白藜芦醇对体外培养的水牛卵丘细胞增殖活力及其相关基因mRNA表达的影响

doi:10.16431/j.cnki.1671-7236.2022.01.022

摘要/Abstract

摘要： [目的] 研究白藜芦醇（resveratrol，RES）对水牛卵丘细胞体外培养过程中细胞增殖活力、激素分泌、卵丘扩展及抗凋亡和抗氧化能力的影响。[方法] 用不同浓度（0（对照组）、1、10、20、30、40、50和60 μmol/L）RES培养卵丘细胞，用CCK-8试剂盒测定细胞增殖活力，筛选最佳RES处理浓度及时间用于后续试验。用ELISA法测定培养液中卵丘细胞分泌的雌二醇（estradiol，E₂）和孕酮（progesterone，P₄）的含量，实时荧光定量PCR法测定卵丘细胞扩展、凋亡和抗氧化相关基因的相对表达量。[结果] 与对照组相比，在体外培养24~36 h内，1、10和20 μmol/L RES组水牛卵丘细胞的增殖活力均有升高趋势，10 μmol/L RES处理36 h细胞活力最强，因此用于后续试验中细胞的处理。体外培养36 h时，10 μmol/L RES组细胞增殖能力和E₂、P₄的分泌显著增加（P<0.05）；10 μmol/L RES组卵丘细胞扩展相关基因穿透素3（PTX3）、前列腺素内过氧化物合酶2（PTGS2）、抗凋亡基因B淋巴细胞瘤-2（Bcl-2）和超氧化物歧化酶1（SOD1）基因mRNA相对表达量显著或极显著增加（P<0.05；P<0.01），而促凋亡基因Bcl-2相关蛋白（Bax）、半胱氨酸蛋白酶-3（Caspase-3）和p21的表达量显著或极显著降低（P<0.05；P<0.01），透明质酸合酶2（HAS2）和过氧化氢酶（CAT）的表达量无显著差异（P>0.05）。[结论] 培养液中添加10 μmol/L RES能够提高水牛卵丘细胞体外培养的增殖活力，促进卵丘细胞激素分泌和卵丘细胞扩展，并增强卵丘细胞抗氧化能力并减少细胞凋亡。

关键词: 白藜芦醇; 水牛; 卵丘细胞; 增殖活力; 基因表达

Abstract: [Objective] The experiment was to study the effect of resveratrol(RES) on the proliferation activity, hormone secretion, cumulus expansion as well as apoptosis and antioxidase of buffalo cumulus cells cultured in vitro. [Method] Cumulus cells were cultured in medium supplemented with different concentrations (0(control group), 1, 10, 20, 30, 40, 50 and 60 μmol/L) of RES, cell proliferation activity was measured using CCK-8 kit. The best RES treatment concentration and time were selected for subsequent tests. The concentrations of estradiol (E₂)and progesterone (P₄)in the culture medium were measured by ELISA method, and the expression level of genes related to cumulus expansion, apoptosis and antioxidase were determined by Real-time quantitative PCR method. [Result] Compared with control group the proliferation activities of cumulus cells cultured in medium supplemented with 1, 10 and 20 μmol/L RES within 24-36 h were significantly promoted. The cell viability was the strongest after 36 h treatment with 10 μmol/L RES, so it was used for cell treatment in subsequent experiments. When cultured in vitro for 36 h, the cell proliferation ability and the secretion of E₂ and P₄ of 10 μmol/L RES group were significantly increased (P<0.05). The relative mRNA expression of PTX3, PTGS2, Bcl-2 and SOD1 genes of 10 μmol/L group was significantly or extremely significantly increased (P<0.05;P<0.01), and the expression of Bax, Caspase-3 and p21 genes was significantly or extremely significantly decreased (P<0.05;P<0.01), while there was no significant difference in the expression of HAS2 and CAT genes (P>0.05). [Conclusion] 10 μmol/L RES could increase the proliferation activity of cumulus cells, promote hormone secretion and the expansion of cumulus cells, and enhance the antioxidant capacity and reduce apoptosis of cumulus cells in buffalo.

Key words: resveratrol; buffalo; cumulus cells; proliferation activity; gene expression

中图分类号:

S823.8⁺3

郑海英, 杨春艳, 李玲玉, 唐丽萍, 郑威, 尚江华. 白藜芦醇对体外培养的水牛卵丘细胞增殖活力及其相关基因mRNA表达的影响[J]. 中国畜牧兽医, 2022, 49(1): 208-215.

ZHENG Haiying, YANG Chunyan, LI Lingyu, TANG Liping, ZHENG Wei, SHANG Jianghua. Effects of Resveratrol on Proliferation of Cumulus Cells Cultured in vitro and mRNA Expression Levels of Related Genes in Buffalo[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 208-215.

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