多器官功能障碍综合征大鼠小肠ICC网状结构损伤机理研究
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摘要
背景：多器官功能障碍综合征(multiple organ dysfunction syndrome, MODS)是指严重创伤(包括休克、重型胰腺炎等)、严重感染、大面积烧伤、外科大手术和病理产科等原发病发生后,在全身炎症反应综合症(systemic inflammatory response syndrome,SIRS)的基础上,同时或序贯发生两个或两个以上脏器功能障碍以致衰竭的临床综合征。当前MODS所致的多脏器衰竭(multiple organ failure,MOF)仍然是腹部外科患者死亡的重要原因。目前研究认为：胃肠道是SIRS的枢纽器官,是炎症介质的扩充器,是MODS的动力部位,也是MODS的靶器官。MODS可引起胃肠运动功能受损,从而导致细菌移位,肠屏障功能受损。胃肠道Cajal间质(interstitial cells of Cajal,ICC)形成网络状结构广泛分布于胃肠道各肌层,与胃肠道的运动密切相关。ICC按照功能分为重要的两种：肌间ICC (ICC-MY),在环形肌和纵行肌之间行成网络,是起搏细胞；另一种是深部肌间神经丛ICC (ICC-DMP),位于内外环形肌层之间,被兴奋性和抑制性肠运动神经元支配。ICC与平滑肌通过缝隙连接紧密相连,并和肠神经丛一起形成肠神经-ICC-平滑肌网络。ICC是肠神经系统(enteric nerve system, ENS)向胃肠道平滑肌细胞(smooth muscle cells, SMC)传送信号的中介。作为胃肠动力基本功能单位,肠神经-ICC-平滑肌网络与许多疾病引起的胃肠功能障碍密切相关。
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,探讨MODS致胃肠运动障碍的产生机制。
方法：健康成年Wistar大鼠60只,雌雄各半,体重200g-250g,随机分为对照组(20只)、MODS模型组(40只)。对照组每只动物予腹腔注射1.5ml生理盐水。MODS模型组每只动物予腹腔注射1.5ml E.coloi.混悬液(血清型O127H6的大肠杆菌8×108cfu/ml混悬液,内含10%的硫酸钡),建立细菌性腹膜炎致MODS模型。造模24小时后,取两组中存活大鼠的上段小肠组织。制作超薄切片用于透射电镜观测；制作石蜡切片,应用免疫组化的方法,比较正常组和MODS组标本小肠肌层缝隙连接蛋白43(connexin43,Cx43)的表达。
结果：
1.大体标本较之对照组,MODS组大鼠胃肠明显扩张。浆膜充血肿胀,网膜有出血斑点。腹腔内大量渗出液,出现血性腹水。大部分肠管呈暗红色,出现淤血斑,肠腔内潴留液为暗红色血水样。肠黏膜面出血,坏死；肠管粘连和腹膜炎征象明显。
2.透射电镜观测①ICC-DMP的超微结构：MODS组ICC-DMP超微结构受损明显,细胞核皱缩,核周胞浆和ICC突起受损,胞膜泡状化。②ICC网络的超微结构：与对照组相比,MODS组ICC网络中的ICC-DMP和平滑肌受到明显破坏；ICC-DMP与平滑肌细胞间,ICC-DMP相互间,平滑肌与平滑肌之间的缝隙连接和桥粒连接显著受损。
3.免疫组化较正常对照组,MODS组小肠肌层Cx43阳性表达显著减少,Cx43平均光密度(mean density=IOD SUM／area)减弱(P＜0.01)。
结论：①细菌性腹膜炎致MODS组大鼠胃肠极度扩张,胃肠道麻痹；②MODS时,ICC-DMP超微结构受到损伤；③MODS时,ICC网络受到明显破坏。
Background:Multiple organ dysfunction syndrome (MODS) is a clinical syndrome,which is induced by the primary disease, such as serious injury (including shock, acute severe pancreatitis, et al), severe infections, extensive burns, surgical and obstetric major operations and oriented from systemic inflammatory response syndrome (SIRS). In MODS there are two or more organs dysfunction simultaneously or successively. Multiple organ failure (MOF)induced by MODS remains the major cause of death in patients with abdominal surgery in current. At present, the study suggests that the gastrointestinal tract is the hub of organs in the SIRS, the device in expansion of inflammatory media, the initiation and target organ. MODS may cause damage to gastrointestinal motility, bacterial translocation and impaired intestinal epithelial barrier function.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 locate 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 lie 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 network associated with the varicosities of enteric motor neuron and have close association with the smooth muscle cells through gap junctions. ICC is the medium of the nerve signal conveying from the enteric nerve system to the smooth muscle in the gastrointestinal tract. As a basic functional unit of gastrointestinal motility, the network of enteric nerve-ICC-smooth muscle is considered to be related to the gastrointestinal dysfunction in many diseases.
