Nitric oxide and endothelin in pathophysiology of Hirschsprung's disease

对切除的先天性巨结肠标本进行bNOS、eNOS和iNOS免疫组织化学染色。在肛门直肠畸形患者结肠造口术的对照标本中，eNOS和iNOS分别定位于血管内皮细胞和粘膜下层。先天性巨结肠无神经节节段与正常节段之间无明显差异，定位结果与对照肠段相同。NADPH免疫组织化学也显示了与以前报道的结果几乎相同。但在无神经节段肌间层的神经束中发现了bNOS的定位，并进行了ET-3和ET-1受体的免疫组织化学染色。在对照肠段，ET-3和ET-1受体在粘膜下神经丛和粘膜肌层以及肌层和肌间神经丛中均有较强的表达。先天性巨结肠症患者病变肠段的免疫组织化学结果与对照组无明显差异。用新鲜冰冻切除的肠段进行内皮素A受体(EDNRA)和内皮素B受体(EDNRB)放射自显影。在对照肠段，Ednra弱定位于粘膜下层，EDNRB不仅存在于正常的肌层，而且存在于肌间神经丛和粘膜下神经丛。在先天性巨结肠病变节段，EDNRB基因定位于肌间层的神经束中，采用聚合酶链式反应-单链构象多态性分析方法对EDNRB基因进行突变分析。EDNRB基因的所有七个外显子都没有发现突变，即使在一名患有先天性中枢性低通气综合征的患者中也是如此。

Immunohistochemistry of bNOS, eNOS, and iNOS was performed on resected specimen of Hirschsprung's disease. Localization of eNOS and iNOS was observed in vascular endothelium and in submucosal layer to mucosa, respectively in the control specimen, which were obtained from colostomy in patients with anorectal malformation. In the Hirschsprung's disease, no significant differences were observed between aganglionic and normoganglionic segment, and findings of localization were as same as those in control bowel. NADPH immunohistochemistry also showed nearly same results as reported before. However, bNOS localization was found in the nerve bundles appeared in the intermuscular layer of the aganglionic segment.Immunohistochemistry endothelin 3 and endothelin 1 receptor was also performed. In the control bowel, endothelin 3 and endothelin 1 receptor were strongly localized in the submucosal plexus and mucosal muscle layer and also in the both muscle layers and intermuscular plexus. Affected bowel of Hirschsprung's disease did not show any differences in immunohistochemical findings as compared with those in controls.Autoradiography of endothelin A receptor (EDNRA) and endothelin B receptor (EDNRB) was performed using freshly frozen section of resected bowels. In the control bowel, EDNRA was localized weakly in the submucosal layer and EDNRB was found not only in the proper muscle layer but also in both the intermuscular and submucosal plexus. In the affected segment of Hirschsprung's disease, EDNRB was localized in the nerve bundles in the intermuscular layer.Mutational analysis of EDNRB gene was performed by PCR-SSCP method using peripheral leucocytes from Hirschsprung's disease. No mutation was found in all seven exons of the EDNRB gene even in a patient with congenital central hypoventilation syndrome.