跨膜蛋白16A在功能性便秘小鼠结肠中的表达及其参与调控便秘的相关机制研究

Expression of transmembrane protein 16A in colon of mice with functional constipation and its related mechanism in regulating constipation

目的探讨跨膜蛋白16A(TMEM16A)在功能性便秘小鼠结肠中的表达情况及其相关调控机制。方法取20只C57BL/6小鼠,分为构建功能性便秘模型小鼠(实验组)和正常小鼠(对照组),两组各10只。采用生物机能测定仪对两组小鼠结肠肌电改变进行检测,并分别采用免疫印迹法和实时荧光定量聚合酶链式反应检测两组小鼠结肠中TMEM16A、钙离子转运ATP酶B2(ATP2B2)蛋白和基因表达情况。结果肌电生理检测发现,实验组小鼠结肠平滑肌的肌电慢波频率较对照组明显下降(5.36±1.17次/min vs.10.75±2.24次/min),慢波振幅较对照组明显升高(105.76±9.43V vs.66.90±5.64V)(P<0.01)。免疫印迹法检测发现,实验组小鼠TMEM16A蛋白表达水平较对照组明显降低(0.35±0.09 vs.1.03±0.15),ATP2B2蛋白表达水平较对照组明显升高(1.41±0.18 vs.0.99±0.11)(P<0.01)。实时荧光定量聚合酶链式反应检测发现,实验组小鼠TMEM16A基因相对表达量较对照组明显降低(0.46±0.06 vs.1.00±0.13),ATP2B2基因相对表达量较对照组明显升高(1.51±0.17vs.1.01±0.09)(P<0.01)。结论TMEM16A表达下调可能与功能性便秘小鼠结肠收缩功能减弱有关,TMEM16A可能通过调节ATP2B2表达而起到调控功能性便秘小鼠发病机制的作用,提示TMEM16A有望成为治疗功能性便秘的潜在靶点。

Objective To investigate the expression and regulatory mechanism of transmembrane protein 16A(TMEM 16A)in the colon of mice with functional constipation.Methods A total of 20 C57BL/6 mice were selected to construct functional constipation mouse models(experimental group)and normal mice(control group),with 10 mice in each group.Bioassay was used to detect electromyography(EMG)changes in the colons of the two groups of mice.And the expression of TMEM 16A,ATPase,Ca 2+transporting,plasma membrane 2(ATP2B2)proteins and genes in the colons of the two groups of mice were detected by immunoblotting and real-time quantitative polymerase chain reaction,respectively.Results Myoelectric electrophysiological examination showed that the frequency of myoelectric slow wave of colonic smooth muscle in the experimental group was significantly lower than that of the control group(5.36±1.17 times/min vs.10.75±2.24 times/min),and the slow wave amplitude was higher than that of the control group(105.76±9.43 V vs.66.90±5.64 V)(P<0.01).Western blot detection showed that the expression level of TMEM 16A protein in the experimental group was significantly lower than that in the control group(0.35±0.09 vs.1.03±0.15),and the expression level of ATP2B2 protein was significantly higher than that in the control group(1.41±0.18 vs.0.99±0.11)(P<0.01).Real-time quantitative polymerase chain reaction showed that the relative expression of TMEM 16A gene in the experimental group was significantly lower than that in the control group(0.46±0.06 vs.1.00±0.13),and the relative expression of ATP2B2 gene was significantly higher than that in the control group(1.51±0.17 vs.1.01±0.09)(P<0.01).Conclusion The down-regulated expression of TMEM 16A may be closely related to the reduced colonic systolic function of functional constipation mice,and TMEM 16A may regulate the pathogenesis of functional constipation mice by regulating the expression of ATP2B2,suggesting that TMEM 16A may be a potential target for the treatment of functional constipation.