Sfrp5/Dkk4基因在ETU诱导大鼠胚胎肛门直肠畸形中表达的初步研究

Preliminary study of expression of Sfrp5/Dkk4 genes with ethylenethiourea-induced congenital anorectal malformations in rats

目的利用分子生物学方法检测肛门直肠畸形(anorectal malformations,ARM)大鼠胚胎发育中Sfrp5/Dkk4基因的表达。方法对Wistar孕鼠进行乙烯硫脲(ethylenethiourea,ETU)致畸,制作ARM动物模型;分别在孕15d(E15)、17d(E17)、19d(E19)和21d(E21),于ETU-无畸形对照组(NE组)、ETU-ARM畸形组(AE组)和生理盐水对照组(NS组)中各选取6只胎鼠末端直肠组织,采用蛋白印迹(Westernblot)和实时定量PCR(qRT-PCR)方法检测Sfrp5/Dkk4的表达,并对其表达进行定量与比较分析。结果Western blot结果显示在E15、E17、E19和E21时,Sfrp5在NE组蛋白表达量分别为63.55±0.35、24.51±0.41、13.28±0.09和49.67±0.12;在AE组分别为21.43±0.18、59.57±0.44、61.23±0.47和32.73±0.51;在NS组分别为52.17±1.08、23.66±0.87、16.21±1.33和50.01±2.03。Dkk4在NE组蛋白表达量分别为20.07±0.09、47.15±0.15、39.88±0.37和13.75±0.47;在AE组分别为54.27±0.61、19.43±0.25、21.09±0.17和57.53±0.49;在NS组分别为23.11±0.15、45.89±0.67、41.44±0.37和17.57±1.99。提示NE组、NS组Sfrp5/Dkk4与AE组E17和E19蛋白表达量比较,差异均有统计学意义(P<0.05);而NE组与NS组在4个时间点比较,差异均无统计学意义(P>0.05)。qRT-PCR结果显示在E17和E19时,Sfrp5在AE组中mRNA高表达,其表达量分别是NE组的1.63倍和1.77倍(P=0.049、0.022)、NS组的2.68倍和3.29倍(P=0.041、0.034);Dkk4在AE组中mRNA高表达,其表达量是NE组的1.75倍和1.87倍(P=0.045、0.024)、NS组的2.04倍和1.36倍(P=0.004、0.028)。结论Sfrp5/Dkk4蛋白与mRNA在胚胎发育过程E17和E19中出现异常表达,可能是导致ARM发病的因素。

Objective To detect the expression of Sfrp5/Dkk4 genes in Wistar embryos with anorectal malformations (ARM) induced by ethylene thiourea (ETU). Methods The Wistar pregnant rats received the injections of ethylene thiourea (ETU) and teratogenic ARM.At E15, E19, E17 and E21, 6 specimens were harvested in each group.Under anatomical microscopic observation of anal location, no anal opening confirmed the presence of anorectal malformations. And terminal rectum tissues of control group received normal saline (NS). The expressions of Sfrp5/Dkk4 were detected by Western blot and quantitative real-time PCR (qRT-PCR) in E15, E17, E19 and E21. And quantitative and comparative analyses were performed. Results For ETU teratogenic ARM, the protein expressions of Sfrp5 were 63.55±0.35, 24.51±0.41, 13.28±0.09 & 49.67±0.12(NE15, NE17, NE19 & NE21)in normal group, 21.43±0.18, 59.57±0.44, 61.23±0.47 & 32.73±0.51 (AE15, AE17, AE19 & AE21) in ARM group and 52.17±1.08, 23.66±0.87, 16.21±1.33 & 50.01±2.03 (NS15, NS17, NS19 & NS21) in NS group;For ETU teratogenic ARM, the protein expressions of Dkk4 were 20.07±0.09, 47.15±0.15, 39.88±0.37 & 13.75±0.47 (NE15, NE17, NE19 & NE21) in normal group, 54.27±0.61, 19.43±0.25, 21.09±0.17 & 57.53±0.49 (AE15, AE17, AE19, & AE21) in ARM group and 23.11±0.15, 45.89±0.67, 41.44±0.37 & 17.57±1.99 (NS15, NS17, NS19 & NS21) in NS group respectively. The protein expressions of Sfrp5/Dkk4 showed significant differences in E17 and E19 in normal and ARM groups(P<0.05)and significant differences existed in E17 and E19 in NS and ARM groups too (P<0.05). The mRNA levels of Sfrp5 was 1.63 folds higher in AE17 group than that in NE17 group (P=0.049), 1.77 folds higher in AE19 group than that in NE19 group (P=0.022), 2.68 folds higher in AE17 group than that in NS17 group (P=0.041) and 3.29 fold higher in AE19 group than that in NS19 group (P=0.034). The mRNA levels of Dkk4 was 1.75 folds higher in NE17 group than that in AE17 group (P=0.045), 1.87 folds higher in NE19 group than that in AE19 group (P=0.024), 2.04 folds higher in AE17 group than that in NS17 group (P=0.004) and 1.36 folds higher in AE19 group than that in NS19 group (P=0.028). Conclusions Abnormal expressions of Sfrp5/Dkk4 protein and mRNA may be two major pathogenic factors of ARM of E17 and E19. This discovery provides a new mechanistic insight of ARM.