双酚A抗孕激素样作用的体外观察

目的 观察双酚A在人类子宫内膜Ishikawa细胞中的抗孕激素样作用.方法 不同浓度孕酮、双酚A/米非司酮＋孕酮混合分别干预细胞48h,RT-qPCR法检测各干预组细胞中PRmRNA表达的情况,且建立S函数量效曲线.Western Blotting检测各组PR蛋白分泌的情况.结果 孕酮单独干预下PRmRNA呈下调性表达,孕酮下调PRmRNA的表达受到双酚A、米非司酮浓度依赖性的拮抗,相同现象在蛋白水平上得到确认.结论 双酚A能产生与米非司酮近似的抗孕激素样作用.     

17β-雌二醇调控大鼠成肌细胞L6GNR4细胞迁移能力的机制研究

目的研究17β-雌二醇对大鼠成肌细胞L6GNR4细胞迁移能力的调控作用及机制。方法L6GNR4细胞培养在含有10%胎牛血清的DM EM高糖增殖培养基中,培养基中添加10 nM雌激素17β雌二醇和/或50 nM雌激素竞争性拮抗剂Tamoxifen。应用细胞划痕试验在药物处理后0、12、24 h观察17β雌二醇对成肌细胞迁移能力的影响。在药物处理24 h后,应用荧光定量PCR技术检测各组细胞中雌激素受体α和β的表达。同时还检测了成肌细胞迁移相关关键基因(Pax3、Fhl1以及Fhl1相互作用蛋白Actg1、Myh10)在各组细胞中的表达情况。结果与对照组比较,17β-雌二醇在划痕24 h时可以明显抑制L6GNR4的细胞迁移。17β-雌二醇能够明显抑制ERβ的表达,而雌激素竞争性拮抗剂Tamoxifen能够部分逆转17β-雌二醇对ERβ的抑制作用。17β-雌二醇能够明显抑制成肌细胞迁移相关的关键因子Pax3、Fhl1以及Fhl1相互作用蛋白Myh10的表达,而雌激素竞争性拮抗剂Tamoxifen能够逆转或部分逆转17β-雌二醇对Pax3、Fhl1和Myh10的转录抑制作用。相同浓度的雌激素对L6GNR4细胞中Actg1的表达改变不明显。结论雌激素17β-雌二醇通过ERβ转录抑制Pax3、Fhl1以及Fhl1的相互作用蛋白Myh10的表达,发挥其抑制大鼠成肌细胞迁移的作用。     

IKCa1在围着床期子宫内膜的表达及其作用

摘要：目的　检测中电导钙激活性钾离子通道（IKCa1）在围着床期子宫内膜的表达及其在胚胎着床过程中的作用。方法　分别取分泌和增生早、中、晚期子宫内膜组织各5例。应用qPCR和免疫组化技术检测各期IKCa1 mRNA和蛋白的表达。体外培养人子宫内膜癌Ishikawa细胞，不同浓度（0、10、20、30 µmol/L）IKCa1阻滞剂TRAM-34干预后，qPCR检测细胞IKCa1表达，CCK-8法检测细胞增殖能力，Transwell实验检测细胞体外胚胎黏附能力，Western blot检测细胞上皮-间质转化（EMT）相关蛋白E盒结合蛋白1（Zeb1）和上皮型钙黏蛋白（E-cadherin）表达。结果　IKCa1在人子宫内膜中呈时序性表达，分泌期表达水平较增生期显著增高，以分泌中期最高。TRAM-34抑制IKCa1表达后，Ishikawa细胞增殖和胚胎黏附能力减弱；EMT标志蛋白E-cadherin表达显著上调，Zeb1表达显著下调（P＜0.05）。结论　IKCa1可能参与围着床期子宫内膜容受性的建立，并通过影响子宫内膜细胞增殖和EMT调控胚胎着床。     

通心络对Aβ_(1-42)损伤人脑微血管内皮细胞HIF-1α、VEGF表达的干预作用

目的通心络对β淀粉样蛋白片段(Aβ1-42)诱导的人脑微血管内皮细胞中VEGF的分泌及VEGF和HIF-1α蛋白表达的干预作用。方法采用人脑微血管内皮细胞,给予不同剂量的通心络预处理,并用20μmol·L-1的Aβ1-42干预24h诱导细胞损伤,经处理后检测细胞形态的变化、VEGF分泌及VEGF和HIF-1α蛋白的表达。结果Aβ1-42可诱导人脑微血管内皮细胞损伤,VEGF分泌减少及VEGF蛋白表达降低,HIF-1α蛋白表达增强,通心络可升高Aβ1-42诱导的VEGF分泌、增强其蛋白的表达,增强HIF-1α蛋白表达,保护内皮细胞。结论通心络可通过HIF-1α途径增强VEGF蛋白的表达,增加上清液中VEGF蛋白的分泌,从而对Aβ1-42损伤的脑微血管内皮细胞起到保护作用。     

