干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19在小鼠围着床期子宫内膜中的表达及其作用

doi:10.16098/j.issn.0529-1356.2015.04.006

摘要/Abstract

摘要：

目的探讨干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19（GRIM-19）在小鼠围着床期子宫内膜中的表达及其与胚胎着床的关系。方法收集早期妊娠第1~6天小鼠子宫组织，采用免疫组织化学法检测GRIM-19蛋白在子宫内膜上皮细胞中的表达及定位情况；收集早期妊娠、假孕和雌孕激素处理小鼠子宫组织，采用Western blotting 和Real-time PCR检测GRIM-19蛋白和mRNA水平；TUNEL方法检测早期妊娠第1~6天小鼠子宫组织细胞凋亡情况；采用pEGFP GRIM-19质粒GRIM-19-siRNA 转染技术使RL95-2细胞系过表达或低表达GRIM-19，检测其对RL95-2-BeWo 共培养体外着床模型球状体黏附率的影响,AnnexinV/PI双染色法检测细胞凋亡,线粒膜电位检测试剂盒（JC-1）检测线粒体跨膜电位；采用Western blotting 和Real-time PCR检测转染后信号转导子和转录激活因子3（STAT3）、肿瘤坏死因子（TNF)-α及白细胞介素（IL)-11蛋白和mRNA水平。结果 GRIM-19在早期妊娠第1~6天小鼠子宫腔上皮和腺上皮均有表达，妊娠第4天GRIM-19蛋白和mRNA水平减低，细胞凋亡减少；假孕第1~6天小鼠子宫内膜GRIM-19表达无明显差异；雌激素处理组小鼠子宫内膜GRIM-19表达减弱；过表达GRIM-19后，RL95-2-BeWo 共培养球状体黏附率降低，细胞凋亡增加，线粒体膜电位减低，RL95-2细胞系中STAT3及IL-11蛋白和mRNA水平减低，TNF-α蛋白和mRNA水平升高。结论 GRIM-19在胚胎着床中发挥重要作用，可能是通过调节细胞凋亡和免疫耐受为胚胎着床提供保障。

关键词: 干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19, 胚胎, 着床, 子宫, 免疫组织化学, 小鼠

Abstract:

Objective To explore the expression of gene associated with retinoic-interferon-induced mortality-19(GRIM-19) gene and the effect of GRLM-19 on the embryonic implantation. Methods The expression of GRIM-19 in the pregnant mouse uterus was detected by immunohistochemistry. The expression of GRIM-19 in the mouse uterus was detected by Real-time PCR and Western blotting with early pregnancy,hormone treatment and pseudopregnancy models. TUNEL method was used to detect apoptosis in uterine tissues of pregnant mice. An optimized model of R95-2 spheroids and a Bewo monolayer co-culture model were employed to study the effect of GRIM-19 in the embryo-endometrial epithelium attachment. Apoptosis was assayed by flow cytometry. Mitochondrial membrane potential was assayed by JC-1. Western blotting and Real-time PCR were used to detect signal transducer and activator of transcription(STAT3), tumor necrosis factor(TNF)-α and interleukin (IL)-11 mRNA and protein level. Results The immunohistochemical positive staining for GRIM-19 was distributed in the luminal epithelium and glandular epithelium in pregnant mice. The intensities of both GRIM-19 protein and mRNA positive staining were decreased to the lowest level at pregnancy day 4 in the pregnancy group, no significant difference in the pseudopregnancy group, and lower in the estrogen treatment group. Overexpression of GRIM-19 made RL95-2-BeWo co-culture spheroid adhesion rate decreased, apoptosis increased, and mitochondrial membrane potential reduced; STAT3 and IL-11 mRNA and protein levels reduced, but TNF-α protein and mRNA levels increased. Conclusion GRIM-19 plays an important role in embryo implantation, possibly through the regulation of apoptosis and immune tolerance to provide protection for the embryo implantation.

Key words: GRIM-19, Embryo, Implantation, Uterus, Immunohistochemistry, Rat

孙艳艳邓晓惠晁岚* 杨阳程来洋杜天奇王静. 干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19在小鼠围着床期子宫内膜中的表达及其作用[J]. 解剖学报, 2015, 46(4): 471-479.

SUN Yan-yan DENG Xiao-hui CHAO Lan* YANG Yang CHENG Lai-yang DU Tiao-qi WANG Jing. Expression of gene associated with retinoic-interferon-induced mortality-19 and its effect on mouse embryo implantation[J]. AAS, 2015, 46(4): 471-479.

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