小鼠孕早期子宫内膜ICAM-1 mRNA的表达及调节

目的：为探讨细胞间粘附分子－１ｍＲＮＡ（ＩＣＡＭ－１）在孕早期的作用和细胞因子对其表达的影响。方法：用ＲＴ－ＰＣＲ法测定孕１～８ｄ小鼠子宫内膜ＩＣＡＭ－１的表达；在小鼠孕２～４ｄ分别腹腔注射细胞因子ＬＩＦ、ＩＬ－１β或ＩＬ－１ｒａ，测定并比较各组孕第４天子宫内膜ＩＣＡＭ－１ｍＲＮＡ表达水平。结果：ＩＣＡＭ－１ｍＲＮＡ孕第１天表达量较低，第２天开始升高，第４天达高峰（Ｐ〈０．０１）。此后缓慢下降。与对照组相比。ＬＩＦ和ＩＬ－１β组ＩＣＡＭ－１ｍＲＮＡ的表达水平升高（Ｐ〈０．０１）。结论：ＩＣＡＭ－１在着床期表达水平较高，可能参与了着床过程中的胚胎粘附。细胞因子ＬＩＦ和ＩＬ－１可能是子宫内膜表达ＩＣＡＭ－１的潜在促调节因子。     

原癌基因产物Ras及c—erbB2在着床前小鼠胚胎中的 …

目的 观察小鼠着床前的胚胎细胞中原癌基因ｒａｓ编码的蛋白产物ｐ２１Ｒａｓ、ｃ０ｅｒｂＢ２的表达，并了解其对着床前的胎发育的可能作用。方法 采用免疫组织化学丰床前小鼠胚胎中ｐ２１Ｒａｓ、ｃ－ｅｒｂＢ２蛋白的表达。结果 小鼠着床前的胚胚ｐ２１Ｒａｓ的免疫反应呈阳性，阳性物质分布于胞质中，胚胎外透明带呈阴性反应；而在各期着床关的胚胎中均未检测到ｃ－ｅｒｂＢ２蛋白的表达。结论 小鼠着床前的胚胎中匀未到ｃ－     

小鼠胚泡植入早期FucT-Ⅶ和sLe(X)寡糖抗原表达

目的探讨妊娠第1～5天(d1～5)小鼠子宫内膜唾液酸化的路易斯寡糖[sLe(X)]合成关键酶α-1,3岩藻糖基转移酶基因(FucT-Ⅶ)的表达和其表面sLe(X)寡糖抗原表达对胚胎植入的影响。方法应用RT-PCR和免疫组化方法检测妊娠第1～5天小鼠子宫内膜FucT-Ⅶ及寡糖抗原的表达规律。用子宫角内注射sLe(X)单克隆抗体的方法检测小鼠胚胎着床数。结果妊娠第1～5天小鼠子宫组织均有FucT-Ⅶ的表达,且在妊娠d4表达更强(P<0.05)。妊娠第1～5天sLe(X)寡糖抗原表达在子宫内膜的腔上皮和腺上皮。单侧子宫角sLe(X)抗体注射后胚胎的着床数量明显较对侧(注射等体积生理盐水)减少。结论在小鼠胚胎着床过程中,FucT-Ⅶ及sLe(X)寡糖抗原可能参与了胚泡的定位、黏附及侵袭过程,在胚泡植入的早期发挥作用。     

凋亡调节基因bcl-2及p53在早期胚胎中的表达

目的 探讨两种重要的细胞凋亡调控基因bcl-2及p53在着床前小鼠胚胎中的表达及其其对着床前胚胎发育的作用。方法 建立超排卵小鼠早孕的模型，获取着床前胚胎，采用免疫组化LSAB法、检测bcl-2及p53蛋白。结果在小鼠2细胞、4细胞、8细胞和桑葚胚中，均检测到bcl-2和p53蛋白。结论 bcl-2和p53凋亡调控基因在小鼠着床前胚胎的表达可能与细胞碎片形成有关，对于维护胚胎正常发育，促进发育异常的胚胎细胞发生凋亡，从而提高胚胎质量有重要作用。体内增殖与凋亡处于一个动态平衡，共同调节胚胎发育。     

