核因子κB受体活化因子配体/骨保护素在骨髓瘤中的表达

目的研究核因子κB受体活化因子配体(RANKL)/骨保护素(OPG)在多发性骨髓瘤(MM)中的表达情况。方法运用反转录聚合酶链反应(RT-PCR)检测人骨髓瘤细胞系(8226、XG1、XG7细胞)、患者MM细胞及MM患者骨髓基质细胞对RANKL/OPG的表达。结果 MM细胞系(8226、XG1、XG7细胞)及患者MM细胞均不表达RANKL及OPG m RNA;伴有骨病的MM患者骨髓基质细胞RANKL表达明显增加,OPG表达减低。结论 MM细胞不直接表达RANKL/OPG,可能通过调节RANKL/OPG的平衡间接参与MM骨病的发生。     

金雀异黄素和槲皮素对人类乳腺癌细胞增殖和细胞周期的影响

目的探讨金雀异黄素(genistein,Gen)和槲皮素(quercetin,Que)对人类乳腺癌细胞株增殖的影响。方法采用噻唑蓝(MTT)比色法测定槲皮素对雌激素依赖性乳腺癌细胞MCF-7、T47D和非雌激素依赖性乳腺癌细胞MDA-MB231的细胞增殖作用,并以雌激素受体拮抗剂ICI182780为工具药来评价金雀异黄素、槲皮素发挥雌激素样作用与雌激素受体的关系,流式细胞术对MCF-7细胞的增殖情况进行分析。结果Gen和Que在一定剂量范围内能促进T47D和MCF-7细胞的增殖,而对雌激素受体阴性MDA-MB231细胞未见增殖作用,并将MCF-7细胞周期由G1期向S期推进,促进DNA合成,提高细胞分裂增殖指数,且Gen和Que促进MCF-7细胞增殖作用被雌激素受体拮抗剂所拮抗。结论金雀异黄素和槲皮素具有雌激素活性,此作用可能是通过雌激素受体(ER)介导的。     

骨碎补总黄酮对骨质疏松模型大鼠OPG/RANKL/RANK轴系统的影响

目的 研究不同剂量骨碎补总黄酮灌胃对骨质疏松模型大鼠雌激素水平及骨保护素(orthopantomography,OPG)、核因子κB受体活化因子(receptor activator of NF-κB,RANK)和核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)表达的影响,探讨其对骨代谢影响的可能机制。方法 通过去卵巢法造成骨质疏松大鼠模型,以戊酸雌二醇片0.1 mg·kg^-1及低、中、高不同剂量骨碎补总黄酮(0.054,0.108,0.216 g·kg^-1·d^-1)喂养3个月后,取动脉血,采用放射免疫法测定雌激素水平,用酶联免疫吸附法(ELISA)检测破骨细胞OPG、RANK和RANKL的表达。结果 与模型组2相比,各给药组雌二醇及OPG的表达量增加(P<0.05),RANK和RANKL的表达量减少(P<0.05),且随着骨碎补总黄酮剂量的增加,雌二醇及OPG表达呈上升趋势,RANK和RANKL表达均呈下降趋势。结论 不同剂量骨碎补总黄酮均影响OPG/RANKL/RANK轴系统,且随骨碎补总黄酮剂量的增加效果越明显,可能是通过调控OPG/RANKL/RANK轴系统使OPG表达增加、RANK和RANKL的表达下降来实现的。     

丹参酮ⅡA抗乳腺癌细胞增殖作用研究

目的利用雌激素受体(estrogenic receptor,ER)阳性乳腺癌T47D细胞和ER阴性乳腺癌MDA-MB-231细胞观察丹参酮ⅡA(TanshinoneⅡA)对细胞增殖活性的影响及其对雌激素受体亚型的调节功能。方法以ER拮抗剂ICI182,780为工具药,采用MTT细胞增殖实验观察1×10-6mol.L-1和1×10-7mol.L-1丹参酮ⅡA对T47D和MDA-MB-231细胞增殖的影响。利用实时荧光定量PCR法及流式细胞术检测其对T47D细胞ERα和ERβmRNA及蛋白表达情况的影响。结果丹参酮ⅡA能够抑制T47D细胞增殖,且该作用可被ICI182,780部分拮抗;对MDA-MB-231细胞增殖的抑制作用较其对T47D细胞的作用更为明显。丹参酮ⅡA可使T47D细胞ERα和ERβmRNA和蛋白表达明显增加,并使ERα/ERβ比值有所上升。结论丹参酮ⅡA具有抑制乳腺癌细胞增殖的作用,抑制强度与对其ER亚型的调节作用相关。     

