Aslam A, Spicer R D, Corfield A P
Department of Paediatric Surgery, Bristol Children's Hospital, England.
J Pediatr Surg. 1999 Feb;34(2):330-3. doi: 10.1016/s0022-3468(99)90202-6.
BACKGROUND/PURPOSE: Mucin glycoproteins (mucins) recently have been shown to be deficient in the colonic mucosa of patients with Hirschsprung's disease (HD). The authors performed a detailed histo- and immunohistochemical analysis of mucins in the colonic mucosa and studied the expression of mucin genes to characterize histologically mucin quality and gene expression in HD compared with controls.
Paraffin-embedded 4-microm thick sections from patients with HD (n = 11 ganglionic, 10 aganglionic) and controls (n = 19) were taken. Slides were stained with mild periodic acid Schiff with and without saponification with KOH (reacts with O-actylated mucins), high iron diamine/alcian blue (differentiates sulphated v nonsulphated mucins), the monoclonal antimucin antibodies, PR3A5 (against di- and tri-O-acetylated sialic acids) and 91.9H (against sulphated mucins). O-acetylation and sulphation both confer an increased resistance of mucins to bacterial degradation and are thought to be important in the defensive function of the colonic mucus gel layer. In situ hybridization was used to study expression of the mucin genes MUC 1, 2, 3, 4, 5AC, 5B, 6, 7, and 8. [35S]-sulphate-labelled antisense oligonucleotide 48mer probes designed to the known tandem repeat domains of MUC genes were used. After hybridization and washing the slides were opposed to Hyperfilm MP for 7 days. The autoradiographs were scored by three independent observers for differences in expression and by image analysis. Those with positive findings were dipped in photographic emulsion, developed, and counterstained for photomicrographs.
There were different patterns of staining dependent on the region of the colon and especially the age of the patient with three reagents. No significant differences in the histological staining pattern was detected between HD patients and controls. The colonic mucins in HD were found to be primarily O-acetylated and sulphated. The MUC gene expression was similar in patients and controls. MUC2 and 4 were strongly expressed, MUC1, 3, and 5B had moderate to weak expression, and MUC 5AB, 6, 7, and 8 had baseline expression.
The mucin glycoproteins in children with HD, although quantitatively deficient, show no qualitative differences on histo- and immunohistochemical staining from normal controls. The expression of all the known mucin genes, the genetic control of mucin secretion, and the quality of mucins, is similar to normal controls.
背景/目的：最近研究表明，黏蛋白糖蛋白（黏蛋白）在先天性巨结肠病（HD）患者的结肠黏膜中存在缺陷。作者对结肠黏膜中的黏蛋白进行了详细的组织学和免疫组织化学分析，并研究了黏蛋白基因的表达，以与对照组相比，从组织学上表征HD中黏蛋白的质量和基因表达。
取自HD患者（神经节阳性11例，神经节阴性10例）和对照组（19例）的石蜡包埋4微米厚切片。玻片用轻度过碘酸希夫氏试剂染色，分别进行或不进行KOH皂化处理（与O-乙酰化黏蛋白反应），高铁二胺/阿尔辛蓝染色（区分硫酸化和非硫酸化黏蛋白），单克隆抗黏蛋白抗体PR3A5（针对二-O-乙酰化和三-O-乙酰化唾液酸）和91.9H（针对硫酸化黏蛋白）。O-乙酰化和硫酸化均使黏蛋白对细菌降解的抵抗力增强，被认为在结肠黏液凝胶层的防御功能中起重要作用。采用原位杂交技术研究黏蛋白基因MUC 1、2、3、4、5AC、5B、6、7和8的表达。使用针对MUC基因已知串联重复结构域设计的[35S] -硫酸盐标记的反义寡核苷酸48聚体探针。杂交和洗涤后，玻片与Hyperfilm MP胶片接触7天。由三名独立观察者对放射自显影片进行评分以评估表达差异，并进行图像分析。对有阳性结果的玻片进行照相乳胶浸片、显影，并进行复染以制作显微照片。
根据结肠区域，特别是患者年龄，三种试剂的染色模式不同。HD患者和对照组之间在组织学染色模式上未检测到显著差异。发现HD中的结肠黏蛋白主要为O-乙酰化和硫酸化。患者和对照组的MUC基因表达相似。MUC2和4强烈表达，MUC1、3和5B中度至弱表达，MUC 5AB、6、7和8为基线表达。
HD患儿的黏蛋白糖蛋白虽然在数量上存在缺陷，但在组织学和免疫组织化学染色上与正常对照组无质量差异。所有已知黏蛋白基因的表达、黏蛋白分泌的遗传控制以及黏蛋白的质量均与正常对照组相似。