类固醇激素在体外对肿瘤细胞bcl-2表达及端粒酶活性影响的研究

探讨类固醇激素对端粒酶活性调节的机制及 bcl- 2蛋白水平表达的影响。采用 PCR- ELISA方法检测类固醇激素作用前后 KS、SGC端粒酶活性的改变 ,免疫组化 S- P方法检测 bcl- 2蛋白表达水平。结果说明乙烯雌酚显著增强 KS、SGC端粒酶的活性 ,丙酸睾丸酮和乙烯雌酚不同程度增强bcl- 2蛋白表达。     

4-取代17β-羟基在-7α-甲基-4-雌烯-3-酮的合成和抗生育活性

为了寻找抗生育药物,从17β-羟基-7α-甲基-4-雌烯-3-酮经17β-羟基-7α-甲基-4β,5β-环氧雌烷-3-酮合成了七个4-位取代的目标化合物:4,17β-二羟基-7α-甲基-4-雌烯-3-酮及其17-乙酸酯、4-甲氧基-17β-羟基-7α-甲基-4-雌烯-3-酮及其17-乙酸酯、4-氯-17β-羟基-7α-甲基-4-雌烯-3-酮及其17-乙酸酯和4-溴-17β-羟基-7α-甲基-4-雌烯-3-酮。这些化合物均可竞争性地与人蜕膜孕酮受体结合,并能抑制蜕膜细胞的发育,对大鼠具有显著的抗着床活性。     

4-取代17β-羟基在-7α-甲基-4-雌烯-3-酮的合成和抗生育活性

为了寻找抗生育药物,从17β-羟基-7α-甲基-4-雌烯-3-酮经17β-羟基-7α-甲基-4β,5β-环氧雌烷-3-酮合成了七个4-位取代的目标化合物:4,17β-二羟基-7α-甲基-4-雌烯-3-酮及其17-乙酸酯、4-甲氧基-17β-羟基-7α-甲基-4-雌烯-3-酮及其17-乙酸酯、4-氯-17β-羟基-7α-甲基-4-雌烯-3-酮及其17-乙酸酯和4-溴-17β-羟基-7α-甲基-4-雌烯-3-酮。这些化合物均可竞争性地与人蜕膜孕酮受体结合,并能抑制蜕膜细胞的发育,对大鼠具有显著的抗着床活性。     

雌二醇对大鼠肝星状细胞TGF-β1、CTGF表达的影响

目的 探讨雌二醇对肝星状细胞(HSC)的影响及其可能的作用途径.方法 培养大鼠HSC,初次传代后,HSC随机分为4组,分别加入不同浓度的雌二醇,加药后72小时,噻唑蓝(MTT)法检测雌二醇对HSC增殖的影响;逆转录-聚合酶链式反应(RT-PCR)分析其对HSC的转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)的mRNA表达的影响;免疫细胞化学分析其对HSC的α-SMA蛋白的表达的影响.结果 MTT法发现雌二醇抑制HSC的增殖;RT-PCR分析雌二醇干预组HSC TGF-β1mRNA、CTGFmRNA的表达明显下降;免疫细胞化学分析雌二醇干预组HSC α-SMA蛋白表达明显下降;并且都随浓度增加抑制作用增强,差异有显著性(P＜0.05).结论 雌二醇具有抑制肝纤维化作用是因为:①可以抑制HSC的增殖、活化;②抑制了致纤维化生长因子TGF-β1及其下游信号CTGFmRNA表达.     

粝固醇激素在体外对肿瘤细胞bcl—2表达及端粒酶活性影响的研究

探讨类固醇激素对端粒酶活性调节的机制及bcl-2蛋白水平表达的影响。采用PCR－ELISA方法检测类固激素作用前后KS、SGC端粒酶活性的改变,免疫组化S－P方法检测bcl-2蛋白表达水平。结果说明了乙烯雌酚显著增强KS、SGC端粒酶的活性,丙酸睾丸酮和乙烯雌酚不同程度增强bcl-2蛋白表达。     

