雌激素对子宫内膜异位症在位内膜间质细胞β-catenin mRNA的影响

目的:探讨Wnt信号通路在子宫内膜异位症(EMs)发病机制中的作用。方法:体外分离培养内异症患者在位内膜间质细胞,应用RT-PCR检测不同浓度(0mol/L,10-14mol/L,10-12mol/L,10-10mol/L,10-8mol/L,10-6mol/L)17β-雌二醇(17β-E2)作用子宫内膜间质细胞48h和17β-E2(10-10mol/L)作用子宫内膜间质细胞不同时间(0h,12h,24h,48h)后β-catenin mRNA的表达水平。同法检测雌激素受体拮抗剂ICI182,780(10-6mol/L)对17β-E2促进β-catenin mRNA表达的影响。结果:17β-E2体外能明显促进在位内膜间质细胞β-catenin mRNA的表达,并呈剂量和时间依赖性,于10-10mol/L17β-E2作用48h最明显。ICI182,780能明显抑制雌激素对在位内膜间质细胞β-catenin mRNA的表达。结论:Wnt信号通路可能参与了EMs的发生及发展。     

TWEAK在子宫内膜异位症在位和异位内膜上的表达

目的:探讨肿瘤坏死因子样凋亡的微弱诱导剂(TWEAK)在子宫内膜异位症(EMs)发病的关系。方法:采用实时定量逆转录-聚合酶链反应(Real-time RT-PCR)和免疫组化、Western Blot方法检测EMs患者在位内膜、异位病灶中TWEAK mRNA和蛋白的表达,并与正常对照子宫内膜比较。结果:TWEAK蛋白表达于子宫内膜的腺上皮细胞和间质细胞的胞浆内。与正常对照组内膜和在位组内膜相比,TWEAK mRNA和蛋白在异位内膜上表达量下调(P<0.05),且无论是EMs在位内膜还是对照组内膜,其增生期TWEAK mRNA表达明显低于分泌期(P<0.05)。结论:TWEAK在子宫内膜中表达,表达量在分泌期明显升高。EMs患者异位子宫内膜TWEAK表达降低,可能导致子宫内膜细胞的凋亡水平下降,参与EMs的发生发展过程。     

抗孕激素抑制异位子宫内膜间质细胞EGF和GM-CSF表达

目的:探讨抗孕激素治疗子宫内膜异位症的作用机理。方法:分离、传代培养卵巢异位子宫内膜,培养液中分别加入不同浓度的米非司酮和利洛司酮。ELISA检测间质细胞培养上清液中表皮生长因子(EGF)和巨噬细胞集落生长因子(GM-CSF)浓度;RT-PCR检测异位间质细胞GM-CSF的表达。结果:培养72 h后,米非司酮组培养上清中EGF和GM-CSF的浓度显著高于利洛司酮组(P<0.05);异位间质细胞中GM-CSF mRNA电泳带光密度比值,米非司酮组也显著高于利洛司酮组(P<0.05);而两种抗孕激素的空白对照组EGF和GM-CSF及GM-CSF mRNA的表达均显著高于实验组的10-6 mol/L组和10-5 mol/L组(P<0.05)。结论:米非司酮和利洛司酮均可降调节异位子宫内膜间质细胞生长因子EGF和GM-CSF的表达,抗孕激素可能通过此环节抑制异位子宫内膜间质细胞的生长。     

HOXA10基因在子宫内膜异位症不孕患者内膜组织中的表达及意义

目的:探讨HOXA10基因在子宫内膜异位症(EMs)不孕中的作用及作用机制。方法:以行腹腔镜手术并经病理确诊为EMs合并不孕症的患者为研究对象(18例,A组),以同期行腹腔镜手术的输卵管性不孕患者(18例,B组)、正常生育组(15例,C组)为对照。分别应用荧光定量PCR、免疫组织化学和Western blotting等方法从mRNA及蛋白质水平检测HOXA10基因在EMs不孕症患者在位、异位子宫内膜以及对照组在位子宫内膜的表达。结果:HOXA10 mRNA及蛋白表达水平在C组的内膜较高;B组稍低于C组,但两者相比差异无统计学意义(P>0.05);A组的在位及异位内膜表达水平较C组明显降低(P<0.01);但A组的在位内膜与异位内膜基因和蛋白的表达水平均无统计学差异(P>0.05)。结论:HOXA10基因在EMs不孕患者在位子宫内膜中的表达显著降低,可能是EMs不孕患者子宫内膜容受性降低,进而引起不孕的重要因素之一。     

