【作者】 高庆敏；

【导师】 刘俊峰；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 硕士

【摘要】 目的:医学界对食管下括约肌(lower esophagealsphincter,LES)是否存在及其结构特点的探讨经历了一个很长的发展过程。直到1979年,Liebermann—Meffert等学者通过对32例尸体标本的解剖研究,详细阐明了食管胃连接部(esophagogastricjunction,EGJ)平滑肌束的宏观排列,进一步提出人类LES的肌肉系统由套索纤维(sling fibers)和钩状纤维(clasp fibers)共同构成。套索纤维是一组不完整的U形肌束悬挂于胃食管交界部,食管下端的左侧,His角上方。它的开口侧位于胃小弯,闭口侧位于胃大弯。钩状纤维位于LES右侧,呈半环形。这两束肌肉共同维持LES的关闭状态,并形成了LES高压带(high-pressure zone,HPZ,1 5～30 mmHg)。这一理论为进一步研究LES的生理、病理和药理学特性奠定了基础。随后学者们主要对不同动物LES的生理学特点和功能调节进行了更进一步的探讨,发现LES具有明显不同于其它平滑肌结构的特性。而且更为重要的是,构成LES的钩状纤维和套索纤维在诸多方面也存在着很大的差异性。神经递质Ach是食管下括约肌的最主要的兴奋性神经递质。体外实验证明套索纤维对乙酰胆碱(Ach)的敏感性更高。钩状纤维有更大的自发张力。造成LES套索纤维和钩状纤维结构和功能的不同的机制还未完全阐明。许多实验证明无论是在离体还是活体情况下,Ach引起食管下括约肌环形肌和纵形肌的兴奋。乙酰胆碱能神经元大部分位于食管下括约肌的左侧壁,右侧壁由轴突支配。这可能是食管下括约肌左侧压力较右侧为高的原因。P物质为脑肠肽类神经递质,P物质免疫相关性神经纤维在猫和豚鼠的食管下端中数量很多,在肠肌丛和黏膜下丛分布也较多,在环型肌和纵形肌及黏膜中也有分布。免疫组化证明,P物质存在于迷走神经末梢中。食管末端P物质可能作为对化学伤害感受刺激的神经递质,加强食管平滑肌的收缩反应。本文试图研究在乙酰胆碱和p物质这两种不同的神经递质作用下套索纤维和钩状纤维功能上的差异是否表现在细胞收缩程度上的不同。本实验应用平滑肌细胞分离技术,在倒置显微镜下通过测量平滑肌细胞长度变化来测定乙酰胆碱、P物质分别对人食管下括约肌钩状纤维、套索纤维、食管环形肌和胃底环行肌的作用,并为将来治疗食管功能性疾病奠定基础。方法:选取2008年12月至2009年12月在河北医科大学第四医院因食管中段癌行食管大部切除术患者30例,其中男性18例,女性12例,平均年龄59.3岁。手术室采集新鲜手术标本,锐性剥去胃贲门部及食管下段的粘膜层及粘膜下层后,制备食管环形肌、胃底环形肌、套索纤维、钩状纤维的肌条,将肌条剪成1 mm3-2mm3的小块,用含胶原酶II的细胞分离液分离出单个平滑肌细胞。确认分离细胞活性在90%以上。然后用37℃Hepes缓冲液悬浮备用。细胞存活率测定:取5滴细胞悬浮液,加1滴0.2%的台盼蓝溶液,混匀3 min内计数,当细胞死亡时将被染成红色,活细胞无色。用以下公式计算细胞存活率:存活率(%)=活性细胞总数/(活性细胞数+死细胞数)×100%。然后在倒置显微镜下分别测定乙酰胆碱、P物质作用于套索纤维和钩状纤维与食管环形肌、胃底环形肌后,这四种肌纤维单细胞分别的收缩情况。每一个反应的细胞悬液被放在载玻片上,加盖玻片后在倒置显微镜下观察,随机检测30个细胞长度。细胞收缩的反应以前后细胞变化的百分数来表示。结果:在倒置显微镜下,游离的平滑肌细胞形态呈梭形,胞质密度高,表面光滑、透明,中间有一圆形或椭圆形的核。(Fig.1-Fig.4)在HEPEs缓冲液孵育的状态下,钩状纤维平均长度为(97.99±12.15)μm (n=210)、套索纤维平均长度为(99.33±10.98)μm (n =210)、食管环形肌纤维平均长度为(97.86±12.27)μm (n =210)、胃底环形肌纤维平均长度为(99.32±11.50)μm (n =210)。四组纤维细胞长度间无统计学差异。(P=6.57)乙酰胆碱可以引起食管下括约肌的钩状纤维、套索纤维及食管环形肌、胃底环形肌四种类型的肌纤维收缩,这四种肌细胞的收缩程度不同,收缩百分比分别为(11.94±2.21)%、(22.62±3.22)%、(16.62±9.71)%和(22.82±2.84)%;四组纤维细胞长度间有统计学差异(P=0) (Table 2)。P物质引起食管下括约肌钩状纤维、套索纤维及食管环形肌、胃底环形肌的收缩百分比分别为(22.48±3.36)%、(24.77±9.01)%、(19.01±4.35)%和(19.11±3.21)%;四组纤维细胞长度间有统计学差异(P=0) (Table 3)。结论:乙酰胆碱可以引起食管下括约肌的套索纤维、钩状纤维、食管环形肌细胞和胃底环形肌细胞产生收缩效应。其中套索纤维产生的收缩效应最大;P物质同样可以引起食管下括约肌的套索纤维、钩状纤维、食管环形肌细胞和胃底环形肌细胞产生收缩效应,其中套索纤维、钩状纤维收缩效应最明显,两者之间无差异。更多还原

