米非司酮对恒河猴母胎界面细胞凋亡和凋亡分子p53表达的影响

胎盘形成过程中发生活跃的细胞增殖、迁移和凋亡等活动。p53蛋白是参与调节细胞周期和凋亡过程的原癌基因。本实验用原位末端标记、蛋白印迹和免疫组织化学方法研究正常和米非司酮(RU486)处理后恒河猴母胎界面绒毛和蜕膜组织细胞凋亡及p53蛋白表达。在正常妊娠的恒河猴母胎界面,凋亡信号主要集中在合体滋养层和细胞柱内的一些滋养层细胞;p53蛋白主要定位于细胞滋养层。在母体蜕膜中,也在部分基质细胞中检测到细胞凋亡和p53蛋白表达。经过RU486处理2d后,胎盘绒毛和母体蜕膜中凋亡细胞数都显著增加,绒毛中增加的凋亡信号集中于细胞滋养层。同时,RU486处理也导致绒毛细胞滋养层和蜕膜基质细胞中p53表达明显增加。以上结果提示,在正常妊娠中,生理性的细胞凋亡和p53表达可能是控制细胞滋养层细胞增殖、保持胎盘组织动态平衡的一个重要机制;RU486终止早孕的可能途径之一是促进母胎界面细胞凋亡,推测p53参与这一过程。     

血立停胶囊对早孕大鼠RU486药流后NO、NOS表达的影响

目的:研究血立停胶囊对早孕大鼠RU486药物流产后的子宫平滑肌一氧化氮(NO)及一氧化氮合酶(NOS)水平变化的影响。方法:选择妊娠Wistar大鼠,随机分为5组,即对照组,米非司酮组,大剂量血立停组,小剂量血立停组,催产素组。于妊娠第7天,开始相应处理,妊娠第14天分别监测早孕大鼠RU486药物流产后的子宫平滑肌一氧化氮(NO)及一氧化氮合酶(NOS)水平后处死。结果:大剂量血立停可明显降低大鼠子宫肌组织匀浆中NO、NOS含量,与对照组比较差异有显著性(P<0.05)。结论:血立停胶囊可降低子宫肌组织匀浆中NO、NOS水平,从而起到对药物流产后阴道出血的治疗作用。     

米非司酮对大鼠恐惧条件化及消退的影响

目的：研究米非司酮（RU486）对大鼠恐惧条件化及消退的影响。方法：大鼠连续4天给药（或生理盐水）后开始行为学实验,1 d适应环境;2 d进行恐惧条件化;3 d恐惧消退训练（也是条件化恐惧的表达检测阶段）;4 d进行消退记忆再现检测。结果：在恐惧表达阶段,两处理组与各自对照组大鼠的僵直水平组间差异都无显著性;在消退再现检测阶段,高剂量RU486组大鼠的僵直水平显著高于对照组,低剂量RU486组与对照组大鼠的僵直水平组间差异无显著性。结论：米非司酮损害消退记忆的再现,且这种损害与剂量有关。     

PTSD样大鼠海马MR和GR变化的研究

目的研究PTSD大鼠海马神经元核受体MR(mineralocorticoid receptor,盐皮质激素受体)和GR(glu-cocorticoid receptor糖皮质激素受体)表达的变化。方法采用国际认定的SPS方法刺激大鼠建立PTSD大鼠模型,分别取SPS处理后24h、7d、14d大鼠脑组织;同时取正常脑组织(非SPS刺激)作为对照,应用免疫组化、Western Blot方法分别进行各组海马神经元GR和MR表达变化的观察及定量检测。结果(1)经SPS处理后,免疫组化和Western Blot结果显示海马神经元MR表达呈现随着24h、7d、14d逐渐下调的趋势;(2)经SPS处理后,免疫组化和Western Blot结果显示海马神经元GR表达于24h时下调,而7d、14d时呈现逐渐回升趋势。结论大鼠经SPS处理后,海马MR表现为持续下调状态;而GR表达为短暂下调,随后回调,揭示PTSD大鼠海马神经元核受体—MR和GR的表达变化可能是引发海马功能降低的重要因素之一。     

