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原花青素对体外培养牛卵泡颗粒细胞增殖及其类固醇合成与分泌功能的影响
杨小峰1,2, 党文庆2, 秦小伟,等2
1.忻州师范学院 生物系;2.山西农业大学 动物科学学院
摘要:
【目的】探究原花青素(proanthocyanidins,PC)对体外培养的牛卵泡颗粒细胞(granulosa cells,GCs)增殖及其类固醇合成与分泌功能的影响。【方法】采集性成熟荷斯坦奶牛卵巢组织,分离卵泡GCs,使用免疫荧光对其进行鉴定。用不同质量浓度(10,30,50,70和90 μg/mL)的PC对牛卵泡GCs作用不同时间(12,24和48 h)后,测定GCs存活率。以β-actin为内参基因,采用实时荧光定量PCR(qRT-PCR)检测10,50和90 μg/mL PC对牛卵泡GCs周期相关基因(CCND1和CCND2)、凋亡相关基因(caspase-3、caspase-9和Bim)以及类固醇激素合成相关基因(CYP19A1、CYP11A1、STAR和3β-HSD)相对表达量的影响,并检测培养上清液中类固醇激素(雌二醇(E2)和孕酮(P4))的浓度。【结果】分离获得了纯度较高的牛卵泡GCs。当PC质量浓度为50 μg/mL且培养24 h时,牛卵泡GCs的存活率最高。当PC质量浓度为50 μg/mL时,牛卵泡GCs周期相关基因CCND1和CCND2相对表达量均极显著升高(P<0.01);凋亡相关基因caspase-3和caspase-9相对表达量均极显著降低(P<0.01),Bim相对表达量显著降低(P<0.05);E2和P4浓度均极显著增加(P<0.01);类固醇激素合成相关基因CYP19A1、CYP11A1、STAR和3β-HSD相对表达量均极显著升高(P<0.01)。当PC质量浓度为10 μg/mL时,牛卵泡GCs凋亡相关基因caspase-3的相对表达量显著降低(P<0.05);P4浓度显著增加(P<0.05);CYP19A1和3β-HSD相对表达量均显著升高(P<0.05),CYP11A1STAR相对表达量均极显著升高(P<0.01)。当PC质量浓度为90 μg/mL时,E2浓度显著增加(P<0.05),P4浓度极显著增加(P<0.01);CYP19A1、CYP11A1、STAR和3β-HSD相对表达量均极显著升高(P<0.01)。【结论】50 μg/mL PC通过促进牛卵泡GCs周期相关基因的表达和抑制凋亡相关基因的表达而促进牛卵泡GCs的增殖。10~90 μg/mL PC通过促进类固醇激素合成相关基因的表达而促进了牛卵泡GCs合成和分泌类固醇激素,进而影响牛卵泡GCs类固醇激素合成与分泌功能。
关键词:  原花青素  牛卵泡颗粒细胞  细胞增殖  细胞凋亡  类固醇
DOI:
分类号:
基金项目:国家自然科学基金项目(31873002);山西省基础研究计划面上项目(20210302123393);山西省青年三晋学者专项(J241644001);忻州市科技计划(专项)项目(20210202-7);“1331工程”提质增效建设计划项目子课题(2023WTS03)
Effect of proanthocyanidins on proliferation of bovine follicular granulosa cells cultured in vitro and their steroid synthesis and secretion functions
YANG Xiaofeng,DANG Wenqing,QIN Xiaowei,et al
Abstract:
【Objective】The aim was to investigate the effects of proanthocyanidins (PC) on proliferation of bovine follicular granulosa cells (GCs) cultured in vitro and their steroid synthesis and secretion functions.【Method】Ovarian tissue was collected from sexually mature Holstein cows and follicular GCs were isolated and characterized using immunofluorescence.Cell viability was determined after acting on bovine follicular GCs for different times (12,24 and 48 h) with different PC concentrations of 10,30,50,70 and 90 μg/mL.The effects of PC at 10,50 and 90 μg/mL on relative expressions of genes related to bovine follicular GCs cycle (CCND1 and CCND2),apoptosis (caspase-3,caspase-9 and Bim) and steroid synthesis (CYP19A1,CYP11A1,STAR and 3β-HSD) were examined using real-time fluorescence quantitative PCR (qRT-PCR) with β-actin as an endogenous reference gene.The concentrations of steroid hormones estradiol (E2) and progesterone (P4) in the culture supernatant were measured.【Result】The purity of follicular granulosa cells obtained by isolation was high.The highest survival rate of bovine follicular GCs was observed when PC concentration was 50 μg/mL and incubation time was 24 h.When PC concentration was 50 μg/mL,the relative expression of CCND1 and CCND2 in bovine follicular GCs was highly significantly increased (P<0.01),the relative expression of caspase-3 and caspase-9 was highly significantly decreased (P<0.01),while the relative expression of Bim was significantly decreased (P<0.05).The concentrations of E2 and P4 were highly significantly increased (P<0.01) and the relative expressions of CYP19A1,CYP11A1,STAR and 3β-HSD were highly significantly elevated (P<0.01).When PC concentration was 10 μg/mL,the relative expression of caspase-3 was significantly decreased (P<0.05),the concentration of P4 was significantly increased (P<0.05),the relative expression of CYP19A1 and 3β-HSD was significantly elevated (P<0.05), and the relative expression of CYP11A1 and STAR was extremely significantly elevated (P<0.01).When PC concentration was 90 μg/mL,the concentration of E2 was significantly increased (P<0.05),the concentration of P4 was highly significantly increased (P<0.01),and the relative expression of CYP19A1,CYP11A1,STAR and 3β-HSD were highly significantly elevated (P<0.01).【Conclusion】PC at 50 μg/mL improved the proliferation of bovine follicular GCs by promoting the expression of cell cycle-related genes and inhibiting the expression of apoptosis-related genes in bovine follicular GCs.PC at 10-90 μg/mL promoted the synthesis and secretion of steroid hormones in bovine follicular GCs by increasing the expression of steroid hormone synthesis-related genes,which in turn affected the function of steroid hormone synthesis and secretion in bovine follicular GCs.
Key words:  proanthocyanidins  bovine follicular granulosa cell  cell proliferation  cell apoptosis  steroids