CART对FSH诱导下水牛颗粒细胞雌二醇分泌及相关基因表达的影响

doi:10.16431/j.cnki.1671-7236.2020.09.021

摘要/Abstract

摘要： 本试验采用无血清培养体系培养原代水牛卵巢颗粒细胞，研究了外源可卡因-苯丙胺调控转录肽（exogenous cocaine amphetamine regulated transcript，CART）对促卵泡激素（FSH）诱导下颗粒细胞产生雌二醇（E₂）的影响及其相关基因的表达情况。采用Long-term培养方法在无血清培养液中分别添加不同浓度的FSH（0、0.25、0.5、1、2、4 ng/mL）培养7 d，利用双抗体夹心法检测各孔的E₂浓度，筛选出最佳添加浓度；将原代颗粒细胞随机分成4组（2组对照组（无FSH）和2组最佳FSH添加组）培养6 d后，其中一组对照组和FSH添加组分别与25 μg/mL CART共作用24 h，检测E₂浓度的变化并对颗粒细胞中CART受体（CARTPT）和芳香化酶基因（CYP19A1）进行定量检测。结果发现：①在培养液中不添加CART时，颗粒细胞中E₂浓度随着FSH添加浓度的升高呈先升高后降低的趋势，当FSH添加浓度为1 ng/mL时，E₂浓度达到最高值116.16 pmol/mL，显著高于对照组和其他浓度组（P<0.05）；②添加外源CART显著降低了FSH作用下颗粒细胞培养液中E₂的浓度（P<0.05），两对照组（0 FSH和0 FSH+CART）差异不显著（P>0.05）；③在添加1 ng/mL FSH时，CART作用24 h后显著降低了FSH诱导下颗粒细胞中CYP19A1 mRNA的表达（P<0.05），而细胞中CARTPT mRNA的相对表达量显著升高（P<0.05）。在0 ng/mL FSH下，添加25 μg/mL CART作用24 h，颗粒细胞中CYP19A1和CARTPT mRNA的表达与不添加CART组相比差异不显著（P>0.05）。由此推测CART在水牛卵巢发育中是一种负向调控信号，抑制卵泡的健康发育，且需一定量的FSH诱导CART对颗粒细胞增殖分化的负向压力。

关键词: CART; FSH; E₂; 水牛; 颗粒细胞; CYP19A1

Abstract: In this study,primary buffalo ovarian granulosa cells were cultured in a serum-free culture medium in vitro to investigate the effect of exogenous cocaine amphetamine regulated transcript (CART) on the production of estradiol (E₂) in granulosa cells induced by follicle stimulating hormone (FSH) and its related gene expression.The granulosa cells was cultured for 7 days by using a long-term method,in each group that was adding different concentration levels of FSH (0,0.25,0.5,1,2 and 4 ng/mL).The concentration of E₂ was assayed by ABC-ELISA kit to screen out the optimal dosage of FSH.Primary granulosa cells were randomly divided into 4 group (2 groups of control (without FSH) and 2 groups of optimal FSH levels) for 6 days culure,one of the control group and FSH group were respectively combined with 25 μg/mL CART for 24 h,to detect the change of E₂ concentration and quantitative detection the gene expression of CART receptors and aromatase in granulosa cell.The results showed as the follows:①Without CART,the E₂ concentration in granulose cells increased first and than decreased with the increased of FSH.When adding 1 ng/mL FSH,the concentration of E₂ reached the hightest (116.16 pmol/mL) and significantly higher than the other groups (P<0.05);②Adding CART significantly reduced the concentration of E₂ in granulosa cells induced by FSH (P<0.05) and the two control groups 0 FSH and 0 FSH+CARD had no significantly difference (P>0.05).③With 1 ng/mL FSH,exogenous CART significantly reduced the expression of CYP19A1 mRNA in granulosa cells after 24 h treated (P<0.05),while the relative expression of CARTPT mRNA in granulosa cells significantly increased (P<0.05).Under the condition of 0 ng/mL FSH,the expression of CYP19A1 and CARTPT mRNA had no significant difference (P>0.05).Therefore,this study was speculated that CART was a negative regulatory signal in the ovarian development of buffalo,which inhibited the healthy development of follicles,a certain amount of FSH was required to induced the negative pressure of CART on granulosa cell proliferation and differentiation.

Key words: CART; FSH; E₂; buffalo; granulose cells; CYP19A1

中图分类号:

S814.4

李玲玉, 郑海英, 于农淇, 尚江华, 黄加祥, 杨春艳. CART对FSH诱导下水牛颗粒细胞雌二醇分泌及相关基因表达的影响[J]. 中国畜牧兽医, 2020, 47(9): 2876-2883.

LI Lingyu, ZHENG Haiying, YU Nongqi, SHANG Jianghua, Huang Jiaxiang, Yang Chunyan. Effects of CART on Estradiol and Related Gene Expression in Buffalo Follicle Granulosa Cells Induced by FSH[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2876-2883.

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