【作者】 张珂；

【导师】 刘俊峰；

【作者基本信息】 河北医科大学 ， 外科学， 2014， 博士

【摘要】 食管胃结合部（Esophageogastric junction, EGJ）一束约2-3cm宽、特殊增厚的环形肌肉被称为食管下括约肌(Lower esophageal sphincter,LES)。Liebermann-Meffert在1979年第一次提出了人LES是由位于胃小弯侧的钩状纤维(clasp fibres)与胃大弯侧套索纤维（sling fibers）共同构成。斜行的套索纤维，半环形的钩状纤维与膈脚一起，在包括神经、体液等多种因素的共同作用下保持持续的收缩状态，因此EGJ形成了一个高压带，从而EGJ发挥了生理性抗返流屏障的作用，能够有效地防止胃向食管的返流。在中枢神经系统的支配下，食管下括约肌收缩和舒张功能的调节由多种神经递质、激素及自身肌源性因素共同完成。以往的研究发现，迷走神经末梢与食管下括约肌的肌层肠道运动神经元构成了调节食管下括约肌收缩和舒张的神经通路，即兴奋性迷走神经通路和抑制性迷走神经通路。节前、节后胆碱能运动神经元二者共同构成了兴奋性运动传导通路。而抑制性运动传导通路则由节前胆碱能运动神经元与节后非胆碱能非肾上腺素能神经元（Nonadrenergic Noncholinergic, NANC）二者共同构成。上述两种神经通路激活后引起胆碱能节后神经元或者肠道神经元各种神经递质的释放。通过这些神经递质的释放，从而实现了神经对食管下括约肌收缩、舒张的调节。根据既往科研结果，目前研究比较明确的是兴奋性神经传导通路激活后，神经末梢主要释放乙酰胆碱和P物质两种神经递质，引起食管下括约肌的收缩。而抑制性传导通路激活后，导致肠道神经元主要释放一氧化氮和血管活性肠肽两种神经递质，使食管下括约肌舒张。同时还存在其他类型的神经递质参与食管下括约肌的收缩与舒张过程，但目前看，他们并不发挥主要作用。研究文献发现，例如贲门失弛缓症、胡桃夹食管和弥漫性食管痉挛等食管运动障碍性疾病，以及其它涉及食管排空及返流性疾病中，绝大部分表现有食管下括约肌舒张收缩功能异常。综上所述，我们认为只有对食管下括约肌调节机制展开深入的研究，才能开展对食管运动障碍性疾病有效的诊断与治疗。目前国内外关于人食管下括约肌的研究主要集中在P物质、血管活性肠肽、乙酰胆碱、一氧化氮以及等神经递质，信号传导通路以及受体等方面。内皮素（Endothelin, ET）是一种由21个氨基酸组成的生物活性物质。它主要由内皮细胞合成，具有非常强烈的收缩血管的生物活性。近年来研究发现除了具有强烈缩血管作用，同时内皮素也具有诱导导血管生成、促分化、促细胞丝分裂和类细胞生长因子的性质。研究文献发现人有3种内皮素亚型（ET-1、ET-2、ET-3），这三个异构肽的生物活性有一定差异，ET-1的作用最强，ET-3最弱。内皮素分子通过内皮素受体(ETRs)发挥其生理和病理作用。内皮素受体被发现广泛地表达于动物的胃肠道之中。本研究采用实时定量聚合酶链反应(Quantitative PCR)测定各受体的表达相对定量，然后以逆转录聚合酶链反应(RT-PCR)验证上述结果，在用蛋白印迹法（Western-blot）分析受体蛋白表达是否与mRNA表达一致。然后以受体激动、拮抗及电场刺激（Electrical field stimulation, EFS）等方法对ETRs在人食管下括约肌中的表达及功能进行研究。探讨了ETRs在人食管下括约肌调节机制中的作用，为进一步研究内皮素及其受体在人食管下括约肌调节机制中的作用奠定基础，从而进一步完善了人食管下括约肌的调节机制，为临床治疗食管运动功能障碍性疾病提供了理论依据。第一部分内皮素受体在人食管下括约肌的表达研究目的：内皮素受体属于G蛋白偶联受体家族，内皮素通过内皮素受体发挥生理和病理学功能。先以实时定量聚合酶链反应(Quantitative PCR)测定各受体亚型在食管下括约肌的套索纤维、钩状纤维以及胃底和食管的环行肌中表达相对定量，然后以逆转录聚合酶链反应(RT-PCR)验证上述结果，在用蛋白印迹法（Western-blot）分析受体蛋白表达是否与mRNA表达一致，探讨内皮素受体在人食管下括约肌中的表达规律。方法：选取2011年12月至2012年11月在河北医科大学第四医院胸外科，罹患高位食管癌行食管大部切除术的患者32例。其中8名女性，24名男性，年龄平均61岁。手术室采集新鲜的手术食管胃结合部标本，实验室以手术剪、镊子锐性剥去包含食管下段、贲门和胃底的标本上所有的粘膜组织及粘膜下层，制备食管、套索纤维、钩状纤维和胃底环行肌的肌条。实验提取组织内的总RNA，以紫外线分光光度计鉴定其纯度后，再以1％琼脂糖凝胶电泳测定其完整性，之后，再分别应用2种内皮素受体亚型的引物行实时定量PCR，测定这两种受体的mRNA在4种肌条表达的ΔCT值，同时测定相应部位组织内β-actinΔCT值，然后以二者计算受体亚型mRNA表达的相对定量。实时定量PCR后的扩增产物以5Xbuffer染色后电泳，验证产物的分子量。再以最初提取的总RNA，分别应用2种内皮素受体亚型的引物行RT-PCR，验证产物分子量。之后测定扩增产物的光密度值(IOD)，再用Gel Pro软件分析，以内皮素受体亚型的IOD与β-actin的IOD的比值代表各个肌条中mRNA的相对表达量。提取各位点的组织总蛋白，以考马斯亮蓝G-250定量蛋白后，将各蛋白样品调至浓度相同，再以电泳仪电泳分离出与内皮素2个亚型相对应的受体蛋白后再转膜，分别以内皮素受体2个亚型的抗体进行孵育，再以荧光二抗染色，经Odyssey软件分析荧光反应条带的光密度值(IOD)。结果：紫外分光度计显示总RNA A260/A280比值为1.8-2.0；1%琼脂糖凝胶电泳验证RNA完整性，可见28S处电泳条带的宽度和亮度均是18S处条带的约两倍。实时定量PCR的内参β-actin于130bp处，在各个位点的扩增样品中，内参β-actin的实时定量PCR扩增结果一致。计算后发现2种内皮素受体亚型的mRNA在各肌条中均有明确的表达，再将扩增的产物以5Xbuffer染色后电泳，验证其分子量。于166bp和128bp可见两条清晰的条带，分别是内皮素受体A(ETA)，内皮素受体B(ETB)的mRNA。ETA受体亚型的mRNA表达最高，在钩状纤维、套索纤维、食管平滑肌和胃底平滑肌肌条的表达水平分别为0.01151±0.00055；0.01207±0.00086；0.01185±0.00031；0.01161±0.00055；ETB表达水平比ETA低，依次为0.001691±0.00020；0.001793±0.00030；0.001616±0.00028；0.001571±0.00013；实时定量PCR扩增各个产物的长度与最初设计长度完全一致。同一肌条中，不同的内皮素受体亚型mRNA的表达水平比较有统计学差异(F=72.291; P=0.000)。同一个受体亚型在不同的位点，mRNA的表达并无差异(F=0.398; P=0.5329)。再以RT-PCR方法验证上述结果，电泳后检测发现内参β-actin在各个位点的扩增结果基本一致，且与设计分子量一样，同为362bp。ETA和ETB这2种内皮素受体的电泳条带的分子量与设计大小一样，分别为263bp和200bp。ETA相对表达量在钩状纤维、套索纤维、食管平滑肌和胃底平滑肌肌条的表达水平分别为0.01163±0.00062；0.01219±0.00049；0.01147±0.00049；0.01148±0.00054；ETB表达水平比ETA低，依次为0.001725±0.00019；0.001700±0.00029；0.001632±0.00027；0.001625±0.00020；同一肌条中，不同的内皮素受体亚型mRNA的表达水平比较有统计学差异(F=102.827;P=0.000)。