mir-30d对人子宫内膜上皮细胞功能和上皮—间质—转化的影响

发布时间：2018-06-08 22:43

本文选题：子宫内膜容受性 + 微小RNA　；参考：《安徽医科大学》2014年硕士论文

【摘要】：胚胎着床是人类生殖的一个关键步骤，成功建立子宫内膜容受性，又是胚胎着床的关键步骤。子宫内膜容受性（endometrial receptivity，ER）是指子宫内膜允许胚胎黏附、侵入并诱导内膜间质发生一系列变化，最终植入内膜的一种状态，这段时间被称为“种植窗”期。容受期在雌孕激素的协同调控下，受细胞因子，粘附分子，生长因子和脂质等诸多分子的调控，其中也包括miRNA。miRNA是一种非编码RNA，高度保守，长度约18-22核苷酸，不仅可以降解其靶基因，抑制蛋白翻译，还参与胚胎着床和ER的建立。之前的研究中， mir-30d在容受期显著上调，且表现出最高的丰度和变化倍数，提示mir-30d在ER的调节中可能是一个重要的调节分子。然而mir-30d在子宫内膜中的分布规律，相关功能和所调控的通路在人类和模式动物中都鲜见报道。本研究通过研究mir-30d在子宫内膜中的分布、功能以及上皮-间质转化的影响，为进一步认识ER的调控提供一个新的角度，为评估内膜容受状态提供依据。目的 1.检测mir-30d在子宫内膜上皮和间质细胞中定性和定量的表达。 2.明确mir-30d体外表达水平的改变对子宫内膜上皮细胞胚泡黏附能力的调节作用。 3.了解mir-30d对子宫内膜上皮间充质转化的影响。方法 1、采用原位杂交法，检测mir-30d在不同周期子宫内膜中分布的情况；利用分离原代子宫内膜上皮和基质细胞，以及Real-time PCR的方法定量分析mir-30d在上皮和基质细胞中的分布。 2.通过转染mir-30d的抑制物（inhibitor）和模拟物（mimic），在子宫内膜上皮细胞Ishikawa中建立mir-30d的得功能和封闭mir-30d的Ishikawa细胞模型。 3.利用上皮细胞-滋养细胞球（Jarspheroid）的体外着床模型分析mir-30d表达水平改变对胚泡侵入和黏附的影响。 4.通过Western blot和real-time PCR检测mir-30d表达水平改变对上皮-间质转化的标志物表达水平的影响，，利用划痕实验验证mir-30d表达水平改变后对内膜上皮细胞迁移能力的影响。结果 1、原位杂交表明mir-30d主要表达与子宫内膜上皮，Real-time PCR表明mir-30d在上皮的表达量多于基质； 2、转染72小时后，当浓度达到50nmol/L时，mir-30d抑制物和模拟物可显著改变Ishikwa中mir-30d的表达；mir-30d Mimic不但可以促进胚泡的粘附，还能够抑制胚泡的侵入； 3、mir-30d抑制物和模拟物，可以显著调节子宫内膜上皮细胞上皮-间质转化（Epitheliai-mesenchymal transition，EMT）的相关基因，同时也具备调节子宫内膜上皮细胞迁移的能力。结论 mir-30d作为一个调控因子，改变容受期的子宫内膜上皮细胞的形态和功能，并参与上皮-间质转化，在子宫内膜容受期的建立中扮演重要的角色。
[Abstract]:Embryo implantation is a key step in human reproduction. Endometrial receptivity is a state in which the endometrium allows the embryo to adhere, invade and induce a series of changes in the stroma of the endometrium, which is called the "implant window" stage. The receptive period is regulated by cytokines, adhesion molecules, growth factors and lipids under the co-regulation of estrogen and progesterone, including miRNA.miRNA is a non-coding RNAs, highly conserved, and is about 18-22 nucleotides in length. It not only degrades its target gene, inhibits protein translation, but also participates in embryo implantation and ER. In previous studies, mir-30d was significantly up-regulated during the receptive period, showing the highest abundance and multiple changes, suggesting that mir-30d may be an important regulatory molecule in ER regulation. However, the distribution of mir-30d in endometrium, related functions and regulated pathways are rarely reported in humans and model animals. By studying the distribution and function of mir-30d in endometrium and the effect of epithelial-interstitial transformation, this study provides a new perspective for further understanding the regulation of ER and provides a basis for evaluating the state of endometrial receptivity. Objective 1. To detect the qualitative and quantitative expression of mir-30d in endometrial epithelium and stromal cells. 2. To investigate the effect of the change of mir-30d expression on the blastocyst adhesion of endometrial epithelial cells in vitro. 3. 3. To understand the effect of mir-30d on the mesenchymal transformation of endometrial epithelium. Methods 1. The distribution of mir-30d in different cycles of endometrium was detected by in situ hybridization, and the primary endometrial epithelium and stromal cells were isolated. Real-time PCR was used to analyze the distribution of mir-30d in epithelial and stromal cells. The function of mir-30d and the Ishikawa cell model of blocking mir-30d in endometrial epithelial cells were established by transfection of mir-30d inhibitor and mimicus. 3. The effects of mir-30d expression on blastocyst invasion and adhesion were analyzed by the implantation model of Jarspheroid. epithelial-trophoblast in vitro. 4. Western blot and real-time PCR were used to detect the effect of mir-30d expression on the expression of markers in epithelial-mesenchymal transformation. Results 1. In situ hybridization showed that the expression of mir-30d was mainly related to the expression of mir-30d in endometrial epithelium and real-time PCR showed that the expression of mir-30d was higher than that of matrix, 2, 72 hours after transfection, the expression of mir-30d was higher than that of matrix. When the concentration reached 50nmol / L, the inhibitor and mimic could significantly change the expression of mir-30d in Ishikwa. Mimic could not only promote the adhesion of blastocyst, but also inhibit the invasion of blastocyst, 3mir-30d inhibitor and mimic, and the expression of Mimic in Ishikwa could not only promote the adhesion of blastocyst, but also inhibit the invasion of blastocyst. Epitheliai-mesenchymal transition (EMT) related genes can be significantly regulated in endometrial epithelial cells. Conclusion mir-30d is a regulatory factor for the migration of endometrial epithelial cells. To change the morphology and function of endometrial epithelial cells and participate in epithelial-interstitial transformation, which plays an important role in the establishment of endometrial receptive phase.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

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