Le^Y寡糖单克隆抗体对小鼠胚泡基质金属蛋白酶表达和分泌的影响

以LeY 寡糖特异性单克隆抗体AH6为工具中和胚泡表面LeY 寡糖后 ,通过RT PCR、明胶酶谱法、免疫印迹法等方法 ,在体外研究了着床前小鼠胚泡表面LeY 寡糖抗原与其基质金属蛋白酶 (MMP)、金属蛋白酶组织抑制因子 (TIMP1)的表达和分泌之间的关系。结果显示 :胚泡表面LeY 寡糖抗原被中和后仅 1.5h ,胚泡MMP2和MMP9基因转录表达明显下降、而TIMP1基因转录表达则略有升高 ;随后抗体中和引起胚泡MMP2、MMP9的分泌减少 ,而TIMP1的分泌则未见明显变化。结果表明胚泡表面的LeY 寡糖抗原对着床前胚泡的MMP的合成和分泌具有调节作用 ,而且这种作用可能主要是通过调节相应的MMP2和MMP9基因的表达而引起的     

胚泡表面Le^Y寡糖对小鼠胚泡表皮生长因子及其受体表达和分泌的影响

在胚胎与子宫内膜间识别、粘附及胚胎植入过程中 ,阶段特异表达的寡糖抗原LeY 起重要作用。经与特异性单克隆抗体AH6预保温 ,中和胚泡表面LeY 寡糖后 ,通过RT PCR和免疫印迹方法 ,观察了LeY 寡糖对着床前小鼠胚泡表皮生长因子及其受体的表达和分泌的影响。结果显示 ,在体外培养中 ,经AH6封闭胚泡表面的LeY寡糖后 1.5h ,胚泡EGF的转录及分泌明显受到抑制 (P <0 .0 1) ,并且这种抑制作用持续到 6h以上 ;而EGF R表达及分泌仅有轻微降低的趋势。结果提示 ,EGF表达和分泌的降低可能是胚泡表面LeY 寡糖调节胚泡自身发育及随后的着床过程的多种途径之一     

白血病抑制因子与哺乳动物的胚胎着床(英文)

胚胎着床是处于活化状态的胚泡与处于接受态的子宫相互作用,最后导致胚胎滋养层与子宫内膜建立紧密联系的过程。已证实白血病抑制因子(LIF)在哺乳动物胚胎着床过程中起着十分重要的调节作用。LIF通过其受体及信号传递亚单位gp130发挥其生物学功能。LIF对胚胎发育到胚泡阶段及以后内细胞团和滋养层细胞的生长和分化有明显的促进作用。 在小鼠中,LIF及其受体和gp130在着床期小鼠子宫内表达量最高,因此LIF可能在小鼠胚胎着床过程中起重要作用。在人中,LIF在子宫内膜中的表达与人胚胎着床的时间一致,提示LIF可能与人的胚胎着床紧密相关。此外,LIF在猪、羊、水貂、兔和臭鼬等动物胚泡着床前和着床期的子宫中也都有表达,并在着床期出现峰值。因此,LIF也可能在这些动物的胚胎发育和着床过程中有重要作用。LIF受体基因敲除小鼠表现为胎盘发育不全,这说明LIF对小鼠胎盘形成和胎盘的功能维持起重要作用。 小鼠子宫中LIF的表达可能受雌激素而上调。美洲长尾猴(绒)及兔子宫中LIF的表达则呈孕酮依赖性。然而孕酮可抑制人着床期子宫内膜腺上皮和蜕膜组织内LIF的表达。在不同种类的动物中,LIF在子宫中的表达有不同的调节机制。 胚泡在LIF基因敲除的雌鼠子宫内不能着床的原因并不是由于胚泡发育异常,而是由于雌鼠不能表     

