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目的免疫组织化学方法检测4种肠道神经标志物在正常结肠、先天性巨结肠症(HD)、神经元发育不良-B(IND-B)及神经节细胞减少症(HG)病变结肠的表达特点,并采用图像分析技术对肠道神经系统进行定量分析。方法组织蛋白酶D、蛋白基因产物9.5、S-100蛋白和外周蛋白4种抗体,对10例正常结肠、20例HD、8例IND-B型及20例HG的患儿结肠石蜡标本切片采用过氧化酶标记的链霉卵白素免疫组织化学染色。ImageJ图像处理软件对所有数码图像(×400)中神经丛、节细胞计数以及节细胞形态进行定量测量。结果组织蛋白酶D仅在神经节细胞表达阳性,不表达于神经纤维;PGP 9.5和外周蛋白在肠壁肌间、黏膜下神经丛及神经节细胞中均有阳性表达;S-100在神经节细胞中表达阴性。定量分析:HD病变肠段缺乏神经节细胞;IND-B病变肠段肌间神经节细胞与正常比较计数明显增多,节细胞直径以及细胞核直径分别为正常的1.22倍和1.34倍(P<0.01);HG病变肠段神经丛内节细胞数量明显减少(P<0.05)。结论免疫组织化学染色图像分析技术使不同病变间的形态差异得到定量分析,有助于对HD和同源病的病理学研究以及临床诊断和鉴别诊断。
Objective To detect the expression of four kinds of intestinal neurological markers in normal colon, Hirschsprung’s disease (HD), neuronal dysplasia-B (IND-B) and ganglion cell hypothyroidism (HG) Features, and the use of image analysis of the intestinal nervous system quantitative analysis. Methods Four kinds of antibodies, cathepsin D, protein gene product 9.5, S-100 protein and periplasmic protein were used in this study. Colonic paraffin specimens from 10 normal colon, 20 HD, 8 IND-B and 20 HG were collected Oxidase-labeled streptavidin immunohistochemical staining. ImageJ image processing software quantifies plexus, ganglion cell count and ganglion morphology in all digital images (x 400). Results Cathepsin D was positive only in ganglion cells and not expressed in nerve fibers. PGP 9.5 and peripherin were positively expressed in intestinal wall, submucosal plexus and ganglion cells. S-100 was expressed in ganglion cells Negative expression. Quantitative analysis showed that the ganglion cells in the intestine of HD patients were lack of ganglion cells. The number of myenteric ganglion cells in IND-B lesion was significantly increased compared with the normal ones, the diameter of the ganglion cells and the diameter of the nucleus were 1.22 and 1.34 times higher than those of the normal controls (P <0.01) The number of intracerebral ganglion cells in HG lesions decreased significantly (P <0.05). Conclusions The immunohistochemical staining technique can quantitatively analyze the morphological differences between different lesions and contribute to the pathological study of HD and homologous diseases as well as clinical diagnosis and differential diagnosis.