HSP70在早期妊娠大鼠子宫中的表达及己烯雌酚的诱导作用

目的探讨在早期妊娠大鼠子宫中热休克蛋白70(hearshockprotelns,HSP70)的表达变化以及己烯雌酚(dithylstilbestrol,DES)对子宫HSP70表达的诱导作用。方法采用免疫组织化学和图像分析技术研究正常大鼠未孕及已孕的子宫。结果①孕鼠子宫HSP70的阳性表达较未孕鼠明显增强,差异有显著性(P<0.01);②小剂量(1.25滋g/50g)大剂量(5滋g/50g)DES均可诱导大鼠子宫HSP70免疫反应阳性细胞数量显著增加(P<0.01)。结论热休克蛋白70在早期妊娠大鼠子宫内膜固有层的基质细胞及蜕膜内蜕膜细胞的表达明显增高,DES对子宫HSP70。表达具有明显诱导作用。     

ATF4基因在小鼠胚胎围着床期子宫内膜的表达

目的 探讨转录激活因子4(ATF4)在小鼠胚胎围着床期子宫内膜中的表达规律及在胚胎植入过程中的作用.方法 妊娠0～7d小鼠子宫内膜,用RT-PCR、免疫组织化学法和Western blot分别检测小鼠内膜ATF4 mRNA及蛋白的表达;用子宫角内注射ATF4抗体进行干预.结果 1)妊娠各组小鼠子宫内膜ATF4 mRNA的表达量均显著高于未孕组d0(P ＜0.0S),且随妊娠天数的增加其表达量逐渐增高,到d4达到峰值,之后逐渐下降;2)ATF4蛋白主要在基质细胞胞质表达,其表达规律与mRNA表达规律基本一致.3)子宫角ATF4抗体注射后与对照组相比着床数明显减少.结论 ATF4在小鼠妊娠早期可能参与子宫内膜蜕膜化过程,有利于着床.     

子宫内膜基质细胞AQP7的表达以及卵巢激素对AQP7的调控作用

目的:探讨水通道蛋白7(Aquaporin 7,AQP7)在小鼠子宫内膜及在体外诱导蜕膜化模型中的表达,卵巢激素对AQP7的调控,以探讨AQP7在子宫蜕膜化中的作用.方法:分离孕4-8天小鼠子宫基质细胞(Uterine stromal cells,ESCs),实时定量PCR(RT-PCR)检测基质细胞AQP7表达.收集孕4～8天小鼠子宫,免疫组织化学染色检测子宫内膜组织AQP7的表达.分离原代小鼠基质细胞,建立体外诱导蜕膜化模型,RT-PCR检测AQP7在ESCs中的表达.在体外培养的孕5天小鼠子宫基质细胞中分别加入雌二醇(E2)、孕酮(P4)以及E2和P4联合处理.用雌激素受体(ER)的拮抗剂ICI 182,780和孕激素受体(PR)拮抗剂RU486预处理基质细胞,再用E2+P4处理.收集各组ESCs,RT-qPCR检测卵巢激素对基质细胞中AQP7的调控作用.结果:AQP7 mRNA在体外分离的孕4～8天的子宫基质细胞中的表达逐渐升高.从孕5～8天,随着蜕膜化进程,子宫基质细胞中AQP7表达也逐渐增加.在体外诱导蜕膜化模型中,小鼠基质细胞中AQP7 mRNA表达显著升高(P<0.01).在E2和P4单独作用下,基质细胞中AQP7表达无明显变化;而与对照组比较,E2和P4联合处理组在E2和P4联合作用24小时后基质细胞中AQP7表达显著升高(P<0.01);且在E2和P4联合作用后、基质细胞中AQP7的升高能够被雌激素受体的拮抗剂ICI 182,780和孕激素受体拮抗剂RU486阻断(P<0.01).结论:随着蜕膜化进程,子宫基质细胞中AQP7表达逐渐增加.卵巢雌、孕激素联合作用可上调基质细胞中AQP7表达.子宫基质细胞中AQP7在蜕膜化和胚胎着床中发挥着重要作用.     

