甲状腺素对猪卵巢卵泡颗粒细胞类固醇激素合成及增殖的影响  

Effects of thyroid hormones on ovarian granulosa cell steroidogenesis and proliferation in pigs

作　　者：宋丹 吴国云[1] 丁威[2] 邢军[1,2] 魏全伟[1] 石放雄[1] SONG Dan1, WU Guoyun1, DING Wei2, XIN Jun1,2, WEI Quanwei1, SHI Fangxiong1(1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China ;2. Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, Chin)

机构地区：[1]南京农业大学动物科技学院,江苏南京210095 [2]江苏农林职业技术学院,江苏句容212400

出　　处：《南京农业大学学报》2018年第2期357-363,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31432075;31572403);中国高校博士后基金项目(2014M561671);江苏省农林职业技术学院科技项目(2016kj001)

摘　　要：[目的]本试验旨在探究甲状腺素对猪卵巢卵泡颗粒细胞类固醇激素合成功能以及增殖的影响。[方法]首先采用免疫组化法鉴定甲状腺素受体(TRα/β)在猪卵巢组织的表达情况,随后体外培养猪卵巢原代颗粒细胞,选取低(10-8mol·L^(-1))、中(10-7mol·L^(-1))、高(10-6mol·L^(-1))3种浓度的甲状腺素(T4)孵育24 h,采用酶联免疫法检测细胞培养液中孕酮(P4)和雌二醇(E2)水平,并采用蛋白印迹法检测颗粒细胞内激素合成相关酶蛋白(3β-HSD、CYP19)的表达情况。采用MTT法检测各浓度甲状腺素对细胞活力的影响,利用增殖细胞核抗原(PCNA)免疫荧光染色验证不同浓度甲状腺素对颗粒细胞增殖的影响。[结果]免疫组化结果显示:TRα/β在猪卵巢不同发育阶段卵泡的颗粒细胞、膜细胞中有广泛表达,在卵母细胞的胞质中也有分布,在闭锁卵泡中也见明显表达。猪卵巢原代颗粒细胞经甲状腺素处理后,细胞培养液中的孕酮和雌激素水平都显著低于空白对照组(P<0.05),并且各剂量处理组间孕酮呈现出剂量依赖性降低趋势;雌激素在低浓度和高浓度甲状腺素处理组较中浓度处理组高。蛋白免疫印迹结果显示:与对照组相比,3β-HSD和CYP19蛋白在较高浓度甲状腺素(10-7和10-6mol·L^(-1))处理组的表达量均显著降低(P<0.05);而在低浓度甲状腺素(10-8mol·L^(-1))处理组,CYP19蛋白表达量显著降低(P<0.05),但3β-HSD蛋白表达无显著差异。此外,MTT细胞毒性和增殖试剂盒检测结果显示:不同浓度甲状腺素处理并不影响细胞活力,这与PCNA免疫荧光强度统计结果一致,进一步证实了甲状腺素对原代颗粒细胞增殖没有影响。[结论]甲状腺素能够作用于卵巢颗粒细胞,干扰类固醇激素的合成,其作用机制可能是通过改变激素合成相关蛋白3β-HSD和CYP19的表达来完成的。而甲状腺素对颗粒细胞激素合成的干扰效应并不影响细胞增殖状态。[Objectives]This study was designed to examine the role of thyroid hormones on ovarian granulosa cell proliferation and steroidogenesis in pigs.[Methods]The expression of thyroid hormone receptor（TRα/β） was detected by immunohistochemistry.Subsequently the primary granulosa cells of porcine ovarian were clutured in vitro,cells were then treated for different dose of thyroxine（T4）from low（10^-8 mol·L^-1）,medium（10^-7 mol·L^-1）and high（10^-6 mol·L^-1）and incubated for 24 h.Serum medium were collected and stored for determining the concentration of hormones（such as progesterone and estradiol） by radioimmunoassay（RIA） examination and the cell sample were collected for detecting the expression of protein involved in the biosynthesis of steroids by Western blot（such as 3β-HSD and CYP19）.Then granulosa cell numbers and viability were evaluated with MTT assay.The effect of thyroid hormones on proliferation of granulosa cells was verified by immunofluorescence staining with proliferating cell nuclear antigen（PCNA）.[Results]The immunohistochemical results indicated that the TRα/β was widely expressed in ovarian granulosa cells and theca cells,and also distributed in the cytoplasm of oocytes.Treatment with T4 resulted in a significant increase in progesterone and estrogen secretion by the cultured primary granulosa cell relative to control（P〈0.05）,and the dose of progesterone in each group showed a dose-dependent trend;the estrogen levels in the treatment groups of low and high concentration significantly increased compared to the group of medium concentration.Expressions level of 3β-HSD and CYP19 decreased significantly when treated with the higher concentrations of L-thyroxine（10^-7 mol·L^-1 and 10^-6 mol·L^-1）（P〈0.05）;and the expression level of CYP19 decreased significantly in the group of10^-8 mol·L^-1 L-thyroxine（P 0.05）,but there was no significantly difference in the expression of 3β-HSD.In addition,the MTTcytotoxicity and proliferation assay showe

关 键 词：猪卵巢 原代颗粒细胞 甲状腺素 类固醇合成 增殖 

分 类 号：S852.21[农业科学—基础兽医学]