摘要
目的研究高浓度雄激素环境对卵巢颗粒细胞(GC)分泌功能和增殖、分化的影响。方法体外培养猪卵巢窦状卵泡GC,先以梯度浓度(500、250、125、62.5、31.25、15.625μmol/L)加入丙酸睾酮,通过CCK-8法检测各组GC相对活力,确定适合继续研究的丙酸睾酮浓度;放射免疫法检测并对比实验组(加入丙酸睾酮)和空白对照组(未加入任何物质)细胞上清液中雌二醇(E2)与孕酮(P)的含量;采用半定量RT-PCR和Western blot分别检测两组的PCNA、FSHR、StAR mRNA与PCNA、FSHR、StAR、IGF-Ⅰ、p-ERK1/2、p-p38、Cyclin D2和CDK4蛋白表达水平。结果实验组E2较对照组显著升高(P<0.001),P则显著降低(P<0.001);GC中PCNA、StAR和FSHR mRNA表达量较对照组均明显下降(P<0.05);同时PCNA、FSHR、StAR、IGF-Ⅰ、p-ERK1/2与p-p38蛋白表达较对照组也明显下降(P<0.05)。结论雄激素对卵泡发育的影响具有双重性,高浓度雄激素抑制GC分泌P的作用可能早于抑制E2的分泌;并影响ERK1/2和p38MAPK信号通路,一方面通过抑制Cyclin D2,使正常的细胞周期调控受阻而抑制细胞增殖,另一方使细胞形态和存活率失调,而负向调控细胞正常分化。
Objective To study the effects of high androgenic environment on the secretory function,proliferation and differentiation of granulosa cells.Methods The pig granulosa cells were cultured in testosterone of different concentrations in vitro,the relative vitality of granulosa cells was tested by CCK-8 method for the determination of the suitable concentration.The E2 and P levels were assessed in the supernatant by radioimmunoassay.Semi-quantitative RT-PCR was applied for assessment of mRNA expression of PCNA,FSHR and StAR;while Western blot was applied for protein expression of StAR,IGF-Ⅰ,p-ERK1/2,p-p38,Cyclin D2 and CDK4.Results Significant elevation of E2 and reduction of P were revealed in experimental group when comparing with those in control group（P0.001）.Furthermore,significant down-regulation of mRNA expression of PCNA,StAR,FSHR and protein expression of PCNA,FSHR,StAR,IGF-Ⅰ,p-ERK1/2 and p-p38 in granulose cells of experimental group were also observed（P0.05）.Conclusion The probable inhibitive effect on P secretion in granulosa cells ahead that on E2 secretion is observed in high concentrations of androgen.Androgen also affects the ERK1/2 and p38MAPK signaling pathways,with down-regulation of Cyclin D2 thus block the cell proliferation and differentiation;and further impairs the cell vitality,thus down-regulates the normal differentiation.
出处
《广东医学》
CAS
CSCD
北大核心
2012年第20期3046-3050,共5页
Guangdong Medical Journal
基金
国家自然科学基金资助项目(编号:30701119)
广东省自然科学基金资助项目(编号:9151008901000050)
广东省自然科学基金重点
自由申请和博士科研启动项目(编号:5300997)
关键词
雄激素
颗粒细胞
增殖
分化
分泌
androgen
granulosa cell
proliferation
differentiation
secrete
