【作者】 李毅；

【导师】 齐清会；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2008， 硕士

【摘要】 背景:多器官功能障碍综合症(multiple organ dysfunction syndrome, MODS)是腹部外科患者死亡的重要原因,以高代谢、高动力循环状态、过度失控的炎症反应和器官功能障碍为特征的临床综合征。胃肠道被认为是促发全身炎症反应综合征(systemic inflammation response syndrome, SIRS)和MODS的始动器官与靶部位,胃肠功能状态被认为是判断危重病人预后的一项重要指标。促进胃肠运动功能的恢复是有效地阻断MODS向多器官衰竭(multiple organ failure, MOF)发展的有效手段。胃肠道Cajal间质细胞(interstitial cells of Cajal, ICC)形成网络状结构广泛分布于胃肠道各肌层。ICC按照功能分为重要的两种:肌间ICC(ICC-MY),是胃肠慢波(slow wave, SW)活动的起搏器和传导者,具有基本节律起搏和传播功能;深部肌间神经丛ICC(ICC-DMP),是肠神经作用的首要靶细胞,肠神经释放的递质主要是通过ICC-DMP来调节胃肠动力。ICC作为信息整合中转站,参与肠神经系统(enteric nerve system,ENS)信号向平滑肌细胞(smooth muscle cells,SMC)的传送,从而在胃肠动力的发生与调控中起重要作用,同时ENS运动末梢、肌间丛ICC、SMC相互联系形成网络,构成胃肠动力基本功能单位(basic functional unit of gastrointestinal motility,BFUGM)。ICC在肠神经向平滑肌的信号传导中起重要的中介作用,肠神经-ICC-平滑肌网络与胃肠运动功能障碍密切相关。目的:本实验以细菌性腹膜炎致MODS大鼠模型为研究对象,观察MODS大鼠模型小肠神经-ICC-平滑肌网络的形态学变化以及大承气汤(Da-Cheng-Qi Decoction, DCQD)治疗的影响,探讨MODS致胃肠运动障碍的产生机制和大承气汤治疗MODS机理。方法:健康成年Wistar大鼠100只,雌雄各半,体重200g-250g,随机分组如下:对照组(20只)、MODS模型组(40只)和DCQD治疗组(40只)。对照组每只动物予腹腔注射1ml生理盐水。MODS模型组和DCQD治疗组每只动物予腹腔注射1ml E.coloi.混悬液,建立细菌性腹膜炎致MODS模型。DCQD治疗组于造模当日即给予DCQD灌胃,2次/日,每次1ml/100g。造模24小时后取存活大鼠上段小肠组织制作肠肌层全厚组织标本,进行c-Kit和囊泡装乙酰胆碱转运体(VAChT)/神经型一氧化氮合酶(nNOS)免疫荧光双标记染色后用于激光扫描共聚焦显微镜检测、分析;制作电镜标本用于透射电镜观测。结果:①大体标本:较之对照组,MODS组大鼠胃肠极度扩张,胃肠梗阻征象明显;DCQD治疗组胃肠扩张、梗阻较MODS组明显减轻。②共聚焦显微镜检测ICC-DMP网络:与对照组相比,MODS组大鼠ICC-DMP数量明显减少(p<0.01),ICC突触的数量减少,相互间的连接不连续,完整的网络样结构消失,细胞荧光强度减弱(p<0.01);DCQD治疗组大鼠ICC-DMP数量比MODS组有所增多(p<0.01),ICC-DMP突触增多,相互保持联系,基本保持网络状结构,细胞的荧光强度比MODS组有所增强(p<0.01)。③共聚焦显微镜检测肠神经-ICC网络:与对照组相比,MODS组大鼠ICC-DMP数量明显减少(p<0.01),突触的数量减少,完整的网络样结构消失,细胞荧光强度减弱(p<0.01);胆碱能/氮能神经神经纤维明显减少(p<0.01/p<0.01)),神经网结构紊乱,彼此间的连接减少,神经网络的荧光强度减弱(p<0.01/p<0.01));ICC与肠神经纤维两者间失去紧密连接,肠神经-ICC网络的完整结构受到破坏。DCQD治疗组大鼠ICC-DMP数量比MODS组有所增多(p<0.01),保持网络状结构,细胞的荧光强度比MODS组有所增强(p<0.01);胆碱能/氮能神经纤维较MODS组多(p<0.01/p<0.01)),维持神经网络样结构,神经节之间的连接较MODS组增多,荧光强度有所增强(p<0.01/p<0.01));ICC与肠神经纤维之间维持连接,肠神经-ICC网络保持连续。④透射电镜观测ICC-DMP超微结构:MODS组ICC-DMP细胞核皱缩,胞浆内细胞器数量明显减少,结构出现异常,基底膜缺乏或不完整,超微结构受损明显。较之MODS组,DCQD治疗组ICC-DMP细胞核形态基本正常,胞浆内细胞器数量较多,形态结构较为清楚,基底膜基本完整,超微结构较MODS组损伤明显减轻。⑤透射电镜观测肠神经-ICC-平滑肌网络的超微结构:与对照组相比,MODS组ICC-DMP细胞突起明显减少或消失,相互间的缝隙连接缺失;ICC-DMP与神经纤维间的突触样连接缺如,间距增加;ICC-DMP相互间及其与平滑肌细胞间缝隙连接消失,存在较大间隙;神经-ICC-平滑肌网络的形态学基础受到明显破坏。较之MODS组,DCQD治疗组部分ICC-DMP细胞突起减少,细胞突起损伤不明显;相互间保持连接;ICC-DMP与神经纤维间保持突触样连接;ICC-DMP与平滑肌细胞间存在缝隙连接,无明显间隙;基本保持神经-ICC-平滑肌网络结构的完整。结论:①细菌性腹膜炎致MODS组大鼠胃肠极度扩张,胃肠梗阻征象明显;DCQD治疗能够显著改善胃肠麻痹梗阻征。②MODS时,ICC-DMP网络受到破坏,ICC-DMP超微结构受到损伤;DCQD治疗可维持MODS状态下ICC-DMP网络结构,减轻ICC-DMP超微结构的损伤。③MODS时,小肠胆碱能/氮能神经和ICC-DMP数量减少,肠神经-ICC网络结构受到破坏; DCQD治疗能够增加神经、ICC的细胞数量,维持肠神经-ICC网络结构的完整。④MODS时,肠神经、ICC-DMP和平滑肌三者间相互连接的超微结构受到损伤,肠神经-ICC-平滑肌细胞网络的超微结构被破坏;DCQD治疗能够减轻肠神经、ICC和平滑肌三者间相互连接间的超微结构的损伤,维持肠神经-ICC-平滑肌细胞网络形态学基础的完整性。更多还原

