本文關(guān)鍵詞：維生素A酸對小鼠囊胚發(fā)育的影響　出處：《武漢大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 全反式維生素A酸 轉(zhuǎn)化生長因子-β1(TGF-β1) Fas 囊胚 小鼠 激光共聚焦顯微鏡

【摘要】：目的:近年來,隨著妊娠早期流產(chǎn)率的不斷增加,有關(guān)著床期和著床前期發(fā)育毒性也越來越受到人們的關(guān)注和探討。有研究發(fā)現(xiàn)著床前期胚胎暴露于某些化學(xué)物,可誘發(fā)胎兒畸形或引起胚胎死亡等發(fā)育毒性,此發(fā)現(xiàn)對胚胎著床前期是實驗致畸不敏感期這一傳統(tǒng)的致畸理論提出質(zhì)疑。而維生素A酸(RA)是近年來研究比較廣泛的一種凋亡誘導(dǎo)劑和致畸因子,用RA處理的發(fā)育中的胚胎可出現(xiàn)面部、神經(jīng)管和四肢等部位的多種畸形。本課題主要研究全反式維生素A酸(ATRA)誘導(dǎo)體外培養(yǎng)小鼠囊胚內(nèi)細胞群(ICM)和滋養(yǎng)層(TE)細胞凋亡,而導(dǎo)致囊胚細胞總數(shù)、內(nèi)細胞群和滋養(yǎng)層細胞數(shù)目的變化,以及由此引起的囊胚細胞中細胞因子TGF-β1蛋白和Fas蛋白表達的改變,并了解這兩種細胞因子對著床前胚胎發(fā)育的作用。從而探討ATRA對著床前期胚胎發(fā)育毒性作用,為其致畸機理提供實驗依據(jù)。 方法:通過促超排卵,獲取妊娠3.5d小鼠囊胚,隨機分成三組,即對照組(空白)、實驗1組(含1μmol/L的ATRA)和實驗2組(含10μmol/L的ATRA),分別培養(yǎng)在含0、1μmol/L和10μmol/L的ATRA的M199培養(yǎng)基中,培養(yǎng)24h,然后再吸出囊胚。72個囊胚用熒光染料propidium iodide(PI)和Bisbenzimide(Hoechst 33258)雙重染色,分別計數(shù)囊胚內(nèi)細胞總數(shù)以及內(nèi)細胞群和滋養(yǎng)層細胞數(shù)。75個囊胚固定干燥后,用帶有熒光(FITC)標記的原位末端標記(TUNEL)檢測法,并通過激光共聚焦掃描顯微鏡觀察ATRA誘導(dǎo)囊胚細胞凋亡效應(yīng),記錄熒光值并分析;其余囊胚用免疫組織化學(xué)S-P法,分別檢測不同劑量組ATRA對小鼠囊胚TGF—β1蛋白和Fas蛋白表達的狀況,利用HPIAS-1000圖像分析系統(tǒng)測定TGF-β1蛋白和Fas蛋白在以上三組中表達的平均光密度和平均陽性面積率。 結(jié)果:(1) 囊胚熒光染色:在激光共聚焦顯微鏡下,滋養(yǎng)層(TE)細胞核呈粉紅色,內(nèi)細胞群(ICM)細胞核呈藍色,實驗2組中細胞總數(shù)和滋養(yǎng)層細胞數(shù)比實驗1組和對照組明顯減少(p0.01);以及內(nèi)細胞群細胞數(shù)較實驗1組和對照組減少(P0.05):其中以滋養(yǎng)層細胞減少為主。但實驗1組與對照組比較無顯著性差異(p0.05)。(2) TUNEL法熒光標記檢測囊胚細胞凋亡:通過激光共聚焦顯微鏡定
[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
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R321

【參考文獻】

相關(guān)期刊論文 前2條

1 雷霆,楊在清;早期胚胎生長因子的基因表達與調(diào)控[J];動物醫(yī)學(xué)進展;1999年02期

2 羅晨玲,王斌,陳清;維生素A、維生素A酸、維生素A酸受體與肺癌[J];疾病控制雜志;2000年03期

，

本文編號：1415767