金宁方中β-谷甾醇增进槲皮素诱导肺癌A549细胞凋亡作用

目的 探讨β-谷甾醇联合槲皮素对A549细胞抑制作用以及机制。方法 将A549细胞分为对照组、槲皮素组、槲皮素+β-谷甾醇组、雌二醇组、雌二醇+槲皮素组以及雌二醇+槲皮素+β-谷甾醇组。采用细胞存活率检测A549细胞的抑制作用。采用流式细胞技术和免疫荧光法检测肺癌细胞的促凋亡情况。应用免疫印迹法检测磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的调控作用。结果 槲皮素可抑制A549细胞生存率且具量效关系(P <0.05);雌二醇可促进A549细胞生长;与槲皮素组或槲皮素+β-谷甾醇比较,雌二醇+槲皮素+β-谷甾醇组更抑制A549细胞增殖,提高细胞凋亡比例及caspase-3的表达水平(P <0.05);促进mTOR和Akt磷酸化,减少PI3K磷酸化。结论 在雌二醇干预下,β-谷甾醇联合槲皮素对A549细胞有抑制作用,其机制可能通过PI3K/Akt/mTOR通路对细胞凋亡起促进作用。     

Potential hazards to embryo implantation: A human endometrial in vitro model to identify unwanted antigestagenic actions of chemicals

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17β-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.     

Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats.

Considerable attention has recently been focused on environmental chemicals that disrupt the reproductive system by altering steroid receptor function. Although numerous in vitro and in vivo methods have been shown to be useful approaches for identifying chemicals that can disrupt reproduction through a direct interaction with the estrogen receptor, it is imperative that the protocols selected be capable of detecting chemicals with a broad range of estrogenic activity. Here we evaluate the reliability of the 3-day uterotrophic assay for detecting chemicals with strong or weak estrogenic activity in both prepubertal and ovariectomized adult Long Evans rats. These data were compared to additional measures of estrogenic activity, which included the age of vaginal opening, the induction of cornified vaginal epithelial cells in ovariectomized adult rats, and estrous cyclicity in intact adult rats. Test chemicals selected for these studies included 17-beta-estradiol, ethynyl estradiol, methoxychlor, 4-tert-octylphenol, 4-nonylphenol and bisphenol A. Data from in vitro receptor binding assays compared the ability of the test chemicals to compete with [3H]-estradiol or [3H]-promegestone for binding to estrogen or progesterone receptors. As expected, the binding affinities for the estrogen receptor ranged from high to low, as reflected by Ki concentrations of 0.4 nM for 17-beta-estradiol and ethynyl estradiol, and 0.05-65 microM for 4-tert-octyphenol, 4-nonylphenol, and methoxychlor. Although none of the test chemicals demonstrated a high affinity for binding to the progesterone receptor, 4-tert-octylphenol and 4-nonylphenol exhibited a weak affinity, with Ki concentrations ranging from 1.2 to 3.8 microM. In vivo studies indicated that the 3-day uterotrophic assay in prepubertal rats was the best method for detecting estrogenic activity when compared with all other end points, based upon the dose-response data for ethynyl estradiol (0.01-0.1 mg/kg), 4-tert-octylphenol (50-200 mg/kg, oral), and 4-nonylphenol (25-100 mg/kg, oral). Although oral doses of ethynyl estradiol (0.01 mg/kg) and 4-nonylphenol (50 mg/kg) induced a significant increase in uterine weight in the prepubertal rats, these doses were ineffective for stimulating a similar response in ovariectomized adult rats. The age of vaginal opening was advanced following oral exposure from postnatal days 21-35 to ethynyl estradiol (0.01 mg/kg), methoxychlor (50 mg/kg), 4-tert-octylphenol (200 mg/kg), and 4-nonylphenol (50 mg/kg). Although bisphenol A (200 mg/kg, oral) induced a significant uterotrophic response within 3 days in prepubertal rats, doses up to 400 mg/kg failed to advance the age of vaginal opening. Monitoring changes in the vaginal epithelium of ovariectomized adult rats was the least effective method for detecting estrogenic activity for 4-tert-octylphenol and bisphenol A. The number of 4-5 day estrous cycles was reduced during a 25-day exposure to ethynyl estradiol (0.01 mg/kg), methoxychlor (50 mg/ kg), 4-tert-octylphenol (200 mg/kg), 4-nonylphenol (100 mg/kg), and bisphenol A (100 mg/kg) by oral gavage. Although long periods of extended diestrus (7-14 days) were generally correlated with exposure to ethynyl estradiol and 4-tert-octylphenol, the cycling patterns following exposure to methoxychlor, 4-nonylphenol and bisphenol A were not as clearly defined, with shorter periods of extended diestrus (4-7 days) and/or estrus (3-5 days) intermittently observed throughout the exposure period. Together these data provide a comparison of the 3-day uterotrophic assay with alternative measures of estrogenic activity for a group of test chemicals with a broad range of affinities for the estrogen receptor. These data can be useful during the assessment and validation of methods for screening environmental chemicals for endocrine disrupting activity.     

