先天性巨结肠症Wnt10b基因表达及其临床意义

Expression and clinical significance of WntlOb gene in Hirschsprung disease

目的 分析Wnt10b基因在先天性巨结肠症（HD）中的表达，探讨其在HD发生中的意义。方法 收集2003年至2010年中国医科大学附属盛京医院经病理检查证实的散发性HD狭窄段和正常段 组织标本各60例。采用HE染色和免疫组织化学染色方法检测标本中Wnt10b的表达情况；再用荧光实时定量PCR（qRT-PCR）、Western blot从其基因和蛋白水平检测HD中Wnt10b的表达情况，并对其表达进行定量与比较分析。数据采用独立样本ｔ检验。 结果 经HE染色检测，60例患儿HD诊断明确，标本符合本研究要求。Wnt10b在ＨＤ狭窄段肠壁的肌间和黏膜下细胞胞质内呈强阳性反应，而在HD正常段肠壁的肌间和黏膜下细胞胞质内表达呈弱阳性或阴性；经免疫组织化学染色检测Wnt10b在HD狭窄段蛋白阳性面积百分率为0.061%±0.014%，正常段为0.006%±0.005%，两者比较，差异有统计学意义（t=2.955，P〈0.05）。经qRT-PCR检测，Wnt10b在HD狭窄段肠管中mRNA相对含量为23.5±1.6，高于正常段肠管中的13.1±1.7（t=1.687，P〈0.05）；60例患儿的HD狭窄段肠管组织标本中Wnt10b mRNA高表达者45例，低表达者15例。经Western blot检测，Wnt10b在HD狭窄段肠管中蛋白相对表达量为35.2±2.3，高于正常段肠管中的19.1±1.3（t=2.046, P〈0.05）；60例患儿的HD狭窄段肠管组织标本中Wnt10b蛋白 高表达者43例，低表达者17例。 结论 Wnt10b mRNA与蛋白在HD肠管组织中的异常表达，与HD的发生有密切关系，可能在先天性消化道畸形的肠道发育中起一定作用。

Objective To study the expression and clinical significance of Wnt10b gene in tissue samples of Hirschsprung disease （HD）.Methods Sixty samples of stenotic intestines and 60 samples of normal intestines were collected from 60 patients with sporadic HD who were admitted to the Shengjing Hospital of China Medical University from 2003 to 2010. Tissue samples were stained by hematoxylin and eosin （HE）, and then the expression of Wnt10b gene in the tissues was detected by immunohistochemical staining. The mRNA and protein expressions of Wnt10b in HD were detected by quantitative real time polymerase chain reaction （qRT-PCR） and Western blot. All data were analyzed by two independent sample t test.Results The results of HE staining confirmed HD in the 60 patients, and the samples met the requirement of the study. The expressions of Wnt10b were strong positive in the plasma of intramuscular and submucosal cells in stenotic intestines, while weak positive or negative in normal intestines. The results of immunohistochemical staining showed that the percentages of positive proteins in the stenotic intestines and the normal intestines were 0.061%±0.014% and 0.006%±0.005%, respectively, with a significant difference （t=2.955, P〈0.05）. The results of qRT-PCR showed that the relative quantification of mRNA of Wnt10b was 23.5±1.6 in the stenotic intestines, which was significantly higher than 13.1±1.7 in the normal intestines （t=1.687, P〈0.05）. High mRNA expressions of Wnt10b were observed in 45 patients with HD, and low mRNA expressions of Wnt10b were observed in 15 patients. The results of Western blot showed that the relative protein expression of Wnt10b was 35.2±2.3 in the stenotic intestines, which was significantly higher than 19.1±1.3 in the normal intestines （t=2.046, P〈0.05）. High protein expressions of Wnt10b were observed in 43 patients,and low protein expressions of Wnt10b were observed in 17 patients.Conclusion There is a close relationship between the abnormal mRNA and protein expressions of Wnt10b in the intestines, which might play a role in the pathogensis congenital malformation of the alimentary tract.