前列腺素D 2对山羊黄体细胞内分泌功能及其凋亡相关基因表达的影响  

Effect of Prostaglandin D 2 on Endocrine Function and Expression of Apoptosis-related Genes in Goat Luteal Cells

作　　者：杨恒[1] 李利财 付琳 胥辉豪[1] 张德志[1] 李前勇[1] YANG Heng;LI Licai;FU Lin;XU Huihao;ZHANG Dezhi;LI Qianyong(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;Chongqing Academy of Animal Sciences,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆402460 [2]重庆市畜牧科学院,重庆402460

出　　处：《畜牧兽医学报》2023年第4期1652-1663,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：中央高校基本科研业务费专项资金(SWU-KT22015);重庆市自然科学基金项目(cstc2020jcyj-msxmX0427)。

摘　　要：旨在研究前列腺素家族中生殖领域鲜有报道的PGD 2成员对单一环境下黄体细胞内分泌功能及其凋亡相关基因表达的影响,并解析其在黄体退化中的作用机理,为全面探析前列腺素家族成员的生物学作用提供新的理论依据。采集空怀母山羊黄体中期的卵巢组织,通过胶原酶II消化和胰酶差速离心法分离纯化以获得山羊黄体细胞。采用DMEM/F12进行离体培养,观察不同离体培养时间的黄体细胞生长状态;采用免疫组化法和细胞形态特征鉴定黄体细胞,将PGD 2设置三个不同梯度确定其对黄体细胞作用效果的剂量依赖性关系。最后,通过PGD 2最佳依赖性剂量处理黄体细胞,利用ELISA法检测黄体细胞的内分泌功能变化,流式细胞术测定黄体细胞的凋亡率及qRT-PCR/Western blot法检测凋亡相关基因mRNA/蛋白表达水平。结果显示,经突触素(synaptophysin,SYP)特异性表达蛋白鉴定,成功分离并获得了山羊原代黄体细胞。细胞形态实时观察和ELISA检测结果显示,离体培养5 d时黄体细胞胞质饱满、外形紧凑、形态清晰可见,并且黄体细胞的内分泌P 4水平最高。此时,细胞生长曲线结果也证实,离体培养5 d时细胞生长到达峰值,细胞生长曲线呈倒置的“S”形。接种对数期峰值点细胞后,经PGD 2处理48 h,发现与对照组相比,不同剂量组别中的黄体细胞分泌P 4水平排序为2μg组<3μg组<1μg组。同时,在2μg剂量处理组中,与对照组相比,培养基中P 4浓度呈极显著下降(P<0.01);类固醇急性应激调节蛋白(steroidogenic acute regulatory protein,StAR)和3β-羟-甾体脱氢酶(3-beta-hydroxysteroid dehydrogenase,3β-HSD)基因表达呈显著性下调(P<0.05)。此外,流式细胞仪检测和Flow Jo软件分析结果显示,黄体细胞凋亡率明显增加(P<0.05);qRT-PCR和Western blot结果证实,抗凋亡因子B淋巴细胞瘤-2(B-cell lymphoma-2,BCL-2)mRNA和蛋白呈显著性下调(P<0.05)、促凋亡因子半�This study aimed to determine the effects of PGD 2 members in the prostaglandin family, which are rarely reported in the reproductive field, on the endocrine function of luteal cells and its apoptosis-related gene expression in a single environment, and to analyze its mechanism in the luteal regression, providing a new theoretical basis for the comprehensive analysis of the biological role of prostaglandin family members. The ovarian tissues of the middle stage of the corpus luteum of the empty pregnant female goat were collected, isolated and purified by collagenase II digestion and pancreatic enzyme differential centrifugation to obtain goat corpus luteal cells. DMEM/F12 was used for in vitro culture to observe the growth status of luteal cells at different time of in vitro culture, and the luteal cells were identified by immunohistochemistry and cell morphological characteristics. PGD 2 was set to three different gradients to determine the dose-dependent relationship of its effect on the luteal cells. Finally, luteal cells were treated with the best dependent dose of PGD 2 , the endocrine function changes of luteal cells were detected by ELISA, the apoptosis rate of luteal cells were measured by flow cytometry, and the expression level of mRNA/protein expression of apoptosis-related genes were detected by qRT-PCR/Western blot. The results showed that the goat primary luteal cells were successfully isolated and obtained after identification of the specific expression protein of synaptophysin (SYP). Real-time observation of cell morphology and ELISA detection showed that luteal cells had a full cytoplasm, compact shape, and clear morphology at 5 days of ex vivo culture, and the endocrine P 4 level of luteal cells was the highest. At this time, the cell growth curve results also confirmed that cell growth peaked at 5 days of ex vivo culture, and the cell growth curve was inverted “S” shaped. After 48 h of logarithmic peak cells seeding and PGD 2 treatment, it was found that the order of P 4 secretion levels o

关 键 词：黄体 前列腺素 内分泌 凋亡基因 

分 类 号：S826.8[农业科学—畜牧学] S852.21[农业科学—畜牧兽医]