ATF6在转基因敲除鼠人工诱导蜕膜化模型中的研究

Study of ATF6 in an artificially induced decidualization model of transgenic knockout mice

目的利用小鼠人工诱导蜕膜化模型,探讨转录活化因子6(ATF6)在小鼠子宫内膜中的作用机制。方法构建繁育野生型(Atf6^(+/+))和ATF6敲除型(Atf6^(-/-))小鼠,并构建小鼠人工诱导蜕膜化模型。雌鼠交配后见阴栓第1天记为Day 1。Day 4进行诱导蜕膜化,蜕膜化后48 h和72 h(即Day 6和Day 7)收集小鼠子宫进行相应指标检测,实时荧光定量PCR方法和蛋白质免疫印迹法检测Atf6 mRNA与蛋白质在蜕膜化子宫中的表达情况;免疫组织化学方法检测野生型(Atf6^(+/+))和ATF6敲除型(Atf6^(-/-))小鼠人工诱导蜕膜化模型中ATF6和催乳素(PRL)在子宫中的阳性信号表达情况。收集人子宫内膜生理周期中不同时相子宫内膜组织,进行石蜡包埋切片,免疫组织化学方法检测ATF6在子宫内膜中的阳性信号表达情况。结果与小鼠未蜕膜子宫相比,Day 6和Day 7蜕膜化子宫中的Atf6 mRNA表达水平显著升高(P<0.05),细胞核内的蛋白水平也显著增高(P<0.05);Day 7时子宫大体形态学显示子宫较粗且均匀。野生型和敲除鼠诱导蜕膜化子宫HE染色显示,绝大部分子宫基质细胞变大变圆,表明诱导蜕膜化的子宫内膜蜕膜化充分;PRL免疫组化染色结果表明,在野生型(Atf6^(+/+))和敲除型(Atf6^(-/-))小鼠蜕膜化子宫内膜中,PRL染色阳性信号分布范围无显著性差异(P>0.05)。不同月经周期时相人子宫内膜组织的免疫组化结果提示,ATF6在月经期和增殖早期的表达水平较高,显著高于其余周期时相(P<0.05)。结论ATF6能够参与子宫内膜蜕膜化,但不是蜕膜化发生的必要条件;ATF6可能通过参与子宫内膜的生理修复过程参与子宫内膜生理周期。

Objective:To explore the mechanism of transcription activating factor 6(ATF6)in the endometrium of artificial induced decidualization mice model.Methods:The wild-type(Atf6^(+/+))and ATF6-knockout(Atf6^(-/-))mice were constructed and bred,and then a mouse model of artificially induced decidualization was constructed.Day 1 is the first day when vaginal plug was seen in female rat after mating.The decidualization was induced on Day 4.The uterus of mice was collected 48 hours and 72 hours after decidualization(i.e.Day 6 and Day 7)for corresponding indexes detection.The expression of Atf6 mRNA and protein in decidualization uterus were detected by using the quantitative real-time PCR method(qRT-PCR)and Western blot.Immunohistochemical methods were used to detect the positive signal expression of ATF6 and prolactin(PRL)in the uterus of wild-type(Atf6^(+/+))and ATF6 knockout(Atf6^(-/-))mice models with artificially induced decidualization.Endometrial tissues were collected from different phases of the human endometrial physiological cycle,paraffin-embedded sections were performed,and the positive signal expression of ATF6 in the uterus was detected by immunohistochemistry.Results:Atf6 mRNA expression levels and ATF 6 protein levels in the nuclei were significantly higher in Day 6 and Day 7 decidualization uterus compared with without decidualization uterus of mice(P<0.05).The gross morphology of the uterus on Day 7 showed that the uterus was thick and uniform.HE staining of wild-type mice and knockout mice induced decidualization uterus showed that the vast majority of uterine stromal cells became larger and rounder,indicating the decidualization of endometrium was sufficient.Immunohistochemical staining of PRL showed that there was no significant difference in the distribution of positive PRL staining signals between the wild-type(Atf6^(+/+))and knockout(Atf6^(-/-))mice(P>0.05).Immunohistochemical results in human endometrial tissues from different phases of menstrual cycles suggested that ATF6 was expressed at higher levels at menstruation and early proliferation phase,which were significantly higher than the rest of the menstrual phases(P<0.05).Conclusions:ATF6 participates in endometrial decidualization,but it is not a necessary condition for decidualization.ATF6 may involve in the endometrial physiological cycle by participating in the physiological repair process of the endometrium.