Wortmannin抑制Wnt/β-Catenin通路对猪卵巢颗粒细胞发育的影响  

Effect of Inhibition of Wnt/β-Catenin Signaling by Wortmannin on Development of Porcine Granulosa Cells

作　　者：杨有福 耿果霞[2] 陈华丽[1] 程建勇[1] 李青旺[1] YANG Youfu;GENG Guoxia;CHEN Huali;CHENG Jianyong;LI Qingwang(College of Animal Science and Technology,Northwest A&F University,Yangling,Shaanxi 712100,China;College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《家畜生态学报》2019年第9期19-25,共7页Journal of Domestic Animal Ecology

基　　金：陕西省重点研发计划(2018ZDCXL-HY-02-06);国家自然科学基金(31372280)

摘　　要：为探究PI3K通路与Wnt/β-Catenin通路在猪卵巢颗粒细胞发育中的互作关系以及调控功能,以不同浓度(DMSO,0.1,1,1.5,3,5μM)PI3K通路抑制剂Wortmannin处理体外培养猪卵巢颗粒细胞48h,CCK-8法检测猪卵巢颗粒细胞细胞活力,Western Blot法检测β-Catenin蛋白水平;5μMWortmannin处理体外培养猪卵巢颗粒细胞48h,实时定量PCR检测猪卵巢颗粒细胞内类固醇生成、激素受体和细胞凋亡相关基因的表达。结果表明,Wortmannin处理猪卵巢颗粒细胞可抑制颗粒细胞细胞增殖活力,其中3μM和5μMWortmannin,相对其他处理组,显著地(P<0.05)降低细胞增殖活力;对应的,不同浓度的Wortmannin也阻遏Wnt/β-Catenin通路,对比其他组,3μM和5μMWortmannin处理组抑制效果最显著(P<0.05);实时定量PCR检测结果表明,5μMWortmannin处理显著下调猪卵巢颗粒细胞CYP19A1(P<0.001)和CYP11A1(P<0.01)mRNA水平,抑制激素受体基因FSHR和LHCGR基因表达(P<0.05),促进Caspase3(P<0.05)和PTSG2(P<0.01)表达。综上所述,Wortmannin抑制猪卵巢颗粒细胞Wnt/β-Catenin通路,诱导细胞活力下降,促进细胞凋亡,并且阻碍激素受体表达和类固醇激素合成。To explore interaction and regulatory function between PI3K signaling and Wnt/β-Catenin signaling on development of granulosa cells,porcine granulosa cells were treated with Wortmannin ( DMSO,0.1,1,1.5,3,5 μM ),the inhibitor of PI3K signaling,for 48 h in vitro in this study.The cell viability of porcine granulosa cells was detected by CCK-8 kit and protein level of β-Catenin was measured by Western Blot.Moreover,granulosa cells were cultured with 5 μM Wortmannin for 48 h in vitro,and the expression of steroidogenesis-related genes,hormone receptor-related genes and apoptosis-related genes of porcine granulosa cells were determined by qRT-PCR.The results showed the cell viability of porcine granulosa cells was inhibited by Wortmannin.Among that,the cell viability of porcine granulosa cells achieved the most significant inhibition ( P <0.05 ) treated by 3 μM and 5 μM Wortmannin compared with other groups.Correspondingly,Wnt/β-Catenin signaling was inhibited by Wortmannin ( DMSO,0.1,1,1.5,3,5 μM ),and the best inhibition was obtained in porcine granulosa cells cultivated with 3 μM and 5 μM Wortmannin in comparison to the others ( P <0.05 ).The results of qRT-PCR indicated that 5 μM Wortmannin significantly lowered mRNA level of CYP19A1 ( P <0.001 ),CYP11A1 ( P <0.01 ) and inhibited hormone receptor-related genes FSHR and LHCGR ( P <0.05 ).And a brightly upregulation of Caspase3 and PTSG2 represented in porcine granulosa cells cultured with 5 μM Wortmannin.In conclusion,Wortmannin inhibited Wnt/β-Catenin signaling,induce the reduction of cell viability,increased cell apoptosis and inhibited expression of hormone receptor and steroidogenesis.

关 键 词：WORTMANNIN WNT/Β-CATENIN通路 PI3K通路 猪 颗粒细胞 细胞活力 凋亡 类固醇合成 

分 类 号：S811.5[农业科学—畜牧学]