同一个受体亚型在不同的位点， mRNA的表达并无差异(F=0.521; P=0.476)。在测定蛋白的表达实验中发现2种内皮素受体的亚型的蛋白在四种肌条内均可见表达，ETA和ETB分子量大小分别为48.73kDa和50kDa。ETA受体亚型的受体蛋白表达最高，在钩状纤维、套索纤维、食管平滑肌和胃底平滑肌肌条的表达水平分别为0.01121±0.00063；0.01145±0.00048；0.01035±0.00068；0.01024±0.00099；ETB表达水平比ETA低，依次为0.001366±0.00022；0.001597±0.00027；0.001289±0.00020；0.001409±0.00021；同一肌条不同的内皮素受体亚型的蛋白表达水平的比较有统计学差异(F=533.841; P=0.000)，其蛋白表达水平的强弱与mRNA表达水平的结果一致。同一内皮素受体亚型的蛋白表达水平在不同的肌条间比较没有统计学差异(F=0.398; P=0.755)。结论：人LES存在两种内皮素受体亚型，分别是ETA、ETB。ETA其表达水平高于ETB，可能在人LES的舒张、收缩功能调节中发挥着作用。第二部分内皮素受体在人食管下括约肌调节机制中的功能目的：研究非选择性内皮素受体激动剂和选择性的内皮素受体拮抗剂对人离体食管下括约肌肌条的套索纤维（sling）、钩状纤维（clasp）的作用，探讨内皮素受体在人食管下括约肌收缩和舒张的过程中所发挥的调节机制。方法：选取2012年10月到2013年12月在河北大学附属医院因高位食管癌行食管大部切除术患者30例。男性患者16例，女性患者14例，平均年龄65岁。手术室采集新鲜标本后剪下食管胃结合部（包含部分食管下端及部分胃底、胃小弯），以手术剪沿胃大弯切开标本全层后，冲洗干净。，将标本粘膜面向上固定于蜡盘中（蜡盘盛Krebs液浸泡过标本表面，并以95％O2和5％CO2的混合气体持续通气）。锐性剥除掉食管下段以及胃贲门部的粘膜层及粘膜下层，可见食管胃结合部的一束增厚、发白的环形肌肉即为食管下括约肌。肉眼识别出位于标本、胃小弯侧的半环形的钩状纤维（clasp）与位于标本两端、斜行走形、胃大弯侧的套索纤维（sling）。分别沿钩状纤维（clasp）和套索纤维（sling）各自肌纤维走行的方向锐性游离肌束，制备用于实验的(2~4) mm×(8~12) mm肌条。将已经用丝线分别扎紧两端的肌条置于37℃恒温、含Krebs液10ml的浴槽中，持续以5%CO2和95%O2的混合气体通气。肌条上端固定于JZ101型肌肉张力换能器，下端固定，肌肉张力大小经Medlab信号采集系统采集后记录肌条的张力变化情况。轻柔、慢速地牵拉肌条，使之张力固定于400mg的稳定状态，将这时肌条保持的长度记录为初始长度L0，接下来多次轻柔、慢速牵拉肌条，每次使其初始长度L0的增加幅度约25％左右，每次肌条在被动张力下稳定15min后才可以再次牵拉。如此反复牵拉（间隔约20min），直至肌条长度被牵拉至初始长度L0的200％，记录肌条长度做为最适初长度。肌条以最适初长度稳定约40分钟后，首先向浴槽中加入10-6mol/L的卡巴胆碱(Carbachol)，待其产生的收缩力达到稳定后采集张力变化数据，洗脱。待肌条恢复原状态后，向浴槽的Krebs液中以浓度累积方式分别加入非选择性内皮素受体激动剂（Endothelin-1, ET-1）。给药浓度为(10-10、10-9、10-8、10-7、10-6mol/L)。观察给药后肌条张力变化情况，待肌条的肌力稳定后才可以继续增加一个浓度，每次加药与上一次加药后浓度稳定的时间间隔为5分钟，测定数值后根据数据绘制累计给药的浓度-反应量效曲线。在观察加入拮抗剂后的效应阶段，均于应用激动剂前30min首先加入拮抗剂，拮抗剂的浓度与激动剂诱导肌条产生最大效应的激动剂浓度相同。药物诱发的肌条舒缩反应，均以肌条舒缩产生的张力变化所占卡巴胆碱(Carbachol)于10-6mol/L产生的收缩张力的百分比均数±标准误(x±SE)来表示。结果：1非选择性内皮素受体激动剂ET-1在10-9mol/L的浓度就可以产生可以检测到的人食管下括约肌钩状纤维（clasp）和套索纤维（sling）肌条的收缩效应。在10-7mol/L时达到最大收缩张力，约为卡巴胆碱于10-6mol/L产生的收缩张力的58%。ET-1在钩状纤维（clasp）和套索纤维（sling）产生的效应无显著性区别(F=0.25P=0.62)。2应用受体阻滞剂后能拮抗激动剂的的收缩效应。(F=67.9P=0.000)应用选择性的ETA阻滞剂BQ-123在（10-9、10-8、10-7、10-6mol/L）浓度下可以部分拮抗ET-1产生的肌肉收缩作用(F=31.3P=0.0000)。选择性的ETB阻滞剂BQ-788也可部分拮抗ET-1产生的肌肉收缩作用(F=5.6P=0.02511)，其拮抗作用与BQ-123相比较明显较小(F=11.5P=0.0021)。结论：1非选择性内皮素受体激动剂ET-1在10-9mol/L的浓度可以诱导人食管下括约肌肌条产生收缩效应。随着其浓度的升高逐渐达到最大收缩效应应。提示内皮素受体A(ETA)、内皮素受体B(ETB)可能参与了对人食管下括约肌收缩作用的调节。2选择性的ETA阻滞剂BQ-123和选择性的ETB阻滞剂BQ-788均可以部分阻断非选择性内皮素受体激动剂ET-1对离体食管下括约肌肌条的收缩作用。进一步说明内皮素受体A(ETA)、内皮素受体B(ETB)不仅参与了对人食管下括约肌的调节，而且ETA和ETB激动后在人食管下括约肌均表现为收缩效应。另外ETA参与收缩效应的比重要大于ETB。第三部分内皮素受体在电刺激诱导人食管下括约肌反应中的作用目的：研究选择性内皮素受体的拮抗剂在电场刺激(EFS)下对人食管下括约肌的套索纤维、钩状纤维的作用，探讨内皮素受体在人食管下括约肌收缩调节机制中发挥的作用。方法：选取2012年10至2013年12月在河北大学附属医院胸外科因高位食管中段癌，而手术食管大部切除术患者30例，其中男性患者21例，女性患者9例，平均年龄61岁。制备套索纤维和钩状纤维肌条，方法同前。将肌条置于保持37℃恒温条件，含10ml Krebs液的浴槽中。并向溶液中以95%O2和5%CO2的混合气体持续通气。将肌条的上端与肌肉张力换能器固定，以带铂金电极的L形的固定架上固定肌条的下端。适当调整肌条位置，使之位于两个平行的环形铂金电极中间，肌条的上下端与电极环间的距离应当在3mm以上，将铂金电极连接于生理药理多用仪行电场刺激(EFS)。肌条张力变化情况利用Medlab信号采集系统记录。将肌肉牵拉至最适初长度方法同前（不同处为：使肌条张力固定于200mg的稳定状态，记录初始长度L0）。肌条以最适初长度稳定约60分钟后，开始行EFS。EFS刺激参数：单脉冲方波、波宽5ms、电压50V、频率1～512Hz以倍数递增。加入L-NNA后频率从小到大行EFS，计算刺激后的最大效应(Emax)。刺激停止后待肌条恢复平衡，先向浴槽分别加入河豚毒素，在加药20分钟后在给予EFS,比较加药前后刺激人LES出现的反应有何变化。再分别加入选择性内皮素受体A拮抗剂BQ-123（10-6mol/L）；选择性的内皮素受体B拮抗剂BQ-788（10-6mol/L）。在加药后20min后再分别行EFS，比较加药前后EFS刺激人食管下括约肌诱发的肌条反应有无变化。