干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19在小鼠围着床期子宫内膜中的表达及其作用

目的 探讨干扰素/维甲酸联合应用诱导细胞凋亡相关基因-19（GRIM-19）在小鼠围着床期子宫内膜中的表达及其与胚胎着床的关系。方法 收集早期妊娠第1~6天小鼠子宫组织,采用免疫组织化学法检测GRIM-19蛋白在子宫内膜上皮细胞中的表达及定位情况;收集早期妊娠、假孕和雌孕激素处理小鼠子宫组织,采用Western blotting 和Real-time PCR检测GRIM-19蛋白和mRNA水平;TUNEL方法检测早期妊娠第1~6天小鼠子宫组织细胞凋亡情况;采用pEGFP GRIM-19质粒GRIM-19-siRNA 转染技术使RL95-2细胞系过表达或低表达GRIM-19,检测其对RL95-2-BeWo 共培养体外着床模型球状体黏附率的影响,AnnexinV/PI双染色法检测细胞凋亡,线粒膜电位检测试剂盒（JC-1）检测线粒体跨膜电位;采用Western blotting 和Real-time PCR检测转染后信号转导子和转录激活因子3（STAT3）、肿瘤坏死因子（TNF)-α及白细胞介素（IL)-11蛋白和mRNA水平。结果 GRIM-19在早期妊娠第1~6天小鼠子宫腔上皮和腺上皮均有表达,妊娠第4天GRIM-19蛋白和mRNA水平减低,细胞凋亡减少;假孕第1~6天小鼠子宫内膜GRIM-19表达无明显差异;雌激素处理组小鼠子宫内膜GRIM-19表达减弱;过表达GRIM-19后,RL95-2-BeWo 共培养球状体黏附率降低,细胞凋亡增加,线粒体膜电位减低,RL95-2细胞系中STAT3及IL-11蛋白和mRNA水平减低,TNF-α蛋白和mRNA水平升高。 结论 GRIM-19在胚胎着床中发挥重要作用,可能是通过调节细胞凋亡和免疫耐受为胚胎着床提供保障。     

ICAM-1在着床期小鼠PBLC及EC/DC中表达的研究

目的：研究细胞间粘附因子－１（ＩＣＡＭ－１）在着床期外周血淋巴细胞（ＰＢＬＣ）、子宫内膜及蜕膜细胞（ＥＣ／ＤＣ）中的不同表达特点及动态变化规律。方法：以着床期小鼠为动物模型,细胞分析采用单抗免疫荧光标主多参数流式细胞术分析技术。结果：发现ＩＣＡＭ－１在ＰＢＬＣ、ＥＣ／ＤＣ中的均存在明显的动态变化;其中ＩＣＡＭ－１的表达在ＰＢＬＣ中于妊娠第２天（Ｄ２）达最低小平,而在ＥＣ／ＤＣ中于妊娠第４天（Ｄ４）     

原癌基因产物Ras及c-erbB2在着床前小鼠胚胎中的表达

目的　观察小鼠着床前的胚胎细胞中原癌基因ras编码的蛋白产物 p2 1Ras及c erbB2的表达 ,并了解其对着床前的胚胎发育的可能作用。方法　采用免疫组织化学ABC法 ,检测着床前小鼠胚胎中p2 1Ras及c erbB2蛋白的表达。结果　小鼠着床前的胚胎细胞中 p2 1Ras的免疫反应呈阳性 ,阳性物质分布于胞质中 ,胚胎外透明带呈阴性反应 ;而在各期着床前的胚胎中均未检测到c erbB2蛋白的表达。结论　小鼠着床前的胚胎中 ,有原癌基因ras编码蛋白的表达 ,p2 1Ras是增殖和分化受体介导的信号传导途径中的重要组成部分 ,可能在小鼠着床前的胚胎发育过程中起重要的调节作用。C erbB2基因对于围着床期子宫的生长发育具有重要的作用 ;而在小鼠着床前的胚胎发育过程中似乎不起重要作用     

围着床期小鼠胚卵L-选择素的表达变化及作用

目的 探讨围着床期小鼠胚卵L-选择素的表达规律及其对胚胎着床的影响.方法 1.分别采用实时荧光定量PCR(FQ-PCR)和免疫荧光组织化学方法检测围着床期小鼠胚卵L-选择素mRNA和蛋白的表达,并用免疫荧光组织化学方法检测L-选择素在细胞的定位;2.用子宫角内抗体干预的方法观察并统计小鼠着床胚胎数.结果 1.随着胚卵分化成熟,L-选择素mRNA和蛋白表达均呈逐渐增加趋势,胚泡期表达最强(P<0.05).L-选择素在单细胞期和4细胞期有少量表达,桑椹胚周边区L-选择素表达显著增强,胚泡滋养层L-选择素表达最强.2.小鼠子宫角实验侧(经抗L-选择素单克隆抗体干预)胚胎着床数较对照侧(经等体积生理盐水干预)明显减少(P<0.05).结论 胚卵可能通过L-选择素信号转导途径及其与子宫内膜的糖蛋白受体结合参与胚胎着床.     

