维生素A酸对小鼠囊胚发育的影响

发布时间：2018-01-12 20:04

本文关键词：维生素A酸对小鼠囊胚发育的影响　出处：《武汉大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:近年来,随着妊娠早期流产率的不断增加,有关着床期和着床前期发育毒性也越来越受到人们的关注和探讨。有研究发现着床前期胚胎暴露于某些化学物,可诱发胎儿畸形或引起胚胎死亡等发育毒性,此发现对胚胎着床前期是实验致畸不敏感期这一传统的致畸理论提出质疑。而维生素A酸(RA)是近年来研究比较广泛的一种凋亡诱导剂和致畸因子,用RA处理的发育中的胚胎可出现面部、神经管和四肢等部位的多种畸形。本课题主要研究全反式维生素A酸(ATRA)诱导体外培养小鼠囊胚内细胞群(ICM)和滋养层(TE)细胞凋亡,而导致囊胚细胞总数、内细胞群和滋养层细胞数目的变化,以及由此引起的囊胚细胞中细胞因子TGF-β1蛋白和Fas蛋白表达的改变,并了解这两种细胞因子对着床前胚胎发育的作用。从而探讨ATRA对着床前期胚胎发育毒性作用,为其致畸机理提供实验依据。方法:通过促超排卵,获取妊娠3.5d小鼠囊胚,随机分成三组,即对照组(空白)、实验1组(含1μmol/L的ATRA)和实验2组(含10μmol/L的ATRA),分别培养在含0、1μmol/L和10μmol/L的ATRA的M199培养基中,培养24h,然后再吸出囊胚。72个囊胚用荧光染料propidium iodide(PI)和Bisbenzimide(Hoechst 33258)双重染色,分别计数囊胚内细胞总数以及内细胞群和滋养层细胞数。75个囊胚固定干燥后,用带有荧光(FITC)标记的原位末端标记(TUNEL)检测法,并通过激光共聚焦扫描显微镜观察ATRA诱导囊胚细胞凋亡效应,记录荧光值并分析;其余囊胚用免疫组织化学S-P法,分别检测不同剂量组ATRA对小鼠囊胚TGF—β1蛋白和Fas蛋白表达的状况,利用HPIAS-1000图像分析系统测定TGF-β1蛋白和Fas蛋白在以上三组中表达的平均光密度和平均阳性面积率。结果:(1) 囊胚荧光染色:在激光共聚焦显微镜下,滋养层(TE)细胞核呈粉红色,内细胞群(ICM)细胞核呈蓝色,实验2组中细胞总数和滋养层细胞数比实验1组和对照组明显减少(p0.01);以及内细胞群细胞数较实验1组和对照组减少(P0.05):其中以滋养层细胞减少为主。但实验1组与对照组比较无显著性差异(p0.05)。(2) TUNEL法荧光标记检测囊胚细胞凋亡:通过激光共聚焦显微镜定
[Abstract]:Objective: in recent years, with the increase of abortion rate in early pregnancy. More and more attention has been paid to developmental toxicity during implantation and pre-implantation. Some studies have found that exposure to certain chemicals in preimplantation embryos can induce developmental toxicity such as fetal malformation or embryo death. The findings question the traditional teratogenic theory that preimplantation is an experimental teratogenic insensitive period. Vitamin A acid RAA is a widely studied apoptosis inducer and teratogenic factor in recent years. A developing embryo treated with RA may appear on the face. The aim of this study was to investigate the induction of apoptosis of mouse blastocyst cells (ICM) and trophoblast cells by all-trans vitamin A (ATRAA) in vitro. The changes of the total number of blastocyst cells, the number of inner cell groups and trophoblast cells, and the expression of cytokine TGF- 尾 1 and Fas protein in blastocyst cells were also found. The effects of these two cytokines on preimplantation embryo development were investigated, and the toxic effects of ATRA on pre-implantation embryo development were discussed, which provided experimental basis for the teratogenicity mechanism. Methods: the blastocysts of 3.5d gestational mice were obtained by superovulation. The blastocysts were randomly divided into three groups: control group (blank). Experimental group 1 (containing 1 渭 mol/L ATRAA) and experimental group 2 (containing 10 渭 mol/L of ATRAA) were cultured in the presence of 0 渭 mol/L. M199 medium containing 1 渭 mol/L and 10 渭 mol/L ATRA was cultured for 24 hours. Then the blastocysts were sucked out. 72 blastocysts were treated with fluorescent dyes propidium iodide Pi and Bisbenzimide(Hoechst 33258). Double stain. The total number of cells in blastocyst and the number of cells in inner cell group and trophoblast layer were counted respectively. After fixed and dried, Tunel was used to detect the number of blastocysts. The apoptosis of blastocyst cells induced by ATRA was observed by confocal laser scanning microscope. The fluorescence value was recorded and analyzed. The expression of TGF- 尾 1 protein and Fas protein in mouse blastocysts were detected by immunohistochemical S-P method in different dose groups of ATRA. The average optical density and average positive area rate of TGF- 尾 1 protein and Fas protein in the above three groups were measured by HPIAS-1000 image analysis system. Results (1) fluorescence staining of blastocyst: the trophoblast cell nucleus was pink and the inner cell group ICM nucleus was blue under confocal laser microscope. The total number of cells and the number of trophoblastic cells in experimental group 2 were significantly lower than those in group 1 and control group (P 0.01). The number of cells in the inner cell group was lower than that in the experimental group 1 and the control group (P 0.05%), but there was no significant difference between the experimental group 1 and the control group in the number of trophoblastic cells. Detection of blastocyst cell apoptosis by TUNEL fluorescent labeling: determination of blastocyst cell apoptosis by confocal laser microscopy
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R321

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