MST1重组腺病毒的制备及对人子宫内膜间质细胞蜕膜化的作用

目的:探讨丝/苏氨酸激酶MST1在人子宫内膜间质细胞蜕膜化过程中的作用。方法:制备Ad-FLAG-MST1重组腺病毒,用于感染人子宫内膜间质细胞;采用荧光定量PCR实验结合腺病毒介导的MST1基因过表达,分析人子宫内膜间质细胞蜕膜化进程中MST1的表达及其对蜕膜化特异性基因的调控。结果:获得滴度为2×1011ifu/ml的重组Ad-FLAG-MST1腺病毒,该腺病毒感染人子宫内膜间质细胞后,可高水平表达FLAG-MST1融合蛋白,过表达的MST1显著增加人子宫内膜间质细胞中PRL、IGFBP1、TIMP3和DCN等蜕膜化特异性基因的表达;体外培养的人子宫内膜间质细胞经8-Br-cAMP(0.5mmol/L)和MPA(1μmol/L)刺激后,细胞中MST1总蛋白水平增加3倍以上,同时磷酸化的MST1(Thr183)显著增加。结论:MST1的活化可能参与子宫内膜间质细胞的蜕膜化相关基因的调控。     

乙酰肝素酶基因在子宫内膜异位症患者在位与异位子宫内膜组织中的表达

目的　研究乙酰肝素酶基因在子宫内膜异位症发病中的作用。方法　应用原位杂交技术 ,检测腹腔镜手术中证实为子宫内膜异位症患者 (EM组 ,2 3例 )的在位和异位子宫内膜组织乙酰肝素酶mRNA的表达 ,并与正常妇女 (对照组 ,2 5例 )子宫内膜组织比较。结果　 ( 1)EM组在位与异位子宫内膜乙酰肝素酶mRNA阳性表达率一致 ,但异位子宫内膜组织中 ,阳性细胞着色较深。在位子宫内膜腺上皮细胞较间质细胞胞浆染色深 ,异位子宫内膜腺上皮细胞与间质细胞胞浆着色基本一致 ;EM组增生期与分泌期子宫内膜均有乙酰肝素酶mRNA的表达 ,增生期阳性表达率为 83 3%( 10 / 12 ) ;分泌期为 72 7% ( 8/ 11) ,两者比较 ,差异也无显著性 (P >0 0 5 ) ;( 2 )对照组增生期子宫内膜乙酰肝素酶mRNA的阳性表达率为 4 1 7% ( 5 / 12 ) ,分泌期子宫内膜阳性表达率为 7 7% ( 1/ 13) ,两者比较 ,差异有显著性 (P <0 0 5 )。 ( 3)EM组子宫内膜乙酰肝素酶mRNA阳性表达率为 78 3% ( 18/2 3) ;对照组为 2 4 0 % ( 6 / 2 5 ) ,两者比较 ,差异也有显著性 (P <0 0 5 )。结论　乙酰肝素酶基因的表达与子宫内膜异位症的发病相关 ,其可能成为子宫内膜异位症治疗的一个有价值的靶点。     

二噁英对子宫内膜异位症患者子宫内膜细胞TNF-α表达的影响

目的:探讨二噁英(TCDD)在子宫内膜异位症(EMs)发病中的作用。方法:通过预实验,加入一定浓度的TCDD分别作用于11例EMs患者离体在位子宫内膜细胞和12例离体对照组子宫内膜细胞24 h,应用Real-time RT-PCR和ELISA法检测TCDD处理前后子宫内膜细胞肿瘤坏死因子-α(TNF-α)mRNA和蛋白的表达。结果:TCDD未处理前,EMs组子宫内膜细胞分泌TNF-α蛋白和mRNA水平均显著高于对照组(P<0.05)。TCDD呈剂量依赖方式促进子宫内膜细胞TNF-α蛋白的分泌,以10 nmol/L TCDD作用最明显(P<0.01),但TCDD对子宫内膜细胞TNF-αmRNA表达均无明显影响(P>0.05)。结论:二噁英可能通过影响子宫内膜细胞TNF-α蛋白水平的表达而参与EMs的发病过程,其主要机制可能不是通过调节其转录途径。     

