摘要目的 通过对DPF3、Smarcd3及MEF2基因在先天性巨结肠(HD)患儿肠道组织内的检测,验证DPF3、Smarcd3及MEF2基因在肠道组织内的表达情况,并探讨DPF3、Smarcd3及MEF2基因在HD发病过程中的可能作用.方法 随机选取28例重庆医科大学附属儿童医院HD患儿手术标本,并根据手术标本取材的位置分为:痉挛段组和吻合口组.利用RealTime-PCR、免疫蛋白印迹法及免疫组织化学染色法从RNA和蛋白水平对DPF3、Smarcd3及MEF2基因在HD患儿肠道组织内的表达进行检测,验证DPF3、Smarcd3及MEF2在肠道组织内的表达,并分析其参与HD发病过程的可能机制.其中,利用BIO-RAD对mRNA数据半定量分析;Genesnap系统及软件对蛋白数据进行分析;免疫组织化学法及光学显微镜数码摄片进行蛋白染色分析;GraphPad Prism5绘图,SPSS(version 17.0)进行数据统计.结果 DPF3 mRNA在患儿吻合口和痉挛段的表达为1.76±1.30和1.07±0.93;蛋白的表达分别为2.31±0.62和1.95±0.57.Smarcd3 mRNA在患儿吻合口和痉挛段的表达为0.68±0.62和0.38±0.42;蛋白的表达分别为0.85±0.43和0.61±0.34;MEF2 mRNA在患儿吻合口和痉挛段的表达为1.01±0.63和0.68±0.39;蛋白的表达分别为2.29±0.38和1.83±0.76.DPF3、Smarcd3及MEF2 mRNA及蛋白在患儿痉挛段的表达较吻合口均降低(P＜0.05),且趋势相一致.DPF3、Smarcd3及MEF2在吻合口神经节细胞处可见阳性的棕色颗粒状染色,而在痉挛段未见明显染色.结论 DPF3、Smarcd3及MEF2可能参与HD的发病过程.

abstractsObjective To detect the expression levels of DPF3,Smarcd3 and MEF2 in proximal anastomosis (control) and stenotic segments of colonic tissues from patients with HD and analyze their potential pathogenic roles in Hirschsprung's disease (HD).Methods All resected colonic tissues were obtained randomly from the HD patients at Children's Hospital of Chongqing Medical University between June 2012 and April 2014.Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used for detecting DPF3,Smarcd3 and MEF2 mRNAs and protein expression in different portions of colonic tissues respectively.Immunohistochemistry and light microscope digital radiography were used for analyzing protein staining.GraphPad Prism 5 (GraphPad Software,Inc,USA) and SPSS statistical software (version 17.0) were used for spreadsheet and statistical analysis.Results The expression levels of DPF3 mRNA and protein were 1.76 ± 1.30,1.07 ± 0.93 and 2.31 ± 0.62,1.95 ± 0.57 in proximal anastomosis and stenotic segments respectively.The expression levels of Smarcd3 mRNA and protein were 0.68 ± 0.62,0.38 ± 0.42 and 0.85 ± 0.43,0.61 ± 0.34 in proximal anastomosis and stenotic segments respectively.And the expressions of MEF2 mRNA and protein in proximal anastomosis and stenotic segments were 1.01 ± 0.63,0.68 ±0.39 and 2.29 ± 0.38,1.83 ± 0.76 respectively.Besides,both mRNA and protein of DPF3,Smarcd3 and MEF2 were remarkably less in stenotic segments than those in proximal anastomosis.And positive staining of DPF3,Smarcd3 and MEF2 was observed in proximal anatomosis.However,no significant staining was found in stenotic segment.Conclusions DPF3,Smarcd3 and MEF2 may be involved in the process of HD.