小鼠胚胎与子宫内膜中整合素α5的表达

目的：观察整合素α5在小鼠早期胚胎和子宫内膜中的表达分布状况，探讨整合素在胚胎着床中的可能作用。方法：用免疫组织化学ABC法染色，对发育不同时期的小鼠胚卵和妊娠2d-6d、8d、13d以及非妊娠期的子宫内膜中整合素内的表达分布进行形态学观察与半定量分析。结果：整合素α5的阳性反应物质在小鼠早期胚卵胞浆内；非妊娠期子宫内膜和妊娠期的脱膜上皮与基质细胞内均有整合素α5表达，植入前表达相对较弱，植入后随妊娠的进展逐渐增强。结论：提示整合素α5在小鼠胚卵着床中起重要作用：推测它不参与胚卵对母体子宫内膜的识别和粘附，而是参与了粘附后的侵入及胚胎发育过程。     

小鼠胚胎正常着床与实验性延缓着床的比较

本实验企图了解小鼠正常着床与实验性延缓着床的规律。将对照组与延缓着床组的怀孕小鼠,分别在1—7天或1—10天处死,取子宫和输卵管,常规固定,石腊包埋,连续切片,HE染色,光镜观察。结果表明,孕1—3天,两组形态特征相似,孕3天之后,延缓组滞育胚胎运行减慢,发育迟缓。雌二醇注射24、48、72小时,与对照组第5、6和7天相同。延缓组着床前胚胎变性较多。除滞育期激素条件外,延缓着床与正常着床过程基本相同。     

外源性LIF和IL-1对围着床期小鼠子宫内膜整合素β3亚基表达的调节

目的研究外源性LIF及IL-1对围着床期子宫内膜整合素β     

p16INK4A在动情周期和早孕期小鼠子宫内膜的表达

目的 检测肿瘤抑制基因p16INK4A在小鼠动情周期和早孕期子宫内膜中的表达,初步探讨p16^INK4A在小鼠胚胎植入过程中的生物学作用。方法 运用反转录-聚合酶链反应(RT-PCR)技术检测动情周期和早孕期小鼠子宫内膜p16^INK4AmRNA的表达水平;用免疫组织化学和Western blotting方法检测p16INK4A蛋白的表达。结果 RT-PCR显示,早孕期比动情周期p16^INK4AmRNA的表达有显著增强。动情周期中动情期比其他3期表达明显增强;早孕期中,随妊娠天数的增加,p16^INK4AmRNA的表达也逐渐增强,孕5d达峰值,之后又逐渐减弱。免疫组织化学和Western blotting的结果也都显示,p16^INK4A蛋白在子宫内膜的表达及规律与RT-PCR的结果一致。结论 p16^INK4A在小鼠的胚泡植入过程中起重要作用。     

Uterine fibronectin mRNA content and localization are modulated during implantation.

Fibronectin mRNA and protein content were examined during embryonic implantation in the rat uterus. Content of total fibronectin mRNA at day 6 of pregnancy increased relative to the non-pregnant uterus. In contrast, fibronectin protein content of the subepithelial stroma was relatively decreased except in the region directly surrounding the lumen, and this fibronectin immunoreactivity was sensitive to hyaluronidase treatment. These changes are likely to reflect the degradation and subsequent remodeling of the previously stable uterine extracellular matrix in preparation for embryonic implantation. A+, B-, V + fibronectin mRNAs were present in both the non-pregnant and day 6 pregnant uterus with increased content of A+ and V+ fibronectin mRNAs in the latter. A + fibronectin mRNA was distributed throughout the endometrial stroma of the non-pregnant uterus and content of the subepithelial stroma increased by day 4 of pregnancy, coincident with progesterone action on the endometrium. On day 6 of pregnancy, fibronectin mRNAs encoding the V95 and A regions were preferentially localized to the mesometrial zone of the subepithelial stroma. Accumulation of these mRNA splicing variants at the mesometrial zone was dependent upon decidualization, but the embryo was not required. Thus, there are two major changes in uterine fibronectin gene expression as a result of pregnancy: increased fibronectin mRNA content and mesometrial localization. These changes suggest a key function for fibronectin in implantation and imply the operation of a regulatory program of fibronectin gene expression which depends on hormonal sensitization and a nidatory stimulus.     

白细胞介素-8在妊娠早期小鼠子宫内膜的表达及对胚泡着床的影响

目的：研究白细胞介素-8（IL-8）在妊娠早期小鼠子宫内膜的表达及其对小鼠胚泡着床的影响。方法：用免疫组化法及图像分析技术对IL-8在妊娠1～6d小鼠子宫内膜的表达进行定位和测定;子宫角注入IL-8抗体,探讨IL-8对胚泡着床的影响。结果：在子宫内膜腔上皮,IL-8主要定位于细胞的游离面,妊娠1d阳性反应最弱,4d表达最强,5d有所下降,6d又开始增强;在子宫内膜腺上皮,妊娠4d表达最弱,5d和6d最强;在子宫内膜的基质细胞,随妊娠天数增加,IL-8的表达逐渐增强。IL-8Ab明显抑制小鼠胚泡着床。结论：IL-8在妊娠早期小鼠子宫内膜持续表达,并参与胚泡的着床调控过程。     

