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

doi:10.11843/j.issn.0366-6964.2023.04.029

摘要/Abstract

摘要： 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2 μg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P₄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 proteinase-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。

关键词: 黄体, 前列腺素, 内分泌, 凋亡基因

Abstract: This study aimed to determine the effects of PGD₂ 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. PGD₂ 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 PGD₂, 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 P₄ 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 PGD₂ treatment, it was found that the order of P₄ secretion levels of luteal cells in different dose groups was 2 μg group < 3 μg group < 1 μg group. At the same time, in the 2 μg dose treatment group, the concentration of P₄ in the medium decreased significantly compared with the control group (P<0.01). The expression of the steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) genes were significantly downregulated (P<0.05). In addition, the results of flow cytometry detection and Flow Jo software analysis showed that the apoptosis rate of luteal cells increased significantly (P<0.05). qRT-PCR and Western blot results confirmed that the anti-apoptotic factor B lymphoma-2 (BCL-2) mRNA and protein were significantly downregulated (P<0.05), and the pro-apoptotic factor cysteine aspartate-specific protease-3 (Caspase-3) mRNA and protein were significantly upregulated (P<0.05). The above results show that PGD₂ can not only inhibit the endocrine function level of luteal cells by downregulating the expression of StAR/3β-HSD gene, but also accelerate the apoptosis process of luteal cells by downregulating the expression of anti-apoptotic BCL-2 gene and upregulating the expression of pro-apoptosis Caspase-3 gene, and finally participate in the molecular regulation of luteal cells in the form of dual pathways. This lays a solid foundation for further improving the biological functional effects of prostaglandin family members and subsequently comprehensively exploring the molecular regulatory network mechanism of luteal maintenance and degradation in domestic animals.

Key words: corpus luteum, prostaglandin, endocrine, apoptosis gene

中图分类号:

杨恒, 李利财, 付琳, 胥辉豪, 张德志, 李前勇. 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[J]. 畜牧兽医学报, 2023, 54(4): 1652-1663.

YANG Heng, LI Licai, FU Lin, XU Huihao, ZHANG Dezhi, LI Qianyong. Effect of Prostaglandin D₂ on Endocrine Function and Expression of Apoptosis-related Genes in Goat Luteal Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1652-1663.

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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

Effect of Prostaglandin D₂ on Endocrine Function and Expression of Apoptosis-related Genes in Goat Luteal Cells

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