The Research Of Sigmoid Colon Enteric Nervous System In Slow Transit Constipation Patients And Normal People

Objective : This research usese cathepsin D(CAD) and S-100,the specific markers of enteric nervous system, to inspect the composition of sigmoid enteric plexus presence and whether or not there are changes in patients of slow transit constipation by immunohistochemical examination. Observe the normal colon tissues and the morphological and quantity change of ganglion cells and nerve fibers change in slow transit constipation tissue. Through the experiment we can explore the pathogenesis of slow transit constipation, thereby to guide clinical treatment.Methods: 1 Specimen collection 1.1 Control group: Take sigmoid colon from surgical specimens of 20 colorectal cancer patients without constipation history and no other disease history of colon as control group, 8 patients are male, the other 12 are female, age range is 29-76 years, the median age is 63 years old, The specimens were taken at least 5 cm from the resection margin in tumour areas. 1.2 Experimental group: Functional constipation diagnosis accord with standard of Roman Ⅲ, no other disease history of colon.All objects were diagnosed with slow transit constipation, sigmoid colon transit abnormal was testified by colonic transit experiment before surgery. Surgical removal of the sigmoid colon specimens of 20 cases, 6 patients are male, the other 14 are female, age range is 32-71 years, the median age is 59 years old. 2 Experiment processCut off full-thickness intestinal tissue immediately after operation, wash by physiological saline, then cut into size of 2×2cm. Use neutral formalin to fixed 24 hours. Fish and trim the specimen, dehydrate through graded alcohol, transparence by xylene, dipping wax, paraffin-embedded specimens. Application manual microtome to slices of thickness 4um, dewax, hydration, incubated with 3% hydrogen peroxide, after dropping an anti-serum close(CAD or S-100)and the second antibody, stain with DAB, stain with hematoxylin, dehydration in graded alcohol, finally seal slice. By an optical microscope, observe submucosal plexus and myenteric plexus, count the submucosa and muscular plexus in five unoverlapping low magnification vision in the same slice randomly, then count ganglion cell within submucosa and muscular plexus under higher magnification of identification, and collect typical image. 3 Data analysisThe experiment’s measure data is expressed by( x±s), adopting SPSS 19.0 statistics software, applying two independent samples t-test, test level α=0.05.Results:1 CAD staining: The nerve plexus were visible in submucosal and myenteric of the control group, ganglion cells appear deep brown staining with uniform size. The nerve plexus were visible in submucosal and myenteric of the experimental group, plexus proportion and the number of ganglion cells were decreased.2 S-100 staining: The plexus, stained claybank, were visible in submucosal and myenteric of the control group, with cell-like “blank area”, which is the ganglion cell. The ganglion cells with normal size distribute uniformly. Nerve fiber is regular. The plexus were visible in submucosal and muscle of the experimental group, the number of the “blank area” decreased compared with the control group, nerve fiber distributes disorderly, distortion and proliferation of nerve fibers can be seen.3 CAD staining analysis result: Compared with the control group, ganglion cells of the experimental group were significantly(P<0.05) decreased both in submucosal and muscle, the plexus is not statistically different(P>0.05).4 S-100 staining analysis result: Compared with the control group, ganglion cells of the experimental group were significantly(P<0.05) decreased both in submucosal and muscle, the plexus number decreased, but there is no statistical difference(P>0.05).Conclusions:1 As ENS ganglion cells and nerve fibers specific markers, CAD and S-100 can reflect the colon profile of the nervous system.2 There are no significant changes in sigmoid colon submucosal and muscle plexus of patients with slow transit constipation, compared with the control group. The number of ganglion cells were decreased, nerve fibers hyperplasia and disorders and other pathological changes are observed.