OPN5对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响  

Effects of OPN5 on Apoptosis,Proliferation and Steroid Hormone Synthesis of Duck Granulosa Cells

作　　者：刘付穗 潘建秋 江丹莉[1,2] 沈栩 许丹宁 田允波[1,2] 黄运茂 LIUFU Sui;PAN Jianqiu;JIANG Danli;SHEN Xu;XU Danning;TIAN Yunbo;HUANG Yunmao(Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;Guangdong Key Laboratory of Waterfowl Health Breeding,Guangzhou 510225,China)

机构地区：[1]仲恺农业工程学院,广州510225 [2]广东省水禽健康养殖重点实验室,广州510225

出　　处：《畜牧兽医学报》2021年第12期3480-3491,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广东省高等学校珠江学者岗位计划资助项目(2018);国家自然科学基金(32072730);广东省教育厅基础研究重大项目(2018KZDXM039);2016国家重点研发计划重点专项(2016YFD0500510);2020年广东省研究生教育创新计划项目(KJCX2020011)。

摘　　要：旨在研究视神经蛋白(neuropsin,OPN5)对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响。本研究分别对鸭颗粒细胞进行OPN 5过表达质粒和OPN 5 siRNA处理72 h(n=6),应用EdU细胞增殖检测技术、流式细胞技术、Annexin V-FITC、RT-PCR、Western blot及ELISA技术系统地检测颗粒细胞的增殖、凋亡情况及生殖相关基因的mRNA、蛋白水平及激素水平的变化规律。结果显示,OPN 5过表达能促进鸭卵泡颗粒细胞增殖,抑制鸭卵泡颗粒细胞凋亡;能极显著地促进GnRHR、FSHR、LHR的表达,并抑制GnIH、GnIHR的表达(P<0.01);能显著或极显著上调StAR、CYP 11A1、3β-HSD、CYP 17A1、CYP 19A1的mRNA水平(P<0.05或P<0.01);显著升高OPN5、3β-HSD及CYP19A1的蛋白表达水平(P<0.05)和极显著升高E2、P4分泌水平(P<0.01),并极显著降低INHβ的水平(P<0.01)。OPN 5 siRNA能够显著降低OPN5的表达水平(P<0.01),抑制卵泡颗粒细胞增殖并促进凋亡,极显著下调GnRH、GnRHR、FSHR、LHR(P<0.01),促进GnIH、GnIHR的表达(P<0.01);并极显著抑制StAR、CYP11A1、CYP17A1的表达(P<0.01);显著降低OPN5、3β-HSD及CYP19A1蛋白表达水平(P<0.05)及极显著降低E2、P4的分泌水平(P<0.01),并能极显著升高INHβ的水平(P<0.01)。研究表明,OPN5能促进鸭卵泡颗粒细胞的增殖,抑制其凋亡,促进颗粒细胞中类固醇激素的生成和分泌。This study aimed to characterize the effects of neuropsin(OPN5)on apoptosis,prolife-ration and steroid hormone production of granulosa cells of duck.The duck granulosa cells were isolated and transfected with OPN 5 overexpression plasmid and OPN 5 siRNA,respectively.After continuous culture for 72 h(n=6),both proliferation and apoptosis of granulosa cell were detected using EdU cell proliferation assay,flow cytometry,Annexin V-FITC.The mRNA and protein expression levels,as well as hormone secretion were investigated by RT-PCR,Western blot and ELISA,respectively.The results showed that overexpression of OPN 5 promoted prolife-ration and inhibited apoptosis of follicular granulosa cells in ducks.Moreover,overexpression of OPN 5 significantly promoted the expression of GnRHR,FSHR,LHR,inhibited the expression of GnIH and GnIHR(P<0.01),significantly or highly significantly upregulated the mRNA levels of StAR,CYP 11A1,3β-HSD,CYP 17A1 and CYP 19A1(P<0.05 or P<0.01),significantly increased the protein expression levels of OPN5,3β-HSD and CYP19A1(P<0.05),highly significantly increased the secretion levels of E2 and P4(P<0.01).However,the levels of INHβwas highly significantly decreased(P<0.01).OPN 5 knockdown with siRNA could effectively downregulate OPN5 expression in transfected cells(P<0.01),inhibit proliferation and promote apoptosis of follicular granulosa cells.siRNA transfection significantly downregulated the expression levels of GnRH,GnRHR,FSHR,LHR(P<0.01),promoted the expression of GnIH and GnIHR(P<0.01),significantly inhibited the expression of StAR,CYP 11A1,CYP 17A1(P<0.01).OPN 5 knockdown significantly decreased the protein expression levels of OPN5,3β-HSD and CYP19A1(P<0.05),highly significantly decreased the secretion levels of E2 and P4(P<0.01),and highly significantly increased the level of INHβ(P<0.01).In conclusion,OPN5 promotes the proliferation and inhibits the apoptosis of duck follicular granulosa cells,and promotes the production and secretion of steroid hormones in granulosa cells.

关 键 词：OPN5 鸭 颗粒细胞 凋亡 增殖 类固醇激素 

分 类 号：S834.83[农业科学—畜牧学]