金雀异黄素和5-FU对人结肠癌细胞株(SW480)的相互作用的体外观察

目的 探讨金雀异黄素（Genistein）和5-氟脲嘧啶（5-FU）合用对人结肠癌细胞株（SW480）的相互作用。方法 采用MTT法利用中效原理判定两药合用的效果。结果两种药物单独应用时随着药物剂量的增加其效应增加，合用时两药大剂量时（大于中效浓度）其拮抗作用，小剂量（小于中效浓度）时其有协同作用；两药给药时间及给药顺序都会影响效应的大小。结论 上述两种药合用时，大剂量时有拮抗作用小剂量时有协同作用且效应大小与给药时间及给药顺序有关。     

金雀异黄素对人乳癌细胞MCF-7增殖和凋亡的影响

目的：采用离体细胞培养的方法，研究大豆异黄酮的主要成分金雀异黄素(GEN)对人乳腺癌MCF-7细胞的增殖抑制作用及其作用机理。方法：MCF-7细胞接受不同浓度的GEN处理，MTT比色法测定GEN抑制MCF-7细胞的量效关系；细胞分裂指数试验用于评价GEN对该细胞的恶性增殖的抑制作用；细胞形态学观察及原位细胞凋亡检测法(TUNEL)用于检测细胞凋亡。结果：GEN的IC30与IC50分别为17．5μmol／L及32．0μmol／L，GEN对MCF-7具有明显的抑制作用，且该抑制作用呈剂量反应关系；各剂量GEN均可抑制MCF-7细胞分裂并且呈剂效关系；细胞形态学观察及TUNEL检测发现GEN处理48h及96h后有明显的凋亡细胞出现，并呈现剂效关系，而本次实验未发现其时效关系。结论：GEN可抑制离体培养MCF-7细胞的增殖，其作用机理可能与GEN诱导细胞凋亡的途径有关。     

Genistein对EJ细胞增殖抑制作用的研究

目的:观察酪氨酸蛋白激酶(TPK)抑制剂Genistein对膀胱癌EJ细胞增殖的抑制作用.方法:以不同浓度的Genistein作用EJ细胞,采用MTT法检测细胞的增殖程度;原位凋亡细胞检测技术和流式细胞仪检测Genistein对EJ细胞凋亡和细胞周期的影响.结果:Genistein引起EJ细胞增殖比明显下降,其程度随Genistein浓度增高而增强;Genistein促进EJ细胞的凋亡,使细胞阻滞于G2/M期.结论:Genistein对EJ细胞的增殖有抑制作用,可以诱导EJ细胞的凋亡.     

染料木素对大鼠成骨细胞增殖与分化的影响

目的:探讨染料木素体外对大鼠成骨细胞增殖与分化的影响。方法:用改良的组织块法分离培养新生大鼠颅骨成骨细胞,染料木素以不同浓度加入细胞培养体系,作用不同时间后,用MTT法检测成骨细胞的增殖情况;用对硝基苯二钠基质动力学法测定细胞内碱性磷酸酶的活性,用改良的Lowry法测蛋白含量。结果:染料木素在1×10~(-5)～1×10~(-9)mol/L浓度范围内作用24h、48h均能促进成骨细胞增殖,在1×10~(-7)～1×10~(-5)mol/L范围内作用72 h可提高成骨细胞内碱性磷酸酶的活性。结论:染料木素体外能促进成骨细胞的增殖与分化。     

Phytoestrogens alter the reproductive organ development in the mink (Mustela vison)

The aim of the present study was to examine the reproductive effects of two perorally applied phytoestrogens, genistein (8 mg/kg/day) and beta-sitosterol (50 mg/kg/day), on the mink (Mustela vison) at human dietary exposure levels. Parental generations were exposed over 9 months to these phytoestrogens and their offspring were exposed via gestation and lactation. Parents and their offspring were sampled 21 days after the birth of the kits. Sex hormone levels, sperm quality, organ weights, and development of the kits were examined. The exposed females were heavier than the control females at the 1st postnatal day (PND). The control kits were heavier than the exposed kits from the 1st to the 21st PND. Phytoestrogens did not affect the organ weights of the adult minks, but the relative testicular weight of the exposed kits was higher than in the control kits. The relative prostate weight was higher and the relative uterine weight lower in the beta-sitosterol-exposed kits than in the control kits. Moreover, the plasma dihydrotestosterone levels were lower in the genistein-exposed male kits compared to the control male kits. This study could not explain the mechanisms behind these alterations. The results indicate that perinatal phytoestrogen exposures cause alterations in the weight of the reproductive organs of the mink kits.     

