【作者】 龙春兰；

【导师】 王应雄；

【作者基本信息】 重庆医科大学 ， 遗传学， 2011， 硕士

【摘要】 目的:研究妊娠过程小鼠子宫内膜DNA甲基转移酶( DNA methyltransferases,DNMTs)DNMT1,DNMT3a和DNMT3b的表达规律以及它们对胚胎着床的影响,初步探讨DNMTs在胚胎着床中的调控作用。方法:1.建立昆明小鼠妊娠模型,随机收集妊娠第一天(Dl)至妊娠第七天(D7)子宫内膜组织,腊块包埋或于-80℃冻存备用。2.荧光定量PCR检测小鼠妊娠D1-D7子宫内膜DNMTs mRNA的表达,统计分析表达规律;3.免疫组织化学检测妊娠D1-D7的小鼠子宫内膜DNMTs蛋白表达水平,并分析其表达规律;4.随机选择妊娠D1小鼠,于腹腔注射200μl浓度为0 mg/kg,0.1mg/kg和0.5 mg/kg的DNMTs抑制剂5-aza-CdR,建立DNMTs表达抑制的药物诱导小鼠模型,用于检测DNMTs对胚胎着床影响的功能实验。5.荧光定量PCR检测不同浓度5-aza-CdR诱导组妊娠D3、D5小鼠子宫内膜的DNMTs mRNA的表达抑制情况。6.免疫组织化学检测不同浓度5-aza-CdR诱导对妊娠D3、D5小鼠子宫内膜的DNMTs蛋白的表达抑制情况;7.荧光定量PCR和免疫组织化学检测不同药物浓度对妊娠D5小鼠子宫内膜蜕膜化标志基因Hoxa10基因和蛋白表达情况。8.统计分析妊娠D5天,不同浓度药物诱导下胚胎植入数。结果:1. 2-△△T法分析荧光定量PCR结果显示:D1-D7各组小鼠子宫内膜均有DNMTs mRNA表达,其中DNMT1在胚胎着床窗口期前,妊娠小鼠子宫内膜的表达随着孕天增加而增高,至妊娠第3天(D3)达最高峰(P<0.05),随后逐渐降低,胚胎着床后(D6,D7)基本恢复至妊娠D1水平;DNMT3a在妊娠小鼠子宫内膜的表达与DNMT1的表达模式相似,即在胚胎着床窗口期前随着孕天增加而增高,妊娠第3天(D3)达最高峰(P<0.05),随后逐渐降低;DNMT3b与DNMT1和DNMT3a不同,妊娠D3达最高峰(P<0.05),但在着床窗口期后(D5,D6,D7),DNMT3b的表达显著低于妊娠第1天(P<0.01)。2.免疫组织化学结果显示:DNMTs蛋白在妊娠小鼠子宫内膜均有表达。DNMT1主要定位于腔上皮、腺上皮以及基质细胞的胞核,在胞浆弱表达; DNMT3a主要定位于腔上皮、腺上皮以及基质细胞的胞核;DNMT3b主要定位于腔上皮、腺上皮细胞紧密处以及基质细胞的胞浆和部分胞核中。DNMTs的蛋白表达规律基本与mRNA一致,即在妊娠D3表达最高,其后表达逐渐降低。3.0.1 mg/kg 5-aza-CdR诱导妊娠小鼠2天(D3)、4天(D5)后,子宫内膜DNMT1、DNMT3a、DNMT3b mRNA水平均低于正常组,但差异不显著(P>0.05);0.5mg/kg 5-aza-CdR诱导妊娠小鼠2天(D3)、4天(D5)后,子宫内膜DNMT1、DNMT3a mRNA水平均低于正常组,差异显著(P<0.05);妊娠D3子宫内膜DNMT3b mRNA水平与正常组相比,无显著差异(P>0.05),妊娠D5子宫内膜DNMT3b mRNA水平显著降低(P<0.05);0.5 mg/kg 5-aza-CdR诱导组妊娠D3、D5小鼠子宫内膜DNMT1、DNMT3a和DNMT3b mRNA水平均显著低于0.1 mg/kg 5-aza-CdR诱导组(P<0.05)。4. 5-aza-CdR诱导后,子宫内膜DNMT1主要定位于腔上皮、腺上皮细胞胞浆以及基质细胞的胞核中;DNMT3a主要定位于腔上皮、腺上皮以及基质细胞的胞核中;DNMT3b主要定位于腔上皮、腺上皮细胞的胞浆中。DNMTs的蛋白表达抑制程度与mRNA的表达抑制具有较高的一致性。5. 0.1 mg/kg 5-aza-CdR诱导组小鼠子宫内膜HOXA10 mRNA、蛋白水平均高于正常组,但差异无显著性(P>0.05);0.5 mg/kg 5-aza-CdR诱导组小鼠子宫内膜HOXA10 mRNA、蛋白水平低于正常组,差异显著(P<0.05)。6.0.1 mg/kg 5-aza-CdR诱导组孕鼠,可见明显胚胎着床点,且着床点数量与药物空白对照组无显著差异(P>0.05);0.5 mg/kg 5-aza-CdR处理孕鼠后,妊娠D5子宫未见任何着床点,药物诱导组与空白对照组之间差异极为显著(P<0.01)。结论:1.DNMTs在妊娠早期子宫内膜组织均有表达;在植入窗口期前高表达,窗口期及着床后表达降低,以及在小鼠子宫内膜的分布变化提示DNMTs可能与胚胎着床相关;2. DNMTs可能通过自身的降低来调节子宫内膜容受性相关的基因表达,从而促进细胞黏附分子、糖蛋白类和细胞因子的表达或分泌,使子宫内膜容受性达最佳状态,以利于胚胎的植入。更多还原