EFS诱发的肌条反应以肌条收缩或舒张的百分比的均数±标准误(x±SE)来表示。结果：1EFS对人食管下括约肌（钩状纤维和套索纤维）的作用：电场刺激可诱导钩状纤维和套索纤维出现频率依赖性收缩，最大舒张时电刺激频率为128Hz，EFS诱导食管下括约肌收缩的收缩的最大收缩百分比为19.3±0.8％。2人LES应用河豚毒素后EFS诱导的频率依赖性收缩能明显降低，比较应用前后，肌条的收缩效应有统计学差异（F=23.3P=0.000）。3人LES因EFS诱导产生的频率依赖性收缩反应，在应用选择性内皮素受体A拮抗剂BQ123（F=0.78P=0.384661）和选择性的内皮素受体B拮抗剂BQ788（F=0.38P=0.542583）前后收缩幅度均无明显减低，差异无显著性。结论：1应用河豚毒素后能显著降低EFS引起的人LES频率依赖性收缩。比较用药前后肌条的收缩效应发现有统计学差异。提示EFS诱导产生的人LES频率依赖性收缩是神经来源的。2选择性ETA拮抗剂BQ123和选择性ETB拮抗剂BQ788对EFS引起的人LES的收缩反应均没有抑制作用，用药前后收缩反应的变化均没有统计学差异。提示EFS诱导产生的人食管下括约肌频率依赖性收缩没有ETA和ETB的参与，即内皮素受体A和B亚型并不参与人LES的神经调节。更多还原

【Abstract】 The lower esophageal sphincter (LES), which is located at thegastroesophageal junction in human, is a specially differentiated thickenedregion of the circular muscle layer, extending over an axial distance about2–3cm. In1979, Liebermann-Meffer first proposed that the human LES consistsof clasp fibers and sling fibers at the lesser curvature and the greater curvaturerespectively. The crural diaphragm, the sling fibers and the clasp fibers are ina state of constant contraction, which is due to both neurogenic and myogenicregulation of the human lower esophageal sphincter and forms a high pressurezone at the esophageogastric junction (EGJ) to prevent the gastric contentsreflux into the esophagus.In the human lower esophageal sphincter, the functional regulation ofcontraction and relaxation is completed by several neurotransmitters,hormones and spontaneous myogenic factors, which are under the control ofthe the central nervous system. Previous studies revealed that the vagalefferent fibers synapse and the enteric motor neurons (EMN), which arelocated in the myenteric plexus (MP), form the inhibitory and excitatorymovement pathway. The preganglionic neurons that form the inhibitory andthe excitatory pathway are cholinergic. Moreover, the inhibitorypostganglionic neurons are nonadrenergic noncholinergic (NANC), whereasthe excitatory postganglionic neurons are cholinergic. Furthermore, both theneurotransmission of the excitatory and inhibitory vagal pathways couldrelease a variety of neurotransmitters at and regulate the tone of the humanLES. The activation of the vagal inhibitory pathway can induce relaxation ofthe LES, whereas the activation of the vagal excitatory pathway can induce thecontraction of the LES.Previous researches show that esophageal motor disorders such as achalasia, nutcracker esophagus and diffuse esophageal spasm demonstratedabnormalities of the human LES. Therefore, it is important for the treatmentand diagnosis of esophageal motility disorders to study the regulationmechanism of the human lower esophageal sphincter. The studies showed thatthe regulatory mechanism of the human lower esophageal sphincter involvesof vasoactive intestinal peptide, nitric oxide, acetylcholine, their signaltransduction pathways and receptors.ETs are composed of21-aminoacids produced by endothelial cells andconsidered to be potent vasoconstrictor. Recent studies found that they can notonly induce the angiogenesis, mitosis and differentiation, but also act the roleof cytokines. Previous studies have identified three isoforms of ET (ET-1,ET-2and ET-3), which are involved in a variety of