【作者】 杨玲蓉；

【导师】 江米足；

【作者基本信息】 浙江大学 ， 儿科学， 2011， 硕士

【摘要】 研究背景：胃食管反流(GER)是指胃内容物反流入食管甚至口咽部的一种生理现象,但如反流频率和持续时间增加到一定程度,达到病理状态和/或给机体带来不适时则称为胃食管反流病(GERD).典型的GERD症状往往认为与胃酸的反流有关,临床上采用抑制胃酸分泌作为常规治疗手段,其中质子泵抑制剂(PPI)常作为首选用药。但15-20%患者症状会持续,甚至增加剂量,许多患者在治疗期间还会发生酸或非酸反流。反流入食管的酸和非酸物质如不能有效清除,会导致食管黏膜的损伤,而引起反流性食管炎(RE)等并发症。GERD属动力异常性疾病,其发病因素较多,发病机制尚未完全阐明。主要相关因素有：下食管括约肌(LES)功能失调、食管清除能力下降、食管黏膜屏障功能受损以及食管内脏高敏性。食管黏膜清除能力取决于食管动力功能,其在反流致食管黏膜损伤中起重要作用。正常食管蠕动功能有赖于食管平滑肌的协调性收缩和松驰,而其生理基础则是食管平滑肌对不同神经递质的正常反应性。有研究表明,酸反流或混合反流引起的RE与食管平滑肌反应性受损有关,认为食管平滑肌反应性受损是食管动力异常的关键因素。因此,食管平滑肌对不同神经递质的反应性在RE发病机制研究中受到重视。研究发现,5-羟色胺(5-hydroxytryptamine,5-HT;亦称血清素serotonin)信号通路异常可导致胃肠道动力及分泌功能异常和内脏高敏感性,认为5-HT是调节胃肠道运动、分泌及感觉功能的重要神经递质。大约90%的5-HT储存于消化道黏膜,其合成、释放、再摄取和受体状态的异常,均可引起一系列病理生理改变。5-HT可通过与5-HT4受体(5-HT4R)结合,激活腺苷酸环化酶(AC),引起环磷酸腺苷(cAMP)增高,从而激活cAMP依赖性蛋白激酶A(PKA),引起平滑肌细胞超极化,Ca2+内流减少,而引起平滑肌松驰。食管平滑肌松驰在维持食管正常蠕动功能中发挥重要作用。5-HT4R部分激动剂替加色罗(tegaserod)可降低餐后食管酸反流；降低食管对机械扩张的疼痛阈值、减少反流发生；改善食管蠕动功能。5-HT转运体(SERT)抑制剂西酞普兰(citalopram),能降低食管对化学和机械刺激的敏感性。表明5-HT信号通路异常可能与GERD发病有关,但机制尚有待于阐明。目的：拟建立GER动物模型(部分贲门成形术),通过食管pH检测评价其反流程度；通过病理切片评估食管黏膜损伤程度；测定食管组织5-HT含量、SERT和5-HT4R mRNA及蛋白质水平的表达,探讨5-HT信号通路在反流致食管黏膜损伤中的作用,为GERD治疗提供新的方向。方法：选用清洁级8周龄雄性SD大鼠50只,随机分为GER模型组30只与假手术对照组20只。GER模型组参照文献采用食管下段喷门肌纵切横缝加胃底韧带结扎术建立GER模型。实验动物饲养至术后4周测定食管pH值后处死,肉眼观察食管黏膜大体变化及显微镜下病理组织学改变。采用高效液相色谱-电化学法(HPLC-ECD)测定食管组织5-HT及其主要代谢产物五羟吲哚乙酸(5-HIAA)的含量,运用荧光定量RT-PCR法检测SERT和5-HT4R mRNA的表达,采用Western-blot方法检测5-HT4R蛋白质的表达。进一步应用SERT抑制剂氢溴酸西酞普兰对GER模型大鼠进行干预,评价其疗效。结果：术后4周GER模型组大鼠存活27只,死亡3只,平均体重增长与食管下端pH值均低于对照组,差异均具有统计学意义。GER模型组食管黏膜大体观可见不同程度黏膜充血、部分可见糜烂,对照组黏膜均光滑完整。食管黏膜病理切片GER模型组中20例可见不同程度的炎症改变(食管炎,RE组),7例未见明显炎症表现(无食管炎,NE组)。对照组食管黏膜均未见炎症改变。食管组织5-HT含量RE组较对照组和NE组升高,差异有统计学意义；NE组和对照组之间差异无统计学。5-HIAA含量RE组和NE组均较对照组显著升高,差异有统计学意义。SERT mRNA表达量RE组和NE组均较对照组显著增加,差异有统计学意义,而RE组又高于NE组,差异有统计学意义。RE组、NE组和对照组之间5-HT4R mRNA表达的差异无统计学意义。5-HT4R蛋白质的表达RE组低于对照组,差异有统计学意义；而NE组和对照组之间差异无统计学意义。氢溴酸西酞普兰20mg/kg组和10/kg组食管黏膜炎症情况均较未给药模型组明显改善,与10/kg组相比,20mg/kg组并未表现出更佳的治疗效果。结论：5-HT信号通路异常在反流致食管黏膜损伤中发挥作用。5-羟色胺转运体抑制剂可望用于治疗反流性食管炎。更多还原