RhoA在小鼠胚胎围着床期子宫内膜的表达及其意义

目的探讨RhoA在小鼠胚胎植入过程中的表达及其生物学作用。方法取胚胎围种植期（D0至D6天）昆明小鼠子宫作切片，用免疫组织化学方法和图像分析方法对RhoA在子宫内膜中的表达进行定位和半定量分析。结果RhoA主要分布于小鼠子宫内膜腺上皮、腔上皮和基质细胞。受精后，RhoA在子宫内膜的含量高于未孕组（P＜0．05）；受精后第4d（植入期），小鼠子宫内膜腔上皮中RhoA的含量明显增多，明显高于未孕及植入前各时间组（P＜0．05），并逐渐向基质细胞延伸，第5d广泛分布在蜕膜细胞和基质细胞，到第6天大部分子宫内膜基质区域及腺上皮的细胞中有大量的RhoA分布。RhoA在小鼠子宫内膜组织中的表达强度在受精后呈逐渐上升趋势。结论①RhoA可能参与了胚胎的种植和子宫内膜容受性的建立；②还可能间接参与植入后胚胎的滋养层扩展、分化及子宫内膜蜕膜化反应。     

高胰岛素增加早孕小鼠子宫内膜蜕膜化进程中血管紧张素Ⅱ的表达

目的探讨高水平胰岛素对早孕小鼠子宫内膜蜕膜化进程中血管标志分子血管紧张素Ⅱ(Ang-Ⅱ)的影响。方法皮下注射胰岛素构建高水平胰岛素动物模型。将其与正常雄鼠交配后收集妊娠第6、7和8天(D6、D7和D8)的子宫组织及血清,并监测血糖。另将其与输精管结扎的雄鼠交配构建人工诱导蜕膜化模型,于假孕D8收集子宫组织。ELISA检测血清中胰岛素及雌、孕激素的变化,real-time PCR检测子宫内膜Ang-ⅡmRNA的表达,Western blot对子宫内膜Ang-Ⅱ蛋白表达进行检测。结果高胰岛素模型中血清胰岛素水平于妊娠D6、D7和D8均明显升高(P0.001)、雌激素水平于妊娠D8出现显著下降(P0.05),孕激素水平于妊娠D6、D8出现明显下降(P0.01)。与对照组比较,高胰岛素模型中子宫内膜组织中Ang-Ⅱ的mRNA及蛋白的表达显著增加(P0.05)。结论高胰岛素增加孕鼠蜕膜化进程中子宫内膜组织Ang-Ⅱ的表达,从而影响子宫内膜蜕膜化进程中的血管生成。     

小鼠子宫内膜基质细胞分离培养、鉴定及体外蜕膜化的诱导

背景：国内已建立许多有关人的子宫内膜基质细胞分离培养及蜕膜化的诱导方法,但人的子宫标本存在取材困难等问题。 目的：建立小鼠子宫内膜基质细胞的分离培养及体外蜕膜化的诱导方法,为进一步研究子宫内膜蜕膜化的机制提供一种简单、有效的模型。 方法：通过胰酶与胶原酶Ⅱ消化及差时贴壁等方法,分离和纯化小鼠妊娠第4天的子宫内膜基质细胞;用细胞免疫化学方法鉴定基质细胞;通过孕酮和雌二醇联合处理诱导子宫内膜基质细胞的蜕膜化,然后用苏木精染核、实时定量PCR和免疫印迹方法检测蜕膜化标志分子的变化。 结果与结论：①分离培养的子宫内膜基质细胞的存活率为(90.1±2.3)%。②细胞免疫化学方法染色显示,分离纯化的子宫基质细胞Vimentin染色呈阳性,而Cytokeratin染色呈阴性,细胞纯度可达(92.3±2.4)%。③孕酮和雌二醇处理48,72 h后,苏木精染色显示细胞呈现多核化,蜕膜化标志分子dPRP、周期蛋白D3、孕酮受体mRNA随着培养时间延长均明显增加(P < 0.05)。结果可见采用酶消化和差时贴壁方法,可获得高纯度的子宫内膜基质细胞;子宫内膜基质细胞在体外通过孕酮和雌二醇联合诱导可产生蜕膜化。     

