钙视网膜蛋白在先天性巨结肠肠壁的表达及其意义

The expression and significance of calretinin in the colon of patients with Hirschsprung＇s disease

目的 本研究应用免疫组织化学染色方法,观察对照组肠壁、先天性巨结肠症（Hirschsprung＇s disease,HD）患儿有神经节细胞段和无神经节细胞段肠壁神经组织中钙视网膜蛋白（Calretinin,CR）的表达结果,目的在于了解HD的病理生理改变以及寻找诊断先天性巨结肠的简便有效的方法.方法 收集苏州大学附属儿童医院小儿外科2005至2008年手术切除HD标本54例,包括HD扩张段与痉挛段.以15例年龄与之相符的无HD患儿的手术切除结肠标本作为对照组.分别对HD痉挛段、扩张段、对照组肠壁组织切片进行CR的免疫组织化学染色和HE染色,计算机成像系统照相存盘,用图像分析软件（Image-Pro-Plus）分别判定CR在HD扩张段与痉挛段神经丛中阳性染色面积百分比.所得数据用SPSS 12.0统计软件包进行处理分析.结果 ①无论是HE或免疫组化染色HD的扩张段神经节细胞皆存在,肌层神经纤维有不同程度的排列改变,神经节细胞大小不等；痉挛段均未见神经节细胞；②在正常结肠及HD扩张段肠壁免疫组化染色可见钙视网膜蛋白在肌间及黏膜下神经丛中对神经节细胞呈强阳性表达,神经纤维也呈阳性反应；而HD痉挛段肠壁的免疫组化染色则可见钙视网膜蛋白在肌间神经丛及黏膜下神经丛大多表达阴性（90.7％）,仅少量呈弱阳性表达（9.3％）；③定量分析：CR分别在HD痉挛段之间神经丛中阳性染色面积百分率（0.00665±（）.00387）与其在HD扩张段神经丛中阳性染色面积百分率（0.26483±0.14626）存在差异,有显著统计学意义（P＜0.01）.结论 钙视网膜蛋白免疫组化染色可很好的显示正常结肠及HD扩张段肠壁的神经节细胞及神经纤维,而在HD痉挛段该指标免疫组化染色结果呈阴性或弱阳性表达.钙视网膜蛋白可能作为诊断HD的神经标志物之一.

Objective To study the possible pathogenesis and find a simple and sensitive diagnostic tool for HD using Calreinin （CR） immunohistochemically.Methods Specimens were collected from resected colon in both aganglionic bowel segments and ganglionic segments of 54 HD patients.The control group colon was resected from 15 age-matched patients without HD.All specimens underwent haematoxylin and eosin （HE） staining,followed by immunostaining for CR.Morphometric analysis of each sample was performed by image analysis program （Image-Pro-Plus）.Positively stained area density was evaluated at 400 magnification by calculating the sum of the areas occupied by the positively stained area of the plexuses.Results 1.The ganglionic cells were detected in the dilated segments of HD by HE or immunohistochemical staining; The array of nerve fibres in the muscle layer was changed; and the ganglionic cells were not detected in the narrow segments.2.Calretinin immunohistochemical staining was strongly positive in ganglionic cells both in the myenteric and submucosal plexuses in the dilated segments of HD and the control group.It was also positive in peripheral nerve fibers,but was negative in most of （about 90.7%） the narrow segments of HD group.3.Quantitative analysis showed that the positive staining area percentage of CR was 0.00665 ± 0.00387 in the narrow segments of HD,and was 0.26483 ± 0.14626 in the dilated segments of HD （P〈0.01 ）.Conclusions 1.Calretinin immunohistochemical staining was strongly positive in ganglionic cells both in the dilated segments of HD group and the normal colon.It was also positive in the peripheral nerve fibers,but was mostly negative in the narrow segments of HD group.It may thus be useful as one of the neuronal markers in the diagnosis of HD.