Objective:To establish the model of MODS in rats induced by bacterial peritonitis, and observe the morphological changes in the network of enteric ICC-smooth muscle cells in rats with MODS.
Methods:sixty Wistar rats of both sexes weighing 200 to 250g were randomly divided into two groups:control group (n=20), Model group of MODS (n=40). The model of MODS was established according to previous study. Briefly,1.5 ml suspension of 8×10 cfu/ml of Escherichia coli strain O127H6,which contained 10% BaSO4 was injected under sterile conditions into the abdominal cavities of the rat in the MODS group. The rats of control group were injected 1.5ml normal saline. Twenty-four hours after injection, short segments of proximal jejunum were removed from alive rats in two groups.Ultrathin sections were cut and studied with transmission electron microscope.Sections were embedded in paraffind and processed according to the procedures for immunohistochemistry.Immunoreactivities to Cx43 were compared in the intestinal muscle of control and MODS rats.
Results:Anatomical changes Compared with those in control group, gastrointestinal tracts were distended significantly in the rats with MODS. Serosa congestioned and swelled. Omentum bleeding spots, a large number of peritoneal exudate,and bloody ascites were observed.Most of the intestines were dark red with congestion spots.Dark red fluid was observed in lumen. Intestinal mucosa bleeded and necrosis; intestinal adhesions and peritonitis were obvious.
Observation under electron microscopy Compared the ultrastructural features of ICC-DMP of the MODS group with the control group, ICC-DMP displayed severe ultrastructural abnormalities.The nuclei of ICC-DMP were shrunken.Injury was displayed in both perinuclear cytoplasm and the ICC-DMP processes.The plasma membrane bleb was observed. Compared the ultrastructural features of ICC network of the MODS group with the control group, the ultrastructural features of ICC-DMP and smooth muscle cells were severely damaged.Gap junctions and desmosomes between ICC-DMP-smooth muscle cells,ICC-DMP-ICC-DMP,smooth muscle cells-smooth muscle cells were destructed significantly.
Immunohistochemistry examination Compared with the control group, immunoreactivities and mean densities of Cx43 of intestine decreased significantly in MODS group (P<0.01).
Conclusions: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. Compared with the control group,ultrastructural features of ICC-DMP were injured in rats with MODS;ICC network of the MODS group were severely damaged.
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,探讨MODS致胃肠运动障碍的产生机制。
方法：健康成年Wistar大鼠60只,雌雄各半,体重200g-250g,随机分为对照组(20只)、MODS模型组(40只)。对照组每只动物予腹腔注射1.5ml生理盐水。MODS模型组每只动物予腹腔注射1.5ml E.coloi.混悬液(血清型O127H6的大肠杆菌8×108cfu/ml混悬液,内含10%的硫酸钡),建立细菌性腹膜炎致MODS模型。造模24小时后,取两组中存活大鼠的上段小肠组织。制作超薄切片用于透射电镜观测；制作石蜡切片,应用免疫组化的方法,比较正常组和MODS组标本小肠肌层缝隙连接蛋白43(connexin43,Cx43)的表达。
结果：
1.大体标本较之对照组,MODS组大鼠胃肠明显扩张。浆膜充血肿胀,网膜有出血斑点。腹腔内大量渗出液,出现血性腹水。大部分肠管呈暗红色,出现淤血斑,肠腔内潴留液为暗红色血水样。肠黏膜面出血,坏死；肠管粘连和腹膜炎征象明显。
2.透射电镜观测①ICC-DMP的超微结构：MODS组ICC-DMP超微结构受损明显,细胞核皱缩,核周胞浆和ICC突起受损,胞膜泡状化。②ICC网络的超微结构：与对照组相比,MODS组ICC网络中的ICC-DMP和平滑肌受到明显破坏；ICC-DMP与平滑肌细胞间,ICC-DMP相互间,平滑肌与平滑肌之间的缝隙连接和桥粒连接显著受损。
3.免疫组化较正常对照组,MODS组小肠肌层Cx43阳性表达显著减少,Cx43平均光密度(mean density=IOD SUM／area)减弱(P＜0.01)。
结论：①细菌性腹膜炎致MODS组大鼠胃肠极度扩张,胃肠道麻痹；②MODS时,ICC-DMP超微结构受到损伤；③MODS时,ICC网络受到明显破坏。
Background:Multiple organ dysfunction syndrome (MODS) is a clinical syndrome,which is induced by the primary disease, such as serious injury (including shock, acute severe pancreatitis, et al), severe infections, extensive burns, surgical and obstetric major operations and oriented from systemic inflammatory response syndrome (SIRS). In MODS there are two or more organs dysfunction simultaneously or successively. Multiple organ failure (MOF)induced by MODS remains the major cause of death in patients with abdominal surgery in current. At present, the study suggests that the gastrointestinal tract is the hub of organs in the SIRS, the device in expansion of inflammatory media, the initiation and target organ. MODS may cause damage to gastrointestinal motility, bacterial translocation and impaired intestinal epithelial barrier function.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 locate 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 lie 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 network associated with the varicosities of enteric motor neuron and have close association with the smooth muscle cells through gap junctions. ICC is the medium of the nerve signal conveying from the enteric nerve system to the smooth muscle in the gastrointestinal tract. As a basic functional unit of gastrointestinal motility, the network of enteric nerve-ICC-smooth muscle is considered to be related to the gastrointestinal dysfunction in many diseases.