小鼠子宫内膜中鸡冠珊瑚树凝集素和双花藕豆凝集素受体在胚泡植入过程中的表达

目的 探讨小鼠子宫内膜鸡冠珊瑚树凝集素(ECL)受体和双花藕豆凝集素(DBA)受体与胚泡植入的关系.方法 以生物素标记的ECL和DBA来检测怀孕侧和输卵管结扎未孕侧小鼠孕早期子宫内膜中两种凝集 素受体的分布状况和变化规律.结果怀孕侧,ECL主要表达于胚胎滋养层细胞及其基膜,蜕膜细胞及其周围的细胞外基质(ECM);未孕侧,主要表达于子宫内膜上皮和腺上皮.在怀孕侧,DBA受体主要表达于胚胎滋养层细胞和子宫内膜血管基膜;在未孕侧,主要表达于子宫内膜血管的基膜.结论 ECL和DBA受体与小鼠胚泡植入过程密切相关.     

自然流产模型小鼠蜕膜细胞凋亡及Bcl-2、Bax表达的研究

目的：通过比较正常妊娠模型小鼠及自然流产模型小鼠蜕膜细胞凋亡及Bcl-2、Bax蛋白的表达，从细胞及分子水平探讨自然流产的发病机制。方法：建立正常妊娠模型CBAXBALB／c和自然流产模型CBAXDBA／2。用免疫组化SABC法测定两组模型孕13天蜕膜细胞Bcl-2和Bax蛋白的表达，并通过MIAS-2000医用彩色病理图像免疫组化测量系统对其表达进行半定量分析，其结果用平均灰度值表示；同时应用DNA缺口原位末端标记技术（TUNEL）测定两组模型孕13天蜕膜细胞凋亡情况。结果：与正常妊娠模型相比，自然流产模型蜕膜细胞Bcl-2蛋白的表达降低（P〈0．01）；Bax蛋白的表达明显升高（P〈0．01）。蜕膜细胞凋亡指数，自然流产模型明显高于正常妊娠模型（P〈0．01）。结论：早孕期蜕膜组织细胞凋亡异常是自然流产的机制之一，Bcl-2／Bax途径可能是诱导早孕期蜕膜细胞凋亡的重要因素。     

CXCR2在妊娠早期小鼠子宫内膜的表达

目的研究CXCR2在妊娠早期小鼠子宫内膜的表达情况.方法应用免疫组织化学和图像分析技术,观察CXCR2在妊娠1d、4d、5d、6d小鼠子宫内膜的表达部位及蛋白水平的变化规律;HE染色后光镜下观察子宫内膜的形态学变化.结果免疫组化染色结果显示:在子宫内膜腔上皮,CXCR2于妊娠4d表达最强,妊娠5d开始逐渐下降,妊娠6d降至妊娠1d水平;在子宫内膜腺上皮和基质,CXCR2的表达水平随妊娠天数的增加而逐渐升高.HE染色可见妊娠1d子宫内膜腔上皮和基质中有大量中性粒细胞浸润;妊娠4d腔上皮中未见中性粒细胞,基质中则有大量中性粒细胞浸润;妊娠5d和妊娠6d腔上皮中未见中性粒细胞,基质中中性粒细胞数量甚少.结论CXCR2可能通过与其配体白细胞介素-8结合而参与小鼠胚泡着床过程.     