【Abstract】 Objective:To investigate the molecular mechanism of DNMTs during the blastocyst implantation, mRNA and protein expression of DNMTs were detected in early pregnancy mouse endometrium in this research.Methods:1. Establishment of mouse model of pregnancy. Pregnant Dl to D7 mice were randomly selected.The endometria of pregnant mice were collected and stored at -80℃immediately or embedded by paraffin.2. FQ-PCR was used to detect the mRNA level of DNMTs in the endometria of pregnant mice.3.Immunohistochemistry was used to analyze the expression of DNMTs protein in the endometria of early pregnant mice.4. Establishment of the induction mouse model by 5-aza-CdR, the pregnant mice on D1 was randomly chosed to inject i.p. (200μl per injection) with the of DNMTs inhibitor, 5-aza-CdR with the concentration of 0, 0.1, or 0.5 mg/kg).5. The mRNA level of DNMTs in the endometria of normal mice and the treated mice by 5-aza-CdR on pregnant D3 and D5 were detected by FQ-PCR.6.Immunohistochemistry was used to analyze the protein expression of DNMTs and Hoxa10 in the endometria of normol and the inducted mice with the treatment of 5-aza-CdR on the pregnant D3 and D5.7. FQ-PCR and Immunohistochemistry were used to analyze the mRNA level and protein expression of Hoxa10 in the endometria of normol and the inducted mice on the pregnant D5.8.The number of implantated blastocysts was evaluated by the way of Trypan blue injection through tail vein on pregnant D5.Result:1. The FQ-PCR data showed that the mRNA levels of DNMT1 in endometria of pregnant mice was gradually elevated,and reached the maximum level on pregnant D3 (P<0.05), then gradually declined to the level of D1 from D4 to D7; The mRNA expression of DNMT3a in endometria of pregnant mice resembling to the DNMT1, gradually increased,and reached a maximum level on D3 (P<0.05), then gradually declined from D4 to D7 in the same. The DNMT3b expression in mRNA gradually increased, and reached a maximum level on D3 ,then remarkably declined from D5 to D7(P<0.01) in the endometria of pregnant mice.2. Immunohistochemistry staining showed that DNMT1 protein was strong stained in the nucleus of luminal epithelia, gland epithelia and stromal cells, weak stained in the cytoplasm; DNMT3a protein was located in the nucleus of luminal epithelia, gland epithelia and stromal cells; DNMT3b protein was mainly located in the cytoplasm of luminal epithelia, gland epithelia and stromal cells, and less in nucleus. The expression patern in protein level in the endometria of pregnant mice was in concordance with mRNA level of of DNMTs, thus the expression of DNMTs protein was increased,and gradually declined when it reached a maximum level on D3 (P<0.05).3. The mRNA levels of DNMTs were down-regulated without significance in endometria of the mice treated for two days (pregnant D3) or four days (pregnant D5) by 5-aza-CdR at concentration of 0.1 mg/kg, comparing with the control group (P>0.05). The expression of DNMT3b in mRNA level of the mice endometria was down-regulated without significance (P>0.05) when treated for two days while with significance (P<0.05)when treated for four days by 5-aza-CdR at concentration of 0.1 mg/kg. The expression of DNMT1, DNMT3a and DNMT3b in mRNA level in the endometria were remarkably down-regulated with significance after two or four days’treatment with 5-aza-CdR at concentration of 0.5 mg/kg, when compared with the control ones.4. After treated with 5-aza-CdR, immunohitochemistry staing showed that DNMT1 was mainly located in the cytoplasm of luminal epithelia, gland epithelia and stromal cells, DNMT3a was located at the nucleus of luminal epithelia, gland epithelia and stromal cells and DNMT3b was located at cytoplasm of luminal epithelia, gland epithelia and stromal cells. The protein level of DNMTs almost the same as the they were in mRNA level.5. No significant difference was observed in the number of implantation sites in the group treated by 5-aza-CdR at concentration of 0.1 mg/kg, when compared with control. No implantation site was observed in the mice when treated by 5-aza-CdR at level of 0.5 mg/kg. The uteri were smaller on the pregnant D5 in the mice treated by 0.5 mg/kg 5-aza-CdR treated mice than the control. No significant difference in size was found between the embryos from 0.1 mg/kg 5-aza-CdR treatment mice and the control.6. The expression of Hoxa10 mRNA and protein were higher than the control group after the four days’treatment of 0.1 mg/kg 5-aza-CdR but had no significant difference(P>0.05). When treated by 0.5 mg/kg 5-aza-CdR, the mRNA and protein level of Hoxa10 in endometria were significant lower than those in the control(P<0.05) .Conclusion:1. mRNA and protein expression of DNMTs in the endometria of pregnant mice were distributed regularly indicated that DNMTs might participated in the implantation process.2. DNMTs might promote expression of some cellular adhesion molecule, glycoproteins, cytokine, making the best reception of endometrium via decreasing methylation of those genes related to reception.更多还原