摘要
目的探讨两面神激酶2-信号传导及转录激活蛋白3(JAK2-STAT3)信号转导通路在先天性巨结肠相关性小肠结肠炎发病中的作用。方法2019年2月至11月选择21 d龄内皮素受体B(Ednrb)基因敲除小鼠(先天性巨结肠模型鼠)作为实验组(12只),相应日龄野生型小鼠作为对照组(12只)。基因敲除小鼠(背景B6:129)由美国俄亥俄州立大学全国儿童医院捐赠,并在华中科技大学同济医学院动物实验中心培育、繁殖。收集结肠标本,依据形态将实验组小鼠结肠分为狭窄段及扩张段,对照组小鼠肠管相应分为结肠远、近段。苏木精-伊红(HE)染色法判断肠管炎症程度;酶联免疫吸附法(ELISA)进行定量分析各组结肠标本中白细胞介素(IL)-10的表达量;蛋白质印迹法(Western blot)检测各组结肠标本磷酸化两面神激酶2(p-JAK2)、JAK2、磷酸化信号传导及转录激活蛋白3(p-STAT3)、STAT3的蛋白表达水平;实时定量反转录聚合酶链反应(RT-qPCR)检测JAK2、STAT3的mRNA水平。组间比较采用t检验。结果Western blot结果发现先天性巨结肠模型鼠(Ednrb-/-小鼠)狭窄段肠管JAK2、p-JAK2、STAT3、p-STAT3表达水平较扩张段明显升高,差异有统计学意义(狭窄段:0.791±0.108、0.438±0.059、0.327±0.028、0.858±0.091,扩张段:0.479±0.109、0.206±0.024、0.297±0.013、0.579±0.102,t=7.074、12.679、3.428、7.096,P值均<0.05)。RT-qPCR检测显示Ednrb-/-小鼠结肠狭窄段JAK2、STAT3的mRNA表达水平高于扩张段,差异有统计学意义(狭窄段:0.427±0.042、0.272±0.044,扩张段:0.234±0.031、0.127±0.014,t=12.820、10.836,P值均<0.05)。ELISA显示Ednrb-/-小鼠扩张段肠管的IL-10的表达量下降,而狭窄段肠管升高,差异有统计学意义(狭窄段:2.540±0.710,扩张段:1.330±0.350,t=5.284,P<0.05)。结论先天性巨结肠小鼠模型扩张段炎症重,而狭窄段炎症较轻,其机制与JAK2-STAT3信号转导通路被抑制和激活有关。
Objective To investigate the role of Janus kinase2-signal transducer and activator of transcription3(JAK2-STAT3)signal transduction pathway in the pathogenesis of hirschsprung’s disease associated enterocolitis(HAEC).Methods Endothelin receptor type B(Ednrb)knockout mice were raised and bred in an specific pathogen free(SPF)laboratory.Ednrb knockout(Ednrb-/+)mice(B6:129 background)were donated by Nationwide Children’s Hospital of Ohio State University and bred in the Animal Experimental Center of Tongji Medical College of Huazhong University of Science and Technology.For experimental group,21-day-old Ednrb-/-mice were selected(n=12),with comparable-age wild type mice controls(n=12).Intestinal samples were collected.The experimental colons were divided into narrow and dilated segments according to morphological changes.The control colons were divided into distal and proximal segments.Colon hematoxylin eosin(HE)staining was used to judge HAEC.Interleukin(IL)-10 levels in colon was measured using enzyme-linked immunosorbent assays.The phosphorylated Janus kinase2(p-JAK2),JAK2,phosphorylated signal transducer and activator of transcription3(p-STAT3)and STAT3 were studied by Western blotting.The mRNA levels of JAK2 and STAT3 were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The measurement data are expressed as Mean±SD.The t-test was used for statistical analysis between groups.P<0.05 was considered statistically significant.Results Colon HE staining indicated that HAEC mainly occurred in the dilated segments of Ednrb-/-mice(EDNRB-P).Western blotting showed that the JAK2,p-JAK2,STAT3 and p-STAT3 levels in the narrow segments(EDNRB-D)were significantly higher than those in EDNRB-P(EDNRB-D:0.791±0.108,0.438±0.059,0.327±0.028 and 0.858±0.091;EDNRB-P:0.479±0.109,0.206±0.024,0.297±0.013 and 0.579±0.102,t=7.074,12.679,3.428,7.096,all P<0.05).The JAK2 and STAT3 mRNA levels in EDNRB-D were significantly higher than those in EDNRB-P(EDNRB-D:0.427±0.042 and 0.272±0.044,EDNRB-P:0.234±0.031 and 0.127±0.014,t=12.820,10.836,all P<0.05).IL-10 content in EDNRB-P was significantly lower than that in EDNRB-D(EDNRB-D:2.540±0.710,EDNRB-P:1.330±0.350,t=5.284,P<0.05).Conclusion During HAEC,the inflammation in the narrow segment is milder than the dilated segment,which is associated with the activation and inhibition of the JAK2-STAT3 signal transduction pathway.
作者
陈锋
魏孝钰
陈小华
向磊
蒙信尧
冯杰雄
Chen Feng;Wei Xiaoyu;Chen Xiaohua;Xiang Lei;Meng Xinyao;Feng Jiexiong(Department of Pediatric Surgery,Fujian Medical University Union Hospital,Fuzhou 350001,China;Department of Pediatric Surgery,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2020年第6期1089-1092,共4页
Chinese Journal of Experimental Surgery
基金
福建省卫生厅中青年骨干培养项目(2018-ZQN-27)。