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目的通过对层粘连蛋白(LN)、层粘连蛋白基因、RET基因在先天性巨结肠(HD)病儿狭窄段、移行段、正常段的表达研究,探讨HD的病因。方法用二步法对27例HD患儿狭窄段、移行段、正常段肠壁组织进行免疫组织化学染色,RSImage成像系统照相,JD801形态学图像分析软件判定阳性染色面积;用RT-PCR技术对LN基因及RET基因在三段肠壁的表达定性研究,美国al-pha9900凝胶图像分析系统进行强度表达;用实时荧光定量PCR技术对LN基因及RET基因在三段肠壁的表达定量分析,采用7000型SDS应用软件相对定量2-??Ct法比较各基因的表达差异;相应数据用SPSS11.5软件统计分析,推断LN的表达与HD的关系,并分析两基因表达的相关性及与HD发生的关系。结果层粘连蛋白及其基因在狭窄段表达明显增高,从狭窄段到正常段逐渐减低;而RET基因在狭窄段表达明显减低,从狭窄段到正常段逐渐增高,两者呈负相关。结论层粘连蛋白在无神经节细胞段表达增高是内在的,它引起肠壁神经嵴细胞的过早成熟定居导致HD的发生。层粘连蛋白基因与RET基因的表达呈负相关关系,对HD的形成两者可能存有某种内在的联系。
Objective To investigate the etiopathogenisis of HD by studying the expression of laminin (LN), laminin gene and RET gene in the stenosis, transitional and normal segments of children with Hirschsprung ’s disease. Methods Two-step method was used to detect the positive staining area in the stenosis segment, the transitional segment and the normal segment of 27 cases of HD children by immunohistochemical staining, RSImage imaging system and JD801 morphological image analysis software. RT-PCR LN gene and RET gene expression in three stages of intestinal qualitative research, the United States al-pha9900 gel image analysis system for the intensity of expression; using real-time fluorescent quantitative PCR technology LN gene and RET gene quantitative expression in the three segments of the intestinal wall, Using 7000 SDS software, the relative quantification 2 - Ct method was used to compare the expression of each gene. The corresponding data were analyzed by SPSS11.5 software to infer the relationship between LN expression and HD, and to analyze the correlation between the two gene expression and HD happened. Results The expression of laminin and its gene in the stenosis increased significantly from the stenosis to the normal, while the expression of RET decreased in the stenosis and gradually increased from the stenosis to the normal, which was negatively correlated. Conclusions The increased expression of laminin in the absence of ganglion cells is intrinsic, which causes premature colonization of neural crest cells in the gut wall leading to the development of HD. Laminin gene and RET gene expression was negatively correlated, there may be some inherent relationship between HD formation.