| Objective: According to the clinical symptoms,imaging findings and the results of surgery in patients with slow transit constipation,the patients can be divided into STC patients group with spastic colon and non-spastic colon group.The enteric nervous system specific markers 5-HT4 and 5-HT7 were examined by immunohistochemical staining of sigmoid colon tissues in patients with STC.Compared with normal control group,abnormal changes in patients with spastic colon were detected.The aim is to explore the mechanism of constipation and to provide a new drug target for the treatment of STC.Methods:1 Specimen Collection1.1 Selection of Specimens:By twice colonic transit test which time is 72 hours before the operation,it can be defined as the slow colonic transit if the residual marker of colon is more than 20% after 72 hours.It excludes organic disease of the colon and other systemic disease and the diagnostic criteria is in conformity with Rome III about slow transit constipation.Collection of the sigmoid colon wall specimens in 10 STC patients with spastic colon confirmed by operation and barium enema as STC spasm group.Collection of the sigmoid colon wall specimens in 10 STC patients without spastic colon as STC non-spasm group.Collection 10 cases of cancer patients with neither past sigmoid colon organic disease,and no history of chronic constipation and other systemic diseases as normal control group.The samples were collected in the normal bowel wall area 5cm away from the tumor edge.1.2 Specimen Collection Methods:In the operation,we first removed the local intestinal specimens,and thenremoved the whole intestinal wall tissue except the clamping sites.The size of specimen is 2cm×2cm.In the STC spasm group,the specimen was a spastic segment of sigmoid colon,and the STC non-spasm group was the distal sigmoid colon wall.The specimen of control group was taken from the normal sigmoid colon tissue of 5cm above the tumor edge.1.3 Specimen Handling:Wash specimen with PBS buffer then placed it into 4% paraformaldehyde to fixed 24 hours late.Fish and trim the specimen,dehydrate through graded alcohol,transparence by xylene,dipping wax,paraffin-embedded specimens.2 Staining Microscopy2.1 Preparation of Paraffin Section Specim:Use rotary microtome to slice the paraffin block,then then get the paraffin sections flat and fish it by warm water flotation,then dry it.2.2 Staining:Dewax,hydration,antigen retrieval,incubated with 3% methanol hydrogen peroxide,after dropping an anti,erum closed(5-HT4,5-HT7)and the second antibody,stain with DAB,stain with hematoxylin,dehydration in graded alcohol,finally seal slice.2.3 Microscopic Examination:With the help of the biological microscope,we observe the dyeing of5-HT4 and 5-HT7 in the sigmoid colon mucosa and muscular layer,which was quantitatively analyzed by computer image analysis system using Image-Pro Plus 6.3 visual fields were chosen from every slice randomly,and the positive area and positive rate were measured.Calculate the optical density,the positive index of each field of vision(positive ratio×optical density×l00)and the average value.2.4 Mining Maps:Use the straw tools to select the positive area,then save the target color files.The same batch of images should be processed by the same target color file.3 Statistical Analysis of Data:The data was analyzed by Statistical software SPSS20.0.The measurement data were expressed by mean±standard deviation or median(P25,P75).The data of each group were compared by t test or non-parametric test,P<0.05 was statistically significant.Results:1 Immunohistochemical staining of 5-HT4 recepto:In the control group,the 5-HT4 receptors in the colonic mucosa and muscularis of the sigmoid colon were positive,especially in the myenteric plexus,while only a small amount of expression was observed in the muscle cells.In the mucosa,the positive staining was mainly localized in the luminal surface of the goblet cell membrane and absorptive epithelial cells.1.1 Comparison between spasm group and non-spasm group:The expression of the 5-HT4 receptor in sigmoid colon mucosa had no significant difference between two groups(P>0.05).But the expression of the5-HT4 receptor in myenteric plexus was significantly lower in the STC spasm group than that in the non-spasm group,the difference was statistically significant(P<0.05).1.2 Comparison between experimental group and control group:The expression of the 5-HT4 receptor in both the mucosa and myenteric plexus was significantly lower in the STC spasm group and non-spasm group than that in control group,the difference was statistically significant(P<0.05).2 Imunohistochemical staining of 5-HT7 recepto:In the control group,the positive staining of the 5-HT7 receptor in the sigmoid colon was found in the mucosa and muscularis,especially the myenteric plexus which was remarkable.2.1 The comparison between non-spasm group and control group:The expression of the 5-HT7 receptor in both the mucosa and muscularis was significantly higher in the STC non-spasm group than that in the spasm group,the difference was statistically significant(P<0.05).2.2 Comparison of spasm group and control group:The expression of the 5-HT7 receptor in both the mucosa and muscularis had no significan difference between two groups(P>0.05).2.3 Comparison of non-spasm group and spasm group:The expression of the 5-HT7 receptor in both the mucosa and muscularis was significantly higher in the STC non-spasm group than that in the spasm group,the difference was statistically significant(P<0.05).Conclusions:1 The expression of the 5-HT4 receptor in both the mucosa and myenteric plexus was significantly lower in STC spasm group and non-spasm group than that in the control group,suggesting that the 5-HT4 receptor agonist could improve the colonic motility.2 The expression of the 5-HT4 receptor in the intestinal mucosa of patients with spastic sigmoid colon was not statistically significant compared with that of patients with STC.But the expression of 5-HT4 receptor in myenteric plexus was significantly lower in spasm group than that in the non-spasm group.This may be due to the lesion of the myenteric plexus in spastic sigmoid colon.3 The decreased expression of 5-HT4 receptor in patients with spastic sigmoid colon may be involved in the pathophysiological basis of simple spastic colon.4 The expression of 5-HT7 receptor in both the mucosa and muscularis was higher in the non-spasm group than that in the control group,and the high expression of 5-HT7 receptor may lead to the relaxation of colonic smooth muscle,which inhibits the normal intestinal peristalsis and secretion of reflection.5 The expression of 5-HT7 receptor in both the mucosa and muscularis was higher in the non-spasm group than that in the spasm group.This may be due to damage to the intestinal wall of the nervous system of the spastic sigmoid colon. |
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