摘要
旨在通过使用恩杂鲁胺探究雄激素受体(AR)对山羊卵泡颗粒细胞增殖凋亡的影响.本研究首先对山羊卵泡颗粒细胞进行体外分离培养,然后利用显微镜观察和CCK8探究颗粒细胞的恩杂鲁胺适合培养浓度;之后将试验分为3个组,即空白对照组(不做处理)、阴性对照组(DMSO处理)和试验组(恩杂鲁胺处理),每个组均设置3个重复;然后利用EDU检测颗粒细胞的增殖情况,使用Caspase3活性与细胞凋亡检测试剂盒检测颗粒细胞的凋亡情况,并利用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)检测颗粒细胞增殖凋亡相关基因和蛋白的表达情况.结果表明,AR抑制剂恩杂鲁胺能够显著地抑制颗粒细胞的增殖和降低颗粒细胞中CCND1、PCNA和MKI67基因的mRNA水平,并且还能够显著抑制CCND1、PCNA和β-catenin蛋白的表达;此外,恩杂鲁胺还能够促进山羊卵泡颗粒细胞的凋亡,显著提高山羊卵泡颗粒细胞中BAX和BCL2的mRNA水平,但不影响BAX和BCL2的蛋白表达,另外恩杂鲁胺虽然没有影响CASP3基因的mRNA水平但能够提高Caspase 3的活性.这些结果表明,恩杂鲁胺抑制AR活性后能够影响颗粒细胞中β-catenin、PCNA、CCND1、Caspase 3等的表达,参与调控颗粒细胞的增殖凋亡.
Abstract
The aim of this study was to investigate the effects of androgen receptor(AR)on goat granulosa cell proliferation and apoptosis using enzalutamide.Firstly,granulosa cells from goats were isolated and subjected to in vitro culture.The suitable concentration of enzalutamide for cell culture was determined by microscopic observation and CCK8 assay.The experiment was then divided into 3 groups:blank control group(no treatment),negative control group(treated with DMSO),and experimental group(treated with enzalutamide).Each group was set up with 3 replicates.Cell proliferation was detected using the EDU assay,while cell apoptosis was assessed using Caspase3 activity and apoptosis detection kits.The expression of genes and proteins related to cell proliferation and apoptosis was evaluated using quantitative real-time PCR(qRT-PCR)and Western blot analysis.The results showed that the AR inhibitor,enzalutamide,significantly inhibited cell proliferation and reduced the mRNA levels of CCND1,PCNA,and MKI67 in granulosa cells.It also suppressed the protein expression of CCND1,PCNA,and β-catenin.Additionally,enzalutamide promoted apoptosis in goat granulosa cells,as evidenced by increased mRNA levels of BAX and BCL2,without affecting their protein expression.Furthermore,enzalutamide,while not affecting the mRNA level of CASP3,enhanced Caspase3 activity.These findings suggest that enzalutamide,by inhibiting AR activity,can modulate the expression of β-catenin,PCNA,CCND1,Caspase 3,and other genes involved in the regulation of granulosa cell proliferation and apoptosis.
基金项目
广东省林业科技创新项目(2023KJCX004)
广东省南方现代草牧业(羊)创新团队项目(2023KJ127)
广东省农业农村厅项目(2022-XPY-00-009)
韶关市仁化县牛羊产业园项目(GDSCYY2021-018)
国家科技期刊平台
NSTL
万方数据