前列腺素D2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
Acta Veterinaria Et Zootechnica Sinica（2023）
西南大学
Abstract
旨在研究前列腺素家族中生殖领域鲜有报道的PGD2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PGD2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PGD2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P4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PGD2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P4水平排序为2μg组＜3 μg组＜1 μg组.同时,在2 μg剂量处理组中,与对照组相比,培养基中P,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)、促凋亡因子半胱氨酸天冬氨酸特异性蛋白酶-3(cysteinyl aspartate-specific pro-teinase-3,Caspase-3)mRNA和蛋白呈显著性上调(P＜0.05).以上结果表明,PGD2不仅可通过下调StAR/3β-HSD基因表达抑制黄体细胞内分泌功能水平,还可通过下调抗凋亡BCL-2基因表达和上调促凋亡Caspase-3基因表达加速黄体细胞凋亡进程,最终以双重途径的形式参与黄体细胞的分子调控作用,这为进一步完善前列腺素家族成员的生物学功能效应及后续全面探析家畜黄体维持与退化的分子调控网络机制奠定了坚实基础.
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Key words
corpus luteum,prostaglandin,endocrine,apoptosis gene
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