【作者】 贾咏存；

【导师】 魏莎莉；

【作者基本信息】 重庆医科大学 ， 遗传学， 2007， 硕士

【摘要】 3背景:胚泡着床是指胚泡通过与子宫内膜的相互作用而侵入子宫内膜的过程。是人类和哺乳类动物生殖过程中的重要环节,是决定妊娠成功与否的关键,然而其机制尚未完全明了。当受精卵发育到胚泡阶段后,胚泡外面的透明带解体,其表面的滋养层细胞与子宫内膜上皮细胞直接接触,而黏附于子宫表面,进而侵入子宫内膜,这一过程包括胚泡与子宫内膜上皮的识别、黏附和侵入子宫内膜等环节。在“着床窗口期”胚泡的成功植入取决于处于容受状态的子宫内膜和具有侵入性胚泡间的同步发育。这一过程受极其复杂而精细的多因素的调节。在“着床窗口期”,由于受到卵巢激素的作用,子宫内膜上皮和基质增殖、分化,子宫内膜变成一个能吸附胚泡的表面,而间质细胞准备向蜕膜细胞转变。胚泡附着于子宫内膜促使胚泡周围的子宫内膜发生蜕膜反应,子宫内膜处于“容受性”状态,利于胚泡的着床。但子宫内膜“容受性”状态产生的分子机制还不清楚。近年来研究发现,HOXA-10基因对子宫内膜容受性变化的形成及胚泡着床可能有一定影响。同源框基因(Homeobox HOX)是McGinnis[1]和Scott[2]等在果蝇体内发现的一类与果蝇体节突变相关的基因,属于多基因家族的转录调节基因,通过和DNA结合激活或抑制目的基因,其重要功能是决定细胞的定向分化与增殖。HOX基因均含有一个约183个核苷酸组成的同源框序列,编码61个氨基酸构成的同源域结构,通过螺旋-转角-螺旋的结构作为DNA的结合域,于下游靶基因的DNA序列(TTAT或TAAT)相识别,从而激活或抑制下游基因的转录[3]。作为一种多效性的转录调节因子, HOXA-10基因在生殖过程中的作用近年来逐渐受到瞩目,如在胚胎的发育与分化、子宫内膜的增殖与分化、子宫内膜蜕膜化的等。然而在妊娠早期中的表达情况及其对胚胎着床的影响国内外报道较少。因此,有必要对HOXA-10基因在早孕小鼠子宫内膜中的表达规律及其在胚胎着床过程中的作用进行研究。目的:本课题旨在通过研究Hoxa-10基因在小鼠子宫内膜表达的规律性变化及其对胚泡植入的影响,初步探讨它在小鼠生殖中的作用。方法:1.收集未孕(记为D0)和早孕1、2、3、4、5、7天小鼠(分别记为D1、D2、D3、D4、D5、D7)子宫内膜,共7组,每组20只小鼠。①运用实时荧光定量PCR (Real-time fluorescence quantitative PCR ,FQ-PCR)分别检测以上各组小鼠子宫内膜HOXa-10基因表达;②免疫组织化学SP法检测以上各组小鼠子宫内膜中HOXa-10蛋白的表达情况;2.用DNA/脂质体复合物分别转染一个可以持续表达HOXa-10基因的质粒和针对HOXa-10基因的反义寡脱氧核苷酸到受孕D2小鼠子宫角内,于孕7天观察小鼠的胚泡着床数。结果:1. FQ-PCR测得随着小鼠妊娠天数的增加,子宫内膜HOXa-10基因表达量也逐渐增高,在孕D4达到峰值,孕D5开始下降,孕D7时表达已接近未孕状态,单因素方差分析结果显示D0、D1和D7各组之间无显著差异(P>0.05),D0、D1和D7三组与其余各组的差异均有显著性差异(P<0.05)。2.免疫组化分析显示HOXa-10蛋白在小鼠子宫内膜中表达规律与FQ-PCR结果一致,但在早孕小鼠不同时期的子宫内膜中HOXa-10蛋白的表达有以下特点:在小鼠未孕D0至孕D1时,HOXa-10蛋白在子宫腔上皮及腺上皮上有表达,在子宫肌层不表达;在孕D2至D3时,HOXa-10在子宫腔上皮及腺上皮中强表达,在子宫肌层中低表达;从孕D4开始,HOXa-10蛋白在子宫内膜的表达发生转移,子宫上皮表达量开始下降,间质开始表达;孕D5时,在子宫上皮中表达转阴,间质细胞上有表达;孕D7时,HOXa-10在蜕膜间质中继续表达。2.用HOXa-10DNA质粒/脂质体转染孕D2小鼠子宫角,以增强HOXa-10基因表达,小鼠的胚泡着床数明显增加,着床率(%)为79.1±7.14;用HOXa-10反义寡脱氧核苷酸/脂质体转染孕D2小鼠子宫角,抑制HOXa-10基因表达,小鼠的胚泡着床数明显减少,胚泡着床率为33.5±3.72;正常对照组小鼠胚泡着床率为70.8±6.65。各组差异具有统计学意义。结论:HOXa-10基因及蛋白在小鼠子宫内膜中的表达从孕D1至孕D7有从逐渐升高到下降的一个过程,且HOXa-10蛋白随着子宫内膜蜕膜化的完成由子宫腔上皮及腺上皮中强表达转移至子宫肌层间质细胞的低表达,这与胚泡着床时子宫内膜的蜕膜化过程完全一致。表明它可能促进了间质细胞向蜕膜细胞转变,为“着床窗口期”子宫内膜的容受性变化及胚胎着床创造条件。用DNA/脂质体复合物分别转染一个可以持续表达HOXa-10基因的质粒和针对HOXa-10基因的反义寡脱氧核糖核酸到受孕D2小鼠子宫角内后,分别增强和抑制了HOXa-10基因的表达,两组孕鼠的胚泡着床率也明显改变,这进一步说明了HOXa-10基因在胚泡着床过程中可能起着重要作用。更多还原