Objective:To establish the model of MODS in rats induced by bacterial peritonitis, and observe the morphological changes in the network of enteric ICC-smooth muscle cells in rats with MODS.
Methods:sixty Wistar rats of both sexes weighing 200 to 250g were randomly divided into two groups:control group (n=20), Model group of MODS (n=40). The model of MODS was established according to previous study. Briefly,1.5 ml suspension of 8×10 cfu/ml of Escherichia coli strain O127H6,which contained 10% BaSO4 was injected under sterile conditions into the abdominal cavities of the rat in the MODS group. The rats of control group were injected 1.5ml normal saline. Twenty-four hours after injection, short segments of proximal jejunum were removed from alive rats in two groups.Ultrathin sections were cut and studied with transmission electron microscope.Sections were embedded in paraffind and processed according to the procedures for immunohistochemistry.Immunoreactivities to Cx43 were compared in the intestinal muscle of control and MODS rats.
Results:Anatomical changes Compared with those in control group, gastrointestinal tracts were distended significantly in the rats with MODS. Serosa congestioned and swelled. Omentum bleeding spots, a large number of peritoneal exudate,and bloody ascites were observed.Most of the intestines were dark red with congestion spots.Dark red fluid was observed in lumen. Intestinal mucosa bleeded and necrosis; intestinal adhesions and peritonitis were obvious.
Observation under electron microscopy Compared the ultrastructural features of ICC-DMP of the MODS group with the control group, ICC-DMP displayed severe ultrastructural abnormalities.The nuclei of ICC-DMP were shrunken.Injury was displayed in both perinuclear cytoplasm and the ICC-DMP processes.The plasma membrane bleb was observed. Compared the ultrastructural features of ICC network of the MODS group with the control group, the ultrastructural features of ICC-DMP and smooth muscle cells were severely damaged.Gap junctions and desmosomes between ICC-DMP-smooth muscle cells,ICC-DMP-ICC-DMP,smooth muscle cells-smooth muscle cells were destructed significantly.
Immunohistochemistry examination Compared with the control group, immunoreactivities and mean densities of Cx43 of intestine decreased significantly in MODS group (P<0.01).
Conclusions: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. Compared with the control group,ultrastructural features of ICC-DMP were injured in rats with MODS;ICC network of the MODS group were severely damaged.
引文
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36. Hanani M, Freund HR. Interstitial cells of cajal-their role in pacing and signal transmission in the digestive system.Acta Physiol Scand,2000;170(3):177-190.
37. Ward SM, Ordog T, Koh SD, Baker SA, Jun JY, Amberg G, Monaghan K,Sanders KM. Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria.J Physiol(Lond),2000;525(pt2):355-361.
38. Pluja L, Albe E, Fernandez E, Mikkelsen HB,Thuneberg L,Jimenez M. Evidence supporting presence of two pacemakers in rat colon.Am J Physiol Gastrointest Liver Physiol,2001;281(1):G255-G266.
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40. Belzer V,Nissan A,Freund HR,Hanani M.Coupling among interstitial cells of Cajal in the human ileum.Neurogastroenterol Motil,2004;16(1):75-80.
41. Kobilo T,Szurszewski JH,Farrugia G,Hanani M.Coupling and innervation patterns of interstitial cells of Cajal in the deep muscular plexus of the guinea-pig. Neuro-gastroenterol Motil,2003;15(6):635-641
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