【Abstract】 Gastroesophageal reflux (GER) is defined as the gastric contents reflux into the esophagus or oropharyngeal area. It is usually known as a physiological phenomenon. However, when the frequency and duration of reflux exceed a certain extent, leading to the pathological condition and (or) discomfort in individuals, it was termed gastroesophageal reflux disease (GERD). Typical GERD symptoms are often considered to be associated with the reflux of gastric acid, therefore decreasing the gastric acid secretion is clinically used as a routine treatment, including proton pump inhibitor (PPI) as the preferred drug. However,15-20% of patients had acid reflux or non-acid reflux during the treatments even after the dose was increased. If the refluent acid and non-acid materials were not effectively removed from the esophagus, it would induce the esophageal mucosal damage and even reflux esophagitis (RE).GERD is a motility disorder disease. Its pathogenesis is still unclear. The main etiological factors related to GERD are lower esophageal sphincter (LES) dysfunction, decreased esophageal clearance capacity, esophageal mucosal barrier dysfunction and esophageal visceral hypersensitivity. Scavenging capacity of esophageal mucosa depends on esophageal motor function, which may play an important role in the reflux-induced esophageal mucosal injury. Normal esophageal peristalsis depends on the coordination of esophageal smooth muscle contraction and relaxation, and its physiological basis is the normal reaction of esophageal smooth muscle to different neurotransmitters. Some studies reported that RE induced by acid reflux or mixed reflux was associated with impaired esophageal smooth muscle reactivity, while the impaired esophageal smooth muscle reactivity is a key factor in esophageal motility disorders. Therefore, much attention should be paid to the role of esophageal smooth muscle reactivity to different neurotransmitters in the pathogenesis of RE.It is found that 5-hydroxytryptamine (5-HT; serotonin) signaling pathway dysfunction might lead to gastrointestinal motility and secretion abnormalities and visceral hypersensitivity. Therefore 5-HT was an important neurotransmitter to regulate gastrointestinal motility, secretion and sensory function. About 90% of 5-HT is stored in the gastrointestinal mucosa, its synthesis, release, reuptake, and receptor status abnormalities would lead to a series of pathophysiological changes.5-HT may bind to 5-HT 4 receptor (5-HT4R), activating adenylyl cyclase (AC) to increase cyclic adenosine monophosphate (cAMP), leading to activation of cAMP-dependent protein kinase A (PKA), which causes the hyperpolarization of smooth muscle cells and Ca2+ influx reduction, resulting in smooth muscle relaxation. The esophageal smooth muscle relaxation plays an important role in the maintenance of normal esophageal motility. 5-HT4R partial agonist tegaserod can reduce the postprandial esophageal acid reflux, lower esophageal mechanical pain threshold and reflux, and improve esophageal peristalsis.5-HT transporter inhibitor citalopram can reduce the esophageal sensitivity to chemical and mechanical stimuli. It indicates that the 5-HT signaling pathway damage may be the key factor in the pathogenesis of GERD, but the underlying mechanism need to be investigated in the future.Objective:To establish an animal model of GER to evaluate the reflux degree by esophageal pH monitor, to assess the extent of esophageal mucosal injury by pathological slides, and to detect 5-HT concentration, SERT and 5-HT4R mRNA transcription and protein expression in the esophageal tissue, so as to explore the role of 5-HT signaling pathway in the esophageal mucosal injury induced by reflux for providing a new strategy in the treatment of GERD.Methods:Fifty 8-week male SD rats were randomly divided into the GER model group (30 cases) and the sham operation control group (20 cases). GER model was established through gastric cardia muscle longitudinal cut, transverse suture and gastric fundus ligament ligation. Animals were executed after esophageal pH value monitor in the age of 12 weeks. The anatomical forms and histopathological changes of esophageal mucosa were observed. High performance liquid chromatography- electrochemical determination (HPLC-ECD) was used to detect 5-HT/5-HIAA concentrations of the esophageal tissue. SERT and 5-HT4R mRNA expression were investigated by real-time RT-PCR. The level of 5-HT4R protein expression was also measured by western blot method. The SERT inhibitor citalopram hydrobromide was used to treat GER and RE induced by reflux.Results:Four weeks after operation,27 rats in GER model group had survivors, and other three cases were dead. The increased average body weight in GER model group were significantly lower than the control group, and the values of esophageal pH were also significantly lower than the control ones. Anatomically, different degrees of esophageal mucosal congestion and erosion were observed in the most of GER model group, while esophageal mucosa was smooth in the control group. Histopathologically, twenty rats in GER model group had esophageal mucosal inflammation (reflux esophagitis, RE group), other seven cases had no obvious inflammation (no esophagitis, NE group). No inflammatory pathological changes were observed in the esophageal mucosa of control group. The 5-HT levels of esophageal tissue in the RE group were significantly higher than those in the control and NE groups. The differences of 5-HT levels in esophageal tissue between the NE group and control group had not reached significance. The 5-HIAA concentrations of esophageal mucosa both in RE group and in NE group were significantly higher than that in control group. The SERT mRNA expressions of esophageal mucosa both in RE group and in NE group were increased significantly than that in the control group, and the SERT mRNA levels in RE group was significantly higher than that in NE group. There were no significant differences in the 5-HT4R mRNA levels among RE group, NE group and control group. The 5-HT4R protein level of esophageal mucosa in RE group was significantly lower than that in the control group, but no significant difference of 5-HT4R protein level was observed between the NE and control group. The positive rate of esophageal mucosal inflammation after citalopram hydrobromide treatment in the GER model was decreased significantly both 20mg/kg group and 10mg/kg group compared with the model group without citalopram hydrobromide administration. However,20mg/kg citalopram hydrobromide-treated model group did not show better therapeutic effect than the 10mg/kg-treated model group.Conclusions:It was concluded that the 5-HT signaling pathway disorder might be a major factor in the pathogenesis of gastroesophageal reflux and reflux esophagitis.5-HT transporter inhibitors could be used for the treatment of reflux esophagitis.更多还原