Assessing combined toxicity of estrogen receptor agonists in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes

The presence of highly complex mixtures of chemicals in the environment challenges our ability to assess single chemical effects and the interaction that occurs with cellular receptor targets and regulation of endocrine processes. In this study concentration addition (CA) and independent action (IA) prediction models were used to assess the combined toxicity of mixtures of environmental relevant estrogen receptor (ER) agonists (hormones and anthropogenic pollutants) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes using the ER-mediated production of vitellogenin (Vtg) as a biological marker (biomarker) for estrogenicity. Nine of the eleven tested chemicals induced the production of Vtg and the parameters from the fitted concentration-response curves were used to model four mixtures containing four (17β-estradiol, estrone, estriol and diethylstilbestrol), five (musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene), seven (17β-estradiol, estrone, estriol, diethylstilbestrol, 4-tert-octylphenol, bisphenol A and o,p′-DDT) and nine compounds (17β-estradiol, estrone, estriol, diethylstilbestrol, musk ketone, 4-tert-octylphenol, bisphenol A, o,p′-DDT and dibenzothiophene). The CA and IA prediction model proved to be a good estimation for the combined effect of mixtures of ER agonists at low relative mixture concentration (e.g. relative to the maximum mixture concentrations used), but a deviation from the prediction models was observed when exposing hepatocytes to high relative mixture concentrations. The CA and IA prediction models’ ability to predict the combined estrogenic effect of complex mixtures, especially in the low concentration-response range, is of ecological relevance since organisms in the environment generally encounter low concentrations of chemicals from a wide array of chemical groups that may not elicit estrogenic effects on their own.     

Modulation by estrogens and xenoestrogens of recombinant human neuronal nicotinic receptors

The effects of estrogens and xenoestrogens on human neuronal nicotinic acetylcholine receptor/channels were examined by expressing recombinant channels in Xenopus oocytes. When functional channels were expressed with 3 and β4 subunits, estrogens (17β-estradiol, 17-estradiol, 17-ethynylestradiol and diethylstilbestrol) and xenoestrogens (bisphenol A, p-nonylphenol and p-octylphenol) inhibited an ionic current activated by acetylcholine at concentrations up to 100 μM. When the subunit combination was changed to 4β2, diethystilbestrol and the xenoestrogens inhibited the acetylcholine-activated current, but 17β-estradiol or 17-estradiol did not. For 17-ethynylestradiol, the current through the 4β2 receptor/channel was inhibited at 1 μM, but it was markedly enhanced at 10 and 100 μM. Tamoxifen (10 μM), an antiestrogen, itself inhibited the acetylcholine-activated current but did not antagonize the current modulations induced by the estrogens and the xenoestrogens. These and additional results suggest that human neuronal nicotinic acetylcholine receptors are the targets of non-genomic actions of estrogens and xenoestrogens.     

Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.     

Assessment of estrogenicity by using the delayed implanting rat model and examples

Endocrine disrupting chemicals have recently drawn increased interest. The delayed implanting rat model is a method that can identify and quantify the estrogenic activity of a chemical. In rats hypophysectomized after breeding, the administration of progesterone delays embryo implantation, and exposure to one dose of an estrogenic substance initiates implantation. Although methoxychlor was ineffective at dosages below 400 mg/kg when given by injection, the administration of the chemical by gavage resulted in an increase in the percent of fertilized rats exhibiting implantation sites. These results were statistically significant at dosages of 50, 100, 200, and 300 mg methoxychlor/kg. When bisphenol A was administered, by subcutaneous injection, dosages of 50, 100, and 200 mg/kg induced implantation. Only the 400 mg/kg dose of 4-tert-octylphenol was effective. Doses of beta-sitosterol up to 30 mg/kg failed to initiate implantation. These data confirm previous evidence of the availability of this model for evaluating estrogenic activity and provide estimates of the estrogenic potencies of several environmentally important chemicals.     

Potentielle Antineoplastica,III. N-Lostderivate des Diäthylstilböstrols und des Hexöstrols mit freien phenolischen Hydroxylgruppen

Potential Antineoplastics, III: N-Mustard Derivatives of Diethylstilbestrol and Hexestrol with Free Phenolic Hydroxy Groups The mustard compounds 8 and 15 are synthesized as potential mammatropic antineoplastics starting from diethylstilbestrol and hexestrol, respectively. Both compounds are irreversibly bound to the cytoplasmic estrogen receptor. The binding affinitiy is 10⁴ times lower than that of 17β-estradiol.