子宫内膜、正常早孕和自然流产蜕膜组织中△Np63的表达

目的观察△Np63蛋白在自然流产蜕膜组织中的表达，探讨△Np63在胚泡植入和早期妊娠维持中的作用。方法收集自然流产的蜕膜27例组织（其中胚胎停止发育23例、习惯性流产1例及不全流产3例），正常早孕蜕膜66例，非妊娠期子宫内膜组织57例（其中增生期33例，分泌期24例），用SP免疫组化染色法观察△Np63的表达。结果△Np63广泛表达于腺上皮和间质细胞中。增生期和分泌期子宫内膜中△Np63主要表达在腺上皮，蜕膜组织中△Np63在间质蜕膜细胞中的有较强的表达。流产组△Np63蛋白阳性率为55．6％，明显低于增生期的90．9％、分泌期的87．5％及正常早孕组的86．4％（P〈0．05）。△Np63高表达率在分泌期组及流产组分别为16．7％和11．2％，明显低于增生期的63．6％和正常早孕组的57．6％（P〈0．01）。结论△Np63可能参与子宫内膜的生理周期变化，并与子宫内膜向早孕蜕膜组织转化及维持早孕关系密切。     

热休克蛋白70、90在子宫内膜癌中的表达

目的:探讨子宫内膜癌组织中热休克蛋白(HSP)70、90的表达及意义。方法:用免疫组化Envision法及图象分析仪,检测 30例正常子宫内膜、30例增生过长子宫内膜和53例子宫内膜癌中HSP70、HSP90的表达。结果:子宫内膜癌中HSP70表达的灰度值为(209.06±5.36),明显高于正常内膜[(145.21±4.09),P<0.01]和增生过长内膜[(148.59±4.23),P<0.01];子宫内膜癌中HSP90表达的灰度值为(166.98±5.71),明显低于正常子宫内膜[(208.57±31.14),P<0.05]和增生过长子宫内膜[(249.73±4.94),P<0.01]。子宫内膜癌中HSP70的表达随肿瘤病理分级的增加而增强(P<0.01),非内膜样癌(229.90±3.77)较内膜样癌表达强[(198.37±3.15),P<0.01];子宫内膜癌中HSP90表达随肿瘤病理分级的升高而表达减弱,非内膜样癌(140.21±3.22)较内膜样癌表达减弱[(176.59±2.79),P<0.01]。子宫内膜癌中HSP70、90的表达与肌层浸润深度、临床分期及淋巴结转移未见显著相关性(P>0.05)。结论:HSP70、90可能与子宫内膜癌的发生及预后有关。     

Expression of interleukin-11 during the human menstrual cycle: coincidence with stromal cell decidualization and relationship to leukaemia inhibitory factor and prolactin

Interleukin-11 (IL-11) is crucial in the decidualization response of the uterine stroma to the implanting blastocyst in the mouse. This study examined the localization and expression of IL-11 in human endometrium throughout the menstrual cycle and of prolactin and leukaemia inhibitory factor (LIF) in secretory phase endometrium. The mRNA expression of IL-11 receptor alpha and the signalling component, gp130, in endometrial tissue were also determined. Immunoreactive IL-11 was highest in the secretory phase and present in decidualized stromal cells, glandular epithelial cells, endothelial and smooth muscle cells, and the mRNA expression was verified by in-situ hybridization. Decidual cells showed the most intense staining. IL-11 receptor alpha and gp130 mRNA were detected throughout the cycle with minimal variation. Expression of IL-11 mRNA and protein preceded that of prolactin. While immunoreactive prolactin was found in stromal, decidual and glandular epithelial cells, prolactin mRNA was confined to decidual cells. In contrast, endometrial LIF expression preceded IL-11 but was largely confined to the glandular epithelium. The sequence of appearance of LIF, IL-11 and prolactin suggests a synchronized role for each in the differentiation of the endometrium. The cyclical changes and cell type specific expression of IL-11 suggests a potential role in the decidualization of stromal cells.     