physiological andpathological processes. ET-1is most potent one among ETs, whereas ET-3isthe weakest once. ET peptides exhibit their functions in a number ofmammalian physiological systems, including gastrointestinal. ETs act via twodistinct isoforms of receptors in human body, i.e. ETA and ETB, which can befound expressed in many mammalian gastrointestinal tract.Firstly, this study used the quantitative polymerase (quantitative PCR) toidentify the relative quantity of each ETR expression. Secondly, reversetranscription-polymerase chain reaction (RT-PCR) and western blotting wereused to verify the conclusions. Finally, measurement of muscle tension in vitro,and electrical field stimulation (EFS) were used to identify the function of theETRs in the human LES. The purpose of present study is to investigate theexpression of endothelin receptors and the role that the endothelin receptorsplay in modulating human lower esophageal sphincter function. In this way,we could further demonstrate the regulatory mechanism of the human loweresophageal sphincter in detail, and propose the theoretical bases for themedication of esophageal motility disorders much more properly.PartⅠ Expression of endothelin receptors in human lower esophagealsphincterObjective: Endothelin receptors (ETRs) belong to the family of the G protein-coupled receptors, mediate the physiological functions of thenonadrenergic noncholinergic (NANC) neurotransmitter endothlin. We usequantitative polymerase chain reaction (quantitative PCR) to identify therelative quantity of each ETR expression in four muscle strips including slingfibers, clasp fibers, circular muscle strips of esophagus and gastric foundus.Then, reverse transcription-polymerase chain reaction (RT-PCR) and westernblotting were used to verify the conclusions, in order to test whether theexpressions of ETRs mRNAs are consistent with the distribution of the ETRsproteins and to investigate the role of ETRs in modulating human LESfunction.Methods: The specimens are obtained from32patients, who underwentesophagectomy for mid-third esophageal carcinoma in the Department ofThoracic Surgery, the Fourth Hospital, Hebei Medical University, betweenDecember2011and November2012. There were7females and23males,with a mean age of61years. Each specimen including part of thegastroesophageal junction, the esophageal body, and the gastric fundus, wasresected en bloc in the operating room. The submucosa and mucosa wereremoved from the specimens, by sharp dissection. The muscle strips of claspand sling fibers of LES, and circular muscle strips of gastric foundus andesophagus were obtained from different positions of the gastroesophagealjunction (EGJ) and adjacent tissues. In laboratory, total RNA was extracted byacid guanidinium thiocyanate-phenolchloroform extraction. Before theexperiment, identify its integrity and purity with the ultravioletspectrophotometer and the electrophoresis in1%agarose gels successively.Then quantitative polymerase chain reaction (quantitative PCR) wasperformed to determine the relative quantities of each ETR expression, at first.Secondly, reverse transcription-polymerase chain reaction (RT-PCR) wasperformed to determine the