大鼠胸腺细胞糖皮质激素受体结合与糖皮质激素效应的定量关系

本文通过糖皮质激素拮抗剂 RU486阻断不同分数的糖皮质激素受体(GR)后,GR 结合与糖皮质激素(GC)效应的比较,研究于两者的量的关系。在地塞米松(Dex)10_(-9)—10_(-5)mol/L浓度范围内,Dex 和大鼠胸腺细胞 GR 的特异结合与 Dex 抑制细胞尿嘧啶核苷(Urd)参入效应呈正向平行趋势。用 RU486阻断5×10_(-8)mol/L 浓度的 Dex与 GR 结合的不同分数,比较 Dex 特异结合与抑制效应的关系,发现两者表现为线性正相关(P<0.01)。将 Dex 结合-效应直线外推,结果提示 GR 有约20%的占领阈但几乎没有备用受体。     

宽频噪音对谷氨酸中毒损伤AD模型大鼠不同脑区NMDAR1(ζ1)、NMDAR2A(ε1)亚基表达的影响

目的:观察AD大鼠模型颞叶和额叶在98 dB宽频噪音暴露5 min后不同脑区NMDAR1(ζ1)、NMDAR2A(ε1) 表达的影响.方法: 采用Western Blot及 RT-PCR技术,结合ABR测定方法.结果:①AD模型组大鼠、空白对照组大鼠在98 dB宽频噪音暴露5 min后额叶、颞区、海马及小脑NMDAR1(ζ1)亚基表达无明显差异,但AD模型组表达明显弱于空白对照组;②生理盐水组加噪音后NMDAR1(ζ1)亚基小脑表达最强,颞叶最弱; NMDAR2A(ε1)表达最强为颞叶,海马最弱.③在海马三组大鼠NMDAR1(ζ1) 、NMDAR2A(ε 1)亚基表达有较明显的下调趋势;④空白对照组大鼠NMDAR1(ζ1)、NMDAR2A(ε1)亚基mRNA表达各区无差异.⑤AD模型组大鼠颞叶、海马NMDAR2A(ε1) 表达明显减弱 ,最弱为小脑,额叶次之.结论:噪音刺激抑制AD大鼠模型海马NMDAR1(ζ1)亚基表达,且不在mRNA水平.噪音刺激抑制AD模型大鼠颞叶、海马NMDAR2A(ε1) 亚基表达,且有部位差异,在mRNA水平已调节.     

海马微量注射神经肽Y通过抑制一氧化氮合酶改善大鼠抑郁样行为

本文旨在探讨在慢性应激性抑郁发生过程中海马神经肽Y(neuropeptide Y,NPY)和一氧化氮合酶(nitric oxide synthase,NOS)的关系。建立慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)抑郁模型,大鼠海马定位并分别微量注射NPY、NPY-Y1受体阻断剂GR231118和NOS抑制剂L-NAME,测量、计算动物体重变化并通过糖水偏爱测试、旷场和强迫游泳实验等方法检测大鼠行为,免疫组织化学方法检测海马内NPY、神经元型NOS(neuronal nitric oxide synthase,nNOS)和诱导型NOS(inducible nitric oxide synthase,iNOS)的表达。结果显示,与对照组相比,CUMS组大鼠表现出明显的抑郁样行为变化,且海马NPY表达下降、NOS表达显著升高;海马微量注射NPY明显改善应激引起的抑郁样行为表现,并降低海马NOS表达;用GR231118选择性阻断NPY-Y1受体后,大鼠的行为学表现能力下降,海马NOS表达升高;而CUMS以及GR231118所导致的行为学表现能力下降的现象均可被海马微量注射L-NAME所反转。以上结果表明,CUMS引起海马NPY表达下降,NOS表达升高,NO过量产生,导致抑郁发生;而NPY通过NPY-Y1受体抑制NOS的过量表达是NPY抗抑郁的一个重要途径。     

穹窿切断后海马神经元GR表达变化的研究

目的实施大鼠穹窿切断术研究海马神经元核受体GR(glucocorticoidreceptor糖皮质激素受体)表达的变化。方法建立大鼠穹窿切断模型,于穹窿切断术后0、4、7、10d取材;同时取材假手术组(非穹窿切断术)作为对照,应用免疫组化、WesternBlotting方法分别进行各组海马神经元GR表达变化的观察及定量检测。结果穹窿切断7d后,免疫组化和WesternBlotting结果显示海马神经元GR表达下调,10d下调更为显著。结论穹窿切断后,海马GR表达下调,提示可能减弱海马对HPA轴的抑制。     

