WNT7A and β-catenin localisations and roles in regulating periimplantation ovine conceptus development under natural estrous conditions have been elaborated.However,their locations and expression patterns have not be...WNT7A and β-catenin localisations and roles in regulating periimplantation ovine conceptus development under natural estrous conditions have been elaborated.However,their locations and expression patterns have not been reported under induction of oestrus.The localisation,expression and function of WNT7A and β-catenin in the uterine tissues of the early pregnant and non-pregnant sheep on days 10,12,14,16 and 18 following artificial induction of oestrus were investigated by means of in situ hybridisation,real-time RT-PCR,immuno-histochemistry and western blotting methods.WNT7A and β-catenin mRNA and protein were both restricted to the apical surfaces of the uterine luminal epithelium(LE)and glandular epithelium(GE).In pregnant sheep,protein localisation of WNT7A and β-catenin was observed both in the endometrial LE and GE.Their staining presented on day 10,increased between day 12 and day 16,and decreased on day 18.WNT7A and β-catenin mRNA and protein expression increased initially and then decreased from day 10 to day 18,peaking on day 16,and β-catenin reaching a peak on day 18 in the uterine tissues of pregnant sheep(p<0.05).By contrast,no significant changes in WNT7A and β-catenin mRNA and protein expression levels were observed from day 10 to day 18 of the oestrus cycle in the uterine tissues of non-pregnant sheep(p<0.05).Additionally,WNT7A and β-catenin mRNA and protein expression levels in the uterine tissues of the early pregnant sheep were significantly higher than those of non-pregnant sheep(p<0.05).Treatment of endometrial epithelial cells with WNT7A increased the mRNA expressions of β-catenin,c-myc and Cyclin Dl.These results provided an underlying mechanism of periimplantation ovine conceptus development under induction of oestrus.展开更多
目的在真核细胞中表达人Wnt7b基因并且观察对大鼠原代软骨细胞的退变作用。方法取出生24 h SD大鼠关节处软骨,Ⅱ型胶原酶多次消化后获得原代软骨细胞,取P1代细胞进行实验。扩增人Wnt7b基因及克隆至PCDH-GFP上,转染PCDH-GFP和PCDH-Wnt7b...目的在真核细胞中表达人Wnt7b基因并且观察对大鼠原代软骨细胞的退变作用。方法取出生24 h SD大鼠关节处软骨,Ⅱ型胶原酶多次消化后获得原代软骨细胞,取P1代细胞进行实验。扩增人Wnt7b基因及克隆至PCDH-GFP上,转染PCDH-GFP和PCDH-Wnt7b至293ft细胞中,48 h后收集细胞上清及转染细胞,Western blot鉴定Wnt7b在293ft细胞中的表达。收集的上清分别稀释10倍和50倍培养大鼠软骨细胞,24 h后观察细胞形态并收集细胞蛋白和抽提RNA,Western blot和定量PCR检测软骨退变指标MMP13、MMP3、Ⅱ型胶原、A-can、ADAMTS5、ColⅩ和SOX9的表达。结果成功克隆人Wnt7b基因至PCDH-GFP载体上且在293ft细胞中得到有效表达。含Wnt7b培养基干预软骨细胞24 h后,软骨细胞形态由原来的多角形变成长梭形,Wnt7b处理组软骨细胞MMP13和MMP3表达显著上调,Ⅱ型胶原的表达下调。PCR结果表明A-can和Sox9表达下降,ColⅩ和ADAMTS5表达增强。结论有效在真核细胞中表达Wnt7b基因并且促进大鼠软骨细胞退变,建立软骨细胞退变的体外模型。展开更多
基金Supported by the National Natural Science Foundation(31470118,31470118)。
文摘WNT7A and β-catenin localisations and roles in regulating periimplantation ovine conceptus development under natural estrous conditions have been elaborated.However,their locations and expression patterns have not been reported under induction of oestrus.The localisation,expression and function of WNT7A and β-catenin in the uterine tissues of the early pregnant and non-pregnant sheep on days 10,12,14,16 and 18 following artificial induction of oestrus were investigated by means of in situ hybridisation,real-time RT-PCR,immuno-histochemistry and western blotting methods.WNT7A and β-catenin mRNA and protein were both restricted to the apical surfaces of the uterine luminal epithelium(LE)and glandular epithelium(GE).In pregnant sheep,protein localisation of WNT7A and β-catenin was observed both in the endometrial LE and GE.Their staining presented on day 10,increased between day 12 and day 16,and decreased on day 18.WNT7A and β-catenin mRNA and protein expression increased initially and then decreased from day 10 to day 18,peaking on day 16,and β-catenin reaching a peak on day 18 in the uterine tissues of pregnant sheep(p<0.05).By contrast,no significant changes in WNT7A and β-catenin mRNA and protein expression levels were observed from day 10 to day 18 of the oestrus cycle in the uterine tissues of non-pregnant sheep(p<0.05).Additionally,WNT7A and β-catenin mRNA and protein expression levels in the uterine tissues of the early pregnant sheep were significantly higher than those of non-pregnant sheep(p<0.05).Treatment of endometrial epithelial cells with WNT7A increased the mRNA expressions of β-catenin,c-myc and Cyclin Dl.These results provided an underlying mechanism of periimplantation ovine conceptus development under induction of oestrus.