mRNA expressions of ETRs. Each integratedoptical density (IOD) of the amplified products electrophoresis bands wascalculated with Gel-Pro software. The expressions of ETRs mRNA weredemonstrated by the ratio of IOD value of ETRs bands to β-actin band. The integral membrane protein receptors, which were extracted from muscle strips,were quantitated and adjusted to the identical concentration. Then, differentETRs proteins were separated by electrophoresis. Using different polyclonalantibody of each ETR after the trarsmembrane, each protein expression wasdetected by ultraviolet detector. The IOD values were calculated with theOddessy software.Results: The total RNA value of A260/280, which was determined bythe ultraviolet spectrophotometry, was between1.8and2.0. The brightnessand width of18S band was one half than28S band in1%agarose gels.Quantitative PCR identified that the band of β-actin mRNA was uniformly130bp in each strip amplified product of the specimen. After calculating thedata, we found that each ETR mRNA expression was identified in eachmuscle strip of the specimen. Next, dyed with5Xbuffer, the samples wereelectrophoresed in agarose gel, in order to verify whether the product wascorrect. After the ultraviolet detection, the band of ETA and ETB was166bpsand128bps respectively, consistent with the expected size. It was found thatthe ETA expression is more than the ETB by calculation. The ETA expressionrelative quantities of the four muscle strips including clasp fibers, sling fibers,circular muscle strips of esophagus and gastric foundus were0.01151±0.00055,0.01207±0.00086,0.01185±0.00031and0.01161±0.00055respectively. The ETB expression relative quantities of the four muscle stripsincluding clasp fibers, sling fibers, circular muscle strips of esophagus andgastric foundus were0.001691±0.00020,0.001793±0.00030,0.001616±0.00028and0.001571±0.00013respectively. Each amplifiedproduct of the quantitative PCR was consistent with the expected size, whichwas designed before the experiment. Comparing the expression of differentETR mRNA in the same muscle strips, significant differences were found(F=72.291; P=0.000). However, there was no significant difference in ETRsmRNA expression among the four muscle strips (F=0.398; P=0.5329). Then,the RT-PCR was performed to verify the conclusion mentioned above. Wefound that the band of β-actin mRNA was uniformly362bp in each strip of the specimens, after electrophoresis. The band of each ETA and ETB wasconsistent with the expected size including263bps and200bps respectively.The ETA expression relative quantities of the four muscle strips includingclasp fibers, sling fibers, circular muscle strips of esophagus and gastricfoundus were0.01163±0.00062,0.01219±0.00049,0.01147±0.00049and0.01148±0.00054respectively. The ETB expression relative quantity is lessthan the ETA. The ETB expression relative quantities of the four muscle stripsincluding clasp fibers, sling fibers, circular muscle strips of esophagus andgastric foundus were0.001725±0.00019,0.001700±0.00029,0.001632±0.00027and0.001625±0.00020respectively. Comparing the expression ofdifferent ETR mRNA in the same muscle strips, significant differences werefound (F=102.827; P=0.000). However, there was no significant difference inETRs mRNA expression among the four muscle strips (F=0.521; P=0.476).Protein expression of each ETR subtype was determined, they were ETA andETB, with respective molecular,48.73kDa and50kDa. In the same musclestrip, there was a significant difference in IOD values for different ETRS(F=533.841; P=0.000). There was no significant difference in IOD valuesamong the four muscle strips.