目的 探讨幽门螺杆菌(Hp)感染患儿胃黏膜紧密连接蛋白[咬合蛋白(occludin)、闭合蛋白-4(claudin-4)、闭合小环蛋白-1(ZO-1)、上皮型钙黏蛋白(E-cadherin)和β连环蛋白(β-catenin)]表达的变化及其临床意义.方法 选择2010年12月至2013年4月因“恶心、呕吐、呕血、反酸、腹痛、腹胀或黑便”等消化道症状在浙江大学医学院附属儿童医院接受胃镜检查的70例患儿为研究对象,根据活检胃黏膜快速尿素酶试验及组织病理Hp检测结果,将70例患儿分为Hp阴性组23例,Hp阳性组47例(无溃疡组24例,有溃疡组23例).对70份胃黏膜标本进行紧密连接蛋白mRNA及蛋白质水平的测定,并进行免疫组织化学检测,比较E-cadherin、β-catenin、ZO-1、occludin和claudin-4的表达变化.结果 Hp阳性组患儿无论有无溃疡其胃黏膜组织E-cadherin、β-catenin、ZO-1mRNA表达水平均低于Hp阴性组(2-△Ct值无溃疡组分别为0.0008、0.0040、0.001 4,有溃疡组分别为0.001 0、0.009 0、0.001 3,Hp阴性组分别为0.013 7、0.042 3、0.019 8),差异有统计学意义(F=36.956、39.893、38.962,P均＜0.05);claudin-4 mRNA在Hp阳性溃疡组表达明显升高,与Hp阳性无溃疡组和Hp阴性组间差异均有统计学意义(2-△Ct值分别为0.143 8、0.092 6、0.078 9;F=11.964,P＜0.05);而occludin mRNA水平在三组间差异无统计学意义(F =0.975,P＞0.05).免疫组织化学结果显示,Hp阳性组患儿的胃黏膜E-cadherin、β-catenin阳性细胞积分(5.2±2.4、9.9±2.3)均低于Hp阴性组(8.6±3.1、11.4±1.5),差异有统计学意义(t=3.981和2.340,P均＜0.05).Western blot结果显示,Hp阳性溃疡组β-catenin蛋白水平明显低于Hp阴性组(Hp阳性溃疡组、无溃疡组和Hp阴性组的灰度比值分别为0.78、1.09、1.05,F=5.07,P＜0.05);Hp阳性组E-cadherin蛋白水平无论有无溃疡均明显低于Hp阴性组(Hp阳性溃疡组、无溃疡组和Hp阴性组的灰度比值分别为0.86、0.75、1.15,F=5.445,P＜0.05).结论 Hp感染患儿胃黏膜紧密连接蛋白E-cadherin、β-catenin、ZO-1表达降低,Hp阳性消化性溃疡患儿claudin-4表达增加,提示儿童Hp感染时胃上皮屏障功能受损可能是Hp相关性胃病的主要致病机制.%Objective To understand the junction protein expression of gastric mucosa including occlusal proteins (occludin),closed protein-4 (claudin-4),zonula occluden-1 (ZO-1),epithelial cadherin (E-cadherin),and β ring protein (β-catenin) and the clinical significance in children with Helicobacter pylori (Hp) infection.Method Seventy patients in whom gastric endoscopy was performed because of nausea,vomiting,abdominal pain,bloating,acid reflux,melena,and other gastrointestinal symptoms were enrolled in this study from Dec.2010 to Apr.2013 in our hospital.Informed consent was signed by their parents,and the study was in accordance with the principles of medical ethics.Hp positivity was confirmed if both respiratory urea test (RUT) and Hp were positive by gastric mucosal pathology.Gastric mucosal samples from 70 patients were enrolled in this study,23 of them were Hp negative,47 of them were Hp positive (24 cases without peptic ulcer,23 cases with peptic ulcer).The mRNA levels and protein expression of tight junction protein of gastric mucosa were measured by RT-PCR and Western blot respectively.The location and semi quantitative content of E-cadherin and β-catenin in gastric mucosa were detected by immunohistochemical staining method.Result The mRNA level of E-cadherin,β-catenin,ZO-1 in the Hp positive group regardless of peptic ulcer was significantly lower than that in the Hp negative group.Hp positive without peptic ulcer group were 0.000 8,0.004 0,0.001 4,respectively;Hp positive with peptic ulcer group were 0.001 0,0.009 0,0.001 3,respectively;Hp negative group were 0.013 7,0.042 3,0.019 8,respectively (F values were 36.956,39.893,38.962,respectively,all P ＜ 0.05).The expression of claudin-4 mRNA in Hp positive group with peptic ulcer increased significantly,the difference among Hp positive group with peptic ulcer,Hp positive group without peptic ulcer and Hp negative group was statistically significant (0.143 8 vs.0.092 6 vs.0.078 9) (F value was 11.964,P ＜ 0.05),while the difference of occludin mRNA levels among the three groups was not statistically significant.Immunohistochemistry results showed that the score of E-cadherin,β-catenin positive cell in the Hp positive patients were also significantly lower than that in the Hp negative group (t values were 3.981 and 2.340,all P ＜ 0.05,respectively).Western blot results showed that the protein levels of β-catenin in Hp positive group with peptic ulcer were significantly lower than that in Hp negative group,while the protein levels of E-cadherin in Hp positive patients regardless of peptic ulcer were decreased significantly in Hp negative group.Conclusion Our results revealed that the tight junction protein E-cadherin,β-catenin,ZO-1 expression of gastric mucosa were decreased in children with Hp infection,while claudin-4 expression was increased in Hp positive patients with peptic ulcer,suggesting that damage to gastric epithelial barrier function may be the main pathogenesis of Hp associated gastric diseases in children.
展开▼
