沙眼衣原体感染对大鼠早期妊娠的影响

目的检测沙眼衣原体(CT)感染后妊娠大鼠子宫内膜热休克蛋白70(HSP70)和整合素α4β1的表达,探讨CT感染对妊娠的影响.方法取正常未孕大鼠、阴道接种CT后妊娠大鼠和正常妊娠大鼠孕4～8d子宫作切片,采用免疫组织化学方法,研究CT感染对早期妊娠子宫HSP70和整合素α4β1表达的影响.结果 HSP70在未孕及妊娠大鼠子宫均有表达,其主要存在于子宫内膜固有层的基质细胞及蜕膜细胞,整合素α4β1存在于内膜上皮、腺上皮和基质细胞.CT感染后(实验组)妊娠大鼠子宫内膜HSP70的阳性表达在孕4～6d较正常妊娠组(对照组)及未孕组明显增强,差异有显著性(P< 0.01);在孕7～8d与对照组相比无明显差异,但强于未孕组.而整合素α4β1的阳性表达在孕4～6d弱于对照组(孕第5d尤为明显),强于未孕组,差异有显著性(P< 0.01).结论 (1)大鼠生殖道感染CT引起胚泡着床期(4～6d)HSP70的高表达,且主要存在于子宫内膜固有层的基质细胞及蜕膜细胞,内膜上皮、腺上皮中未见表达,推测CT生殖道感染可能影响母体子宫内膜蜕膜细胞的增殖,干扰妊娠,引起不孕或流产.(2)生殖道感染CT引起胚泡着床期整合素α4β1的低表达,并与HSP70表达的变化趋势相反,推测CT生殖道感染可影响孕早期胚泡植入,其不良妊娠结果可能通过HSP70与整合素α4β1的共同作用导致.     

沙眼衣原体感染对大鼠着床期间子宫内膜上皮及其β-连接素表达的影响

目的研究沙眼衣原体(Chlamydiatrachomatis,Ct)感染后大鼠着床期间子宫内膜形态学改变及β-连接素(β-catenin,β-cat)的变化。方法大鼠雌雄合笼建立早孕模型,取正常妊娠和Ct感染后妊娠大鼠子宫,采用扫描电镜(SEM)观察子宫内膜超微结构改变及免疫组化检测β-cat的表达。结果SEM显示对照组子宫内膜腔上皮细胞排列紧密,细胞间隙较窄;实验组腔上皮细胞排列疏松,间隙变宽;免疫组化定性结果示β-cat妊娠4-6d主要定位于内膜上皮细胞膜,妊娠7d在蜕膜细胞膜;半定量结果示在感染组与正常组妊娠第5d其表达量达到高峰,但感染组弱于正常组,且两者之间有显著性差异(P<0·05)。结论Ct感染后可能通过影响子宫内膜黏附分子的表达,破坏着床的微环境;同时影响上皮细胞之间的连接,从而干扰早期妊娠。     

CT感染大鼠子宫内膜植入窗期基质金属蛋白酶的表达

目的 通过大鼠生殖道沙眼衣原体（chlamydia trachomatis,CT）感染模型研究CT初次感染后妊娠大鼠在子宫内膜植入窗期基质金属蛋白酶-9（MMP-9）及金属蛋白酶组织抑制物-1（TIMP-1）的表达对胚胎着床的影响。方法选择成年雌性SD大鼠30只,随机均分为对照组和感染组,感染组通过阴道接种CTD型株,而后在妊娠大鼠的植入窗期（即妊娠第5、6、7天）分别处死大鼠,采用链菌生物素蛋白一过氧化酶连接法和免疫组化测定两组子宫内膜种植窗期MMP-9及TIMP-1。采用HPIAS-1000高清晰度彩色病理图文小报告管理系统测定感染组和对照组中MMP-9＼T1MP-1在单位面积中各项免疫组织化学反应阳性颗粒的平均光密度值,并通过SPSS软件对数据差异显著性进行分析。结果在两组大鼠的子宫内膜中MMP-9均呈阳性表达。腺上皮细胞、腔上皮细胞和基质细胞胞浆内均可见棕黄色颗粒沉着,上皮细胞染色强度大于基质;感染组的表达较对照组弱,差异有显著性（P〈0．05）。TIMP-1表达特点和MMP-9相似,感染组的表达也较对照组弱,差异有显著性（P〈0．05）。结论生殖道感染CT后,妊娠大鼠子宫内膜植入窗期MMP-9及TIMP-1的低表达,可能是影响胚胎着床、导致不孕的重要因素。     

