卵巢激素作用下极性蛋白PAR3介导beta1整合素调节胚胎着床的机制研究

During the"implantation window",the endometrial luminal epithelial cells transform from "polarity" status to "non-polarity" status with the reconstruction of tight junction ,and possess the most receptivity to the embryos. However, the reports about the role of polarity proteins in regulation of embryo implantation have not be seen till now. Par3 is the key polarity protein in the establishment of the epithelial cell polarity and the function of tight junction. β1 integrin is temporally and spatially expressed during the "implantation window", and is proved to be the upstream factor of Par3 in regulation of the epithelial cell polarity. Based on the references and results from our preliminary study, we hypothesis that: under the influence of ovarian steriod hormones, β1 integrin serves as the upstream factor of Par3 to regulate embryo implantation via function in the transformation of endometrial luminal epithelial cell polarity and reconstruction of tight junction. Our study plan to adopt the clinical cases、cell culture experiments and animal models, to apply the "gain of function"、"lose of function" and "function rescue"strategies, and to use in situ hybridization、immunohistochemistry、RT-PCR、western-blot、cell-staining、calcium-switch and transepithelial resistence assay(TER)、transwell assay、JARs spheriods assay 、injection of overexpression-plasmids and antibodys into mouse uterine horns、and other molecular/cell biology methods to explore the mechanism of Par3-mediated β1-integrin cell polarity signaling pathway in regulation of embryo implantation under the influence of ovarian steriod hormones. To clarify the mechanism of polarity proteins in regulation of embryo implantation has great significance in the further study of the role of polarity protein networks in regualtion of embryo implantation and in the treatment of infertile and sterile diseases.

围着床期时，子宫内膜腔上皮由"极化"向"非极化"转变、紧密连接（TJ）重构，表现出对胚胎最大的容受性。然而极性蛋白信号通路调控胚胎着床的机制研究未见报道。Par3是调节细胞极性和TJ功能的核心蛋白；β1整合素是围着床期内膜特异性时空表达的蛋白，并在上皮细胞中作为Par3上游调节细胞极性。结合文献及前期结果，我们提出假设：类固醇激素作用下Par3介导β1整合素，通过调节腔上皮细胞极性和TJ功能，调控胚胎着床。本课题从临床调查、细胞实验、动物实验三个层面；运用"gain and lose of function"、 "function rescue"策略；采用原位杂交、免疫组化、RT-PCR、免疫印迹、免疫荧光、TER、Transwell、胚胎黏附、宫角注射等方法，验证假设阐明极性蛋白调控胚胎着床的机制，为进一步研究极性蛋白网络调控胚胎着床的机制奠定基础。

胚胎着床是子宫内膜接受胚泡侵入，建立妊娠的关键环节。胚胎着床时，子宫内膜腔上皮在胚胎着床时由“极化状态”向“非极化状态”改变，细胞间紧密连接（TJ）重构，细胞表面某些粘附分子表达改变，表现出对胚泡最大的容受性。鉴于极性蛋白 Par3 是参与细胞极性建立和TJ维持的关键蛋白，a2β1 整合素是腔上皮胚胎着床时特异性时空表达的粘附分子，本研究通过RT-PCR、免疫印迹、免疫荧光、TER、transwell、JARs等方法等手段发现： 1. 极性蛋白Pae3在人月经周期的时空表达；2. 极性蛋白PAR3在自然流产/人工流产蜕膜中的表达差异；3. 极性蛋白PAR3在小鼠子宫的时空表达；4. 子宫上皮细胞中极性蛋白与β1整合素的相互作用；5. 极性蛋白PAR3对子宫内膜上皮极性调节作用；6. 小鼠腔上皮极性蛋白PAR3与β1整合素的表达。上述结果证实了我们提出的“a2β1 整合素作为 Par3 上游信号，调控细胞极性和 TJ 功能，从而调节子宫内膜容受性”假说，丰富了子宫内膜容受性机制探索，对生殖医学乃至生命科学都的发展都提供了十分重要的理论和实践意义。