Precision-cut liver slices as a model for the early onset of liver fibrosis to test antifibrotic drugs

Induction of fibrosis during prolonged culture of precision-cut liver slices (PCLS) was reported. In this study, the use of rat PCLS was investigated to further characterize the mechanism of early onset of fibrosis in this model and the effects of antifibrotic compounds. Rat PCLS were incubated for 48 h, viability was assessed by ATP and gene expression of PDGF-B and TGF-β1 and the fibrosis markers Hsp47, αSma and Pcol1A1 and collagen1 protein expressions were determined. The effects of the antifibrotic drugs imatinib, sorafenib and sunitinib, PDGF-pathway inhibitors, and perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone, TGFβ-pathway inhibitors, were determined. After 48 h of incubation, viability of the PCLS was maintained and gene expression of PDGF-B was increased while TGF-β1 was not changed. Hsp47, αSma and Pcol1A1 gene expressions were significantly elevated in PCLS after 48 h, which was further increased by PDGF-BB and TGF-β1. The increased gene expression of fibrosis markers was inhibited by all three PDGF-inhibitors, while TGFβ-inhibitors showed marginal effects. The protein expression of collagen 1 was inhibited by imatinib, perindopril, tetrandrine and pirfenidone. In conclusion, the increased gene expression of PDGF-B and the down-regulation of fibrosis markers by PDGF-pathway inhibitors, together with the absence of elevated TGF-β1 gene expression and the limited effect of the TGFβ-pathway inhibitors, indicated the predominance of the PDGF pathway in the early onset of fibrosis in PCLS. PCLS appear a useful model for research of the early onset of fibrosis and for testing of antifibrotic drugs acting on the PDGF pathway.     

Engineered cardiac tissues: a novel in vitro model to investigate the pathophysiology of mouse diabetic cardiomyopathy

Rodent diabetic models, used to understand the pathophysiology of diabetic cardiomyopathy (DCM), remain several limitations. Engineered cardiac tissues (ECTs) have emerged as robust 3D in vitro models to investigate structure–function relationships as well as cardiac injury and repair. Advanced glycation end-products (AGEs), produced through glycation of proteins or lipids in response to hyperglycemia, are important pathogenic factor for the development of DCM. In the current study, we developed a murine-based ECT model to investigate cardiac injury produced by AGEs. We treated ECTs composed of neonatal murine cardiac cells with AGEs and observed AGE-related functional, cellular, and molecular alterations: (1) AGEs (150 µg/mL) did not cause acute cytotoxicity, which displayed as necrosis detected by medium LDH release or apoptosis detected by cleaved caspase 3 and TUNEL staining, but negatively impacted ECT function on treatment day 9; (2) AGEs treatment significantly increased the markers of fibrosis (TGF-β, α-SMA, Ctgf, Collagen I-α1, Collagen III-α1, and Fn1) and hypertrophy (Nppa and Myh7); (3) AGEs treatment significantly increased ECT oxidative stress markers (3-NT, 4-HNE, HO-1, CAT, and SOD2) and inflammation response markers (PAI-1, TNF-α, NF-κB, and ICAM-1); and (4) AGE-induced pathogenic responses were all attenuated by pre-application of AGE receptor antagonist FPS-ZM1 (20 µM) or the antioxidant glutathione precursor N-acetylcysteine (5 mM). Therefore, AGEs-treated murine ECTs recapitulate the key features of DCM’s functional, cellular and molecular pathogenesis, and may serve as a robust in vitro model to investigate cellular structure-function relationships, signaling pathways relevant to DCM and pharmaceutical intervention strategies.     

