CAJ | 학술논문

目的：观察Wnt/β-catenin信号通路是否在体外以外源性Wnt3a持续作用小鼠胚胎干细胞后被激活，并进一步调控该通路下游基因的表达。方法应用外源性Wnt3a持续作用ES-E14TG2a小鼠胚胎干细胞21 d，通过细胞免疫荧光及Western Blotting检测细胞内β-catenin蛋白，以观察该蛋白的胞内积聚情况；同时QRT-PCR检测WNT下游靶标基因的表达量，采用完全随机F检验并用LSD法进行两两比较，来确定经典WNT/β-catenin信号通路是否被激活。结果 ES-E14TG2a小鼠胚胎干细胞经Wnt3a连续培养21 d后，β-catenin蛋白的细胞荧光明显较强，而对照组中的荧光强度较弱，说明细胞内β-catenin蛋白没有被降解而是在胞内大量积累；Western Blotting检测结果显示Wnt3a连续培养21 d后ES-E14TG2a细胞内β-catenin蛋白条带明显比空白对照的蛋白条带粗；ES-E14TG2a细胞经wnt3a培养后Pitx2、Frizzled、Sox17的表达量均持续上升，Pitx2在培养7 d、14 d、21 d分别为4.17±0.20、7.27±0.35、8.59±0.21（F=222.757，P=0.000）；Frizzled在培养7 d、14 d、21 d分别为1.01±0.06、2.93±0.22、5.44±0.30（F=302.703，P=0.000）；Sox17在培养7 d、14 d、21 d分别为8.45±0.41、18.35±0.17、34.93±0.16（F=7217.083，P=0.000）；Oct4培养到7 d、14 d的表达量持续增加分别为1.22±0.21、1.56±0.04，而连续培养21 d后Oct4基因的表达量下降为1.15±0.07（F=8.827，P=0.016）。结论 Wnt3a持续作用可激活Wnt/β-catenin信号通路，并调控下游基因的表达。
목적：관찰Wnt/β-catenin신호통로시부재체외이외원성Wnt3a지속작용소서배태간세포후피격활，병진일보조공해통로하유기인적표체。방법응용외원성Wnt3a지속작용ES-E14TG2a소서배태간세포21 d，통과세포면역형광급Western Blotting검측세포내β-catenin단백，이관찰해단백적포내적취정황；동시QRT-PCR검측WNT하유파표기인적표체량，채용완전수궤F검험병용LSD법진행량량비교，래학정경전WNT/β-catenin신호통로시부피격활。결과 ES-E14TG2a소서배태간세포경Wnt3a련속배양21 d후，β-catenin단백적세포형광명현교강，이대조조중적형광강도교약，설명세포내β-catenin단백몰유피강해이시재포내대량적루；Western Blotting검측결과현시Wnt3a련속배양21 d후ES-E14TG2a세포내β-catenin단백조대명현비공백대조적단백조대조；ES-E14TG2a세포경wnt3a배양후Pitx2、Frizzled、Sox17적표체량균지속상승，Pitx2재배양7 d、14 d、21 d분별위4.17±0.20、7.27±0.35、8.59±0.21（F=222.757，P=0.000）；Frizzled재배양7 d、14 d、21 d분별위1.01±0.06、2.93±0.22、5.44±0.30（F=302.703，P=0.000）；Sox17재배양7 d、14 d、21 d분별위8.45±0.41、18.35±0.17、34.93±0.16（F=7217.083，P=0.000）；Oct4배양도7 d、14 d적표체량지속증가분별위1.22±0.21、1.56±0.04，이련속배양21 d후Oct4기인적표체량하강위1.15±0.07（F=8.827，P=0.016）。결론 Wnt3a지속작용가격활Wnt/β-catenin신호통로，병조공하유기인적표체。
Objective To study the regulation of Wnt pathway in the mouse embryonic stem cells after sustained exposure to Wnt3a. Methods The ES-E14TG2a mouse stem cells were cultured with the exogenous Wnt3a(100ng/ml) for 21 days, the expression ofβ-catenin was detected by immunofluorescence and western blotting assay. RT-PCR analysis was performed to assess the expression of Wnt pathway downstream genes. F text and LSD were used to compare the difference between the groups. Results Activation of the Wnt pathway led to accumulation of dephosphorylated catenin in the cytoplasm after 21 days. The expression of Wnt downstream target genes such as Pitx2, Frizzled, Sox17 and Oct4 tended to increase at the same time. The mean values of Pitx2, Frizzled and Sox17 on days 7, 14 and 21 were 4.17 ± 0.20, 7.27 ± 0.35 and 8.59 ± 0.21(F = 222.757,P= 0.000); 1.01 ± 0.06, 2.93 ± 0.22 and 5.44 ± 0.30(F =302.703,P=0.000);8.45 ± 0.41, 18.35 ± 0.17 and 34.93 ± 0.16(F=7217.083,P=0.000). respectively. Conclusion Wnt pathway can be activated by exogenous Wnt3a in mouse embryonic stem cells, which can further upregulate Wnt downstream genes.