子宫内膜异位症异位及在位子宫内膜基质细胞RANTES mRNA及蛋白表达

目的探讨子宫内膜异位症(EMS)患者异位及在位子宫内膜基质细胞在白细胞介素-1β(IL-1β)诱导培养条件下RANTES mRNA和蛋白表达模式的变化情况。方法对2006年9月至2007年6月南京医科大学第一附属医院收治的11例EMS患者的异位、在位内膜基质细胞和同期5例正常内膜基质细胞行分离培养,分别加入5、10、20μg/L的IL-1β刺激培养异位内膜基质细胞,选择最佳浓度的IL-1β刺激培养异位、在位和正常内膜基质细胞,采用RT-PCR及ELISA法从转录及转录后水平观察趋化因子RANTES表达模式的变化情况。结果10μg/LIL-1β诱导异位内膜基质细胞分泌RANTES蛋白能力强于5μg/L组,稳定性优于20μg/L组。异位内膜基质细胞于IL-1β刺激12h开始RANTES mRNA表达较在位及正常内膜显著增强,在位内膜基质细胞自48h开始RANTES mRNA表达较正常内膜显著增强。异位内膜自刺激培养24h开始RANTES蛋白分泌量明显高于在位内膜和正常内膜,96h可高达(926.52±97.19)ng/L,在位内膜自刺激培养60h开始RANTES蛋白分泌量较正常内膜显著增加,正常内膜基质细胞RANTES分泌量维持于(39.19±14.29)~(56.88±17.28)ng/L。结论EMS患者的异位、在位子宫内膜与正常子宫内膜相比,其RANTES表达特性已经发生了改变;异位内膜基质细胞可能是EMS患者腹腔液中高浓度RANTES的来源之一。     

米非司酮和利洛司酮诱导异位子宫内膜间质细胞Fas及Annexin V表达的研究

目的 :观察抗孕激素米非司酮和利洛司酮在不同浓度和作用时间下 ,对异位子宫内膜间质细胞Fas和钙磷脂结合蛋白V表达的影响。方法 :应用ELISA法检测培养基上清液中Fas蛋白的浓度。应用流式细胞仪检测AnnexinV表达。结果 :抗孕激素各药物浓度组Fas表达显著高于对照组 (P <0 .0 5 ) ,且Fas浓度呈时间 剂量依赖性升高。抗孕激素10 - 6 mol/L和 10 - 5mol/L浓度组异位间质细胞AnnexinV表达显著高于对照组 (P <0 .0 5 ) ,并呈时间 剂量依赖性增加。抗孕激素 10 - 5mol/L时培养异位子宫内膜间质细胞 4 8h和 72h ,利洛司酮组AnnexinV表达显著高于米非司酮组 (P <0 .0 5 )。结论 :米非司酮和利洛司酮以时间和剂量依赖性方式促进异位子宫内膜间质细胞凋亡。这两种药物可能通过上调Fas蛋白表达促进异位间质细胞凋亡。利洛司酮诱导异位内膜细胞凋亡的作用高于米非司酮     

TNF-α对子宫内膜间质细胞VEGF表达的影响

目的:探讨肿瘤坏死因子-α(TNF-α)对体外培养的子宫内膜异位症(EMs)在位内膜间质细胞生长及血管内皮生长因子(VEGF)表达的影响。方法:应用MTS法分析TNF-α对来自EMs和非EMs症各10例患者的在位子宫内膜间质细胞生长的影响;并以荧光定量PCR、Western blot和定量ELISA检测不同浓度TNF-α作用下间质细胞VEGF的表达情况。结果:TNF-α呈剂量依赖方式促进EMs患者子宫内膜间质细胞的生长,并显著上调VEGF的表达;但对非EMs患者的子宫内膜间质细胞无上述作用。结论:TNF-α能明显促进EMs患者在位内膜间质细胞的生长并促进其VEGF的表达。     