沙眼衣原体感染后妊娠大鼠子宫组织C3 和Crry mRNA的表达变化

目的通过大鼠生殖道沙眼衣原体(Chlamydia trachomatis,CT)感染模型研究CT初次感染后妊娠大鼠在胚胎着床期子宫局部补体C3和补体调节蛋白Crry的mRNA表达量的变化。方法选择成年雌性SD大鼠36只,随机均分为正常组和感染组,感染组通过阴道接种CT D型株。而后在妊娠大鼠的胚胎植入期(即妊娠第5、6、7d)分别处死大鼠,取子宫组织计数胚胎植入数,通过逆转录扩增(RT-PCR)方法半定量检测在胚胎植入期C3和Crry的mRNA的表达量的变化,并通过SPSS软件对数据差异显著性进行分析。结果1.感染组在植入期C3的mRNA比正常组高表达,差异有显著性(P<0.001);2.Crry的mRNA虽比正常组表达有所增加,但两组数据无显著性差异(P>0.05);3.感染组较正常组平均胚胎植入数下降(P<0.01),并且感染组C3mRNA的表达量与对应的胚胎植入数存在显著的负相关(r=(0.638,P<0.05)。结论大鼠生殖道感染CT后,妊娠大鼠胚胎植入数降低,而这种变化很可能与子宫局部补体C3的高表达及过量的补体活化有关。     

沙眼衣原体感染诱导小鼠生殖道局部促炎性细胞因子的表达情况

建立小鼠生殖道沙眼衣原体感染模型,观察小鼠生殖道局部促炎性细胞因子的表达。将小鼠生物型沙眼衣原体C. muridarum 1＆#215;104 IFU阴道接种于C57B6背景雌性小鼠,取感染后阴道拭子做沙眼衣原体培养,计算IFU,监测小鼠感染和病原体清除情况;80 d后处死小鼠,检测子宫输卵管病理改变;ELISA检测感染过程中小鼠生殖道促炎性细胞因子IL-1α、IL-6、MIP-2和TNF-α产生情况。小鼠感染在第3至第15天维持较高水平,然后病原体被逐渐清除,整个病程约3~5周;病理检测显示子宫输卵有严重炎症、管腔扩张积水,狭窄等;于感染后第3天检测到局部IL-1α、IL-6、MIP-2分泌,第7天达高峰,然后逐渐下降至正常水平（ IL-6于11 d恢复正常,IL-1α和 MIP-2于15 d恢复正常）。 TNF-α仅在第7天检测到高水平表达。相对于TNF-α和IL-6,IL-1α和MIP-2维持时间较长。成功建立沙眼衣原体感染小鼠生殖道模型,沙眼衣原体急性感染可诱导小鼠生殖道局部分泌IL-1α、IL-6、MIP-2和TNF-α。     

大鼠输卵管沙眼衣原体感染后体内树突状细胞的变化

目的观察大鼠输卵管沙眼衣原体(CT)感染后体内树突状细胞(DC)的变化.方法选择成年雌性Wistar大鼠,从卵巢囊接种沙眼衣原体E型株.于感染后第1/2d、7d、14d、21d、28d和第35d处死动物取材,显示输卵管和脾内DC的变化.结果 1. 实验1/2d组输卵管未出现S-100 DC,在实验第7d、14d、21d、28d和第35d组输卵管均有S-100 DC出现;2.感染后脾淋巴小结数量增多,体积较大,淋巴小结内S-100 DC密集,动脉周围淋巴鞘内S-100 DC增多.结论输卵管CT感染后,输卵管和脾内S-100 DC增加,提示在体内DC对CT具有抗原提呈和启动机体免疫应答的作用,DC具有作为CT治疗性疫苗基础构建的潜力.     

冰片提取物对沙眼衣原体感染HeLa细胞CT703及CT259表达的影响

目的:探究冰片提取物对沙眼衣原体感染后的He La细胞模型CT703和CT259表达的影响。方法:将成功建立的40例感染L2血清型沙眼衣原体的人宫颈癌上皮(He La)细胞模型随机分成A、B两组,分别添加冰片提取物和等剂量生理盐水。观察感染后He La细胞模型的包涵体数目及大小、RNA抽提结果完整性以及CT70与CT259的表达变化。结果:染色后的40例受沙眼衣原体感染的He La细胞模型体内均发现包涵体,在同一时间点B组细胞内包涵体比A组大,且数目比A组多(P0.05);两组提取的总RNA的OD值均在1.8~2.0之间,通过RNA凝胶电泳结果可清楚发现28S、18S及5S条带;同一时间点CT259和CT703扩增产物的平均灰度值比较,A组感染后He La细胞模型样本基因表达量低于B组,差异具有统计学意义(P0.05)。结论:冰片提取物能够有效降低经沙眼衣原体感染的He La细胞模型中CT703与CT259基因表达量。     