己酮可可碱通过Hedgehog信号通路对日本血吸虫病肝纤维化形成的抑制作用

目的：探讨己酮可可碱（PTX）通过Hedgehog信号通路抑制日本血吸虫病肝纤维化的作用机制。方法：PMA诱导单核细胞THP-1分化成巨噬细胞,用可溶性日本血吸虫虫卵抗原（SEA）刺激活化THP-1源性巨噬细胞,留取培养上清用于检测其活化状况。CCK-8检测培养上清及PTX对肝星状细胞LX-2的活性影响。设空白对照组、上清刺激组（加入活化的巨噬细胞培养上清）、PTX干预组（加入活化的巨噬细胞培养上清和PTX）。收集以上3组LX-2培养上清及细胞,上清用于ELISA检测TGF-β蛋白表达,细胞用于提取总RNA后RT-PCR检测TGF-β,shh,gli-1的基因表达。结果：SEA可活化THP-1源性巨噬细胞,其活化后培养上清可活化LX-2细胞。上清刺激组TGF-β,shh,gli-1的基因表达空白对照组明显上调,而PTX干预组相对于上刺激组则明显降低。结论：Hedgehog信号通路可能在血吸虫肝纤维化形成过程中起重要作用,己酮可可碱可通过该通路对血吸虫肝纤维化的形成起抑制作用。     

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.     

抗肝纤维化药物研究进展

HF是各种慢性肝病的共同病理学基础,也是肝硬化的必经阶段。积极的抗HF治疗可以控制多种肝组织疾病,尤其是肝硬化的病理进程。靶点明确、疗效显著的抗HF药物是治疗的首选。本研究从HF药物作用靶点、药物研究的体内外模型及药物研究3个方面展开阐述抗HF药物的最新研究进展。     

In vitro metabolism of 10-(3-chlorophenyl)-6,8,9,10-tetrahydrobenzo[b][1,8]naphthyridin-5(7H)-one,a topical antipsoriatic agent. Use of precision-cut rat,dog, monkey and human liver slices,and chemical synthesis of metabolites

The metabolism of SCH 40120, which is the clinically effective antipsoriatic drug 10-(3-chlorophenyl)-6,8,9,10-tetrahydrobenzol[b][1,8]naphthyridin-5(7H)-one, was determined in vitro. Rat, dog, cynomolgus monkey, and human liver slices hydroxylated the aliphatic, cyclohexenyl ring of the drug and conjugated the resulting carbinol. The identified metabolites comprised the corresponding 6-, 7-, and 9-carbinols, the glucuronide of the 6-carbinol, and the 6-ketone derived from the parent drug. Although the three carbinols appeared in the liver isolates of all species studied, the relative amounts of these metabolites varied across species. With a high, non-physiological ratio of substrate to liver, the 6-carbinol and its glucuronide were the major metabolites in human and monkey, whereas the 6-ketone was a minor metabolite in dog. Containing a stereogenic axis and center, the 6-carbinol existed as diastereomeric atropisomers. Its structure was established by ¹³C and ¹H NMR spectroscopy, mass spectrometry, and comparison to an authentic sample. © 1998 John Wiley & Sons, Ltd.     

Assessment of hepatocytes and liver slices as in vitro test systems to predict in vivo gene expression.

The use of in vitro systems to predict in vivo responses to chemical agents provides the benefits of requiring fewer animals, reducing variability between samples, requiring less test material, and enabling higher throughput. In the present study rat tissue slices and primary hepatocytes were compared as in vitro systems to predict in vivo changes in gene expression in response to treatment with known liver toxicants or inducers. Five compounds (phenobarbital, carbon tetrachloride, Wy-14,634, alpha-napthylisothiocyanate, and tacrine) were chosen for their established and diverse mechanisms of hepatoxicity or microsomal induction. Expression profiles from male Sprague-Dawley rats or in vitro systems treated for 24 h were measured by DNA oligonucleotide microarrays containing 8700 probe sets. Qualitative comparison of expression revealed a >80% concordance between in vivo liver and both in vitro systems; however, the responsiveness of both in vitro systems to compound-induced changes in gene expression was far less than that of in vivo. Furthermore, both in vitro systems appeared similar in their ability to reproduce compound-induced changes in gene expression observed in vivo.     

In vitro kinetics of coumarin 3,4-epoxidation: application to species differences in toxicity and carcinogenicity.