Comparative responses of three rat strains (DA/Han,Sprague-Dawley and Wistar) to treatment with environmental estrogens

The rat uterotrophic assay is a widely used screening test for the detection of estrogenic, endocrine-disrupting chemicals. Although much attention has been paid to identifying protocol variables and reproducibility between laboratories the question whether toxicodynamic and toxicokinetic variations of different strains may affect their sensitivity to estrogenic stimuli has been rarely addressed. We have compared the estrogenic activity of the environmental chemicals genistein (GEN), bisphenol A (BPA) and p-tert-octylphenol (OCT) in DA/Han (DA), Sprague-Dawley (SD) and Wistar (WIS) rats after repeated oral application. Rats were treated per os for 3 days with different doses of these weakly estrogenic compounds and the potent reference estrogen ethinylestradiol (EE). Then uterine wet weight, thickness of the uterine epithelium, uterine gene expression of clusterin (CLU), and thickness of the vaginal epithelium were examined as parameters for estrogenic potency of the test compounds in the three strains of rats. The uterotrophic response to treatment with BPA, OCT and GEN was similar in the three strains, and allowed us to rank them as GEN being more potent than OCT, and BPA being the weakest estrogen. This was confirmed by analysis of other biological endpoints, despite some differences in the magnitude of their response among strains and to distinct compounds. For instance, the uterus wet weight response to EE treatment indicated lower sensitivity of SD rats than that of DA and WIS rats, but this was not observed for responses of the uterine or vaginal epithelium. Moreover, blood concentrations were assessed at the time of killing and related to biological responses: plasma levels of total and unconjugated BPA and GEN depended upon the dose administered and varied to some extent within treatment groups and among the three rat strains. However, there was no good correlation in the three strains between individual compound concentrations analysed 24 h after the last dose and the uterotrophic wet weights. Summarising our results, we conclude that the sensitivity of various biological endpoints can differ slightly between strains of rats. On the other hand, our data demonstrate that the choice of the rat strain does not lead to pronounced differences in the evaluation of estrogenic activities of chemicals, especially when different biological endpoints are included in the analysis.     

Genistein抑制人卵巢癌细胞系SKOV3增殖和诱导凋亡发生的研究

背景与目的:许多研究表明 Genistein对多种肿瘤细胞有抑制作用,但有关 Genistein对卵巢癌细胞作用的报道很少.本研究旨在通过观测 Genistein对人卵巢癌细胞系 SKOV3的抑制增殖和诱导凋亡作用,探讨其抗癌作用的机理.方法:应用四甲基偶氮唑盐( MTT)法检测不同浓度 Genistein对 SKOV3的生长抑制作用;吖啶橙 /溴乙锭( AO/EB)荧光染色法及电镜观察凋亡细胞及凋亡小体;流式细胞仪分析细胞周期及凋亡率;琼脂糖凝胶电泳检测凋亡特征性 DNA梯形带;免疫细胞化学法及 RT- PCR法分别检测细胞增殖凋亡调控相关蛋白及其 mRNA的表达。结果： Genistein对 SKOV3细胞的增殖抑制作用呈时间及浓度依赖性, 20 μ mol/L和 40 μ mol/L Genistein作用 72 h后,细胞的生长抑制率达 72. 07％和 74. 93％。 20 μ mol/L Genistein作用 SKOV3细胞 48 h后出现细胞周期的 G2/M期阻滞。荧光显微镜和电镜均观察到用药后凋亡细胞典型的形态学特征。细胞凋亡率以 Genistein 20 μ mol/L组最高,达 23. 7％。凝胶电泳观察到特征性 DNA梯形带。 20 μ mol/L Genistein作用 SKOV3 48 h后, bcl- 2基因表达水平降低,而 p21WAF1/CIP1和 bax基因表达增加（ P< 0. 05）; PCNA、Bcl- 2及 cyclin B1蛋白表达水平降低, Bax、p21WAF1/CIP1蛋白表达增加（ P< 0. 01）。结论： Genistein可抑制卵巢癌细胞系 SKOV3的增殖并诱导其发生凋亡, Genistein可能通过上调 p21WAF1/CIP1基因及蛋白水平、下调 cyclin B1及 PCNA蛋白表达水平抑制增殖;通过下调 bcl- 2基因及蛋白表达、上调 bax基因及蛋白表达诱导卵巢癌细胞凋亡.     

