophila period gene. Cell, 1997, 90 : 1003-1011
47. Tei H, Okamura H, Shigeyoshi Y, et al. Circadianoscillation of a mammalian homologue of the Drosophila period gene. Nature, 1997, 389: 512-516
48. Veleri S, Brandes C, Helfrich-Forster C et al. A self-sustaining, Light-entrainable circadian oscillator in the drosophila brain. Current Biology, 2003, 13: 1758–1767.
49. Vitaterna M H, King D P, Chang A M, et al. Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior. Science, 1994, 264: 719-725
50. Whitmore D, Foulkes NS, Sassone-Corsi P. Light acts directly on organs and cells in culture to set the vertebrate circadian clock. Nature. 2000 Mar 2; 404(6773):87-91.
51. Weinstein IB. Disorders in cell circuitry during multistage carcinogenesis: the role of homeostasis.Carcinogenesis. 2000 May; 21(5):857-64.
52. 吴晓红.疼痛按“时间”论治.新疆中医药杂志 1995;(3):82
53. Yamazaki S, Numano R, Abe M, et al. Resetting central and periphera lcircadian oscillators intransgenic rats. Science, 2000, 288(5466): 682-685
54. Yan L, Takekida S, Shigeyosi Y et al. Per1 and Per2 gene expression in the rat suprachiasmatic nucleus: circadian profile and the compartment-specific response to light. Neuroscience, 1999, 94: 141-150
55. 杨普选,李长荣.运用中医时间医学治疗慢性气管炎486例.陕西中医 1998;19(4):1482
56. 杨一民.中医时间医学临床应用举隅.河北中医学院学报 1998;13(4):132
57. 张莉莎,刘燕华.因时制宜临证举隅.中国医药学报 1999;27(6):552
58. Zheng B H, Albrecht U, Kaasik K, et al. Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell, 2001, 105(5): 683-694
59. 周汉清.运用《内经》时间医学治疗五更泻临床观察.新中医 1994;26(11):202
60. ZylkaM J, Shearman L P,W eaver D R, et al. Three period homo logs in mammals: differential ligh t responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron, 1998, 20: 1103- 1110.
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