槲皮素的植物雌激素作用及其受体机制研究

目的:探讨槲皮素(quercetin,Que)的植物雌激素作用及其可能的受体作用机制.方法:采用噻唑蓝(MTT)比色法测定槲皮素对雌激素依赖性乳腺癌细胞T47D和非雌激素依赖性乳腺癌细胞MDA-MB231的细胞增殖作用,流式细胞术对T47D细胞的增殖情况进行分析.Western blot方法测定槲皮素对T47D细胞雌激素受体两种亚型ERα和ERβ表达的影响,并以雌激素受体拮抗剂ICI182,780为工具药来评价槲皮素发挥雌激素样作用与雌激素受体的关系.结果:与溶剂对照组相比较,槲皮素(10,20,40 μmol·L^-1) 能显著促进T47D细胞的增殖,而抑制雌激素受体阴性MDA-MB231细胞,并将T47D细胞周期由G₁期向S期推进,促进DNA合成,提高细胞分裂增殖指数;且槲皮素促进T47D细胞增殖作用被雌激素受体拮抗剂所拮抗,槲皮素在10 μmol·L^-1可明显诱导T47D细胞ERα蛋白表达,而对ERβ表达没有影响.当与ICI 182,780共孵育T47D细胞ERα表达被拮抗.结论:槲皮素具有雌激素活性,此作用是通过影响雌激素受体ERα蛋白表达介导的.     

磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶信号通路参与核因子-κB受体活化因子配体诱导的MDA-MB-231乳腺癌细胞迁移

目的探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)信号通路在核因子-κB受体活化因子配体(RANKL)诱导的乳腺癌细胞迁移中的作用。方法 Transwell法测定RANKL刺激后MDA-MB-231细胞迁移能力的改变。蛋白印迹法检测MDA-MB-231细胞表面RANK蛋白的表达及RANKL刺激后pAkt及Akt的表达;检测数据用x珚±s表示,采用SPSS 16.0软件进行t检验。结果 MDA-MB-231细胞表达RANK蛋白,RANKL诱导MDA-MB-231细胞迁移能力增强,RANKL的圈套受体骨保护素(OPG)可阻断RANKL诱导的细胞迁移(P<0.01)。RANKL刺激后MDA-MB-231细胞p-Akt表达升高,PI3K抑制剂LY294002抑制RANKL诱导的细胞迁移(P<0.01)。结论 PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞MDA-MB-231迁移。     

Med19在乳腺癌中的表达及其与临床病理特征的关系

目的分析中介体19亚基(Med19)表达与乳腺癌临床病理特征的关系。方法用RT-PCR检测Med19基因在6株人乳腺癌细胞ZR-75-30、MDA-MB-231、SK-BR-3、T-47D、HCC-1937及MCF-7中的表达。用免疫组化方法检测Med19蛋白在150例临床乳腺癌组织及癌旁正常组织中的表达并分析其与临床病理特征的关系。结果 Med19基因在人乳腺癌细胞株ZR-75-30、MDA-MB-231、SK-BR-3、T-47D、HCC-1937及MCF-7中均有较高丰度的阳性表达。乳腺癌组织中Med19蛋白阳性表达率为52.0%,明显高于癌旁正常组织的9.3%(P<0.01)。Med19蛋白的阳性表达与乳腺癌病理高分级相关(P<0.01),未发现与患者年龄、肿瘤大小、病理组织类型、TNM分期、雌激素受体(ER)、孕激素受体(PR)、p53有统计学意义的相关性(P>0.05)。结论 Med19在乳腺癌组织中高表达,可能在乳腺癌的发生发展中起重要的作用。     

