CTSD对黔北麻羊卵泡颗粒细胞的调控机制及功能分析  

Regulatory Mechanism and Functional Analysis of CTSD on Follicular Granulosa Cells of Qianbei Ma Goat

作　　者：周志楠 陈祥[1,2] 张艳[1,2] 杨沛方[1,2] 惠茂茂 唐文 洪磊 ZHOU Zhinan;CHEN Xiang;ZHANG Yan;YANG Peifang;HUI Maomao;TANG Wen;HONG Lei(Guizhou Key Laboratory of Animal Genetics,Breeding and Reproduction,Key Laboratory of Plateau Mountain Animal Genetics,Breeding and Reproduction of Ministry of Education,Guizhou University,Guiyang 550025,China;College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室,贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [2]贵州大学动物科学学院,贵阳550025

出　　处：《畜牧兽医学报》2021年第5期1278-1292,共15页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31760652);贵州省科技重大专项(黔科合重大专项字[2016]3002号)。

摘　　要：旨在分析组织蛋白酶D(cathepsin D,CTSD)对黔北麻羊卵泡颗粒细胞的调控机制并初步探究其对产羔性状的影响机理。本研究以36周龄、健康、多胎黔北麻羊母羊(n=5)为研究对象,屠宰后采集卵巢组织分离培养卵泡颗粒细胞,构建真核表达载体pEGFP-N3-CTSD,将其导入细胞后在转录与翻译水平验证真核表达效率;通过CCK-8法检测在不同时间段内重组质粒对颗粒细胞增殖的影响;通过流式细胞仪检测重组质粒对颗粒细胞凋亡及周期的影响;随后使用RT-qPCR法在细胞水平检测重组质粒对细胞凋亡相关基因Bcl-2、Bax、Caspase-3,细胞周期相关因子Cyclin A1、Cyclin D2、Cyclin E mRNA表达水平的影响,最后,以多胎性状候选基因BMPR-IB、FSHR、INHA为功能基因,在转录与翻译水平上验证重组质粒对其mRNA与蛋白表达的影响。双酶切及测序结果证实,黔北麻羊CTSD基因真核表达载体pEGFP-N3-CTSD构建成功,且在转录与翻译水平极显著上调CTSD在颗粒细胞中的表达(P<0.01);细胞增殖检测结果表明,颗粒细胞中上调CTSD后能够抑制细胞的增殖,其中在12、24、48、72 h对细胞增殖的抑制效率达到极显著(P<0.01);细胞凋亡检测结果表明,CTSD的过表达能够极显著促进颗粒细胞的凋亡(P<0.01),且能够显著下调细胞抗凋亡基因Bcl-2的表达(P<0.05),极显著上调细胞促凋亡相关基因Bax、Caspase-3的表达(P<0.01);此外,细胞周期检测发现,pEGFP-N3-CTSD在转染后能够极显著上调G0/G1期与G2/M期的细胞数量(P<0.01),极显著下调S期细胞数量(P<0.01),同时能够极显著提高细胞周期相关因子Cyclin A1的表达(P<0.01),极显著降低Cyclin D2的表达(P<0.01)。RT-qPCR及Western blot检测结果表明,在颗粒细胞中上调CTSD后,能够极显著的下调多胎性状候选基因与蛋白BMPR-IB、FSHR、INHA在转录和翻译水平中的表达(P<0.01)。本研究发现,CTSD的高表达能抑制细胞的增殖,促进细胞凋亡,改变颗粒�The aim of this study was to analyze the regulatory mechanism of cathepsin D(CTSD)on the follicular granulosa cells of Qianbei Ma goat and to explore its effect mechanism on litter size traits.In this study,36-week-old,healthy,polytocous female Qianbei Ma goat(n=5)were used as the research object,follicular granulosa cells from ovarian tissues collected after slaughter were isolated and cultured.The eukaryotic expression vector pEGFP-N3-CTSD was constructed,and the eukaryotic expression efficiency at the transcription and translation levels after introducing it into cells was examined;CCK-8 assay was used to detect the effect of recombinant plasmid on the proliferation of granulosa cells in different time periods.The effects of recombinant plasmid on the apoptosis and cycle of granulosa cells were detected by flow cytometry;Subsequently,RT-qPCR was used to detect the effect of recombinant plasmids on the expression levels of apoptosis-related genes Bcl-2,Bax,Caspase-3,cell cycle-related factors Cyclin A 1,Cyclin D 2,and Cyclin E mRNA at the cellular level.Finally,the prolificacy traits candidate genes BMPR-IB,FSHR,INHA were used as functional genes to verify the effect of recombinant plasmids on their mRNA and protein expression at the transcription and translation levels.The results of double enzyme digestion and sequencing confirmed that the eukaryotic expression vector pEGFP-N3-CTSD with CTSD gene of Qianbei Ma goat was successfully constructed,and the expression of CTSD in granulosa cells could be extremely significantly increased at the transcription and translation levels(P<0.01);The cell proliferation test results showed that the up-regulation of CTSD in granulosa cells could inhibit cell proliferation,and the inhibition efficiency on cell proliferatio n was extremely significant at 12,24,48 and 72 h(P<0.01);The results of apoptosis detection showed that the overexpression of CTSD could significantly promote the apoptosis of granulosa cells(P<0.01)and significantly down-regulate the expression of anti-apop

关 键 词：CTSD 黔北麻羊 颗粒细胞 调控机制 产羔性状 

分 类 号：S827.3[农业科学—畜牧学]