| IntroductionGastroesophageal reflux disease ( GERD) is a very common disorder in infants and children, the incidental rate is increasing gradually. Therefore, the e-tiology, pathology and treatment in gastroesophageal reflux disease continue to attract attention. The etiology of GER is very complex, speak basically is the disorder dynamic of esophagus and gastric. Though a number of new developments in the diagnosis, clinical management, and medical, endoscopic, and surgical therapies were described, further etiologic mechanisms about GERD remain incompletely understood up to now. The animal model can study the pathologic physiology and provide the objective basis for the new treatment method, but the animal models of the GERD are very few. According to literatures, we have created GER animal models, inquirying into the molecular etiology of GER.Interstitial cells of Cajal (ICC) are the pace maker cells in the intricate gastrointestinal neuronal system. ICC play important functions in the gastrointestinal tract: The electrical slow wave appears to originate in ICC and mediate and amplify neuronal input to gastrointestinal smooth muscle cells. The function of ICC is controlled by gastrointestal inherent nerve, excitable and restraintable nerve are also play an important part in nerve mediating. The change of the conformation and confound configuration in ICC is relate to the disease of the turbulence of motivity. In some patients who have gastrointestinal disease, part oftheir bodies the number and structure of ICC are all change, which is approved in animal experiment and clinical research, this change may be related to abnormality of pathologic physiology.It has been demonstrated that nitric oxide ( NO) is a major inhibitory nona-drenergic, noncholinergic ( NANC ) neurotransmitter in the gastrointestinal ( GI) tract. NO released in response to nerve stimulation of the myenteric plexus causes relaxation of the smooth muscle. NO is synthesized by the activation of neuronal NO synthase (nNOS) in the myenteric plexus. Released NO plays an important physiological role in various parts of the GI tract. NO regulates the muscle tone of the sphincter in the lower esophagus, gastric, also regulates the accommodation reflex of the fundus and the peristaltic reflex of the intestine. The reduction of nNOS expression, associated with impaired local production of NO, may be responsible for motility disorders in the GI tract. There is accumulated evidence that dysfunction of NO neurons in the myenteric plexus may cause various GI diseases.A - smooth muscle action (a - SMA) is now wildly used as a marker for smooth muscles. It not only may reflect the change of smooth muscle quantity and contractibility, but also plays an important role in keeping structures and activities of smooth muscle cells.At present, the distributes of NOS only see few articles, but ICC network of pathological changes with GER not yet is seen in the report . In this experiment, we establish the GER animal model with the rabbit, applying the immunhistolog-ical, RT - PCR method, ultrastructural analysis measurement to clarifiy the etiology of GER animal models.Medical conservative and surgical treatment are the only two standards to a-meliorate symptoms of patients, but therapeutic effect still remain controversial, and we have no objective standards to assess. The aim of this study is to investigate gastric myoelectric characters, gastric motility disorders and the features of reflux in animal models and children with GER by EGG and 24 - h gastroesophageal pH monitoring, for further understanding of the pathogenesis and path-physiological features of GER. It will contribute to our clinical diagnosis and therapy of gastroesophageal reflux. According to the relationship between gastricdysfunction and GER, the gastric pacing feedback with music, application of biological reflux and motivate gastric dynamics accelerate gastric emptying, and enhance the coordination of gastric pyloric and duodenal peristalsis. The obtained result will provide more valuable information with how to improve clinical symptoms and EGG results. It can objectively assess therapeutic effect and show a new method to cure GER in children.MaterialsExperimental animalWhite big ear rabbit in Japanese were provided by Medical Animal Center, the Second Affiliated Clinical hospital of China Medical University.Reagents and instruments 24 - h gastroesophageal double pH monitor ( Type: MK III) , made in the company of Sweden CTDSYNECTICS. SABC immunohistochemical dyeing box. The c - kit antibody, the nNOS antibody was bought from the Boshide biological company in Wuhan. The SMA antibody was bought from the Zhongsan biological technique Ltd in Peking. The total RNA separate box was bought from the Hua-mei biological engineering company in