维甲酸对人子宫颈癌细胞系HeLa细胞间隙连接蛋白转导途径调控作用的研究

目的：探讨维甲酸对人子宫颈癌细胞系HeLa细胞间隙连接蛋白（Cx）43信号转导途径的调控作用。方法：应用特异性钙指示剂（Fluo-3 AM)在激光扫描共聚焦显微镜下动态观察维甲酸作用后的细胞质内信号转导分子游离钙的分布及强度变化。采用流式细胞仪、结合蛋白印迹技术分析，检测外源信号分子对Cx43的表达以及蛋白酪氨酸磷酸化状态的影响。结果：HeLa细胞内游离钙经维甲酸作用后明显超载，细胞内游离钙浓度（[Ca^2 ]i)由静息状态下的35.73μmol/L上升至58.16μmol/L。流式细胞仪分析，Cx43阳性细胞表达率由1.9%上升至26.3%。蛋白印迹技术分析，HeLa细胞出现Cx43酪氨酸磷酸化。结论：维甲酸对HeLa细胞Cx43信号转导途径的调控是在细胞质内游离钙的参与下，使Cx43阳性细胞表达率上升，并在酪氨酸位点出现明显的磷酸化作用下实现的。     

Expression of gap junctional connexins 26, 32 and 43 in bovine placentomes during pregnancy

Gap junctional connexins (Cx) are induced in the endometrium during implantation in rodents, the human receptive window, and in the decidua Cx26 and Cx43 expression increases in response to trophoblast invasion. In contrast, this gap junctional response and decidualization is absent in non-invasive epitheliochorial placentae of pigs and horses. Bovine (syn)epitheliochorial placentation represents an intermediate type of trophoblast invasion, since it is characterized by the continuous migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells. Therefore the objective of the present study was to investigate the expression of Cx26, Cx32, and Cx43 in placental tissues during bovine pregnancy, to determine if Cx expression patterns correlate with the depth of trophoblast invasion. Cx26, Cx32, and Cx43 proteins were detected by immunohistochemistry and corresponding specific mRNAs were shown by RT-PCR and localized in tissue sections by in situ hybridization. Cx26 protein was detected at the feto-maternal contact interface and as cytoplasmic staining in TGC. Cx26 mRNA was located in maternal epithelium and in TGC. Cx32 protein expression was observed in the maternal epithelium exclusively on the tips of maternal septa, whereas Cx32 mRNA was detected in all maternal epithelial cells and single TGC. Cx43 protein and mRNA were coexpressed in TGC. Cx43 protein was present in maternal septal stroma and to a lesser extent in chorionic villous mesenchyme, while Cx43 mRNA was associated with the vasculature. In the course of gestation, expression of Cx26, Cx32, and Cx43 did not change. In conclusion, the intermediate invasive status of bovine trophoblast is supported by the fact that TGC coexpress Cx26, Cx32, and Cx43, which may be important for trophoblast migration (invasion), and fusion with maternal epithelial cells. Cx32 could be involved in the control of invasion.     

CON43、CON26蛋白在子宫内膜异位症中的表达及意义

目的:探讨间隙连接蛋白(CON)43、CON26在子宫内膜异位症中的表达及意义。方法:对20例子宫内膜异位症(内异症)内膜用免疫组化法检测CON43和CON26蛋白的表达,并与21例其他良性疾病子宫的内膜进行比较。结果:CON43蛋白主要在内膜腺上皮细胞膜和细胞浆中表达,而CON26阳性染色出现在子宫内膜腺上皮细胞和基质细胞的细胞浆中,而在细胞核中无阳性染色。内异症组中CON43和CON26蛋白表达均较对照组显著降低。结论:由CON43和CON26蛋白介导的子宫内膜各种细胞间的间隙连接,在细胞间的物质和信号的传递中起重要作用。子宫内膜异位症中CON43和CON26蛋白表达的降低,可能导致腺上皮间、基质细胞间以及腺上皮与基质细胞间的物质和信号传递减低或缺失,可能在内膜细胞的游走、粘附于腹膜中起一定作用。     

Effects of hormones on uPA,PAI-1 and suPAR from cultured endometrial and ovarian endometriotic stromal cells