Characterization of IK cytokine expression in mouse endometrium during early pregnancy and its significance on implantation

The expression of IK cytokine was investigated in the mouse endometrium during early pregnancy (D1-D7 of pregnancy) and pseudopregnancy using real-time PCR, western blotting and immunohistochemical analysis, and the effects of IK cytokine on embryo implantation were observed by injection with antisense IK cytokine oligodeoxynucleotides in the uterine horn. Our data showed that the expression of IK cytokine mRNA increased gradually from D1 to D4 of pregnancy and reached a peak level at D4 of pregnancy (P<0.05). Western blotting and immunohistochemical analysis revealed that the expression of IK cytokine protein increased gradually from D1 to D5 of pregnancy and reached a peak level at D5 of pregnancy (P<0.05). The expression of IK cytokine in the pseudopregnant uterus was significantly lower compared to that in the normal pregnant uterus and the level of the protein never showed a high peak during the whole pseudopregnancy. The expression of IK cytokine at the implantation site was much stronger than that in the peri-implantation site on Day 5 of pregnancy. After 24 and 48 h of injection with antisense IK cytokine oligodexynucleotides in the uterine horn on D3 of pregnancy (i.e. implantation window), the expression of IK cytokine in the uterus was remarkably inhibited, while the expression of major histocompatibility complex II (MHC II) increased and the number of implanted embryos significantly decreased in the site of uterine horns receiving antisense IK cytokine (P<0.05). These results suggested that IK cytokine may play a crucial role in implantation.     

溶血磷脂酸受体3在小鼠胚胎围着床期子宫内膜的表达及意义

目的 探讨溶血磷脂酸受体3(LPA-R3)nRNA和蛋白在小鼠胚胎着床过程中表达的动态变化. 方法 将78只雌性昆明小鼠随机分为真孕组(36只)、假孕组(36只)和对照组(6只),应用反转录.聚合酶链反应(RT-PCR)和Western blotting以及免疫组织化学SP法,检测胚胎围着床期(0～6d)LPA-R3 mRNA和蛋白在小鼠子宫内膜的表达和定位. 结果 LPA-R3主要分布于小鼠子宫内膜腔上皮、腺上皮和部分基质细胞的胞质.LPA-R3 mRNA和蛋白在小鼠胚胎着床过程中,3d时开始上升,4d时达峰值.5d、6d骤然下降至基础水平.假孕组子宫内膜LPA-R3mRNA和蛋白表达水平及变化规律与相应同期真孕组一致. 绪论 LPA-R3可能参与了胚胎的着床和子宫内膜容受性的建立过程.     

白血病抑制因子在小鼠子宫内膜不同部位的表达研究

目的探讨白血病抑制因子(LIF)在着床窗口期小鼠子宫内膜不同部位的表达。方法运用半定量逆转录聚合酶链反应(RT-PCR)和免疫组化技术,分别从mRNA水平和蛋白质水平检测LIF在孕4d小鼠子宫内膜着床位点及着床旁组织表达量及位置的分布。结果LIFmRNA及蛋白在胚泡着床位点较着床旁组织表达明显增高(P<0.05)。免疫组化检测结果显示LIF蛋白表达于子宫内膜间质细胞及腺上皮细胞胞桨。结论在着床窗口期,LIF作用的发挥主要是通过在着床位点高表达而促进胚泡着床、胚胎发育。     

Lefty is expressed in mouse endometrium in estrous cycle and peri-implantation period

BACKGROUND: Reproductive tissues are unique structures that exhibit cyclic stromal remodelling during menstrual cycles in humans. Ebaf/lefty participates in tissue remodelling of human endometrium by induction of matrix metalloproteases (MMP). METHODS: We describe the temporal expression and spatial distribution of lefty and tissue remodelling events in mouse endometrium. RT-PCR and real-time PCR were used to identify mRNA expression and western blots to analyse Lefty protein. Immunolocalization was performed with specific antibodies and horseradish peroxidase staining. RESULTS: Lefty was expressed in endometrium throughout the estrous cycle. Expression of MMP (MMP-2, -3, -7 and -14) was higher at estrus, metestrus and/or diestrus while collagen content of endometrium decreased in these phases. During pregnancy, lefty levels were higher on days 3-5 and were minimal by day 9. Similarly, expression of endometrial MMP was higher on days 3 and 5 of pregnancy and was low on day 9. During pregnancy, loss of collagen was initiated on day 3, persisted to day 5, and led to a significantly reduced collagen on day 9. Immunoreactive lefty decorated basal laminae, and was associated with extracellular matrix in stroma. CONCLUSIONS: Regulated expression and spatial distribution of lefty in mouse endometrium confines its biological impact on tissues that undergo remodelling during estrous cycle and pregnancy.