【摘要】 藏羊（Ovis aries）是中国青藏高原地区的主要经济动物，由于其繁殖能力弱，受孕率和妊娠率低，以致种群发展受到限制。因此，进行藏羊繁殖相关问题的研究具有重要意义。本研究旨在研究柚皮素（naringenin, NAR）对藏羊颗粒细胞（granulosa cells, GCs）增殖、凋亡和雌二醇（estradiol, E₂）合成的影响。采集并培养藏羊原代GCs至对数生长期，添加不同浓度NAR（0, 5, 10, 15和20μmol/L）培养12 h，采用qPCR和Western blot方法检测其增殖（PCNA, CCND1）、凋亡（Bcl-2, BAX, Caspase-3）和E₂合成（CYP19A1,CYP17A1, CYP11A1, STAR）相关基因和蛋白的表达；采用CCK-8检测GCs细胞增殖水平，免疫荧光（immunofluorescence, IF）检测GCs的凋亡；酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）检测培养液中E₂的浓度。结果显示，添加10μmol/L NAR使CCND1 mRNA表达显著上调（P<0.05）；不同浓度NAR处理后，处理组增殖相关蛋白（PCNA, CCND1）表达显著高于对照组（P<0.05）；处理组凋亡相关基因及蛋白（BAX, Caspase3）表达均显著降低（P<0.05）。10μmol/L NAR处理组E₂合成相关基因及蛋白（CYP11A1, CYP17A1和CYP19A1）表达水平显著上调（P<0.05）,STAR mRNA及蛋白表达水平显著下调（P<0.05）。综上，NAR可通过上调PCNA、CCND1、Bcl-2 mRNA及下调BAX、Caspase3蛋白表达量，促进GCs增殖，并抑制其凋亡；通过上调CYP11A1、CYP17A1、CYP19A1和下调基因STAR mRNA及蛋白促进E₂合成。本结果为进一步研究NAR对哺乳动物卵巢机能的影响和改善动物生殖潜力提供理论依据。更多还原

【Abstract】 As the main economic animal in the Qinghai-Tibet plateau of China, the development of Tibetan sheep（Ovis aries） population is limited due to its weak reproductive ability, low conception rate andlow pregnancy rate. Therefore, it is of great significance to study the problems related to Tibetan sheep reproduction. This study was designed to investigate the effects of naringenin（NAR） on proliferation,apoptosis and estradiol（E₂） synthesis of granulosa cells（GCs） in Tibetan sheep. The primary GCs of Tibetan sheep were collected and cultured in vitro to logarithmic growth phase, different concentrations of NAR（0, 5,10, 15 and 20 μmol/L） were added to culture GCs for 12 h. qPCR and Western blot were used to detect the expressions of genes and proteins related to proliferation（PCNA, CCND1）, apoptosis（Bcl-2, BAX, Caspase-3） and E₂ synthesis（CYP19A1, CYP17A1, CYP11A1, STAR）. The proliferation of GCs was detected by CCK-8, and the apoptosis of GCs was detected by immunofluorescence. The concentration of E₂ in the culture medium was detected by enzyme-linked immunosorbent assay（ELISA）. The results showed that adding 10μmol/L of NAR significantly up-regulated the expression of CCND1 mRNA（P<0.05）. And the expressions of proliferation-related proteins（PCNA, CCND1） in NAR treatment groups were significantly higher than those of the control group（P<0.05）. The expressions of genes and proteins of BAX and Caspase3 in the treatment groups decreased significantly（P<0.05）. The expression levels of E₂ synthesis-related genes and proteins（CYP11A1, CYP17A1 and CYP19A1） in the 10 μmol/L NAR treatment group rose significantly（P<0.05）,while those of STAR mRNA and protein decreased significantly（P<0.05）. In summary, NAR of 10 μmol/L promoted the proliferation of GCs and inhibited their apoptosis by up-regulating PCNA, CCND1, Bcl-2 mRNA and down-regulating BAX and Caspase3 protein expression; E₂ was synthesized and secreted by up-regulating CYP11A1, CYP17A1, CYP19A1 and down-regulating STAR mRNA and proteins. This study provides a foundation for further study on the effects of NAR on functions of mammalian ovaries and improvement the animal reproductive potential.更多还原