子宫内膜接受性与金属蛋白酶及其抑制因子表达之间的关系

为了研究RU486抗着床的机理和子宫内膜接受性与金属蛋白酶MMP-2及其抑制剂TIMP-1,3之间的关系, 用注射RU486的小鼠作为模型, 研究了注射不同剂量RU486对小鼠胚胎着床率的影响, 以及在着床窗口期子宫内膜中MMP-2和TIMP-1,3表达量的变化. 结果显示, 注射RU486能明显抑制胚胎的着床, 而且有剂量依赖性. 在着床窗口期, 子宫内膜中MMP-2表达量的升高和TIMP-1,3表达量的降低不利于胚胎着床. 提出RU486的抗着床作用可能是通过诱导MMP-2的表达和抑制TIMP-1,3的表达实现的.     

糖皮质激素对人绒毛膜滋养层细胞11β-HSD1的调节

目的：研究糖皮质激素对11β-羟基类固醇脱氢酶I型(11β-hydroxysteroid dehydiogenase type1,11β-HSD1)还原酶活性和mRNA表达的调节。方法：利用原代培养的人类绒毛膜滋养层细胞,运用免疫细胞化学染色方法、放射性酶活性测定及Northem印迹杂交技术,结合图像分析技术检测11β-HSD1 mRNA的表达量。结果：11β-HSD1和糖皮质激素受体(glucocorticoid receptor,GR)免疫活性样物质共存于原代培养的人绒毛膜滋养层细胞中,人工合成的糖皮质激素——地塞米松对绒毛膜滋养层细胞11β-HSD1还原酶活性和mRNA表达具有诱导作用,此诱导作用可以被GR阻断剂RU486所阻断。结论：糖皮质激素与GR结合后上调绒毛膜滋养层细胞11β-HSD1酶活性和mRNA表达。     

Recycling and desensitization of glucocorticoid receptors in v-mos transformed cells depend on the ability of nuclear receptors to modulate gene expression

In v-mos transformed cells, glucocorticoid receptor (GR) proteins that bind hormone agonist are not efficiently retained within nuclei and redistribute to the cytoplasmic compartment. These cytoplasmic desensitized receptors cannot be reutilized and may represent trapped intermediates derived from GR recycling. We have used the glucocorticoid antagonist RU486 to examine whether v-mos effects can be exerted on any ligand-bound GR. In the rat 6m2 cell line that expresses a temperature-sensitive p85gag-mos oncoprotein, RU486 is a complete antagonist and suppresses dexamethasone induction of metallothionein-1 mRNA at equimolar concentrations. Using indirect immunofluorescence, we observe efficient nuclear translocation of GR in response to RU486 treatment in either the presence or absence of v-mos oncoproteins. However, in contrast to the redistribution of agonist-bound nuclear receptors to the cytoplasm of v-mos-transformed cells, RU486-bound GRs are efficiently retained within nuclei. Interestingly, withdrawal of RU486 does not lead to efficient depletion of nuclear GR in either nontransformed or v-mos transformed cells. It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm. Thus, only nuclear GRs that are agonist-bound and capable of modulating gene activity can be subsequently processed and recycled into the cytoplasm.     

Glucocorticoid receptor blockade inhibits brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus

When animals are under stress, glucocorticoids commonly inhibit adult neurogenesis by acting through glucocorticoid receptors (GRs). However, in some cases, conditions that elevate glucocorticoids promote adult neurogenesis, and the role of glucocorticoid receptors in these circumstances is not well understood. We examined the involvement of GRs in social enhancement of brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus. In this species, long-term social interaction simultaneously elevates plasma cortisol, enhances brain cell addition and increases production of aggressive electrocommunication signals (“chirps”). We implanted isolated and paired fish with capsules containing nothing (controls) or the GR antagonist, RU486, recorded chirp production and locomotion for 7 d, and measured the density of newborn cells in the periventricular zone. Compared to isolated controls, paired controls showed elevated chirping in two phases: much higher chirp rates in the first 5 h and moderately higher nocturnal rates thereafter. Treating paired fish with RU486 reduced chirp rates in both phases to those of isolated fish, demonstrating that GR activation is crucial for socially induced chirping. Neither RU486 nor social interaction affected locomotion. RU486 treatment to paired fish had a partial effect on cell addition: paired RU486 fish had less cell addition than paired control fish but more than isolated fish. This suggests that cortisol activation of GRs contributes to social enhancement of cell addition but works in parallel with another GR-independent mechanism. RU486 also reduced cell addition in isolated fish, indicating that GRs participate in the regulation of cell addition even when cortisol levels are low.     