Peking. The RT - PCR box and DNA Marker ( DL 2000) were bought from the Bao biological engineering Ltd in Dalian. Lead was bought from the Aoke biological technique Ltd in Peking. The stereomicroscope and picture analytical system are made in Olympus company. The WD - 5 double pathway gastric bioelectricity treatment instrument is made in the medicine graduate school in Xiamen.Methods1. GER animal models:The animals were randomly divided into four groups: 1 group was used the resection of gastric seromuscular of stomach wall; 2 group was used esophagus -cardia myotomy; 3 group was used esophagus - cardia myotomy combining with the resection of gastric seromuscular of stomach wall; 4 group was the contrastgroup and only used operation, X - ray Barium clairvoyance was performed a week after operation, 6 hours pH was monitored, isotope gastric monitor were carried out, in order to observe GER and gastric emptying change.2. Immunohistochemistry and measurement using PT-PCR method:The animals were killed at two weeks and a month after operation respectively, took out lower esophagus, gastral body and gastral antrum, and kept in 70 Celsius refrigerator. Immunohistochemistry applied SABC method, the result was judged with picture analytical system measurement, the mean value of integral light density showed result intensity. The more value is higher, the more positive result intensity is stronger, it showed that the protein content is more higher. The ratio between absorbency cubage of purposive gene strip and absorb-ency cubage of β - actin strip in RT - PCR method was regarded as relative content of purposive gene mRNA, and judged genie expression. Observing ICC ul-trastructure by electron microscope can know the relation between genie expression with GER.3. The experimental and clinical study of treating GER with Chinese medicine and gastric pacing:The animals confirmed GER were randomly divided into three groups; No treatment group; The Chinese medicine group was that Chinese medicine decoction was infused by gastric tube, twice a day, it is total two weeks; The gastric pacing group were treated two weeks, twice a day. Then gastric emptying and EGG were performed, RT - PCR method measured ICC and nNOS to observe the remedial effect. The 42 cases were diagnosed through X - ray Barium clairvoyance and double pH monitoring. The 20 cases of Contrast group were performed nuclide scanning of gastric emptying and EGG. The GER children patients were paid gastric pacing treatment outside body, and choiced proper frequency to start cure at 20 minutes after meal, 20 minutes each time, twice a day, a period of treatment is 7 days. Finally it would compare with the prethera-peutic and posttherapeutic effect.Results1. GER animal models:41 survived and 7 died in 48 White big ear rabbit of Japanese. All animals have no reflux before operation, the first and fourth group have no reflux after operation also. 14 survived and 7 have reflux in the second group. 14 survived and 10 have reflux in the third group. Gastric emptying reduced in GER group compared with contrast group and the gastric emptying time was accelerated.2. Histological and molecular examinationICC in normal rabbits are found throughout the distal esophagus, the body and antnun of gastric interspersed within the circular and longitudinal muscle layers and also forming a dense network at the level of the neuronal myenteric plexus. While in GER animals, the area and degree of ICC - positive stain were significantly less than those of controls. ICC has fusiform cell body with two longer processes morphologically. Electroscopic observation showed ICC with intact basement membrane, large nuclei, less cytoplasm, and abundant organelles including mitochondria, ribosome, endoplasmic reticulum and Golgi body in control rabbits. In GER rabbits, ICC were observed that intercellular gap increased, gap junction decreased, basement membrane ruptured, cytoplasm lysed and organelles degenerated. NOS distributed in neuronal myenteric plexus of esophagus, the body and antrum of stomach. The shape of NOS positive neurons was basically similar, most of them being round and oval in shape. The size and staining intensity of NOS positive neurons in GER rabbits were larger than those in controls, a - SMA positive staining was nearly absent in both control and GER animals. RT - PCR analysis showed that decreased expression of c -kit, increased NOS and similar SMA expression in GER rabbits compared with contrast group.3. Outcome of gastric pacing and Chinese medicine;GER rabbits showed increased gastric emptying and shorter half - time of emptying both in groups of Chinese medicine preparation and gastric pacing treatment. And EGG showed nearly normal gastric electric rhythm. The expres-...
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