【Abstract】 Objective: :The existence and structural equivalent of the lower esophageal sphincter (LES) in human beings have been a matter of speculation for many years. After studying the anatomy of 32 cadavers, Liebermann-Meffert et al described the arrangement of the smooth muscles at the esophagogastric junction (EGJ) in detail.They demonstrated that the musculature of the human LES consisted of sling fibers and clasp fibers.LES clasp fibers is a semi-circular in the right side,while sling fibers is U-shaped band hoisted in the gastroesophageal junction.Both muscle muscles maintain the closure status of the LES,and form a high-pressure zone (HPZ,15-30mmHg).This theory laid the foundation for further studies on the physiology,pathology and pharmacology of the LES.From then on,some reseachers have investigated the physiology and regulation mechanism of LES from different animals,and found that LES had diffemt property different from other smooth muscle structure.And more importantly, there are also great differences in the sling fibers and clasp fibers.Therefore,the abnormal changes of LES structure and function may be the pathological basis of a variety of esophageal diseases. Acetylcholine (Ach) is the most important excitatory neurotransmitter in the lower esophageal sphincter. Sling fiber is more sensitive to Ach in vitro. Clasp fibers have greater spontaneous tension. The different mechanisms of structure and function of the sling and clasp fibers in LES are not well known yet. Many experiments have proved that Ach could induce contraction of circular muscle and longitudinal muscle of the lower esophageal sphincter either in vitro or in vivo. Acetylcholine neurons mostly in the left of the lower esophageal sphincter, and in the right is the axon. This might be the right reason for the higher pressure on left side of the lower esophageal sphincter. P substance is one of the brain-gut peptide neurotransmitters, and most of its immune nerve fibers distributed in the lower esophageal muscles, intestinal submucosal plexus and plexus, and the ring and longitudinal muscles and mucosa of cats and guinea pigs. Immunohistochemical study demonstrayed that substance P was distributed in nerve endings of the vagus. The substance P might be a chemical neurotransmitters for nociceptive stimulation of the end of esophagous to induce esophageal smooth muscle contraction. In this experiment, we measured the contraction of the clasp fibers, sling fibers, circular muscle of the gastric fundus, circular muscle of the esophagus which was induced by acetylcholine and substance P, for the puepose of understanding and future treatment of esophageal motility disorders.Methods: Smooth muscles of the sling fibers, clasp fibers, circular muscles of the esophagus and gastric fundus were obtained from 30 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital,Hebei Medical University from December 2008 to December 2009. There were 18 males and 12 females with a mean age of 59.3 years.Fresh specimens of EGJ was collected from operating room. Sharp dissection of mucosal and submucosal layers were performed.The muscle strips of sling and clasp fibers were seperated and then cut to small pieces of 1mm3 ~2 mm3.Single cells was isolated using cell separation solution containing collagenase II.After more than 90 percent viability of the cells was confirmed,the cells were resuspended in Hepes buffer at 37℃for subsequent use.Five drops of cell suspention and 1 drop of 0.2%the trypan blue was mixed for 3 min for cell viability determination.Dead cells were dyed red,and living cells colorless.The percentage of cell viability was calculated with the following formula:survival percentage=number of viable cells/total number of cells (viable cell+dead cell)×100%.Cells from clasp fiber, sling fiber, circular muscles of the esophagus and gastric fundus were contracted by exposure to acetylcholine, substance P. Then we measured the contraction of the sling fibers, clasp fibers, circular muscles of the esophagus and gastric fundus which induced by acetylcholine and substance P. A drop of the cell-containing medium was placed on a glass slide and covered with a cover slip. The length of 30 consecutive intact cells encountered at random in each slide was measured with a phase-contrast microscope. The average length of 30 cells measured in the absence of agonists was taken as“control”length. In addition, average cell length was measured after addition of test agents.Results: Under photomicrograph,the smooth muscle cells were spindle and had a high density of nucleus . Its surface was smooth and transparent, and there was a round or oval nucleus in the middle of the cell . In HEPEs buffer incubation, the average length of clasp fibers was 94.09±17.53μm (n = 210) in length, sling fibers was 97.05±11.79μm (n = 210), esophageal circular muscle fibers was 97.21±10.76μm (n = 210), and gastric circular muscle fiber was 97.25±13.719μm (n = 210). There was no significant difference between the length of four groups fiber cells (F=2.67, P=0.07).Acetylcholine could induce contraction of the clasp fibers, sling fibers, circular muscles of the esophagus and gastric fundus, the percentage of muscle cell contraction was (11.94±2.21)% , (22.62±3.22)%, (16.62±9.71)% and (22.82±2.84)%, respectively. There was statistical significance of the length of four smooth musle cells groups ((F=196.97, P = 0). Substance P could induce contraction of clasp fibers, sling fibers, circular muscles of the esophagus and gastric fundus and the percentage of contraction was (22.48±3.36)%, (24.77±9.01)%, (19.01±4.35 )% and (19.11±3.21)% respectively. There was statistical significance for four groups of fiber cells (F=152.27,P = 0).Conclusions: Acetylcholine could cause the contraction of sling fibers, clasp fibers, circular muscles of the esophagus and gastric fundus, and the strongest contraction is on the sling fibers. Substance P could also cause contraction of the four kinds of fibers, and sling fibers and clasp fibers contract gtrongest.更多还原