哌嗪雌酚酮对新生大鼠头盖骨成骨细胞的影响

目的 了解新合成化合物——哌嗪雌酚酮对体外培养成骨细胞的影响。方法 应用MTT法、对硝基苯磷酸盐法、von Kossa染色法、流式细胞术及RT-PCR观察哌嗪雌酚酮对体外培养成骨细胞的增殖、分化、矿化结节形成、细胞周期及I型胶原蛋白表达的影响。结果 哌嗪雌酚酮可刺激成骨细胞增殖,刺激骨结节形成(10-7mol/L组,P<0.05)。结论 哌嗪雌酚酮具有刺激体外培养成骨细胞增殖和晚期分化成熟的功能。     

VALIDITY AND USE OF A NON-PARALLEL INSULIN ASSAY FOR PHARMACOKINETIC STUDIES OF THE RAPID-ACTING INSULIN ANALOGUE,INSULIN ASPART

A radioimmunoassay (RIA) for insulin was validated for reliable measurement of the human insulin analogue, insulin aspart, by correction of non-linear measurements. Specificity was equivalent for several species of insulin, except insulin aspart. A non-linear hyperbolic model fitted insulin aspart with a correction formula for non-linearity of: z = 1503y/(1398 - y), where y denotes measured concentration and z denotes true concentration.

Matrix-effects were insignificant for human, porcine, and canine heparin-plasma and for human and porcine serum. The coefficient of variation was below 15% for 80–800 pmol/L human and porcine insulin and for 80–600 pmol/L insulin aspart. The limit of detection for insulin aspart was 11.5 pmol/L with a lower limit of quantification of 17.5 pmol/L. Dilution of serum with Pharmacia dilution media introduced no significant error. In conclusion, this paper demonstrates that a non-parallel radioimmunoassay can be used to estimate accurate concentrations of insulin aspart.     

Characterization of a membrane-associated estrogen receptor in a rat hypothalamic cell line (D12)

The ability of estrogens to produce rapid changes in cellular function has been firmly established. The question remains whether these changes are mediated by a modified form of the nuclear estrogen receptor (ER) that is associated with the plasma membrane (mER) or by a completely novel membrane receptor. Therefore, we characterized the biochemical properties of the nuclear and membrane-associated ERs expressed endogenously in a rat hypothalamic endothelial cell line (D12). Radioligand binding experiments using D12 membrane fractions showed that these cells exhibit properties consistent with a binding site specific for estrogens (mER). Equilibrium binding assays using [¹²⁵I]16-α-iodo-3,17-β-estradiol revealed saturable binding to mER, an affinity value similar to nuclear ER, with differing receptor expression levels. Competition assays revealed that 9 of 12 ER ligands tested had comparable affinities for mER and ER. For example, 17-α-estradiol and estrone had similar binding characteristics for both receptors while differences were noted for raloxifene, 17β-estradiol (E2), and genistein. Western blot and immunocytochemical analyses using antibodies specific for ERα confirmed that D12 cells expressed a membrane-associated protein with a molecular mass (67 kDa) similar to that of ERα that colocalized with caveolae-enriched membranes. A rapid increase in intracellar Ca²⁺ levels in the presence of E2 suggests that mER can mediate physiologic changes through calcium mobilization. These data support the expression of mER in these brain-derived endothelial cells that is similar to, but biochemically distinguishable from, nuclear ERα.     

Estradiol-delivering vaginal rings for hormone replacement therapy

OBJECTIVES: This study was undertaken to determine the relief of climacteric symptoms by vaginal rings delivering estradiol and to monitor estrogen levels. STUDY DESIGN: Rings releasing in vitro either 60 or 140 microg/d estradiol were used by 35 women who had undergone hysterectomy for each dose level. Hot flash and night sweat incidences, vaginal conditions, and complaints were recorded at clinic visits pretreatment and at 1 week, 2 weeks, 1 month, and monthly thereafter through 6 months. Serum samples were assayed for estradiol, estrone, and estrone sulfate. RESULTS: Hot flash incidence was reduced by about 80% with either ring. Vaginal conditions and mood were improved. Fourteen of 70 women discontinued ring use during the trial, 5 because of ring expulsions. Mean (+/-SD) estradiol levels were 123 +/- 48 and 307 +/- 93 pmol/L for the low and high dosage levels, respectively. Mean estrone levels exceeded estradiol levels by 1.7-fold for the higher dosage ring and 2.6-fold for the lower dosage ring. Increases in estrone sulfate concentrations were many times greater than those of estradiol or estrone. CONCLUSIONS: Vaginal rings are an acceptable method of delivery for periods of >/=6 months of doses of estradiol that reduce vasomotor symptoms and improve vaginal conditions. There was little difference in these responses between the 2 dosage levels.     