RANK/RANKL/OPG系统及其相关疾病和抗RANKL疗法的研究进展

骨骼是一个动态组织,在生物的整个生命周期中不断地进行构建、吸收、重建,核因子(nuclear factor,NF)κB受体活化因子(receptor activator of NF-κB,RANK)/NF-κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)/护骨素(osteoprotegerin,OPG)系统是调控这一过程的关键系统.RANK/RANKL/OPG属于肿瘤坏死因子及其受体超家族,三者通过调控破骨细胞的分化和活化来影响骨吸收和重建的过程.另外,免疫细胞也参与和调节RANK/RANKL的表达和分泌,而免疫细胞本身亦受到该系统的影响,因此,RANK/RANKL/OPG系统成为骨和免疫之间的重要关联.RANK/RANKL/OPG系统的失衡与多种骨代谢性疾病及免疫系统疾病引发的继发性骨病密切相关,该系统的发现为研究相关疾病的病理机制和治疗方法提供了基础.由此建立的抗RANKL疗法已经开始应用于临床试验和研究.此文对RANK/RANKL/OPG系统、系统相关疾病以及抗RANKL治疗的进展做一综述.     

玉米赤霉烯酮对MCF-7和T47D细胞凋亡的抑制作用

玉米赤霉烯酮(ZEA)是一种霉菌毒素，在奶制品及霉变谷类食物中含量较高。据报道，ZEA在结构上与内源性雌激素有相似之处，通过与雌激素受体结合而模拟雌激素效应，增加女性体内雌激素负荷，与乳腺癌发病率的升高有关。本实验室前一阶段的研究表明，ZEA可模拟雌激素作用，促进雌激素依赖性乳腺癌细胞MCF-7及T47D细胞增殖，     

组织蛋白酶B在SAHA诱导乳腺癌MCF-7细胞凋亡过程中的调控作用

目的阐明组织蛋白酶B(cathepsin B,Cat B)在SAHA诱导的乳腺癌雌激素受体阳性细胞系MCF-7细胞凋亡中的调控作用。方法采用MTT法检测SAHA对乳腺癌MCF-7细胞生长状况的影响;应用ELISA方法测定SAHA作用于MCF-7细胞后相关蛋白表达变化的情况;通过Bio Station~(IM)活细胞工作站实时收集各种处理因素对乳腺癌MCF-7细胞增殖干预的形态学影响;并通过自动细胞分析仪Muse Cell Analyzer分析SAHA对MCF-7细胞活力和细胞凋亡的影响。结果 SAHA能明显抑制乳腺癌MCF-7细胞的增殖,其最佳作用浓度为10μmol·L~(- 1),最佳作用时间为24 h。ELISA结果表明,SAHA能诱导乳腺癌MCF-7细胞中Cat B的表达。实时活细胞工作站成像实验从形态学上证明,Cat B抑制剂Cystatin C和SAHA联合应用可有效阻止SAHA对MCF-7细胞生长的抑制作用。细胞学实验结果表明,SAHA能使MCF-7细胞活力下降,细胞凋亡发生率明显增高;然而经过Cystatin C处理后的MCF-7细胞活力增加,细胞凋亡发生率下降。结论 Cat B在SAHA诱导乳腺癌雌激素受体阳性细胞MCF-7凋亡过程中具有重要的调控作用。     

Interaction of estrone and estradiol with DNA and protein of liver and kidney in rat and hamster in vivo and in vitro