Glucocorticoid receptors take part in the apoptotic process of human lens epithelial cells, but the glucocorticoid receptor antagonist RU486 does not rescue the cells fully

To identify an agent with specific activity against human lens epithelial cells (HLECs), we confirmed the presence of glucocorticoid receptors (GRs) and GR-α genes and evaluated whether GRs have a relationship with the apoptotic process in cultured HLECs. We also determined whether the inhibitor RU486 could rescue the cells from apoptosis when the HLECs were exposed to dexamethasone (Dex), a steroid, in 4 concentrations for 4 periods, or were co-treated with the antagonist RU486. We found that Dex, which has been used as a medical agent for a long time, resulted in increased expression of GRE-luciferase, the GR-α gene and GR-protein and, in contrast, decreased the viability of HLECs. The expression of Bax protein was increased in an earlier stage in contrast to the expression of Bcl-2 protein, which was increased in a later stage. Caspase-3 activity was significantly increased under lower concentrations of Dex in the last stage. The nuclear morphology of HLECs showed an obvious apoptotic phenomenon under greater concentrations of Dex in the last stage. However, RU486, a GR antagonist, could partially inhibit GR and Bax expressions and the expression of caspase-3 was increased so that there was not a decrease in the ratio of apoptotic cells and an increase in the viability of HLECs. Our data showed that GRs had a partial relationship to the apoptotic process of HLECs when exposed to Dex and RU486 did not rescue the cells fully. Because of its toxicity, RU486 did not provide a therapeutic benefit in a glucocorticoid induced cataract (GIC) for the in vitro model, however, its activity and pathway targeting should still be studied further with appropriate drug combinations.     

Cell cycle regulation of glucocorticoid receptor function.

Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR.     

血立停胶囊对早孕大鼠RU486药流后子宫收缩的实验研究

目的:研究血立停胶囊对早孕大鼠Ru486药物流产后对子宫平滑肌收缩频率、收缩幅度及活动力变化的影响,旨在探讨血立停胶囊治疗药物流产后出血的作用机制。方法:选择妊娠Wistar大鼠,随机分为6组,即对照组,米非司酮组,大剂量血立停组,小剂量血立停组,催产素组,止血敏组。于妊娠第7天,开始相应处理,妊娠第14天分别监测以下指标后处死:在体子宫平滑肌收缩力,收缩幅度、收缩频率。结果:大剂量血立停可明显增强大鼠在体子宫平滑肌活动力、提高子宫平滑肌收缩频率、收缩幅度,与对照组比较差异有显著性(p<0.05)。结论:血立停胶囊可增强大鼠子宫平滑肌兴奋性,从而起到对药物流产后阴道出血的治疗作用。     

Role of activation function domain-1, DNA binding, and coactivator GRIP1 in the expression of partial agonist activity of glucocorticoid receptor-antagonist complexes

The determinants of the partial agonist activity of most antisteroids complexed with steroid receptors are not well understood. We now examine the role of the N-terminal half of the glucocorticoid receptor (GR) including the activation domain (AF-1), the DNA binding site sequence, receptor contact with DNA, and coactivator binding on the expression of partial agonist activity in two cell lines for GRs bound by five antiglucocorticoids: dexamethasone mesylate (Dex-Mes), dexamethasone oxetanone (Dex-Ox), progesterone (Prog), deoxycorticosterone (DOC), and RU486. Using truncated GRs, we find that the N-terminal half of GR and the AF-1 domain are dispensable for the partial agonist activity of antiglucocorticoids. This contrasts with the AF-1 domain being required for the partial agonist activity of antisteroids with most steroid receptors. DNA sequence (MMTV vs a simple GRE enhancer) and cell-specific factors (CV-1 vs Cos-7) exert minor effects on the level of partial agonist activity. Small activity differences for some complexes of GAL4/GR chimeras with GR- vs GAL-responsive reporters suggest a contribution of DNA-induced conformational changes. A role for steroid-regulated coactivator binding to GRs is compatible with the progressively smaller increase in partial agonist activity of Dex-Mes > Prog > RU486 with added GRIP1 in CV-1 cells. This hypothesis is consistent with titration experiments, where low concentrations of GRIP1 more effectively increase the partial agonist activity of Dex-Mes than Prog complexes. Furthermore, ligand-dependent GRIP1 binding to DNA-bound GR complexes decreases in the order of Dex > Dex-Mes > Prog > RU486. Thus, the partial agonist activity of a given GR-steroid complex in CV-1 cells correlates with its cell-free binding of GRIP1. The ability to modify the levels of partial agonist activity through changes in steroid structure, DNA sequence, specific DNA-induced conformational changes, and coactivator binding suggests that useful variations in endocrine therapies may be possible by the judicious selection of these parameters to afford gene and tissue selective results.     