白细胞介素1β对胚泡植入前后子宫基质金属蛋白酶2(MMP2)表达的影响

采用半定量RT-PCR、免疫组化等方法,对处于胚泡植入前后时期SD妊娠大鼠体内注射超生理剂量的白细胞介素1β(IL-1β),检测处理前后妊娠子宫中基质金属蛋白酶2(MMP2)的表达变化,探讨IL-1β对MMP2表达的影响。统计分析结果,妊娠D4及妊娠D9处理组与对照组无明显差异,妊娠D6处理组MMP2表达明显低于对照组(P<0.01);免疫组织化学检测结果,妊娠D4,MMP2主要表达于子宫腔上皮,基质及腺体表达较少;妊娠D6,对照组MMP2表达主要分布于子宫腔上皮,处理组在子宫腔上皮处MMP2表达降低;妊娠D9,MMP2主要表达于子宫蜕膜层。结果表明,妊娠D6,即胚泡植入时,IL-1β可明显抑制子宫腔上皮MMP2的表达。     

NOD1配体抑制人早孕期滋养细胞的侵袭功能

目的:明确固有免疫受体NOD1对人早孕期滋养细胞侵袭功能的调控及对侵袭相关因子分泌的影响。方法:采用免疫细胞化学法鉴定原代滋养细胞NOD1的表达,用transwell侵袭实验检测激活NOD1后滋养细胞侵袭功能的改变,ELISA检测配体刺激后滋养细胞MMP2和MMP9的分泌情况。结果:免疫细胞化学结果显示滋养细胞分离鉴定成功,且原代滋养细胞可以表达固有免疫受体NOD1。使用NOD1的特异性配体及非特异性配体,发现激活NOD1可以抑制滋养细胞的侵袭,且非特异性配体LPS可以下调侵袭相关金属基质蛋白酶分子MMP2和MMP9的分泌,特异性配体i E-DAP仅下调MMP9的分泌而对MMP2的分泌无影响。结论:固有免疫模式识别受体NOD1可以在早孕期滋养细胞表达,可调控滋养细胞的侵袭功能,其激活会导致侵袭相关分子MMP2和MMP9的分泌下降。     

促黄体素释放激素类似物(LH-RH-A)对妊娠大鼠子宫代谢的直接作用

本实验结果表明,胚泡着床点对~3H-尿嘧啶和~3H-亮氨酸的摄取明显高于非着床点子宫部位。LH-RH-A可显著抑制胚泡着床点对这两种同位素的摄取;且对非着床子宫组织也有抑制作用,但抑制程度较弱。进一步证实,着床的胚泡确能产生一种或几种因子,对子宫内膜的分化起重要作用;同时证实,LH-RH-A可通过对RNA和蛋白质合成的抑制作用而直接影响妊娠大鼠子宫的代谢,对胚泡着床点部位的影响尤为明显。     

双炔失碳酯对兔移植胚泡着床的影响

本文用胚泡移植技术验证了双炔失碳酯对子宫内膜的影响是胚泡不能着床的重要原因。将 29只胚龄4d的兔胚泡移植到给药后假孕4d的子宫,结果没有一只胚泡着床;而对照组的5只受卵兔,移植了41只胚泡,有26只着床成功。 为了了解双炔失碳酯对黄体的影响,在5只妊娠兔上,从交配后1—7d与对照组比较血清孕酮浓度的变化,结果表明:对照组的血清孕酮浓度随着妊娠天数显著升高,妊娠第7天时达 17.35±2.12ng/ml,而给药组升高不明显,第7天仅 1.83±1.03ng/ml,为对照组的1/9。已知足够的孕酮浓度对妊娠的维持和胚泡的成功着床具有十分重要的作用。由此推测,双炔失碳酯抑制孕酮的分泌势必影响子宫内膜的发育,从而阻碍胚泡的着床。     