[6,7-³H] Estrone (E) and [6,7-³H]estradiol-17 (E₂) have been synthesized by reduction of 6-dehydroestrone and 6-dehydroestradiol with tritium gas. Tritiated E and E₂ were administered by oral gavage to female rats and to male and female hamsters on a dose level of about 300 g/kg (54 mCi/kg). After 8 h, the liver was excised from the rats; liver and kidneys were taken from the hamsters. DNA was purified either directly from an organ homogenate or via chromatin. The radioactivity in the DNA was expressed in the units of the Covalent Binding Index, CBI = (mol chemical bound per mol DNA-P)/(mmol chemical administered per kg b.w.). Rat liver DNA isolated via chromatin exhibited the very low values of 0.08 and 0.09 for E and E₂, respectively. The respective figures in hamster liver were 0.08 and 0.11 in females and 0.21 and 0.18 in the males. DNA isolated from the kidney revealed a detectable radioactivity only in the female, with values of 0.03 and 0.05 for E and E₂, respectively. The values for male hamster kidney were < 0.01 for both hormones. The minute radioactivity detectable in the DNA samples does not represent covalent binding to DNA, however, as indicated by two sets of control experiments. (A) Analysis by HPLC of the nucleosides prepared by enzyme digest of liver DNA isolated directly or via chromatin did not reveal any consistent peak which could have been attributed to a nucleoside-steroid adduct. (B) All DNA radioactivity could be due to protein contaminations, because the specific activity of chromatin protein was determined to be more than 3,000 times higher than of DNA. The high affinity of the hormone to protein was also demonstrated by in vitro incubations, where it could be shown that the specific activity of DNA and protein was essentially proportional to the concentration of radiolabelled hormone in the organ homogenate, regardless of whether the animal was treated or whether the hormone was added in vitro to the homogenate.Carcinogens acting by covalent DNA binding can be classified according to potency on the basis of the Covalent Binding Index. Values of 10³–10⁴ have been found for potent, 10² for moderate, and 1–10 for weak carcinogens. Since estrone is moderately carcinogenic for the kidney of the male hamster, a CBI of about 100 would be expected. The actually measured limit of detection of 0.01 places covalent DNA binding among the highly unlikely mechanisms of action. Similar considerations can be made for the liver where any true covalent DNA binding must be below a level of 0.01. It is concluded that an observable tumor induction by estrone or estradiol is unlikely to be due to DNA binding.Paper presented at the Satellite Symposium of the European Society of Toxicology, Rome, March 29, 1983     

Penetration and action of edoxudine in vitro and in vivo

The penetration of 5-ethyl-2'-deoxyuridine (edoxudine, Aedurid) from gel base with and without the addition of urea and other adjuvant has been studied in an in vitro model using guinea pig skin. The formulation of 3% edoxudine gel with 5% urea showed the best results. In vivo experiments on hairless mice infected intracutaneously with herpes simplex virus type 1 also showed this formulation's good efficacy as compared to other formulations.     

Mechanisms of antiplatelet and antithrombotic activity of midazolam in in vitro and in vivo studies

Dopamine regulates various physiological functions in the central nervous system and the periphery. Dysfunction of the dopamine system is implicated in a wide variety of disorders and behaviors including schizophrenia, addiction, and attention-deficit hyperactivity disorder. Medications that modulate dopamine signaling have therapeutic efficacy on the treatment of these disorders. However, the causes of these disorders and the role of dopamine are still unclear. Studying the dopamine system in a model organism, such as Caenorhabditis elegans, allows the genetic analysis in a simple and well-described nervous system, which may provide new insight into the molecular mechanisms of dopamine signaling. In this review, we summarize recent findings on pharmacological and biochemical properties of the C. elegans dopamine receptors and their physiological role in the control of behavior.     

Extraction and determination of prednisolone in drugs and excipients in ointments and creams and the uniformity of distribution in prednisolone ointment

For the purpose of measuring the contents of prednisolone in low concentrated ointments and creams an instruction was elaborated that includes several steps of extraction, in the resulting solution of which the assay of the steroid by Blue Tetrazolium reaction will be done. The procedure permits the determination of prednisolone in presence of most of usual ingredients of ointment bases except wool alcohols. Also no influence is given by some remedies combined with prednisolone for topical application except coal tar solution. The results confirm a correct reflection of the steroid contents declared respectively the recovery of the steroid added to various ointment bases. Introducing discussion to content uniformity concerning low concentrated ointments is made, and some deviations are shown.     

Comparative in vivo and in vitro studies of phenytoin protein binding and in vitro lipolysis in plasma of pregnant and nonpregnant rats

This investigation was designed to determine the cause of the changes in drug protein binding that occur in rat plasma, particularly in plasma from pregnant animals, during in vitro drug-protein binding measurements. In vivo estimates of phenytoin binding in plasma were obtained from steady-state CSF-plasma concentration ratios in pregnant and nonpregnant rats. Immediate ultrafiltration of heparin- or EDTA-anticoagulated plasma yielded phenytoin free fraction values that were in good agreement with in vivo estimates for nonpregnant rats but that were about one-third higher than in vivo estimates for pregnant animals. In vitro free fraction values tended to increase during incubation of plasma and/or during equilibrium dialysis. The concentrations of the four major endogenous free fatty acids were similar in plasma of pregnant and nonpregnant rats if determined immediately after blood collection. Six hours of incubation at 37 degrees C caused fatty acid concentrations to increase about fivefold and twofold in heparin-anticoagulated plasma from pregnant and nonpregnant animals, respectively. The corresponding increases in EDTA-anticoagulated plasma were only about twofold and 1.14-fold, respectively. These changes were associated with decreased plasma protein binding of phenytoin. The in vivo differences between pregnant and nonpregnant rats with respect to phenytoin binding in plasma are not due to differences in fatty acid concentrations, but the in vitro differences are due primarily to corresponding differences in free fatty acid concentrations if extensive in vitro lipolysis occurs.     