【Abstract】 Background:The blastocyst implantation is the process of fertilized egg grows to the blastocyst and hatches from the transparent belt stage, then it accomplish adherency and invasion in endometrium. It including the embryo trophoderm and the endometrium epidermis recognition,cell adherency and the germinal vesicle to the endometrium invasion and so on, that determine whether pregnancy will develop successfully or not. In‘window of implantation’, ovarian hormone stimulate endometrium epidermis and matrix multiplication and differentiation: The cavity epidermis turns the condition which is suitable for to stick extremely, but intersititial cell preparation to decidua cell transformation. The pouch embryo adherency urges around the embryo in the cavity epidermis to have the decidua to respond that, the endometrium is at‘acceptivity’the condition, the blastocyst this time very is easy to complete implantation. But the acceptive endometrium produces the molecular mechanism is not clear. In recent years studied the discovery, HOXA-10 gene all is having certain influence to the acceptive endometrium formation and blastocyst implantation. Homeobox gene (HOX) is McGinnis[1] and Scott[2] which discovered in fruit fly in vivo the related with the fruit fly body festival changes a kind gene, belongs to the multi-gene family nuclear transcription regulatory gene, through activates or the suppression goal gene with the DNA union, its important function is decides the cell the directional differentiation and the multiplication. The HOX gene includes an approximately 183 nucleotide composition homologous frame sequence, codes 61 amino acid constitutions the homologous territory structure, through the screw - corner - screw structure took DNA the union territory, (TTAT or TAAT) distinguishes in the downstream target gene DNA sequence, thus activates or suppression downstream gene copying[3]. HOXA-10 gene gradually received in recent years in reproduction process function focuses attention on, is considered one of for implantation regulatory genes. Therefore, Hoxa-10 gene in the pregnant mouse endometrium expression rule is regard having the necessity to research the influence of the blastocyst implantation.Objective:Studies the rule of Hoxa-10 gene and protein in early pregnant mouse endometrium expression, and discusses it function in the process of blastocyst implantation. Methods:1.Collecte the no pregnant D0 and in early pregnant D1,D2,D3,D4,D5 and D7 mice endometrium, altogether 7 groups, each group of 10 mouse. Real-time fluorescence quantitative PCR (FQ-PCR) and immunohistochemical analysis separately examine various issue of mouse endometrium HOXa-10 gene and protein expression;2.Transfection with DNA/ liposome compound distinction extension to be possible to expression HOXa-10 gene continually the material particle and to becomes in D2 pregnant mouse uterus angle in view of HOXa-10 antisense oligonuleotides/ liposome in pregnancy D2 mouse horn of uterus respectively, and detected the variety of quantity in comparison with normal pregnancy D2 mouse blastocyst nidation. We take normally pregnant D2 mouse as the comparison, contrasts three group of mouse’s blastocyst nidation numbers.Result:FQ-PCR obtains along the mouse pregnancy days number with increase, the endometrium HOXa-10 gene expression quantity also gradually advances, is being pregnant D4 to achieve the peak value, is pregnant D5 start to droping, weather in pregnant D7 its expression closing the no pregnant condition, D0,D1 and D7 ampho-compare P>0.05, other group ampho-compare P<0.05. Immunohistochemical analysis showed the same results as FQ-PCR, but it have different organization distributed characteristic in the different time.Transfection with HOXa-10 DNA / liposome compound in D2 pregnant mouse uterus angle, the rate of implantation obvious increased; Transfection with HOXa-10 antisense oligonuleotides / liposome compound in D2 pregnant mouse uterus angle, the rate of implantation is reducing obviously. The result analysis made use of the statistical analysis software SPSS12.0. Research has revealed each group of differences significance. Conclusion:HOXa-10 gene from is pregnant D1 in mouse endometrium expression in pregnant to have a drop process D5 gradually, it with blastocyst implantation when the endometrium decidualization process is completely consistent. It possible is HOXa-10 promoted the intersititial cell to the decidua cell transformation, Then is advantageous in enhancing implantation‘the window time’endometrium receptivity.It creating the condition for the blastocyst implantation. Transfection with DNA/ liposome compound distinction extension to be possible to express HOXa-10 gene continually the material particle and after becomes in D2 pregnant mouse uterus angle in view of HOXa-10 gene antisense oligonuleotides, Separately strengthened and has suppressed HOXa-10 gene expression, two groups were pregnant mouse’s the rate of implantation also obviously different. It is explained HOXa-10 gene was having the vital role in the blastocyst implantation process.更多还原