| Objective : In normal circumstances of fasting, gastroesophageal junctionin is closing.This allows food to pass through the esophagus and into stomach without flowing backward. The lower esophageal sphincter (LES) is a thickened region of the circular muscle layer located at the gastroesophageal junctionin human, extending over an axial distance of 2–3 cm. It plays an important role in preventing and curing the GERD. Liebermann-Meffer et al proposed that musculature of equivalent of the LES consists of semicircular or clasp fibers at the lesser curvature and sling fibers at the greater curvature. These two muscle fibers and the crural diaphragm form a high-pressure zone at the gastroesophageal junction and maintain sphincter closure by which the LES forms an uniqe one-way valve.The functional regulation of contraction and relaxation of the LES is very complicated, it involves nervous system, humoral factor and spontaneous myogenic factors. VIP, an peptide consists of 28 kinds of amino acid, was purificated from chatterling by Said in 1902. This matter was named VIP (vasoactive intestinal peptide) because of its obvious vasodilatation effects. We find that it distributes widely in circulation,immunity,procreation,digestive systems and centre and peripheral nervous system in our research, so that it is one kind of brain gut peptides. Recently more and more research have shown that VIP is very important in adjusting normal and tumor cells. VIP is passed by a series of signal pipeline to make physiological activity when is binding with receptors in human body, VIP receptor distributes widely in many human and animal's organ tissues. According to the distribution of receptor and the difference of ligand, it will be divided into two categories: VIP1 receptor and VIP2 receptor. Most VIP1 receptor distributes in periphery tissues, such as the lung,small intestine, liver,spleen,pancreas and aorta. Moreover, it disributes in deutocerebrum on centre nerve system, otherwise, it disributes in other encephalic region rarely. VIP2 receptor distrbutes wider than VIP1 receptor, it distributes in most tissues, such as small intestine,spleen,islet,kidney,spermary,and ovary. However, there is no VIP2 receptor in liver and aorta. There is much VIP2 receptor in centre nerve system, especially in hippocampus,pituitary, epiphysis,brain stem. So someone believes that VIP2 receptor should have call endocrine neuroceptor. At present, the reseaches on typing and adjusting of human LES receptor almost are confined to work on cholinoceptor, but it is seldom to study in VIP and VIP receptor. The reported studies on VIP and VIP receptor in esophagus are on animals, which may be different from the results from human because species difference. There are few reseach in the regulatory mechanisms and expression regular of VIP and VIP receptor. This studyt used reverse transcript PCR to examine the expression of VIP receptor in human LES. The aim of the study is to future expound regulation machanism of the human LES, and to set up theoretical basis for the understanding and treatment of esophageal motility disorders.Methods:1 Thirty patients including twenty-two males and eight females undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in our hospital between May 2009 and January 2010 were selected in this study. Fresh specimens were obtained from the gastroesophageal junction in the operating room. After the mucosa and submucosa were removed by sharp dissection, the sling and clasp fibers of LES, and circular muscle strips of esophagus and gastric were obtained from various regions of the gastroesophageal junction and adjacent structures. The specimen was freezed in liquid nitrogen, and then stored in icebox in -80℃.2 One ml of Trizol Reagent per hundred mg of tissue was homogenated thoroughly with the tissue homogenizer under 4℃,followed by the abstraction of Chloroform. The supernatant was precipitated with Isopropyl alcohol and washed with seventy-five percent Ethanol, and then the RNA was obtained from tissue. At the end of the procedure, the RNA was dissolved in Rnase-free DEPC water. After the identification of its purity and integrity with the use of ultraviolet spectrophotometer and 1% agarose gels, we carried out RT-PCR reactions using VIP& its receptors primers, in order to detect the expression of mRNA in the four muscle strips. The amplified products were analyzed by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining. The unit of integrated optical density (IOD) of the gel was calculated with the Gel-Pro software. The relative values of mRNA were expressed by the ratio of IOD value of VIP& it's receptor subtypes toβ-actin.Results:1.According to ultraviolet spectrophotometer and 1% agarose gels, the value of A260/280 of total RNA was between 1.8 to 2.0, the width and brightness of 28S band were double than 18S band. VIP and VIPR1 expressed in sling fibers, clasp fibers, circular muscle of the esophagus and gastric fundus.β-actin has even brightness at 250bp and the results of amplification were similar in each reaction.The length of PCR products were shown in reasonable agreement with those of predicted value. They were 199bp and 394bp. VIPR2 could not be found expression in every strip.2.The data of VIP expression in the four strips shows skewness distribution. The expression of VIP in the four strips have obvious difference (Chi-square=25.700,p=0.000<0.05). Through the Nemenyi test, the result is that there is no statistical significance of VIP in the strip of S, E and G. but the three strips have significance compared with C strip. The mean rank of VIP in C strip is 86.93, S strip is 45.97, E strip is 59.83 and G strip is 49.27. It shows that the expression of VIP in C strip higher than the others. The expression of VIP in the S, E and G strip does not have obvious difference.3.The data of VIPR1 expression in the four strips shows skewness distribution. The difference had no statistical significance in the four strips (Chi-square=1.540,p=0.673>0.05).4.The expression of mRNA of VIPR2 had no expression in the four muscles. Conclusions:1. There are VIP and VIPR1 expression in sling fibers, clasp fibers, and circular muscles of the esophagus and gastric fundus.2.The expression of VIP in C strip are more than other strips. The expression of VIP in the S, E and G strip does not have obvious difference.3. The expression of mRNA of VIPR1 have no obvious difference in the four strips.4. There are no VIPR2 expression in the four strips.
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