Mathematical modelling of in situ and in vitro efflux of ciprofloxacin and grepafloxacin

The efflux process due to p-glycoprotein-like mechanisms of ciprofloxacin (CIP) and grepafloxacin (GRX) has been studied "in situ" in rats and "in vitro" in Caco-2 cells. The results were modelled by a curve fitting procedure which allowed the characterization of the passive (Pd) and carrier mediated parameters (Vm and Km) from the raw data without initial velocities estimation. CIP absorption in rat was characterized as a passive diffusion at the assayed concentrations. Although the involvement of an efflux transporter cannot be ruled out, its relevance in the transport of the fluoroquinolone is negligible. In GRX absorption, an efflux process is implicated and it is detected in both absorption models. GRX permeability depends on the intestinal segment, reflecting the previously reported different expression level of the efflux transporters along the gut in rat. A first attempt to correlate the "in vitro" and the "in situ" data has been done. The mathematical model has been constructed using very simplistic assumptions and it will require further refinement but, nevertheless, the results are promising and demonstrate that a good modelling approach helps to identify the system critical parameters and how the system behaviour change when the parameters are modified as it happens when we move from the "in vitro" to the "in situ" level. Predicted versus experimental permeability values show a good correlation, demonstrating that the relevance of the secretion process "in situ" in rat can be predicted from the "in vitro" cell results.     

Comparison of in vitro and in vivo developmental toxicity and pharmacokinetics of phenytoin in the rat

The rat whole embryo culture was compared to an in vivo experiment with regard to embryotoxicity as well as exposure characteristics, using phenytoin as a model compound. Intra-embryonic concentrations and their embryotoxic effects were determined on gestation day 11 after in vitro administration of 50-150 microg/ml or in vivo gavage of 500-1500 mg/kg body-weight on gestation day 10. In addition, exposure kinetics were studied in vivo after a single oral dose on gestation day 10, and developmental defects on gestation day 21 were scored. The embryotoxic effects observed on gestation day 11 were more pronounced after in vitro exposure in comparison to in vivo exposure at similar intra-embryonic concentrations. Exposure of phenytoin on gestation day 10 in vitro via the culture medium resulted in general embryotoxicity on gestation day 11, whereas in vivo effects as determined on gestation day 11 were minimal. Plasma concentrations of phenytoin increased and plateaued around 35 microg/ml during the 48 hr monitoring period. Plasma concentration curves and pharmacokinetic parameters did not show remarkable differences between the dose groups, indicating that absorption is the limiting factor at the dose range used. Although the developmental effects were minimal as observed in vivo on gestation day 11, specific malformations (defects encompassing the urogenital. craniofacial and skeletal systems) were observed on gestation day 21. These findings show that with similar intra-embryonic concentrations of phenytoin the embryotoxicity in rat whole embryo culture was not comparable with the in vivo embryotoxicity as determined on gestation day 11. This discrepancy may at least partly be explained by differences in exposure characteristics.     

Comparison of pulmonary and hepatic glucuronidation and sulphation of ethanol in rat and rabbit in vitro

1. Pulmonary and hepatic UDP-glucuronyltransferase and sulphotransferase activities in subcellular fractions from rats and rabbits were determined, comparing ethanol with known substrates for these enzymes.

2. No ethyl glucuronide formation was detected with either hepatic or pulmonary microsomal incubations.

3. Chromatographic, autoradiographic and scintillation counting analysis indicated that ethanol is sulphated by rat and rabbit pulmonary cytosol, although this activity was approx. 2–6% of that in liver.

4. Rat hepatic and pulmonary sulphotransferase activities with β-naphthol were approx. 13 and 60 times higher than with ethanol, respectively.

5. Rabbit hepatic and pulmonary sulphotransferase activities with both substrates were higher than those in rat.