【Abstract】 Background: Multiple organ dysfunction syndrome (MODS) is the main cause of the death of patients with abdominal surgical diseases, which characterized with hyper-metabolizability, hyper-circulation, immoderate and out of controlled inflammatory response and organ dysfunction. The gastrointestinal tract is considered to be the key organ to originate and to trigger systemic inflammation response syndrome (SIRS) and MODS, and the condition of gastrointestinal motility is also thought to be the criterion of evaluation the prognosis of severely injured patients. Improving the recovery of gastrointestinal motility function could effectively prevent MODS from deteriorating to multiple organ failure (MOF). Interstitial cells of Cajal (ICC) show a highly branched morphology and form unique networks in gastrointestinal tract which coordinate gastrointestinal motility. Two populations of ICC are known by their functions: one located in the area of the myenteric plexus (ICC-MY), which form a network between the circular and longitudinal muscle layers, and serve as electrical pacemakers; the other in the area of the deep muscular plexus (ICC-DMP), which lies between the outer and the inner subdivisions of the circular muscle layer, and are densely innervated by excitatory and inhibitory enteric motor neurons. These ICCs form the enteric nerve-ICC-smooth muscle cells network associated with the varicosities of enteric motor neuron and have close association with the smooth muscle cells through gap junctions. As a unique unity, enteric nerve-ICC-smooth muscle cells network is considered to be related to the gastrointestinal dysmitility in many diseases.Objective: To observe the morphological changes of enteric nerve- ICC-smooth muscle cells network in rats with MODS, and to investigate the therapeutic effects of Da-Cheng-Qi Decoction (DCQD).Methods: One handred Wistar rats of both sexes weighing 200 to 250g were randomly divided into three groups:control group(n=20), MODS group (n=40)and DCQD treated group(n=40). The model of MODS was established according to previous study. Briefly, 1 ml suspension of 8 x 108cfu/ml of Escherichia coli strain O127 H6, which contained 10% BaSO4, was injected under sterile conditions into the abdominal cavity of the rats in the MODS and DCQD group. The rats of control group were injected 1ml of normal saline. The rats of DCQD treated group were administrated by gavage with DCQD (twice a day) after the suspension was injected. Twenty-four hours after injection, the proximal 10cm segment of jejunum beginning 2cm distal to the pylorus from each group, was studied using c-Kit and vesicular acetylcholine transporter (VAChT)/ neuronal nitric oxide synthase (nNOS) immunohistochemical double-staining with whole-mount preparation technique and confocal laser scanning microscopy and transmission electron microscope.Results:1. Anatomical changes: Compared with those in control group, the gastrointestinal tracts were distended significantly in the rats with MODS; Compared with the MODS group, the distention was markedly alleviated in DCQD group.2. Immunohistochemistry: (1) Compared with the control group, the distributions and densities of ICC-DMP of intestine in MODS group were significantly decreased (P<0.01), the ICC-DMP network was disrupted. After treatment with DCQD, the ICC-DMP network was significantly recovered. Compared with MODS group, the distributions and densities of ICC-DMP of intestine in DCQD