蛋白酪氨酸磷酸酶Shp2在小鼠胚胎植入中的生理作用及机制

发布时间：2018-12-21 10:03

【摘要】：胚胎植入是哺乳动物妊娠建立过程的关键环节之一。在人中有大约75%的早期妊娠失败是由胚胎植入的异常所造成的。而处于接受态的子宫和有植入能力的囊胚是植入的必要前提。子宫接受态的建立是在卵巢雌孕激素及其核受体ER和PR的主导下,通过下游的转录因子、生长因子等的共同作用,引导子宫细胞进行有序的增殖与分化,获得对胚胎有容受能力的过程；因此雌孕激素及其受体的功能调控对子宫接受态的建立有着至关重要的作用。但到目前为止,关于雌孕激素及其受体在子宫及胚胎植入过程中作用的分子机制还有诸多未知的问题。本研究中,我们发现一个机体中广泛表达的胞质蛋白酪氨酸磷酸酶Shp2,在围植入期子宫中呈现时空特异性的动态表达模式,其蛋白主要定位于子宫组织的细胞核中。利用PR-cre工具鼠,我们建立了子宫特异的Shp2敲除小鼠模型,发现子宫中的Shp2缺失导致胚胎植入失败,雌性小鼠不育。进一步的分析表明,Shp2的敲除导致子宫基质细胞增殖减弱,上皮细胞增殖增加以及孕酮响应基因表达下降等一系列孕激素信号响应不足的现象。深入的研究表明,Shp2的敲除影响了雌激素受体的活性,导致基质细胞中雌激素的靶基因PR表达受到抑制,子宫对雌孕激素的响应异常。而生化实验分析发现Shp2通过和ERα的直接相互作用调节ERα的转录活性,而且这种作用并不依赖于ERK信号通路的激活。我们利用CRISPR/Cas9技术建立了Shp2敲除的子宫内膜细胞系,并结合不同突变类型的Shp2表达载体证明细胞核中的Shp2,而非细胞质中的Shp2,通过N端的两个SH2结构域与ERα的相互作用促进ERα的转录活性。最终我们发现Shp2和ERa的相互作用促进ERα与靶基因PR启动子区域的结合及后续转录激活因子的募集过程,对调控子宫ERa的转录活性起着重要作用。本研究揭示了Shp2在胚胎植入过程中的重要作用以及ERa转录激活过程的新机制。这一发现将有助于更好地理解妊娠建立的调控过程,并对妊娠建立过程相关疾病有很好的借鉴意义,也为子宫中雌激素受体功能异常相关的疾病的诊断和治疗提供了理论基础。另外,这是首次揭示了Shp2核定位的生理功能,为Shp2的分子功能研究提供了更多的参考。
[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 伪 through direct interaction with ER 伪, 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 伪 was promoted by the interaction of two N-terminal SH2 domains with ER 伪. Finally, we found that the interaction between Shp2 and ERa promotes the binding of ER 伪 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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714.462

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