Coumarin, a natural product and fragrance ingredient, is a well recognized rat liver toxicant, and dietary administration at toxic dosages increased the incidence of rat cholangiocarcinomas and parenchymal liver-cell tumors in a chronic bioassay. Hepatotoxicity in rats is site- and species-specific, and is thought to result from the formation of coumarin 3,4-epoxide and its rearrangement product, o-hydroxyphenylacetaldehyde (o-HPA). The goals of the current study were to describe the in vitro kinetics of the metabolic activation of coumarin, and determine whether species differences in susceptibility to liver injury correlate with coumarin bioactivation determined in vitro. Coumarin 3,4-epoxidation was quantified via the formation of o-HPA in pooled hepatic microsomes from female B6C3F1 mice, male F344 rats, and individual humans (n = 12 subjects), and the apparent kinetic constants for o-HPA production were calculated using nonlinear regression and fitting to either a one-enzyme or two-enzyme model. Eadie-Hofstee analyses indicated that o-HPA formation was biphasic in both rat and mouse liver. Although the apparent high affinity K:(m) in rat and mouse liver microsomes was 38.9 and 47.2 microM, respectively, the overall rate of o-HPA formation was far greater in mouse than in rat liver microsomes. Furthermore, the total clearance (CL(int)) of coumarin via o-HPA formation in mouse liver microsomes was 4-fold greater than in rat liver microsomes. Since mice are relatively resistant to hepatotoxicity, the data indicated that rates of o-HPA formation in rat and mouse liver microsomes were not directly predictive of liver toxicity in vivo, and further suggested that o-HPA detoxification played a role in modulating coumarin-mediated toxicity. The current studies also indicated that coumarin 3,4-epoxidation in human hepatic microsomes was minimal. In human liver microsomes (n = 12), the kinetics of o-HPA formation were best described by a single enzyme model, with the K(m) for o-HPA formation ranging from 1320-7420 microM. In the most active human sample, the intrinsic clearance of coumarin via the 3,4-epoxidation pathway was 1/9 and 1/38 that of the rat and mouse, respectively. The in vitro kinetics of o-HPA formation, and in particular, the large quantities of coumarin required for o-HPA production in human liver microsomes, strongly suggest that humans are unlikely to produce toxicologically relevant concentrations of this metabolite following low level coumarin exposures.     

Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu.

Hepatic stellate cell (HSC) activation is a key event in the natural process of wound healing as well as in fibrosis development in liver. Current in vitro models for HSC activation contribute significantly to the understanding of HSC biology and fibrogenesis but still fall far short of recapitulating in vivo intercellular functional and anatomic relationships. In addition, when cultured on uncoated plastic, HSC spontaneously activate, which makes HSC activation difficult to regulate or analyze. We have examined whether the use of precision-cut liver slices might overcome these limitations. Liver slices (8 mm diameter, 250 microm thickness) were generated from normal rat liver and incubated for 3 or 16 h with increasing doses of carbon tetrachloride (CCl4). Rat liver slices remained viable during incubation, as shown by minimal enzyme leakage. Expression of markers for HSC activation and the onset of fibrogenesis in the liver slices was studied using real-time PCR and Western blotting. In unstimulated liver slices, mRNA and protein levels of desmin, heat shock protein 47, and alpha B-crystallin remained constant, indicating quiescence of HSC, whereas Krüppel-like factor 6 expression was increased. In contrast, incubation with CCl4 led to a time- and dose-dependent increase in mRNA expression of all markers and an increased alpha B-crystallin protein expression. In conclusion, we have developed a technique to induce activation of quiescent HSC in rat liver slices. This model permits the study of toxicity-induced HSC activation within a physiological milieu, not only in animal but ultimately also in human tissue, and could contribute to the reduction of animal experiments.     

