本文關(guān)鍵詞：人子宮內(nèi)膜窗口期基因表達(dá)平臺(tái)的建立及特異基因的篩選　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 子宮 內(nèi)膜 窗口 基因 表達(dá) 平臺(tái) 建立 特異 篩選

【摘要】： 胚胎著床是哺乳動(dòng)物獨(dú)有的涉及胚胎和母體子宮內(nèi)膜相互作用的生殖過(guò)程。此過(guò)程在時(shí)間和空間上都是高度有序的。哺乳動(dòng)物的子宮內(nèi)膜只有在著床窗開(kāi)放時(shí),才能接受胚泡,最終完成著床。人類的子宮內(nèi)膜僅在月經(jīng)周期很短的一段時(shí)期可以接受胚胎著床。大量研究證實(shí)這一時(shí)期主要受激素調(diào)控,接受態(tài)子宮內(nèi)膜的形成是這一時(shí)期的一個(gè)重要事件。為了解著床窗口期事件包括接受態(tài)子宮內(nèi)膜形成及胚胎著床等過(guò)程中的分子機(jī)制的全貌并深入研究著床窗口期的分子調(diào)控機(jī)制,本研究采用抑制消減雜交技術(shù),將人著床窗口期和早泌期子宮內(nèi)膜組織cDNA互相消減,構(gòu)建了這兩個(gè)時(shí)期的雙向消減cDNA文庫(kù),并對(duì)篩選出來(lái)的部分已知和未知基因進(jìn)行了深入探索。 主要結(jié)果如下: 1.為了構(gòu)建人子宮內(nèi)膜窗口期基因表達(dá)平臺(tái),我們從最初的實(shí)驗(yàn)材料選擇就嚴(yán)格把關(guān)。我們以同一位志愿者的不同時(shí)相的子宮內(nèi)膜作為自身對(duì)照,結(jié)合掃描電鏡觀察結(jié)果、B超檢測(cè)結(jié)果、HE染色結(jié)果和PR免疫組化染色結(jié)果從多方面驗(yàn)證了材料的可靠性,為后續(xù)的分子生物學(xué)實(shí)驗(yàn)打下堅(jiān)實(shí)的基礎(chǔ)。 2.SMART~(TM) PCR cDNA Synthesis技術(shù)和抑制性消減雜交技術(shù)結(jié)合起來(lái),構(gòu)建了人窗口期子宮內(nèi)膜和早泌期子宮內(nèi)膜雙向cDNA抑制消減文庫(kù)。 3.在人窗口期子宮內(nèi)膜cDNA文庫(kù)(HWE)和人早泌期子宮內(nèi)膜cDNA文庫(kù)(HEW)911個(gè)PCR純化產(chǎn)物進(jìn)行斑點(diǎn)雜交分析,有354個(gè)在人窗口期和早泌期子宮內(nèi)膜存在4倍以上表達(dá)差異,其中192個(gè)在窗口期差異表達(dá),162個(gè)在早泌期差異表達(dá)。對(duì)斑點(diǎn)雜交篩選出的354個(gè)克隆中的130個(gè)克隆進(jìn)行測(cè)序,共產(chǎn)生了93個(gè)質(zhì)量滿意的ESTs,總測(cè)序成功率達(dá)到71.54%。 4.通過(guò)在互聯(lián)網(wǎng)上的比對(duì),在HWE文庫(kù)中已知基因、已知ESTs和全新ESTs分別為52.46%、40.98%和6.56%:在HEW文庫(kù)中分別為48.57%、48.57%和2.86%。 5.對(duì)代表已知基因的ESTs按照功能分類,發(fā)現(xiàn)主要涉及細(xì)胞分裂/細(xì)胞凋亡、蛋白合成和分解、物質(zhì)轉(zhuǎn)運(yùn)、分子伴侶、轉(zhuǎn)錄調(diào)控因子、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞骨架、粘附分子幾類,并初步探討了人子宮內(nèi)膜圍著床過(guò)程中差異表達(dá)基因的作用和意義。 6.通過(guò)序列比對(duì)發(fā)現(xiàn)在HWE文庫(kù)中編號(hào)為82G7的克隆序列與已知基因NID-1同源性很高,用原位雜交技術(shù)檢測(cè)在窗口期和早泌期人子宮內(nèi)膜的表達(dá),結(jié)果顯示NID-1在窗口期子宮內(nèi)膜的基質(zhì)細(xì)胞中高表達(dá),在早泌期的子宮內(nèi)膜組織中表達(dá)很弱。根據(jù)文獻(xiàn)推測(cè),NID-1基因的表達(dá)產(chǎn)物的作用可能在人類的胚胎發(fā)生、著床和蛻膜化反應(yīng)中發(fā)揮重要作用。 7.將人子宮內(nèi)膜窗口期特異表達(dá)的克隆編號(hào)為82F4的EST序列進(jìn)行RACE擴(kuò)增獲得一個(gè)新的cDNA序列,命名為PPI-82-F4,登錄GenBank,獲得登錄號(hào)為EF063108。 8.應(yīng)用Northern雜交技術(shù)檢測(cè)PPI-82-F4在窗口期和早泌期人子宮內(nèi)膜的表達(dá),結(jié)果顯示PPI-82-F4在窗口期子宮內(nèi)膜的表達(dá)顯著高于早泌期。應(yīng)用原位雜交技術(shù)檢測(cè)PPI-82-F4在窗口期和早泌期人子宮內(nèi)膜的表達(dá),結(jié)果顯示PPI-82-F4在窗口期子宮內(nèi)膜的基質(zhì)細(xì)胞和腺上皮細(xì)胞中高表達(dá),在早泌期的子宮內(nèi)膜組織中表達(dá)很弱。 綜上所述,我們采用抑制性消減雜交構(gòu)建了雙向人子宮內(nèi)膜窗口期消減早泌期cDNA文庫(kù),在這兩個(gè)文庫(kù)中各有192和162個(gè)陽(yáng)性差異的克隆,我們所得到的基因包括轉(zhuǎn)錄調(diào)控因子、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞骨架幾類。母體子宮內(nèi)膜從排卵到著床的過(guò)程中發(fā)生劇烈的變化,在孕激素的調(diào)控下,為了使著床窗開(kāi)放做了充分準(zhǔn)備,有很多相關(guān)基因表達(dá)。我們發(fā)現(xiàn)了一些基因的表達(dá)上調(diào),同時(shí)還有一些基因表達(dá)降低或受到抑制,為我們發(fā)現(xiàn)潛在的新基因提供可能性。
[Abstract]:Embryo implantation is exclusive to mammals to the reproductive process of embryo and maternal endometrium interactions. This process is highly ordered in time and space. Only in the mammalian endometrial implantation window open, to accept the final completion of the blastocyst implantation. A period of human endometrium during the menstrual cycle is very short. Can accept embryo implantation. Many studies have confirmed that this period is mainly affected by hormone regulation, the formation of the receptive endometrium is one of the most important events in this period. In order to understand the molecular mechanism of implantation window events include the receptive endometrium formation and embryo implantation in the process of the whole and in-depth study of the molecular regulation of implantation window mechanism of suppression subtractive hybridization technique used in this study, the endometrial tissue cDNA implantation window period and early stage secretion cancel each other, constructed the two periods The cDNA library was subtracted bi-directional, and some known and unknown genes were explored in depth.
The main results are as follows:
1. in order to construct the expression gene in human endometrium during the implantation window, we from the initial materials selection is strict. We use the same volunteers at different phases of endometrial as control group, observed with scanning electron microscope, ultrasonic test results, the results of HE staining and PR immunohistochemical staining results from many aspects of verification the reliability of the material, the solid foundation for the subsequent molecular biology experiment lay.
2.SMART~ (TM) PCR cDNA Synthesis technology and suppression subtractive hybridization technology were used to construct a bidirectional cDNA suppression subtractive library in endometrial and early secretory endometrium of human window stage.
3. people in the window period of endometrial cDNA Library (HWE) and early stage endometrial secretion of cDNA Library (HEW) 911 PCR purified products were dot blot analysis, there are 354 people in the window period and early stage endometrial secretion are more than 4 times the difference expression of 192 differentially expressed in the window period, 162 expression in the early period. The difference of 130 clones secreting 354 clones of dot blot hybridization were sequenced, produced a total of 93 ESTs with good quality, the total success rate of 71.54%. sequencing
4., based on the comparison on the Internet, the known genes in the HWE library, known ESTs and new ESTs are 52.46%, 40.98% and 6.56% respectively: in HEW library, they are 48.57%, 48.57% and 2.86%..
5. on behalf of ESTs according to the known gene function classification, found mainly related to cell division / apoptosis, protein synthesis and degradation, material transport, molecular chaperone, transcription factor, signal transduction, cell skeleton, adhesion molecules of several kinds, and discusses the function and significance of human endometrial peri implantation process of gene expression differences.
6. by sequence alignment found in the HWE library and cloning sequence numbers for known genes NID-1 82G7 have high homology with in situ hybridization, the expression in human endometrium during the implantation window and early urinary period, results showed that the high expression of NID-1 in endometrial stromal cells in the window period, in the early stage of endometrial tissue secretion the expression is very weak. It suggested that the expression product of NID-1 gene function may occur in human embryos, play an important role in implantation and decidualization.
7., the human endometrium window period specific expression clone is numbered as 82F4 EST sequence, and RACE is amplified. A new cDNA sequence is obtained, named PPI-82-F4, login GenBank, and get the accession number EF063108..
8. application of Northern hybridization technique to detect the expression of PPI-82-F4 in human endometrium during the implantation window and early urinary period, the results showed that PPI-82-F4 expression in the window period of endometrial was significantly higher than that of early urinary period. In situ hybridization was used to detect PPI-82-F4 in human endometrium during the implantation window period and early urinary expression showed higher expression of PPI-82-F4 in stromal cells and the glandular epithelial cells of endometrium during the window period, in the early stage of endometrial tissue secretion expression is very weak.
In summary, we used to build a two-way inhibition in human endometrium during the implantation window period of urinary cDNA library early subtractive subtractive hybridization, in which two clones in each library 192 and 162 positive difference, we obtain the gene transcription, signal transduction, cell skeleton types. The endometrium from ovulation the dramatic changes occurred in the process of implantation, the progesterone regulation, in order to make the implantation window open made full preparations, many genes were up-regulated. We found some genes expression, and gene expression was reduced or inhibited, potentially new genes provide the possibility for our findings.

【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

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本文編號(hào)：1390779