着床期家兔子宫内膜糖蛋白的动态研究

本文以~3H-glucosamine(~3H-GlcNH_2)为前体,观察了着床期家兔子宫内膜糖蛋白的生物合成及分泌功能的变化,并利用Triton X-100提取膜蛋白,采用凝集素亲和电泳技术对三种不同性质糖蛋白进行了动态分析。结果表明,着床期家兔子宫内膜糖蛋白的合成与分泌功能均于妊娠第六天(D_6)明显增强,其中合成糖蛋白的变化以Triton溶解蛋白部分为主;井发现着床期WGA、RCA-Ⅰ、SBA结合蛋白均于着床前(D_6)明显增加,着床后(D_9)WGA结合蛋白略有降低,RCA-Ⅰ、SBA结合蛋白明显降低,接近未孕水平。着床期子宫内膜总体及不同种类糖蛋白的阶段特异性变化表明,在子宫内膜由对胚泡的非接受状态转变为接受态的过程中,阶段特异性糖蛋白可能具有重要作用。     

哺乳动物胚泡着床及影响因素

哺乳动物胚泡着床是生殖过程中的关键环节。受精卵经过早期发育形成了胚泡，胚泡脱去透明带后，经定位、粘附、滋养层侵入，植入到子宫内膜中，同时母体子宫内膜发生蜕膜化控制植入的程度，最终完成着床过程。着床过程受多种因素的影响，主要因素有：母体子宫内膜和胚泡发育的同步化，母体的激素环境，胚泡分泌的激素，母体子宫的接受性及局部免疫保护作用。     

大鼠子宫内膜存在人绒毛膜促性腺激素样(hCG-like)物质结合部位的初步探讨

在胚泡着床过程中,胚泡可以产生hCG样物质,它对于着床过程可能有某种调节作用。本实验结果表明:大鼠胚泡着床前(交配后第四天)的子宫内膜存在hCG特异性结合部位。对子宫内膜及睾丸组织~(125)I-hCG结合性质测定的平行实验中,证明子宫内膜hCG的结合部位与睾丸组织hOG受体有很相似的特性,其亲和常数(Ka)值分别为9.0×10~9M~(_1)和7.7×10~9M~(_1)。子宫内膜存在hCG结合部位可能与着床过程中胚泡与子宫内膜的同步化调节有一定关系。     

小鼠早期胚胎发育期间LIF基因表达的研究(简报)

白血病抑制因子(Leukemia inhibitory fac-tor,LIF)是近年来研究较为广泛的细胞生长调节因子之一。最初发现LIF能够在体外诱导小鼠髓样白血病细胞株M1细胞向正常细胞分化,进一步分离纯化蛋白以及克隆基因后发现LIF在体外还具有多种功能,作用于不同的靶细胞时引起的生理效应也各不相同。目前已知的功能有:刺激肝脏细胞急性期反应蛋白的     

Effects of leukaemia inhibitory factor on embryo implantation in the mouse

Leukaemia inhibitory factor (LIF) is a pleiotrophic cytokine. Recent reports indicate that LIF is relevant to murine embryo implantation. In this work, results of indirect immunofluorescence under a confocal microscope illustrated that LIF was mainly located in the uterine lumen and uterine epithelial cells in pregnant mice on day 4. The number of embryos implanted in pregnant mice on day 8 decreased significantly after injection of 3 microg LIF antibodies into a uterine horn (P<0.001), which demonstrated again that LIF is a critical factor for embryo implantation. In a co-culture system, LIF (0.1 ng/ml, 1 ng/ml, 10 ng/ml and 100 ng/ml) significantly enhanced the blastocyst outgrowth after 24, 48 or 72 h of co-culture, and outgrowth areas after 72 h of co-culture. Conversely, 5 microg/ml and 10 microg/ml, but not 1 microg/ml, LIF antibodies decreased the percentage of blastocysts with outgrowth; only 10 microg/ml LIF antibody inhibited blastocyst outgrowth area significantly (P<0.001). However, neither LIF nor its antibodies changed embryo attachment. Analysis of correlation showed that the effects of LIF or its antibodies on the blastocyst outgrowth were dose-dependent. In summary, different pathways may exist to regulate the blastocyst attachment and outgrowth on a monolayer of uterine epithelial cells. LIF protein from the maternal uterus exerts an essential role in embryo implantation in the mouse, which is mediated by stimulating trophoblast outgrowth, but not by promoting the attachment.     

Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice

Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(-/-) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(-/-) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(-/-) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.     

Leukaemia inhibitory factor and the regulation of pre-implantation development of the mammalian embryo

This paper reviews the evidence that certain growth factors, particularily leukaemia inhibitory factor (LIF), play a crucial role in regulating the development of the pre-implantation mammalian embryo. LIF was originally implicated in regulating the early development of the mouse embryo because it inhibited the differentiation of embryonic stem (ES) cells, pluripotential cells derived from the inner cell mass of the blastocyst. Subsequent studies on its role in vivo revealed, surprisingly, that it is essential for the growth rather than the differentiation of the blastocyst. In vivo, overtly normal blastocysts can be produced in a LIF-deficient environment that are capable of forming viable fertile adults. However, in the absence of LIF, they fail to implant and enter into a state resembling that exhibited by blastocysts undergoing delayed implantation, which is characterized by a cessation of cell proliferation. This failure to implant occurs because the principle sites of LIF production are the endometrial glands of the uterus. These synthesize and secrete LIF at implantation, with LIF synthesis essential for implantation. Preliminary evidence indicates that LIF synthesis is required both by the uterus for it to undergo decidualization and by the blastocyst for implantation. These data indicate that the maternal environment plays a crucial role in the development and growth of the pre-implantation embryo, by supplying factors that regulate these processes in the embryo. © 1994 Wiley-Liss, Inc.     

妊娠小鼠子宫内膜LIF基因表达的研究

本文对妊娠第4天(Ⅰ组)、第7天(Ⅱ组)、第10天(Ⅲ组)的小鼠(各20只)子宫内膜LIF基因表达进行了研究。Ⅰ组20只小鼠子宫内膜全部存在LIF基因的表达、Ⅱ组有5只小鼠表达、Ⅲ组仅有1只小鼠表达。文中对不同孕期LIF基因的表达程度与胚胎着床的关系进行了讨论。 Abstract:Leukemia inhibitory factor(LIF)is a glycoprotein with multiple activities and is essential for blastocyst implantation in mouse.We have examined LIF gene expression in mice endometrium on day 4(group Ⅰ),day 7(group Ⅱ),day 10(group Ⅲ)of pregnancy.In group Ⅰ all had LIF gene expression,5 mice had LIF gene expression in group Ⅱ,only one mouse had LIF gene expression in group Ⅲ.We discussed the relation between level of LIF gene expression and embryonic implantation.     

In vivo effect of leukemia inhibitory factor (LIF) and an anti-LIF polyclonal antibody on murine embryo and fetal development following exposure at the time of transcervical blastocyst transfer

Leukemia inhibitory factor (LIF) enhances in vitro murine preimplantation development in a time- and dose-dependent fashion. Knockout experiments have demonstrated that endometrial LIF is essential for in vivo murine implantation. We assessed the impact of LIF and an anti-LIF polyclonal antibody (pab) on in vivo development and developed a novel and successful nonsurgical method of embryo transfer for this species, a transcervical blastocyst transfer technique. The objectives of this study were to evaluate the effects of LIF and the anti-LIF pab on 1) implantation, resorption, pregnancy, and viability rates and 2) the overall structural and skeletal development. Two-cell embryos were recovered from superovulated mated donors, cultured to the expanded blastocyst stage, and transferred transcervically into pseudopregnant recipients. Exposure to 5000 U/ml LIF resulted in significant increases in implantation, pregnancy, and viability rates compared with controls. A similar dose of pab produced overall inhibitory effects with a significant decrease in implantation rate. Paradoxically, lower pab doses resulted in significantly increased viability rates. Exposure to LIF had no effect on fetoplacental development. However, pab treatments had variable but significant negative effects on placental length, ossification of the exoccipital bone, and vertebral space width compared with controls. Exposure of murine blastocysts to LIF at the time of transcervical transfer resulted in pronounced positive effects on implantation and pregnancy rates without affecting fetal development. A similar pab dose dramatically reduced implantation and pregnancy rates; at high and low doses, pab produced deleterious effects on placental and skeletal development.