【摘要】：胚胎植入是哺乳動(dòng)物妊娠建立過程的關(guān)鍵環(huán)節(jié)之一。在人中有大約75%的早期妊娠失敗是由胚胎植入的異常所造成的。而處于接受態(tài)的子宮和有植入能力的囊胚是植入的必要前提。子宮接受態(tài)的建立是在卵巢雌孕激素及其核受體ER和PR的主導(dǎo)下,通過下游的轉(zhuǎn)錄因子、生長(zhǎng)因子等的共同作用,引導(dǎo)子宮細(xì)胞進(jìn)行有序的增殖與分化,獲得對(duì)胚胎有容受能力的過程；因此雌孕激素及其受體的功能調(diào)控對(duì)子宮接受態(tài)的建立有著至關(guān)重要的作用。但到目前為止,關(guān)于雌孕激素及其受體在子宮及胚胎植入過程中作用的分子機(jī)制還有諸多未知的問題。 本研究中,我們發(fā)現(xiàn)一個(gè)機(jī)體中廣泛表達(dá)的胞質(zhì)蛋白酪氨酸磷酸酶Shp2,在圍植入期子宮中呈現(xiàn)時(shí)空特異性的動(dòng)態(tài)表達(dá)模式,其蛋白主要定位于子宮組織的細(xì)胞核中。利用PR-cre工具鼠,我們建立了子宮特異的Shp2敲除小鼠模型,發(fā)現(xiàn)子宮中的Shp2缺失導(dǎo)致胚胎植入失敗,雌性小鼠不育。進(jìn)一步的分析表明,Shp2的敲除導(dǎo)致子宮基質(zhì)細(xì)胞增殖減弱,上皮細(xì)胞增殖增加以及孕酮響應(yīng)基因表達(dá)下降等一系列孕激素信號(hào)響應(yīng)不足的現(xiàn)象。深入的研究表明,Shp2的敲除影響了雌激素受體的活性,導(dǎo)致基質(zhì)細(xì)胞中雌激素的靶基因PR表達(dá)受到抑制,子宮對(duì)雌孕激素的響應(yīng)異常。而生化實(shí)驗(yàn)分析發(fā)現(xiàn)Shp2通過和ERα的直接相互作用調(diào)節(jié)ERα的轉(zhuǎn)錄活性,而且這種作用并不依賴于ERK信號(hào)通路的激活。我們利用CRISPR/Cas9技術(shù)建立了Shp2敲除的子宮內(nèi)膜細(xì)胞系,并結(jié)合不同突變類型的Shp2表達(dá)載體證明細(xì)胞核中的Shp2,而非細(xì)胞質(zhì)中的Shp2,通過N端的兩個(gè)SH2結(jié)構(gòu)域與ERα的相互作用促進(jìn)ERα的轉(zhuǎn)錄活性。最終我們發(fā)現(xiàn)Shp2和ERa的相互作用促進(jìn)ERα與靶基因PR啟動(dòng)子區(qū)域的結(jié)合及后續(xù)轉(zhuǎn)錄激活因子的募集過程,對(duì)調(diào)控子宮ERa的轉(zhuǎn)錄活性起著重要作用。 本研究揭示了Shp2在胚胎植入過程中的重要作用以及ERa轉(zhuǎn)錄激活過程的新機(jī)制。這一發(fā)現(xiàn)將有助于更好地理解妊娠建立的調(diào)控過程,并對(duì)妊娠建立過程相關(guān)疾病有很好的借鑒意義,也為子宮中雌激素受體功能異常相關(guān)的疾病的診斷和治療提供了理論基礎(chǔ)。另外,這是首次揭示了Shp2核定位的生理功能,為Shp2的分子功能研究提供了更多的參考。
[Abstract]:Embryo implantation is one of the key steps in the establishment of mammalian pregnancy. About 75% of early pregnancy failures in humans are caused by abnormal embryo implantation. The receiving uterus and blastocyst with implantation ability are the necessary prerequisite for implantation. The establishment of uterine receptive state is a leading role of estrogen and progesterone, its nuclear receptors ER and PR, and induces the orderly proliferation and differentiation of uterine cells through the interaction of downstream transcription factors and growth factors. The process of obtaining receptivity to an embryo; Therefore, the regulation of estrogen and progesterone receptor function plays an important role in the establishment of uterine receptive state. However, there are still many unknown questions about the molecular mechanism of estrogen and progesterone receptor in uterus and embryo implantation. In this study, we found that a widely expressed cytosolic protein tyrosine phosphatase (Shp2,) showed a time-space specific dynamic expression pattern in the peri-implantation uterus, and its protein was mainly located in the nucleus of uterine tissue. Using PR-cre tool mice, we established a womb specific Shp2 knockout mouse model. It was found that the absence of Shp2 in the uterus led to the failure of embryo implantation and the infertility of female mice. Further analysis showed that the knockout of Shp2 resulted in a series of insufficient progesterone signal responses, such as decreased proliferation of uterine stromal cells, increased proliferation of epithelial cells and decreased expression of progesterone responsive genes. Further studies have shown that the knockout of Shp2 affects the activity of estrogen receptor, resulting in the inhibition of estrogen target gene PR expression in stromal cells, and the abnormal response of uterus to estrogen and progesterone. Biochemical analysis showed that Shp2 regulated the transcriptional activity of ER 偽 through direct interaction with ER 偽, and this effect did not depend on the activation of ERK signaling pathway. We established endometrial cell lines with Shp2 knockout by using CRISPR/Cas9 technique, and combined with Shp2 expression vectors of different mutation types to prove Shp2, in the nucleus rather than Shp2, in the cytoplasm. The transcriptional activity of ER 偽 was promoted by the interaction of two N-terminal SH2 domains with ER 偽. Finally, we found that the interaction between Shp2 and ERa promotes the binding of ER 偽 to the PR promoter region of the target gene and the recruitment of subsequent transcriptional activators, which plays an important role in regulating the transcriptional activity of uterine ERa. This study revealed the important role of Shp2 in embryo implantation and the new mechanism of ERa transcriptional activation. The findings will help to better understand the regulation process of pregnancy establishment, and have a good reference value for diseases related to pregnancy establishment, and also provide a theoretical basis for the diagnosis and treatment of diseases related to abnormal estrogen receptor function in the uterus. In addition, this is the first time to reveal the physiological function of Shp2 nuclear localization, which provides more reference for the study of Shp2 molecular function.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R714.462

【共引文獻(xiàn)】

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