group were significantly increased (P<0.01). (2)Compared the MODS group with the control group, the distributions and densities of cholinergic nerves (P<0.01)/ nitrergic nerves (P<0.01) and ICC-DMP (P<0.01) of intestine were significantly decreased respectively, the network of cholinergic/nitrergic nerve-ICC was disrupted. After treatment with DCQD, the damage in network of cholinergic nerve/ nitrergic-ICC were significantly recovered. Compared with MODS group, the distributions and densities of cholinergic nerves (P<0.01)/ Nitrergic nerves (P<0.01) and ICC-DMP (P<0.01) of intestine were significantly increased respectively.3. Electron microscopy: (1) Compared the ultrastructural features of ICC-DMP of the MODS group with the control group, ICC-DMP displayed severe ultrastructural abnormalities. Injury was displayed in both the perinuclear cytoplasm and the ICC-DMP processes; the nuclei of ICC-DMP were shrunken, and the plasma membrane bleb was observed. Compared the ultrastructural features of ICC-DMP of the DCQD group with the MODS group, the shape of ultrastructural features of ICC-DMP were better than the MODS group, the nuclei was nearly normal. (2) Compared the ultrastructural features of intestinal nerve-ICC-smooth muscle cells network of the MODS group with the control group, the ultrastructural features of ICC-DMP, nerve, smooth muscle cells were severely damaged, the gap junction between ICC-DMP and neighbor smooth muscle cells and the other ICC was enhanced or disappeared, the synaptic-like contact between the varicosities of nerve and ICC-DMP was discontinued. Compared the ultrastructural features of intestinal nerve-ICC-smooth muscle cells network of the DCQD group with the MODS group, the ultrastructural features of ICC-DMP, nerve, smooth muscle cells were significantly recovered, the gap junction between ICC-DMP and neighbor smooth muscle cells and the other ICC could be visualized, the synaptic-like contact between the varicosities of nerve and ICC-DMP was reappeared.Conclusions: (1) Gross morphological observation of the gastrointestinal tracts revealed a marked intestinal distention in the rats with MODS; this distention was due to the paralysis of gastrointestinal tracts. DCQD could alleviate the distention significantly. (2) ICC-DMP network was severely damaged and the ultrastructural features of ICC-DMP were injured in rats with MODS. DCQD could protect the ultrastructural features of ICC-DMP and maintain the ICC-DMP network. (3) The number of ICC-DMP, cholinergic nerves and nitrergic nerves were reduced in MODS, the enteric nerve-ICC network was damaged significantly. DCQD could enhance the number of the ICC-DMP, cholinergic nerves and nitrergic nerves, and repair the enteric nerve-ICC network. (4)The ultrastructural features between ICC-DMP and smooth muscle cells, ICC-DMP and terminal nerve were damaged significantly in MODS, and the enteric nerve-ICC-smooth muscle cells network was discontinued. DCQD could protect the ultrastructural features, and restore the enteric nerve-ICC- smooth muscle cells network.更多还原