热休克蛋白在子宫内膜异位症中的表达及意义

目的 探讨热休克蛋白27（HSP27）和HSP70在子宫内膜异位症发病中的作用。方法 采用免疫组化链霉菌抗生素蛋白－过氧化物酶染色法（SP法），检测子宫内膜异位症中56例异位内膜与在位内膜（30例）中HSP27和HSP70的表达。采用原位杂交，检测HSP70mRNA的水平，并与正常子宫内膜相比较。结果 免疫组化结果显示，异位内膜组织中HSP27与HSP70均呈高表达，与正常内膜组的表达差异显著（P＜0．01），而且失去其在正常内膜组织中表达的周期性变化。卵巢子宫内膜异位症组（OEM）与子宫腺肌症组（AM）组中，HSP27和HSP70的表达无显著差异（P＞0．05）。原位杂交结果显示，HSP70 mRNA（分别为P＜0．01和P＜0．05）。HSP27与HSP70的表达水平，同OEM的严重性无关。结论 异位内膜组织中的HSP70基因被激活，转录增加。HSP27和HSP70呈高表达，可能在EM的发病中起重要作用。     

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。     

Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization

The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.     

Heat shock proteins in human endometrium throughout the menstrual cycle

Human endometrium is a steroid-sensitive tissue and there isevidence that supports the viewpoint that heat shock proteins(HSP) are implicated in the regulation of steroid function.Therefore, in this study we examined the expression of variousmembers of the heat shock family of proteins in the steroid-responsivehuman endometrium. Western blot analysis revealed that the expressionof HSP90 showed minimal changes throughout the menstrual cycle.When normalized to the amount of HSP90, the expression of HSP27,HSP60 and the constitutive form of heat shock protein 70 (HSC70)increased progressively during the late proliferative and earlysecretory phases, and diminished in the mid- to late secretoryand menstrual phases. In contrast, the inducible form of heatshock protein 70 (HSP70) did not undergo these changes. Thecellular and subcellular localizations of these proteins wereexamined in human endometria by immunohistochemical staining.With the exception of HSP70, which was found primarily in theepithelial cells, the immunoreactivity for other heat shockproteins was found in both the stroraa and the epithelium. Immunoreactivityfor HSP27 was found in the lymphoid aggregates within endometrialstroma, and both HSP27 and HSP90 were found in endothelial cells.The immunoreactive heat shock proteins were found in the nucleiand/or cytoplasm of cells. However, no consistent nuclear versuscytoplasmic staining emerged, and such localization was irrespectiveof the site, the cell type or the phase of the menstrual cycle.Our findings show that endometrium has a full complement ofheat shock proteins. The menstrual cycle-dependent changes inthe amounts of heat shock protein suggest regulation by steroidhormones.     

The effect of hormone replacement therapy on the immunoreactive concentrations in the endometrium of oestrogen and progesterone receptor, heat shock protein 27, and human beta-lactoglobulin

We determined the expression of oestrogen receptor (ER), progesterone receptor (PR), heat shock protein 27 (HSP27) and human beta-lactoglobulin in the endometrium under hormone replacement therapy (HRT). The immunohistochemical expression during the late progestogenic phase of sequential HRT was compared semi-quantitatively and using image analysis, to the early, mid-, and late luteal phase of the physiological cycle. Under sequential HRT, smaller glands were positive for the ER but larger glands with more advanced secretory features were negative. ER expression was lower in the stroma under HRT, and the difference was statistically significant compared with the early luteal phase (P < 0.05). Expression of HSP27 under HRT was lower in the epithelium but higher in the stroma compared with the physiological luteal phase. Epithelial PR expression was lower under HRT compared with the early, but not the mid- or the late luteal phase. The number of PR-positive stromal cells under HRT was lower compared with the physiological cycle, and the difference was statistically significant in comparison with the early luteal phase (P < 0.05). The glandular area expressing human beta-lactoglobulin during the late progestogenic phase was statistically significantly higher compared with the early, but lower in comparison with the mid- or the late luteal phase (P < 0.05). The study demonstrates a sub-physiological progestogenic response superimposed on evidence of a hypo-oestrogenism, and a differential response in the epithelium and stroma.