Fetal exposure to phytoestrogens--the difference in phytoestrogen status between mother and fetus

The goal of this study was to investigate fetal exposure to phytoestrogens, estrogenic compounds derived from plants, by measuring serum concentrations of phytoestrogens in maternal and cord blood. This study included 51 mothers scheduled for cesarean section (C-section), to obtain the serum of mother and fetus at almost the same time. Serum concentrations of phytoestrogens, including genistein (Gen), daidzein (Dai), coumestrol (Cou), equol (Equ; a metabolite of Dai), and sulfate-conjugated Gen, were measured in maternal and cord blood samples by LC-MS/MS and HPLC. It was found that phytoestrogens were transferred from mother to fetus. The detection rates of Gen, Dai, Equ, and Cou in cord serum were 100%, 80%, 35%, and 0%, respectively. Levels of Gen and Dai were higher in cord than in maternal serum (mean=19.4 ng/ml vs.7.2 ng/ml and 4.3 ng/ml vs.1.8 ng/ml for Gen and Dai, respectively). However, a reverse pattern was seen for Equ (cord mean=0.9 ng/ml, maternal mean=2.0 ng/ml). The correlations were significant between the concentration levels of Gen and Dai, Gen and Equ, and Gen and Dai plus Equ in cord serum. However, in maternal serum, the correlations were weak. Also, in 8 of 10 cord serum samples, sulfate-conjugated Gen was detected (mean=5.2 ng/ml, standard deviation=4.7), but it was detected from only one maternal serum (8.7 ng/ml). This study demonstrates placental transfer of phytoestrogens from mother to fetus. It is suggested that the metabolic and/or excretion rates of phytoestrogens are different between mother and fetus and once phytoestrogens are transferred to the fetus, they tend to stay in the fetal side longer than in the maternal side. While the implications for the health and development of the newborn are not known, these data suggest that the effects of fetal exposure to phytoestrogens should be studied further.     

金雀异黄素抑制大鼠膀胱肿瘤作用的实验研究

目的探讨金雀异黄素对N-甲基亚硝基脲(MNU)诱导的SD大鼠膀胱肿瘤的抑制作用。方法将60只大鼠随机分成3组,用MNU诱导成膀胱肿瘤,实验1组和实验2组分别经腹腔注射金雀异黄素0.5、1.0mg/kg,每两日1次;对照组用同样的方式注射生理盐水。利用统计方法和病理分级来分析金雀异黄素对MNU诱导的SD大鼠膀胱肿瘤的抑制作用。结果实验1组和实验2组与对照组比较,在体质量减少值、肿瘤个数、抑瘤率等方面,差异有统计学意义(P<0.05);实验1组和实验2组的癌变率相比,差异有统计学意义(P<0.05)。结论金雀异黄素对大鼠膀胱肿瘤有抑制作用,剂量越大,对肿瘤癌变抑制作用越强。     

金雀异黄酮对糖尿病大鼠心肌损伤的保护作用

目的 观察金雀异黄酮（genistein, Gen）对糖尿病大鼠心肌损伤的保护作用,并探讨其机制。 方法 雄性SD大鼠随机分为正常（N）组、糖尿病（D）组、Gen 5 mg/kg治疗（L）组和Gen 25 mg/kg治疗（H）组,每组8只。采用链脲佐菌素55 mg/kg腹腔注射诱导1型糖尿病大鼠模型。造模成功4周后,L组和H组大鼠开始分别灌胃给予Gen溶液5 mg/kg和25 mg/kg。干预4周后,测定各组大鼠血流动力学指标和空腹血糖（FBG）;通过HE染色和透射电镜下分别观察大鼠心肌病理形态结构和超微结构改变;测定心肌组织肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）、白介素-6（IL-6）、丙二醛（MDA）、谷胱甘肽（GSH）含量和Caspase-3活性;RT-PCR检测心肌组织Bcl-2和BaxmRNA表达;Western blot检测心肌硫氧还蛋白（Trx）、Trx相互作用蛋白（TXNIP）和凋亡信号调节激酶1（ASK1）的蛋白表达。 结果 与N组比较,D组FBG、TNF-α、IL-1β、IL-6、MDA含量和Caspase-3活性增高（P＜0.01）,血流动力学指标和GSH含量下降（P＜0.01）;心肌病理形态结构和超微结构明显损伤,心肌Bcl-2 mRNA和Trx蛋白表达下降（P＜0.01）,BaxmRNA、TXNIP和ASK1蛋白表达增高（P＜0.01）。与D组比较,L和H组中,FBG差异无统计学意义,TNF-α、IL-1β、IL-6、MDA含量和Caspase-3活性下降（P＜0.05,P＜0.01）,血流动力学指标和GSH含量增高（P＜0.05,P＜0.01）;心肌病理形态结构和超微结构明显改善;心肌Bcl-2 mRNA和Trx蛋白表达增加（P＜0.05,P＜0.01）,Bax mRNA、TXNIP和ASK1蛋白表达下降（P＜0.05,P＜0.01）。 结论 金雀异黄酮对糖尿病大鼠心肌损伤具有保护作用,其机制可能与减轻心肌炎症反应,调节Trx系统表达,抑制氧化应激和细胞凋亡相关。