MUC16对子宫内膜容受性的影响及其意义

目的：多囊卵巢综合征（Polycystic Ovary Syndrome,PCOS）系由于体内复杂的内分泌和代谢环境影响了子宫内膜稳态,导致子宫内膜容受性下降,造成患者生育力减弱和不良妊娠结局。通过测定多囊卵巢综合征患者子宫内膜与正常生育力妇女增生期、分泌早、中、晚期子宫内膜中MUC16的相对表达量,本文探讨MUC16与PCOS患者子宫内膜容受性下降的关系,为临床上改善PCOS患者子宫内膜容受性,提高PCOS患者的妊娠率,降低流产率提供一条新的可能途径。方法：选择PCOS患者子宫内膜、正常生育力妇女增生期、分泌早、中、晚期子宫内膜各20例,用免疫组化SP法检测MUC16在各组的表达情况。结果：（1）MUC16在月经周期各期均有表达,在分泌中期表达最强。（2）PCOS组MUC16的表达较分泌中期组弱,差异有显著性（P〈0.05）。结论：（1）在子宫内膜中的表达呈周期性变化。（2）PCOS患者子宫内膜中MUC16表达异常可能使子宫内膜容受性下降,推测其与胚胎不能正常着床或着床后发育不良、导致流产有关。     

肥大细胞在大鼠输卵管急性沙眼衣原体感染中的作用

目的　为研究肥大细胞在大鼠输卵管急性沙眼衣原体 (Chlamydialtrachomatis,CT)感染中的作用。方法　选择成年雌性SD大鼠 6 0只 ,通过手术从一侧卵巢囊接种沙眼衣原体D型株 ,对照组接种 2 -SPA缓冲液。分别于感染后第 1/ 2d、第 7d、第 14d将大鼠处死 ,取手术侧的输卵管常规固定、脱水、包埋。结果　S -P法显示 :输卵管局部的CD4 + T细胞和血管内皮细胞粘附分子 (VCAM - 1)的表达均较对照组明显增强 (P <0 0 1)。改良的甲苯胺蓝染色法显示 :感染组肥大细胞较对照组数量有显著性增高 (P <0 0 5 ) ,并且其变化趋势与CD4 + T细胞和VCAM - 1表达的变化趋势一致。结论　可以推测 ,沙眼衣原体感染引起急性输卵管炎时 ,肥大细胞通过促进炎症局部小静脉内皮细胞上VCAM -1的表达 ,诱导CD4 + T细胞的浸润 ;然后分泌IL - 4等细胞因子促进CD4 + T细胞向TH2 细胞方向转化 ,不利于机体清除沙眼衣原体 ,从而使发生局部输卵管病理损伤的可能性增加。     

假定蛋白CT440在沙眼衣原体感染细胞中的定位研究

包涵体膜蛋白在沙眼衣原体致病过程中发挥重要的作用.为确定假定蛋白CT440在沙眼衣原体感染细胞中的定位及特征,本研究采用PCR方法从D型沙眼衣原体的基因组中扩增Ct440基因,克隆入pGEX-6p原核表达载体构建pGEX-6p/Ct440原核表达重组体,重组体转化到XL1-blue大肠杆菌,IPTG诱导表达融合蛋白GST-CT440.纯化后的CT440融合蛋白免疫小鼠制备抗体,间接免疫荧光(IFA)和Western blot测定抗体的特异性.特异性抗体用于分析CT440蛋白在衣原体感染细胞内的定位、表达时相特征及其对衣原体感染的影响.结果表明,CT440蛋白定位于沙眼衣原体包涵体膜上,为沙眼衣原体包涵体膜蛋白;该蛋白在衣原体感染12h后开始表达,直至持续到整个感染周期;转基因在胞浆表达的CT440融合蛋白不影响其后的衣原体感染.本实验为深入研究衣原体与宿主细胞间的相互作用,阐明衣原体致病机制提供了重要的实验依据.     

Calcitonin down-regulates E-cadherin expression in rodent uterine epithelium during implantation

Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium homeostasis, is transiently expressed in the receptive rat and human endometrial epithelia within the window of implantation. Attenuation of uterine CT expression using antisense methods severely impaired implantation in the rat. The molecular pathway of CT in the pregnant uterus, however, remains unknown. In the present study, we investigated the cellular events following the binding of CT to its membrane receptors in human endometrial epithelial cell line Ishikawa. We observed that CT treatment triggers a transient rise in intracellular calcium in these cells. Most interestingly, CT treatment also led to the disappearance of E-cadherin, a critical cell adhesion molecule, from cell-cell contact sites. Blockade of intracellular calcium release by BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl) prevented the CT-induced disappearance of E-cadherin. Our studies further revealed that CT treatment markedly down-regulates the level of E-cadherin mRNA in Ishikawa cells. We also examined whether CT influences the expression of E-cadherin mRNA in intact rat uterine tissue during implantation. In pregnant rats, high levels of E-cadherin mRNA were expressed during the first 3 days of gestation when the CT mRNA in uterine epithelial cells is undetectable. Concomitant with a transient burst of CT expression during days 4-5 of pregnancy, the level of E-cadherin mRNA declined sharply. Furthermore, administration of exogenous CT to animals on day 2 of pregnancy led to a premature suppression of E-cadherin mRNA level on day 3, indicating a direct link between elevated levels of uterine CT and the down-regulation of E-cadherin expression in the surface epithelium. Collectively, our results are consistent with the hypothesis that CT-induced reduction in E-cadherin expression may remodel the adherens junctions between epithelial cells, and this change in epithelial cell phenotype might be a critical event during the implantation of the blastocyst.     

PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.     

Hormonal and local factors control the immunohistochemical distribution of immunocytes in the rat uterus before conceptus implantation: effects of ovariectomy, fallopian tube section, and injection.

The distribution of immunocytes in the rat uterus undergoes profound changes during early pregnancy. This study was designed to evaluate the respective contributions of hormonal and local factors to regulation of the distribution and number of MCA341+ monocyte-macrophage antigen-bearing cells and T-lymphocyte-polymorphonuclear leukocyte (PMN) antigen-bearing cells before and during implantation of the fertilized ovum. Immunohistological data in normal rat pregnancy were compared to those found in cycling rats, ovariectomized rats, pseudopregnant rats (the oviducts of which had been sectioned on Day 0.5 of pregnancy), and pregnant rats injected with the antiprogesterone RU-486 on Day 0.5 of pregnancy. Four major events were observed: (1) transient accumulation of T-lymphocyte-PMN antigen-bearing cells in the endometrium close to the lumen and occurring only in the pregnant state 12 h after mating; (2) accumulation of an MCA341+ antigen-bearing monocyte-macrophage subset in the uterus, especially the luminal endometrium, 12 h after ovulation in pregnant as well as cycling rats; (3) progressive disappearance of these labeled cells starting 1 day after ovulation in the pregnant and nonpregnant states and influenced by RU-486 injection; (4) relative persistence of labeled cells in the deep endometrium before the implantation of the conceptus--which requires the presence of fertilized ovum in the genital tract. In conclusion, a complex multifactorial and sequential control of the distribution and number of cells bearing MCA341+ monocyte-macrophage or T-lymphocyte antigens appears to be at work before and during implantation of the rat conceptus, and may involve hormonal factors as well as local factors produced by the embryo or trophoblastic cells.     

E-cadherin在人输卵管妊娠蜕膜组织中的表达

目的探讨E-cadherin在人输卵管妊娠蜕膜组织中的表达及其临床意义。方法应用免疫组织化学方法检测输卵管妊娠蜕膜组织中E-cadherin的表达,同时以正常输卵管粘膜为对照。结果E-cadherin在输卵管妊娠组中的表达低于正常输卵管组,差异有显著性(P<0.05)。结论E-cadherin的低表达可能是引起人输卵管妊娠的因素之一。     

Changes in equine endometrial oestrogen receptor alpha and progesterone receptor mRNAs during the oestrous cycle, early pregnancy and after treatment with exogenous steroids

Two experiments were performed to determine changes in the abundance of oestrogen and progesterone receptor (ER alpha and PR) mRNAs in equine endometrium during the oestrous cycle and early pregnancy, and under the influence of exogenous steroids. In Expt 1, endometrial biopsies were obtained from non-mated mares during oestrus and at days 5, 10 and 15 after ovulation, and from pregnant mares at days 10, 15 and 20 after ovulation. There were overall effects of day on the abundance of ER alpha (P = 0.0001) and PR (P = 0.0014) mRNAs. The amount of ER alpha mRNA decreased at day 10 of pregnancy, and PR mRNA was reduced at day 5 in non-mated mares and at day 15 of pregnancy, compared with oestrous values. Experiment 2 was conducted to determine the effects of exogenous steroids on endometrial ER alpha and PR mRNAs. Endometrial biopsies were obtained from 19 anoestrous mares that had been treated with vehicle, oestradiol, progesterone, or oestradiol followed by progesterone for either a short or a long duration. The steroid treatment affected the abundance of ER alpha mRNA (P = 0.0420), which was higher (P < 0.05) in the oestradiol group than in the group treated with oestradiol followed by long duration progesterone. The steroid treatment did not affect the abundance of PR mRNA. These results demonstrate that the amount of steroid receptor mRNA changes with the fluctuating steroid environment in the uterine endometrium of cyclic and early pregnant mares, and that the duration of progesterone dominance may affect ER alpha gene expression. In addition, factors other than steroids may regulate ER alpha and PR gene expression in equine uterine endometrium.     

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.     

Gene expression of WNTs, β-catenin and E-cadherin during the periimplantation period of pregnancy in pigs--involvement of steroid hormones

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E₂) and progesterone (P₄) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E₂ on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E₂ and P₄ level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.