抗黄体酮联合Aromatase抑制剂或iNOS终止恒河猴妊娠

[目的]评价抗黄体酮(mifepristone)联合Aromatase抑制剂(letrozole或aminoglutethimide)或iNOS抑制剂(aminoguandine)是否能有效终止恒河猴早期妊娠。[方法]将30只猴子随机分为5组(治疗组每组6只,对照组6只),并在妊娠30,31和32天进行如下处理:对照组,每只动物1ml安慰剂;A组,Mifepristone(1mg/kg,sc.);B组,Mifepristone(sc.)+Letrozole(2.5mg/只sc.);C组,Mifepristone(1mg/kg,sc.)+aminoglute-chimide(50mg/kgsc.,bid);D组,Mifepristone(1mg/kg,sc.)+aminoguanidine(150mg/kg,sc.,bid)。所有妊娠猴在妊娠29天通过超声波确认。[结果]在B、C、D组,所有的动物的妊娠都在妊娠早期被终止(6/6)。A组和对照组的妊娠终止率分别为3/6和2/6。同时,联合用药能够有效排空子宫腔和减少出血。[结论]该处理能有效地终止恒河猴早期妊娠。联合用药比用于女人的妊娠治疗更有效,并减少了流血时间,或许可以代替目前的终止妊娠的医疗方法。     

Induction of labor and cervical maturation using mifepristone (RU 486) in the late pregnant rat. Influence of a cyclooxygenase inhibitor (Diclofenac).

The mechanism of action of RU 486 (Mifepristone), an antiprogesterone compound, on labor induction and on cervical maturation, is still not well documented. We have investigated the effect of RU 486, alone and in association with a cyclooxygenase inhibitor (Diclofenac) on the induction of preterm delivery and on concomitant changes in the distribution of cervical glycosaminoglycans (GAGs) in pregnant Wistar rats: a control group (n = 18), a RU 486 treated group (n = 36), and a RU 486 and Diclofenac treated group (n = 15). The results of this study confirm the ability of this antiprogesterone treatment to induce preterm delivery in the rat. This effect was antagonized by cyclooxygenase inhibition, suggesting that the action of RU 486 on labor induction could be mediated by prostaglandins. The absence of an increase in plasma prostaglandin E2 (PGE2) levels in RU 486 treated animals could be explained by local uterine changes in prostaglandin concentrations. Mifepristone also induced some of the biochemical features of cervical maturation (i.e. increased hydration and hyaluronic acid concentration). This effect was not inhibited in Diclofenac treated animals suggesting that factors other than prostaglandins play a role in this phenomenon.     

Induction of labor and cervical maturation using Mifepristone (RU 486) in the late pregnant rat. Influence of a cyclooxygenase inhibitor (Diclofenac)

The mechanism of action of RU 486 (Mifepristone), an antiprogesterone compound, on labor induction and on cervical maturation, is still not well documented. We have investigated the effect of RU 486, alone and in association with a cyclooxygenase inhibitor (Diclofenac) on the induction of preterm delivery and on concomitant changes in the distribution of cervical glycosaminoglycans (GAGs) in pregnant Wistar rats: a control group (n = 18), a RU 486 treated group (n = 36), and RU 486 and Diclofenac treated group (n = 15). The results of this study confirm the ability of this antiprogesterone treatment to induce preterm delivery in the rat. This effect was antagonized by cyclooxygenase inhibition, suggesting that the action of RU 486 on labor induction could be mediated by prostaglandins. The absence of an increase in plasma prostaglandin E2 (PGE2) levels in RU 486 treated animals could be explained by local uterine changes in prostaglandin concentrations. Mifepristone also induced some of the biochemical features of cervical maturation (i.e. increased hydration and hyaluronic acid concentration). This effect was not inhibited in Diclofenac treated animals suggesting that factors other than prostaglandins play a role in this phenomenon.