外源性Wnt3a持续作用对小鼠胚胎干细胞Wnt/β-catenin信号通路的调控作用  

Regulation of mouse embryonic stem cell Wnt pathway after sustained exposure to Wnt3a

作　　者：金静君 林芳[2] 张韬 陈泳[3] 陈金烟 王坤 韩俊永 薛士杰 

机构地区：[1]福建省医学科学研究院免疫研究所 福建省医学测试重点实验室,福州30025 [2]福州大学生物科学与工程学院 [3]福建师范大学生命科学学院

出　　处：《中华细胞与干细胞杂志（电子版）》2013年第4期1-5,共5页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：福建省省属公益类科研院所基本科研专项(2010R1034-1);福建省自然科学基金(2012J01325);福建省医学创新课题(2012-cx-13)

摘　　要：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。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 of β-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.

关 键 词：胚胎干细胞 WNT3A WNT Β-CATENIN信号通路 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]