人子宫内膜窗口期基因表达平台的建立及特异基因的筛选

发布时间：2018-01-07 03:23

本文关键词：人子宫内膜窗口期基因表达平台的建立及特异基因的筛选　出处：《中国协和医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 胚胎着床是哺乳动物独有的涉及胚胎和母体子宫内膜相互作用的生殖过程。此过程在时间和空间上都是高度有序的。哺乳动物的子宫内膜只有在着床窗开放时,才能接受胚泡,最终完成着床。人类的子宫内膜仅在月经周期很短的一段时期可以接受胚胎着床。大量研究证实这一时期主要受激素调控,接受态子宫内膜的形成是这一时期的一个重要事件。为了解着床窗口期事件包括接受态子宫内膜形成及胚胎着床等过程中的分子机制的全貌并深入研究着床窗口期的分子调控机制,本研究采用抑制消减杂交技术,将人着床窗口期和早泌期子宫内膜组织cDNA互相消减,构建了这两个时期的双向消减cDNA文库,并对筛选出来的部分已知和未知基因进行了深入探索。主要结果如下: 1.为了构建人子宫内膜窗口期基因表达平台,我们从最初的实验材料选择就严格把关。我们以同一位志愿者的不同时相的子宫内膜作为自身对照,结合扫描电镜观察结果、B超检测结果、HE染色结果和PR免疫组化染色结果从多方面验证了材料的可靠性,为后续的分子生物学实验打下坚实的基础。 2.SMART~(TM) PCR cDNA Synthesis技术和抑制性消减杂交技术结合起来,构建了人窗口期子宫内膜和早泌期子宫内膜双向cDNA抑制消减文库。 3.在人窗口期子宫内膜cDNA文库(HWE)和人早泌期子宫内膜cDNA文库(HEW)911个PCR纯化产物进行斑点杂交分析,有354个在人窗口期和早泌期子宫内膜存在4倍以上表达差异,其中192个在窗口期差异表达,162个在早泌期差异表达。对斑点杂交筛选出的354个克隆中的130个克隆进行测序,共产生了93个质量满意的ESTs,总测序成功率达到71.54%。 4.通过在互联网上的比对,在HWE文库中已知基因、已知ESTs和全新ESTs分别为52.46%、40.98%和6.56%:在HEW文库中分别为48.57%、48.57%和2.86%。 5.对代表已知基因的ESTs按照功能分类,发现主要涉及细胞分裂/细胞凋亡、蛋白合成和分解、物质转运、分子伴侣、转录调控因子、信号转导、细胞骨架、粘附分子几类,并初步探讨了人子宫内膜围着床过程中差异表达基因的作用和意义。 6.通过序列比对发现在HWE文库中编号为82G7的克隆序列与已知基因NID-1同源性很高,用原位杂交技术检测在窗口期和早泌期人子宫内膜的表达,结果显示NID-1在窗口期子宫内膜的基质细胞中高表达,在早泌期的子宫内膜组织中表达很弱。根据文献推测,NID-1基因的表达产物的作用可能在人类的胚胎发生、着床和蜕膜化反应中发挥重要作用。 7.将人子宫内膜窗口期特异表达的克隆编号为82F4的EST序列进行RACE扩增获得一个新的cDNA序列,命名为PPI-82-F4,登录GenBank,获得登录号为EF063108。 8.应用Northern杂交技术检测PPI-82-F4在窗口期和早泌期人子宫内膜的表达,结果显示PPI-82-F4在窗口期子宫内膜的表达显著高于早泌期。应用原位杂交技术检测PPI-82-F4在窗口期和早泌期人子宫内膜的表达,结果显示PPI-82-F4在窗口期子宫内膜的基质细胞和腺上皮细胞中高表达,在早泌期的子宫内膜组织中表达很弱。综上所述,我们采用抑制性消减杂交构建了双向人子宫内膜窗口期消减早泌期cDNA文库,在这两个文库中各有192和162个阳性差异的克隆,我们所得到的基因包括转录调控因子、信号转导、细胞骨架几类。母体子宫内膜从排卵到着床的过程中发生剧烈的变化,在孕激素的调控下,为了使着床窗开放做了充分准备,有很多相关基因表达。我们发现了一些基因的表达上调,同时还有一些基因表达降低或受到抑制,为我们发现潜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.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

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