【摘要】：山羊?qū)儆诜雌c動(dòng)物,著床方式為表面著床,胚胎與子宮肉阜上皮細(xì)胞相融合、增生,最后形成的胎盤為子葉狀胎盤。胚胎著床是成功建立妊娠的關(guān)鍵環(huán)節(jié),涉及到胚胎與母體子宮之間復(fù)雜的相互作用。整合素作為細(xì)胞黏附分子家族成員,是一種細(xì)胞表面結(jié)合蛋白,主要介導(dǎo)粘附和行使信號(hào)傳遞功能,在胚胎著床過程發(fā)揮重要作用。我們通過轉(zhuǎn)錄組測(cè)序發(fā)現(xiàn):整合素ITGAV(integrinαv,ITGAV)、ITGB3(integrinβ3,ITGB3)和ITGA5(integrinα5,ITGA5)mRNA在山羊圍著床期(D6、D16和D19)子宮中表達(dá)變化特異。因此本文詳細(xì)研究整合素亞基ITGB1(integrinβ1,ITGB1)、ITGAV、ITGB3、ITGA5蛋白在山羊圍著床子宮中的表達(dá)和激素調(diào)節(jié),為深入研究整合素在反芻動(dòng)物胚胎著床中的作用提供實(shí)驗(yàn)依據(jù)。本實(shí)驗(yàn)的目的是檢測(cè)整合素亞基ITGB1、ITGAV、ITGB3和ITGA5在山羊發(fā)情周期、早期妊娠和激素處理子宮中的表達(dá),來確定這些整合素亞基是否在山羊胚胎著床中起作用,是否受類固醇激素調(diào)控。本實(shí)驗(yàn)材料為黑龍江本地山羊,采用Western blot方法檢測(cè)ITGB1、ITGAV、ITGB3和ITGA5在發(fā)情周期、早期妊娠和激素處理山羊子宮中的表達(dá)和類固醇激素調(diào)控;通過免疫組織化學(xué)技術(shù)研究ITGB3和ITGA5蛋白在山羊著床階段子宮中的定位。本實(shí)驗(yàn)所得結(jié)果如下:(1)在發(fā)情周期中,Western blot檢測(cè)到ITGB1、ITGAV、ITGB3和ITGA5在山羊整個(gè)發(fā)情周期的子宮中均表達(dá),ED0表達(dá)較高,排卵后(ED6、ED12)表達(dá)降低,ED16表達(dá)升高。(2)在山羊早期妊娠過程中,Western blot結(jié)果顯示ITGB1在山羊胚胎粘附和胎盤形成早期(D16-D30)高表達(dá),ITGAV、ITGB3和ITGA5在妊娠期表達(dá)基本一致,均在山羊胚胎粘附期(D16)高表達(dá);免疫組織化學(xué)結(jié)果顯示ITGB3在胚胎粘附期(D16、D19)子宮腔上皮和基質(zhì)中有表達(dá),ITGA5在子宮腔上皮、腺上皮和基質(zhì)中有表達(dá)。(3)在類固醇激素處理模型中,Western blot顯示孕酮上調(diào)ITGB1的表達(dá),雌激素?zé)o明顯影響;雌激素上調(diào)ITGAV、ITGB3和ITGA5的表達(dá),孕酮無明顯影響。本實(shí)驗(yàn)結(jié)果表明ITGB1的表達(dá)在山羊子宮中呈現(xiàn)胚胎著床特異性,在胚胎著床和胎盤形成早期,ITGB1蛋白一直處于高水平。推測(cè)ITGB1參與山羊胚胎著床和胎盤形成的過程。孕酮上調(diào)ITGB1的表達(dá),雌激素?zé)o明顯影響。ITGAV、ITGB3和ITGA5在山羊胚胎粘附期(D16)處于高水平,并且ITGB3表達(dá)于著床期子宮腔上皮和基質(zhì)中,ITGA5表達(dá)于著床期子宮腔上皮、腺上皮和基質(zhì)中,推測(cè)ITGAV、ITGB3和ITGA5在山羊胚胎粘附過程發(fā)揮著重要作用。雌激素上調(diào)ITGAV、ITGB3和ITGA5的表達(dá),孕酮無明顯影響。山羊胚胎著床過程中,不同的整合素表達(dá)方式不同,類固醇調(diào)控機(jī)制不同,可能發(fā)揮不同作用。本實(shí)驗(yàn)為研究整合素在反芻動(dòng)物胚胎著床和胎盤形成中作用機(jī)制提供了依據(jù)。
[Abstract]:Goat belongs to ruminant, the implantation mode is surface implantation, embryo and uterine caruncle epithelial cells merge and proliferate, the last placenta is cotyledon placenta. Embryo implantation is the key to the successful establishment of pregnancy, involving the complex interaction between embryo and womb. Integrin, as a member of cell adhesion molecule family, is a cell surface binding protein, which mediates adhesion and signal transduction, and plays an important role in embryo implantation. By transcriptome sequencing, we found that the expression of ITGB3 (integrin 尾 3ITGB3) and ITGA5 (integrin 偽 5ITGA5) mRNA were specific in the uterus of goat (D6D16 and D19). Therefore, the expression and hormone regulation of integrin subunit ITGB1 (integrin 尾 1 ITGB1) ITGAVG3ITGA5 protein in goat surrounding bed uterus were studied in detail, which provided experimental basis for further study on the role of integrin in implantation of ruminant embryos. The purpose of this study was to detect the expression of ITGB3 and ITGA5 in goat estrous cycle, early pregnancy and hormone treated uterus to determine whether these integrin subunits play a role in goat embryo implantation and whether they are regulated by steroid hormones. In this study, the expression of ITGB3 and ITGA5 in estrous cycle, early pregnancy and hormone treated goat uterus were detected by Western blot method. The localization of ITGB3 and ITGA5 proteins in goat uterus during implantation was studied by immunohistochemical technique. The results were as follows: (1) in estrous cycle, ITGB1, ITGB1, ITGB3 and ITGA5 were detected to be highly expressed in the uterus of goat estrous cycle. After ovulation (ED6 + ED12), the expression of ED16 was decreased. (2) during early pregnancy, the expression of ITGB1 in goat embryo adhesion and placental formation (D16-D30) was highly expressed. The expression of ITGAV TGB3 and ITGA5 was basically the same during pregnancy, and both expressed in goat embryo adhesion phase (D16). Immunohistochemical results showed that ITGB3 was expressed in uterine cavity epithelium and matrix during embryonic adhesion phase (D16 / D19). (3) in steroid treated model, the expression of ITGB1 was up-regulated by progesterone by Western blot. Estrogen did not affect the expression of ITGB3 and ITGA5, but progesterone did not. The results showed that the expression of ITGB1 was specific to embryo implantation in goat uterus, and the expression of ITGB1 protein was always at a high level in the early stage of embryo implantation and placenta formation. It is speculated that ITGB1 is involved in the process of implantation and placenta formation of goat embryos. Progesterone upregulated the expression of ITGB1, estradiol had no significant effect on the expression of ITGB3 and ITGA5 in goat embryo adhesion phase (D16), and ITGB3 was expressed in uterine cavity epithelium and matrix in implantation stage, glandular epithelium and matrix. It is inferred that ITGAV ITGB3 and ITGA5 play an important role in the process of goat embryo adhesion. Estrogen upregulated the expression of ITGB3 and ITGA5, but progesterone had no effect. Different integrin expression patterns and steroid regulation mechanisms may play different roles in goat embryo implantation. This study provides a basis for the study of the role of integrin in embryo implantation and placental formation in ruminants.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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