PROBLEM: To investigate whether endometriotic stromal cells release the urokinase plasminogen activator, soluble urokinase plasminogen activator receptor and plasminogen activator inhibitor-1. If so, to establish if there are any differences between endometrial stromal cells from women with and without endometriosis, and whether the release is hormonally regulated. METHOD: Biopsies were obtained from endometriotic tissue and from endometrium from women with and without endometriosis. Stromal cells were isolated, incubated and treated with estradiol-17beta, progesterone or raloxifen. Incubation media collected at 0, 24, 48 and 72 h were analyzed by enzyme-linked immunosorbent assay. RESULTS: All these types of stromal cells released the urokinase plasminogen activator, soluble urokinase plasminogen activator receptor and urokinase plasminogen inhibitor-1. There was a significantly higher release of urokinase plasminogen inhibitor-1 and lower release of urokinase plasminogen activator and soluble urokinase plasminogen activator receptor in endometriotic cells. The release of urokinase plasminogen activator from endometrial stromal cells decreased during the study period both in control cultures and in cultures treated with progesterone or estradiol-17beta, but not in cultures treated with raloxifen nor in endometriotic cultures. The given hormones did not influence the release of the soluble urokinase plasminogen activator receptor. Progesterone significantly increased the urokinase plasminogen inhibitor-1 release in endometrial cells from both patient categories, and raloxifen significantly reduced the urokinase plasminogen inhibitor-1 release from stromal cells from both tissue categories from endometriotic patients. Estradiol-17beta had no effect. CONCLUSIONS: This study shows that stromal cells from endometrium and endometriotic tissues release the urokinase plasminogen activator, soluble urokinase plasminogen activator receptor and urokinase plasminogen inhibitor-1. The release is partly hormonally regulated, but differently in endometriotic than in endometrial cells.     

Altered gene expression and secretion of interleukin-6 in stromal cells derived from endometriotic tissues

OBJECTIVE: To compare the expression of interleukin-6 (IL-6) in endometrial and endometriotic cells. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. PATIENT(S): Twenty patients who underwent either hysterectomy or laparoscopic surgery. INTERVENTION(S): Endometrial and endometriotic stromal cells were obtained from normal endometrium and from chocolate cyst linings of the ovary. Peritoneal macrophages were isolated from peritoneal fluids. Cells were cultured in the presence or absence of tumor necrosis factor-alpha. MAIN OUTCOME MEASURE(S): Gene expression of IL-6 was examined by Northern blot analysis. Interleukin-6 protein production was examined by immunocytochemical staining and ELISA. RESULT(S): A single IL-6 messenger RNA band of approximately 1.3 kilobases was detected in endometriotic stromal cells. Tumor necrosis factor-alpha increased the expression of IL-6 messenger RNA in endometriotic cells in a dose-dependent manner. In endometrial stromal cells, IL-6 messenger RNA signals were much weaker. Endometriotic stromal cells produced significantly larger amounts of IL-6 compared with endometrial stromal cells under basal conditions and after stimulation with tumor necrosis factor-alpha. Interleukin-6 protein was detected in cells isolated from endometriotic tissues by immunocytochemical staining. Interleukin-6 production by cultured macrophages from patients with endometriosis and endometriotic stromal cells was comparable. CONCLUSION(S): Altered gene expression and protein secretion of IL-6 in patients with endometriosis may contribute to the pathogenesis of the disease and/or to endometriosis-associated infertility.     

Impact of epidermal growth factor and transforming growth factor beta-1 on the release of fibrinolytic factors from cultured endometrial and ovarian endometriotic stromal cells

We have investigated whether there are any differences in the release of urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) from cultured endometrial and endometriotic stromal cells, and whether the release is regulated by epidermal growth factor (EGF) or transforming growth factor beta1 (TGFbeta1). The cells were isolated from endometriomas and endometrium from women with and without endometriosis. After treatment with EGF or TGF and in untreated controls, incubated media collected at 0, 24, 48 and 72 h were analyzed by ELISA. Stromal cells from all three types of tissues released uPA and PAI-1, but the soluble receptor of uPA was not measurable in any group. The basal release of uPA and PAI-1 from endometriotic cells was higher than from endometrial cells. The uPA release in endometriotic cells was reduced with and without the addition of EGF (p < 0.05) or TGFbeta1 (p < 0.05). EGF increased the release of PAI-1 from stromal cells from women without endometriosis (p < 0.05) but decreased the release of PAI-1 from stromal cells from endometriotic women (p < 0.05). TGFbeta1 increased the release of PAI-1 from endometriotic cells (p < 0.05) but had no effect in endometrial cells.