毛喉素诱导猪卵泡颗粒细胞体外分化机制的研究  

Mechanisms of in vitro differentiation induced by forskolin in porcine follicular granulosa cells

作　　者：于昊 李曼曼 薛洋 姜志洋 茆达干[1] YU Hao;LI Manman;XUE Yang;JIANG Zhiyang;MAO Dagan(Animal Reproduction Laboratory,College of Animal Science and Technology/National Experimental Teaching Demonstration Center of Animal Science,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物科技学院家畜繁殖研究室/动物科学类国家级实验教学示范中心,江苏南京210095

出　　处：《南京农业大学学报》2021年第1期160-168,共9页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31501956)。

摘　　要：[目的]本试验旨在研究毛喉素(FSK)对体外培养的猪卵泡颗粒细胞分化的作用及机制。[方法]体外分离培养猪原代卵泡颗粒细胞,预培养24 h。用10 nmol·L^-1 FSK处理细胞48 h,检测细胞形态、脂滴积聚、标记基因和周期相关基因表达状况;处理96 h后检测孕酮(P 4)水平及类固醇合成相关蛋白的表达水平。[结果]与对照组相比,FSK改变了细胞形态,使细胞直径增大,细胞内脂滴含量增加(P<0.01);FSK降低细胞的增殖活性和增殖细胞核抗原(PCNA)基因表达水平(P<0.01);FSK降低颗粒细胞标记基因促卵泡素受体(FSHR)基因表达水平,提高黄体细胞标记基因前列腺素受体(PTGFR)和促黄体素受体(LHCGR)基因表达水平(P<0.01);FSK促进P 4分泌,提高了类固醇合成相关蛋白StAR、P450scc和3β-HSD的表达水平(P<0.01);从细胞周期看,FSK降低了细胞周期素B1(CCNB 1)、细胞周期素D1(CCND 1)和细胞周期蛋白依赖性激酶1(CDK 1)、细胞周期蛋白依赖性激酶2(CDK 2)基因表达水平(P<0.01),而使细胞周期蛋白依赖性激酶抑制剂1A/B(P 21 cip1/P27 kip1)基因表达水平上调(P<0.01),并将细胞周期阻滞在G0/G1期。[结论]FSK能够通过抑制细胞增殖、增强脂滴积聚和孕酮合成代谢以及退出细胞周期来促进颗粒细胞向黄体细胞转化。[Objectives]The aim of this study was to investigate the effect and mechanisms of forskolin(FSK)on the differentiation of porcine follicular granulosa cells(GC)in vitro.[Methods]Primary porcine follicular GC were isolated and cultured for 24 h,and then 10 nmol·L^-1 FSK was added into the culture media.48 h later,the cell morphology,lipid drops(LD)and the expression level of marker and cell cycle relative genes were evaluated;96 h later,the progesterone(P 4)level and steroidogenic proteins were measured.[Results]Compared with the control group,FSK changed the morphology of GC,enlarged cells size and increased LD content(P<0.01).FSK decreased the cell viability of proliferation and the gene expression level of proliferating cell nuclear antigen(PCNA)(P<0.01).FSK decreased the gene expression level of follicle-stimulating hormone receptor(FSHR)(P<0.01)which was the marker gene of GC,while FSK up-regulated the gene expression levels of prostaglandin receptor(PTGFR)and luteinizing hormone receptor(LHCGR)(P<0.01),which were the marker genes of luteal cells.The expression levels of P 4,steroidogenic acute regulatory protein(StAR),cytochrome P450 side-chain cleavage enzyme(P450scc)and 3β-hydroxysteroid dehydrogenase(3β-HSD)significantly increased after FSK treatment(P<0.01).For cell cycle,the gene expression levels of cyclin B1(CCNB1),cyclin D1(CCND1),cyclin-dependent kinase 1(CDK1)and cyclin-dependent kinase 2(CDK2)significantly decreased(P<0.01),while gene expression levels of cyclin-dependent kinase inhibitor 1A/B(P 21 cip1/P27 kip1)significantly increased in the FSK group(P<0.01).At the same time,FSK treatment induced cell cycle arrest in the G0/G1 phase.[Conclusions]FSK could change the morphology of GC,inhibit cell proliferation,enhance LD aggregation and P 4 anabolism,and promote GC exit from cell cycle and then differentiate into luteal cells.

关 键 词：毛喉素 猪 卵泡 颗粒细胞 分化 细胞周期 

分 类 号：S814.1[农业科学—畜牧学]