| ObjectiveTo investigate the expression of RET proto-oncogene, glial cellline-derived neurotrophic factor (GDNF), SOX10, and Calretinin (CR), β1integrin(ITGB1) in intestinal segments in children with Hirschsprung’sdisease(HD), and to explore its possible significance role in thedevelopment and for the diagnosis and differential diagnosis of HD inclinical practice.Methods①Affected and normal segments in intestinal specimens of42patientswith HD,10with intestinal neuronal dysplasia (IND-B), and5withhypoganglionosis (HG), and intestinal specimens of10normal controlsdied of non-gastrointestinal disease were also collected.Immunohis-tochemical staining for expression of CR, RET, GDNF, and SOX10wasfollowed by analysis using color pathological image analysis software.②Forty individuals were recruited in this study, the stenotic, transitionaland norrnal appearance intestines from HD patients were collected duringoperation. For comparative gene expression studies, the expression levels of β1integrin was studied using immunohistochemistry, Real-time PCR(RT-PCR) and western blotting.Results①Extensive expression of neural markers CR, RET, GDNF, andSOX10was observed in the affected segments in HD. The area of the nerveplexus, the diameter of the ganglion cells, and the number of ganglion cellswas increased in the affected intestinal segments in IND-B. The area of thenerve plexus, the diameter of the ganglion cells, and the number ofganglion cells was decreased in the intestinal wall in HG. In normalintestinal segments and intestinal tissue in the control group, CR expressionwas observed mainly in mature ganglion cells and nerve fibers; however,CR was weakly expressed or not expressed in immature ganglion cells.Immature ganglion cells and mature neurons had strong RET expression.Ganglion cells and smooth muscle cells expressed GNDF. SOX10wasweakly expressed in ganglion cells and nerve fibers.②Loss of neurons and glial cells was observed in the nerve plexus ofHD lesions, and marked hyperplasia of the nerve plexus was observed,which was3-4times the size of that of the normal group. The area of themyenteric plexus in the normal segment of HD was4952.29+1227.3um2,while the area of the myenteric plexus of HG was2996.79+1162.14um2(p<0.01), which was significantly smaller than that of the the normalmyenteric plexus. Additionally, the area of the myenteric plexus of IND-B was7991.3+1188.19um2(p<0.01), which was significantly greater thanthat of the normal myenteric plexus.Integrated ganglion cells containing anucleus were identified and counted in histologic sections. In the normalintestine,4.25+1.16integrated ganglion cells were observed within eachmyenteric plexus. Only1.53+0.64cells were found in HG, and in somecases there was no integrated ganglion cell at all. Moreover,7.54+2.49ganglion cells were observed in the myenteric plexus of IND-B lesion; thisnumber was significantly higher than that of the normal group (p<0.01).③Immunohistochemistry shows that β1integrin immunoprecipitationproduct was mainly localized to the cytoplasm and membrane of theganglion cells. The mean optical density (OD) value in normal, transitionaland stenotic segment of HD (0.86±0.16,0.61±0.15,0.35±0.07)(give indescending order), the difference was statistically significant (P <0.05).The mRNA of β1integrin in stenotic and transitional segment weresignificantly lower than those in the normal bowel segment (the relativequantification of mRNA0.77±0.17,1.08±0.15,2.26±0.29, P <0.05). Theprotein expressions of β1integrin in these patients changed in the samemanners with what were found in the mRNA.ConclusionsCombined immunohistochemical detection of neural markers CR andRET facilitates the diagnosis and differential diagnoses of HD andHAD.The expression of β1integrin changes in the tissue samples from the patients with HD, which may play a role in the pathogenesis of congenitalanomalies of the alimentary tract. |
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