Muscle performance,sex hormones and training in peri-menopausal and post-menopausal women

Age-related deterioration in muscle performance is one of the major reasons for decreased functional capacity and disability in older people. In women, impaired muscle performance has already been observed during peri-menopause in concert with rapid and dramatic decrease in ovarian hormone production. This observation suggests that female sex steroids may have an important role among other agents in regulating muscle performance in middle-aged and older women. Previous experimental studies have shown that hormone replacement therapy (HRT) and intensive physical training have positive effects on muscle force and explosive power in healthy post-menopausal women. Hormone replacement therapy in combination with physical training may exert even greater gains in muscle performance than HRT and training alone. Despite the significant mean increase in muscle force and power by HRT and/or training, a considerable variability in the individual responses is observed. The mechanism, by which female sex steroids act on muscle performance, is still unclear. Therefore, more research is needed in order to explore all the pathways, by which these steroids could act on skeletal muscle in peri-menopausal and post-menopausal women.     

Estrone sulfatase activity and effect of antiestrogens on transformation of estrone sulfate in hormone-dependent vs. independent human breast cancer cell lines

Summary The effect of the anti-estrogens ICI 164,384 and tamoxifen on the estradiol (E₂) concentration after incubation of estrone sulfate (E₁-S) with different hormone-dependent (MCF-7 and T-47D) and hormone-independent (MDA-MD-231 and MDA-MB-436) mammary cancer cells, as well as the estrone sulfatase activity in these various cell lines, are presented. The anti-estrogen ICI 164,384 decreased very significantly the concentration of E₂ after incubation of E₁-S with MCF-7 (control, mean ±SE: 100±24 pg/mg DNA; + ICI 164,384 [10^–6M]: 7±2 pg/mg DNA). This effect was much more intense than with tamoxifen. A similar effect was observed with T-47D cells. However, no significant effect was observed in the hormone-independent cells. In the intact cell, estrone sulfatase activity was very intense in the hormone-dependent cells, but very small in the hormone-independent cells. However, this activity became very strong after homogenization in the hormone-independent cells. The data suggest that estrone sulfate can play an important role on the bioavailability of E₂ in hormone-dependent breast cancer, and that understanding the control of estrone sulfatase activity can open new knowledge of the estrogen responses and new possibilities of therapeutic application in breast cancer.     

高效液相色谱法检测中成药中4种雌激素的方法研究

目的:建立中成药中雌二醇、炔雌醇、雌酮和己烯雌酚4种激素的高效液相检测方法。方法:采用岛津ODS色谱柱(4.6 mm×250 mm,5μm),以乙腈-水(50:50)为流动相,流速1.0 mL.min-1,检测波长为220 nm,柱温为30℃。结果:4种雌激素均在0～50μg.mL-1范围内呈良好的线性关系(r=0.9999);雌二醇、炔雌醇、雌酮和己烯雌酚的平均回收率(n=4)分别为100.0%(RSD=2.4%),99.8%(RSD=1.9%),100.0%(RSD=2.6%),100.0%(RSD=1.2%)。结论::建立的HPLC方法具有专属性强、灵敏度高、操作简便、准确性好等特点,可以作为中成药中非法添加激素类化学药品检查的有效方法。     

新型雌激素哌嗪雌酚酮对雌激素受体表达的影响及其对靶基因的转录激活作用

目的探讨哌嗪雌酚酮对雌激素受体表达的影响及其对靶基因ERE的转录激活作用。方法小鼠去卵巢后,每天灌服不同剂量的哌嗪雌酚酮(0.5、1、2mg.kg-1)以及雌酚酮(0.71mg.kg-1),连续42d,采用免疫组织化学的方法观察哌嗪雌酚酮对去卵巢小鼠子宫中两种雌激素受体(ERα和ERβ)蛋白表达的影响;采用Tfx-50脂质体共转染目的基因和报告基因于MCF-7细胞株,探讨哌嗪雌酚酮对ERE的转录激活作用。结果与去卵巢模型组比较,哌嗪雌酚酮各剂量组能够上调子宫组织中两种雌激素受体亚型的表达(有剂量依赖性),对ERα亚型作用明显于ERβ;哌嗪雌酚酮能够激活雌激素受体的经典信号途径,与等摩尔剂量的雌酚酮相比仅表现出弱雌激素活性。结论哌嗪雌酚酮能够上调子宫组织中ERs的表达;与雌酚酮比较,其对靶基因ERE的转录激活作用非常微弱。