(F=0.398; P=0.755).Conclusion: ETA and ETB can be identified in the human loweresophageal sphincter, the rank order of ETR expression is ETA>ETB.Probably these two ETRs contribute to the regulation of human LES function.PartⅡ The role of endothelin receptors in modulating human loweresophageal sphincterObjective: To determin the effects of non-selectinve endothelin receptoragonist and selective endothelin receptor antagonists on the LES complexmuscle strips, and investigate the role of endothelin receptor subtypes inregulating relaxation and contraction of the human LES.Methods: The specimens were abtained from30patients who underwentesophagectomy for mid-third esophageal carcinoma in the Department ofThoracic Surgery, the Affiliated Hospital, Hebei University, from October2012to December2013in this study. There were14females and16males, with a mean age of65years. After surgical excision in the operating room,each specimen was resected en bloc and was placed immediately in4℃Krebs solution in the laboratory successively. If there were any part of thetissue prepared for experiment contained macroscopically visible tumour,specimen was not included in this study. In the laboratory, specimens werefixed on a wax plate, which contained TBS, bubbled with continuous mixedgas of95%O2and5%CO2, washed with37℃Krebs solution, with themucosal surface facing upside to sustain its approximate in situ dimensions.Then the submucosa and mucosa were removed by sharp dissection gently.There was a thickened band of cirlular smooth muscle at the esophageogastricjunction (EGJ), namely the clasp and sling fibers of LES, adjacent to thelesser and greater curvature of the stomach, respectively. The clasp and slingmuscle strips of LES were prepared by the method described previously. Theclasp fibers, sling fibers were separated and prepared into (8~12) mm×(2~4)mm muscle strips.With each end tied with silk, the muscle strips were placed in bath, whichwas bubbled with continuous mixed gas (5%CO2and95%O2) and containinga10ml Krebs solution maintained37℃. The upper end of the muscle stripwas fixed to JZ101(an isometric force transducer) and the tension changes ofthe muscle strips was recorded by the software of MedLab6.0. The musclestrips were stretched gently and slightly to make the tension of the musclestrips maintaining at400mg, then the length of the muscle strips at thattension is the initial length L0. Then sequentially, at the increments of25%L0each time, the muscle strips were stretched to200%L0, which was recordedas most suitable initial length.The muscle strips with the most suitable initial length were equilibratedfor40minutes, then add10-6mol/L Carbachol into the bath, and record thetesion data, wash. When the strip was recoverd，then