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促胰液素对早期妊娠小鼠子宫内膜基质细胞cPLA2和mPGEs-1表达的影响

黄祝, 戈一峰, 靖俊, 吴林, 周铮钰, 朱庆丰, 孙廷哲

安庆师范大学生命科学学院皖西南生物多样性研究与生态保护安徽省重点实验室，安庆 246011；南京军区南京总医院生殖医学中心，南京 210002

摘要

促胰液素(secretin)属于一种胃肠肽，已证实它在早期妊娠小鼠子宫内膜基质细胞中有表达。本文旨在进一步研究促胰液素在胚胎着床过程中的功能。通过real-time PCR、ELISA和原位杂交方法检测促胰液素、促胰液素受体、胞质型磷脂酶A2 (cytosolic phospholipase A2, cPLA2)和膜相关的前列腺素E合成酶-1 (membrane prostaglandin E synthase 1, mPGEs-1)在小鼠妊娠第4到8天子宫中的表达。体外培养妊娠第4天小鼠子宫基质细胞，转染促胰液素表达质粒，使用PKA抑制剂H89处理，24 h收集细胞。Real-time PCR和Western blot 检测cPLA2、 mPGEs-1和环磷腺苷效应元件结合蛋白(cAMP responsive element-binding protein, CREB)的表达，ELISA方法分析细胞上清液中前列腺素E2 (prostaglandin E2, PGE2)的水平。结果显示促胰液素、cPLA2和mPGEs-1的mRNA在妊娠第5到7天着床点表达量逐渐增加，表达趋势一致。促胰液素在妊娠第6、7、8天血清中的含量显著高于第5天的含量，并且在第6到7天着床点中的含量明显高于第5天非着床点含量。转染促胰液素表达质粒，促进小鼠子宫基质细胞cPLA2、磷酸化cPLA2 (p-cPLA2)和mPGEs-1的表达，但对PGE2没有明显的诱导。H89 能明显降低促胰液素对CREB、磷酸化CREB (p-CREB)、cPLA2和p-cPLA2的诱导。以上结果表明在小鼠胚胎着床过程中促胰液素在子宫内膜基质细胞中表达量增加，促进p-cPLA2、cPLA2和mPGEs-1的表达，并通过PKA信号通路调节cPLA2/p-cPLA2的水平。

关键词： 促胰液素; 胞质型磷脂酶A2; 膜相关的前列腺素E合成酶-1; 早期妊娠

分类号：Q95

[Effect of secretin on the expression of cPLA2 and mPGEs-1 in mouse endometrial stromal cell during early pregnancy.] [Article in Chinese]

HUANG Zhu, GE Yi-Feng, JING Jun, WU Lin, ZHOU Zheng-Yu, ZHU Qing-Feng, SUN Ting-Zhe

The Province Key Laboratory of the Biodiversity Study and Ecology Conservation in Southwest Anhui, Life Science College of Anqing Normal University, Anqing 246011, China； Reproductive Medicine Center of Nanjing General Hospital, Nanjing 210002, China

Abstract

Secretin, a gastrointestinal peptide, has been found to be expressed in mouse endometrial stromal cells (mESCs) during early pregnancy. In order to further investigate the function of secretin during embryo implantation, the expression levels of secretin, secretin receptor, cytosolic phospholipase A2 (cPLA2) and membrane prostaglandin E synthase 1 (mPGEs-1) were detected in the mice uterus from day 4 to 8 of pregnancy by real-time PCR, ELISA and in situ hybridization. mESCs isolated and cultured from day 4 of pregnancy were transfected with secretin expression vectors or treated with H89, a PKA inhibitor. Then the expression levels of cPLA2, mPGEs-1 and cAMP responsive element-binding protein (CREB) were detected by real-time PCR and Western blot. The concentration of prostaglandin E2 (PGE2) in the supernatant was determined by ELISA. The result showed that secretin, cPLA2 and mPGEs-1 mRNA expression increased gradually in implantation sites from day 5 to day 7 of pregnancy with the same tendency. The secretin levels in serum were significantly higher on days 6, 7 and 8 of pregnancy than that on day 5 of pregnancy. The concentration of secretin was significantly higher in implantation sites on days 6, 7 than that in non-implantation site on day 5. Transfection of secretin expression vector promoted cPLA2, p-cPLA2 and mPGEs-1 expressions in mESCs, but not PGE2 level in the supernatant. H89 could effectively inhibit the expression of CREB, p-CREB, p-cPLA2 and cPLA2 induced by secretin. The results showed that the increased secretin expression in mESCs during embryo implantation may promote p-cPLA2, cPLA2 and mPGEs-1 expression, and the promotion may be through PKA signaling pathway.

Key words: Secretin; cPLA2; mPGEs-1; early pregnancy

收稿日期：2016-03-21　　录用日期：2016-09-18

通讯作者：黄祝　　E-mail: huangzhu@xmu.edu.cn

引用本文：

黄祝, 戈一峰, 靖俊, 吴林, 周铮钰, 朱庆丰, 孙廷哲. 促胰液素对早期妊娠小鼠子宫内膜基质细胞cPLA2和mPGEs-1表达的影响[J]. 生理学报 2016; 68 (6): 725-732.

HUANG Zhu, GE Yi-Feng, JING Jun, WU Lin, ZHOU Zheng-Yu, ZHU Qing-Feng, SUN Ting-Zhe. [Effect of secretin on the expression of cPLA2 and mPGEs-1 in mouse endometrial stromal cell during early pregnancy.] [Article in Chinese]. Acta Physiol Sin 2016; 68 (6): 725-732 (in Chinese with English abstract).