本文選題：子宮內膜容受性 + 微小RNA�。� 參考：《安徽醫(yī)科大學》2014年碩士論文

【摘要】：胚胎著床是人類生殖的一個關鍵步驟，成功建立子宮內膜容受性，又是胚胎著床的關鍵步驟。子宮內膜容受性（endometrial receptivity，ER）是指子宮內膜允許胚胎黏附、侵入并誘導內膜間質發(fā)生一系列變化，最終植入內膜的一種狀態(tài)，這段時間被稱為“種植窗”期。容受期在雌孕激素的協(xié)同調控下，受細胞因子，粘附分子，生長因子和脂質等諸多分子的調控，其中也包括miRNA。miRNA是一種非編碼RNA，高度保守，長度約18-22核苷酸，不僅可以降解其靶基因，抑制蛋白翻譯，還參與胚胎著床和ER的建立。之前的研究中， mir-30d在容受期顯著上調，且表現出最高的豐度和變化倍數，提示mir-30d在ER的調節(jié)中可能是一個重要的調節(jié)分子。然而mir-30d在子宮內膜中的分布規(guī)律，相關功能和所調控的通路在人類和模式動物中都鮮見報道。本研究通過研究mir-30d在子宮內膜中的分布、功能以及上皮-間質轉化的影響，為進一步認識ER的調控提供一個新的角度，為評估內膜容受狀態(tài)提供依據。 目的 1.檢測mir-30d在子宮內膜上皮和間質細胞中定性和定量的表達。 2.明確mir-30d體外表達水平的改變對子宮內膜上皮細胞胚泡黏附能力的調節(jié)作用。 3.了解mir-30d對子宮內膜上皮間充質轉化的影響。 方法 1、采用原位雜交法，檢測mir-30d在不同周期子宮內膜中分布的情況；利用分離原代子宮內膜上皮和基質細胞，以及Real-time PCR的方法定量分析mir-30d在上皮和基質細胞中的分布。 2.通過轉染mir-30d的抑制物（inhibitor）和模擬物（mimic），在子宮內膜上皮細胞Ishikawa中建立mir-30d的得功能和封閉mir-30d的Ishikawa細胞模型。 3.利用上皮細胞-滋養(yǎng)細胞球（Jarspheroid）的體外著床模型分析mir-30d表達水平改變對胚泡侵入和黏附的影響。 4.通過Western blot和real-time PCR檢測mir-30d表達水平改變對上皮-間質轉化的標志物表達水平的影響，，利用劃痕實驗驗證mir-30d表達水平改變后對內膜上皮細胞遷移能力的影響。 結果 1、原位雜交表明mir-30d主要表達與子宮內膜上皮，Real-time PCR表明mir-30d在上皮的表達量多于基質； 2、轉染72小時后，當濃度達到50nmol/L時，mir-30d抑制物和模擬物可顯著改變Ishikwa中mir-30d的表達；mir-30d Mimic不但可以促進胚泡的粘附，還能夠抑制胚泡的侵入； 3、mir-30d抑制物和模擬物，可以顯著調節(jié)子宮內膜上皮細胞上皮-間質轉化（Epitheliai-mesenchymal transition，EMT）的相關基因，同時也具備調節(jié)子宮內膜上皮細胞遷移的能力。 結論 mir-30d作為一個調控因子，改變容受期的子宮內膜上皮細胞的形態(tài)和功能，并參與上皮-間質轉化，在子宮內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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.8

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相關期刊論文 前6條

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本文編號：1997500