the non-selective agonistof the ETRs, namely ET-1, was added into the bath from10-10to10-6mol/L, with a cumulative manner. Not until the response of the previousconcentration stabilized, successive concentrations of the agonists were added in the bath. The time interval is5minutes. Based on the determined tensiondata above, the cumulative administration concentration-responsedose-response curves were constructed. Next, the antagonist was added30min before adding the agonist to observe the effect. And the concentration ofthe antagonist is same to the agonist, which induced maximum effectproduced by the muscle strips. In this study, we use carbachol(1×10-9mol/L)-induced contraction as a reference to express the contractileresponse to the non-selective agonist.Results:1The non-selective endothelin receptor agonist ET-1could induce thedetectable contraction of the clasp and sling fibers of the human LES at theconcentration of10-9mol/L. The maximal tension of contraction (about58%of the tension induced by1×10-9mol/L carbachol) was produced at theconcentration of10-7mol/L. There was no significant difference between thetension of clasp fibers and sling fibers, caused by ET-1.(F=0.25P=0.62)2The selective antagonist could block the contractile effect produced bythe non-selective endothelin receptor agonist ET-1.(F=67.9P=0.000) Theselective ETA antagonist BQ-123could partly block the contractile effectproduced by the non-selective endothelin receptor agonist ET-1, at theconcentration of (10-9、10-8、10-7、10-6mol/L).(F=31.3P=0.0000) Though theselective ETB antagonist BQ-788could also partly block the contractile effectproduced by the non-selective endothelin receptor agonist ET-1at theconcentration mentioned above (F=5.6P=0.02511), the antagonistic effect ofBQ-788is significantly less than BQ-123.(F=11.5P=0.0021)Conclusion:1The non-selective dopamine receptor agonist ET-1could induce thedetectable contraction of human LES at minimal concentration of10-9mol/L.With the sequential increase of ET-1concentration, the muscle strips graduallyreached the maximum contractile effect. It suggests that endothelin receptor A,and endothelin receptor B may be involved in the contractile regulation of thehuman lower esophageal sphincter. 2The selective ETA antagonist BQ-123and the selective ETB antagonistBQ-788could both partly inhibit the contractile effect produced by thenon-selective endothelin receptor agonist ET-1in human lower esophagealsphincter. The conclusion mentioned above further demonstrate that theendothelin receptor A (ETA) and endothelin receptor B (ETB) are involved inthe regulation of the human lower esophageal sphincter (contractile function)and the contribution of ETA is larger than ETB.PartⅢ The contribution of endothelin receptors in the response ofhuman lower esophageal sphincter under the electical fieldstimulationObjective: To identify the role, which the endothelin receptor antagnostsplay, in regulating the clasp fibers and sling fibers of the human loweresophageal sphincter (LES) under the electical field stimulation (EFS), andinvestigate the effect of endothelin receptors in modulating human LESfunction.Methods: The specimens were abtained from thirty