钙库操纵性钙通道在先天性巨结肠结肠平滑肌层表达改变的初步研究  

Expression of store-operated calcium channel in colonic smooth muscle layer of Hirschsprung's disease:a preliminary study

作　　者：刘俊 王建峰 陆士蛟 王彭 王倩倩 陈杰[2,3] Liu Jun;Wang Jianfeng;Lu Shijiao;Wang Peng;Wang Qianqian;Chen Jie(Department of Pediatric Surgery,Jiaxing Maternity&Children's Healthcare Center,Jiaxing 314001,China;Department of Pediatric Surgery,Affiliated Xinhua Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China;Department of Pediatric Surgery,Jiaxing Branch,Affiliated Xinhua Hospital,Shanghai Jiao Tong University Medical School,Jiaxing 314001,China)

机构地区：[1]嘉兴学院附属妇女儿童医院小儿外科,314001 [2]上海交通大学医学院附属新华医院小儿外科,200092 [3]上海交通大学医学院附属新华医院小儿外科嘉兴分部,314001

出　　处：《中华小儿外科杂志》2021年第1期39-44,共6页Chinese Journal of Pediatric Surgery

基　　金：浙江省自然科学基金(LY20C110003);上海市卫生健康委员会卫生行业临床研究专项计划(201940508);嘉兴市科技计划项目公益性研究计划民生科技创新专项(2018AY32019)。

摘　　要：目的观察在先天性巨结肠(Hirschsprung's disease,HSCR)结肠平滑肌上钙库操纵性钙通道(store-operated calcium channel,SOCC)的相关的基因及蛋白Orai1和Stim1表达,初步探索SOCC与HSCR结肠动力障碍机制的相关性。方法选择2017年4月至2018年7月在上海交通大学医学院附属新华医院及嘉兴市妇幼保健院接受手术治疗的HSCR患儿24例。其中,男17例,女7例,均为常见型巨结肠;年龄为(8.6±3.6)个月,范围为6~14个月。采用蛋白质印迹法、荧光定量PCR、免疫组织化学等技术,在24例HSCR患儿手术切除的近端切缘段(正常段)、狭窄段和扩张段结肠平滑肌标本中检测与SOCC相关的基因及蛋白Orai1和Stim1的表达情况。结果蛋白质印迹法结果显示24例HSCR患儿结肠狭窄段Orai1及Stim1蛋白的表达水平与扩张段相比明显增加,差异具有统计学意义(t=2.746,P=0.012;t=2.779,P=0.011);扩张段Orai1及Stim1蛋白的表达水平与近端切缘段相比也明显增加,差异具有统计学意义(t=2.296,P=0.031;t=2.403,P=0.025);狭窄段Orai1及Stim1蛋白的表达水平显著高于近端切缘,差异具有统计学意义(t=5.915,P<0.01;t=7.726,P<0.01)。荧光定量PCR结果显示和扩张段相比,狭窄段Orai1和Stim1的mRNA的表达明显增加,差异具有统计学意义(t=2.330,P=0.029;t=2.698,P=0.013);扩张段Orai1和Stim1的mRNA的表达也较近端切缘明显增加,差异具有统计学意义(t=2.425,P=0.024;t=2.186,P=0.039);狭窄段Orai1及Stim1的mRNA的表达显著高于近端切缘,差异具有统计学意义(t=7.332,P<0.01;t=18.052,P<0.01)。免疫组织化学结果也显示与扩张段相比,Orai1和Stim1蛋白在狭窄段的表达明显增加,扩张段Orai1和Stim1蛋白的表达较近端切缘段亦明显增加。结论HSCR患儿结肠病变部位平滑肌层的SOCC的表达明显增强,说明该信号通路的异常可能与HSCR的结肠动力障碍机制相关。Objective To explore the relationship between abnormal contractility of colonic smooth muscle and store-operated calcium channel(SOCC)by examining the expression of Orai1 and Stim1 genes and proteins in Hirschsprung's disease(HSCR)so as to elucidate the mechanism of colonic motility disorder in HSCR.Methods From April 2017 to July 2018,a total of 24 HSCR children of common megacolon undergoing operations were recruited.There were 17 boys and 7 girls with an average age of(8.6±3.6)(6-14)months.Western Blot,real-time quantitative reverse trascription polymerase chain reaction(RT-PCR)and immunohistochemistry were employed for determining the expressions of Orai1 and Stim1genes and proteins related to SOCC in colonic smooth muscle of HSCR children.Results Western blot indicated that the expressions of Orai1 and Stim1 protein in narrow segment were higher than those in dilated segment(t=2.746,P=0.012;t=2.779,P=0.011),dilated segment was higher than proximal incisional margin(t=2.296,P=0.031;t=2.403,P=0.025)and narrow segment was significantly higher than proximal incisional margin(t=5.915,P<0.01;t=7.726,P<0.01).RT-PCR showed that the expressions of Orai1 and Stim1mRNA were higher in narrow segment than those in dilated segment(t=2.330,P=0.029;t=2.698,P=0.013),dilated segment was higher than proximal incisional margin(t=2.425,P=0.024;t=2.186,P=0.039)and narrow segment was significantly higher than proximal incisal margin(t=7.332,P<0.01;t=18.052,P<0.01).Immunohistochemical results showed that the expressions of Orai1 and Stim1 proteins in narrow segment were markedly up-regulated as compared with dilated segment while dilated segment increased sharply as compared with proximal incisional margin.Conclusions The expression of SOCC is markedly up-regulated in lesions of colonic smooth muscle layer of HSCR children.It implies that an abnormality of signal pathway may be correlated with colonic dyskinesia of HSCR.

关 键 词：巨结肠 先天性 钙库操纵性钙通道 结肠平滑肌 肠动力 

分 类 号：R726.5[医药卫生—儿科]