patients whounderwent esophagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Affiliated Hospital, Hebei University,from October2012to December2013in this study. There were9females and21males, with a mean age of61years. Each muscle strip of the LES wasprepared by using same method described previously. With each end tied withsilk, the muscle strips were placed in bath, which was bubbled withcontinuous mixed gas (5%CO2and95%O2) and containing a10ml Krebssolution maintained37℃. The upper end of the muscle strip was fixed toJZ101(an isometric force transducer) and the tension changes of the musclestrips was recorded by the software of MedLab6.0. The muscle strips of LESwere sequentially stretched to most suitable initial length using similarmethods mentioned previously (The difference was that muscle stripsmaintaining at200mg, record the length as the initial length L0) and had a37℃bath for60min in Krebs solution, which bubbled with continuous mixedgas (95%O2and5%CO2). EFS stimulation parameters: single-pulse square wave, pulse frequency1-512Hz, voltage50V, width5ms. After added L-NNA, the muscle strips issubjected to electrical stimulation according to the frequency from small tobig and the maximum effect to EFS was assessed. The muscle strip wasstimulated again after20min of administration of Tetrodotoxin, BQ-123(aselective ETA antagonist). Then, we repeated the procedure and replacedBQ-123with BQ-788(a selective ETB antagonist). The responses in all of theexperiments were quantified based upon a percentage of the baseline value ofmuscle strip tone relative to the nadir of the response. The data wereexpressed as means±standard error.Results:1The EFS could induce a frequency-dependent contraction in the slingfibers and the clasp fibers of human LES, and the maximum contractileelectrical stimulation frequency was128Hz. The maximum contractionpercentage of the lower esophageal sphincter by EFS-induced was about19.3±0.8%.2After the administration of tetrodotoxin, the frequency-dependentcontraction in the sling and clasp fibers of human LES, which was induced byEFS, significantly decreased.(F=23.3P=0.000)3The selective antagonist BQ-123(F=0.78P=0.384661) and BQ-788(F=0.38P=0.542583) could not inhibit the contraction of human LES musclestrips, which was induced by EFS. There was no significant differencebetween before and after administration.Conclusion:1After the administration of tetrodotoxin, the frequency-dependentcontraction in the sling and clasp fibers of human LES, which was induced byEFS, was inhibited. There was significant difference between the contractionof the muscle strips before and after the administration of tetrodotoxin. Theoptimum frequency leading to maximum contraction was128Hz. It suggeststhat the frequency-dependent contraction of LES induced by EFS is nerve origin.2The selective antagonist BQ-123and BQ-788could not inhibit thefrequency-dependent contraction induced by the EFS in the sling fibers andthe clasp fibers of human LES.(BQ-123F=0.78P=0.384661, BQ-788F=0.38P=0.542583) It suggests that the ETA and ETB was note involed inthe frequency-dependent contraction induced by EFS, and ETA, ETB werenot involved in the nerve regulation.更多还原