TMP通过miR-145调控胆道闭锁动物模型肝纤维化及Smad3信号通路的研究

胆道闭锁(biliary atresia,BA)的肝纤维化呈进行性发展的趋势,最终会发展为终末期肝硬化。有研究表明,miR-145靶向Smad3可能是调控BA发病过程中肝纤维化的重要机制。川芎嗪(tetramethylpyrazine,TMP)对刀豆蛋白A、四氯化碳诱导的肝纤维化具有改善作用[1-2],但TMP在BA治疗中的作用未见报道。本研究将以miR-145为切入点、在BA动物模型中观察TMP对肝纤维化及Smad3信号通路的调控作用。     

体外释放拟合模板编程及应用实例

目的:探索药物体外释放数据的处理方法及规范相应模板的编程.方法:以VBA语言自编体外释放模型拟合软件模块,加载于EXCEL平台,并予以剖析及实例应用.结果:获得中华人民共和国药典2000年版规范要求的3种释放模型的拟合结果,并经拟合优度检验,可优选优度最高的释放模型并予以输出.结论:本法运用方便,可正确使用公认准则进行数据处理.     

Gene expression in two hepatic cell lines, cultured primary hepatocytes, and liver slices compared to the in vivo liver gene expression in rats: possible implications for toxicogenomics use of in vitro systems.

Microarray technology allows the simultaneous analysis of mRNA expression levels of thousands of genes. In the field of toxicogenomics, this technology could help to identify potentially unsafe compounds based on the changes in mRNA expression patterns they induce. Rodent in vivo and in vitro systems are currently the experimental models of choice for predictive toxicology, especially in early phases of development. This study characterizes several hepatic in vitro systems based on mRNA expression profiles, comparing them to gene expression in liver tissue. The in vitro systems investigated comprise two rat liver cell lines (BRL3A and NRL clone 9), primary hepatocytes in conventional monolayer or in sandwich culture, and liver slices. The results demonstrate that liver slices exhibit the strongest similarity to liver tissue regarding mRNA expression, whereas the two cell lines are quite different from the whole liver. We were able to identify genes with strong changes in expression levels in all or at least one of the in vitro systems relative to whole liver. In particular, for some cytochrome P450s the differences observed on the mRNA expression level were paralleled by protein expression and enzymatic activity. In addition, the effect of time in culture was assessed. We were able to show a profound effect of the duration of culture. Expression patterns change most rapidly soon after cell isolation and culture initiation and stabilize with time in culture. The findings are discussed with respect to the usefulness of the various hepatic in vitro systems for microarray-based toxicological testing of compounds.     

Evaluation of an in vitro screening model to assess phosgene inhalation injury

Therapeutic development against exposure to toxic gases is hindered by the lack of appropriate models to evaluate candidate compounds prior to animal efficacy studies. In this study, an in vitro, air-liquid interface exposure model has been tested to examine its potential application for screening treatments for phosgene (carbonyl chloride)-induced pulmonary injury. Epithelial cultures on Transwell^® inserts, combined with a Vitrocell^® exposure apparatus, provided a physiologically relevant exposure environment. Differentiated human bronchial epithelial (16HBE) cultures were exposed for 8?min to phosgene ranging from 0 to 64?ppm and assessed for changes in transepithelial electrical resistance (TEER, epithelial barrier integrity), cellular viability (XTT) and post-exposure (PE) cellular metabolic energy status. Exposure to phosgene concentrations ≥8?ppm caused dose-dependent and significant decreases in TEER and XTT which did not recover within 24-h PE. In addition, at 64?ppm the rate of oxidative glutamine metabolism was significantly inhibited at 6 and 24?h after exposure. Glycolytic activities (glucose utilization and lactate production) were also inhibited, but to a lesser extent. Decreased glycolytic function can translate to insufficient energy sources to counteract barrier function failure. Consistent and sensitive markers of phosgene exposure were TEER, cell viability and decreased metabolism. As such, we have assessed an appropriate in vitro model of phosgene inhalation that produced quantifiable alterations in markers of lung cell metabolism and injury in human airway epithelial cells. Data indicate the suitability of this model for testing classes of anti-edemagenic compounds such as corticosteroids or phosphodiesterase inhibitors for evaluating phosgene therapeutics.     

Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments.

The rapid growth in the use of in vitro methods for nanoparticle toxicity assessment has proceeded with limited consideration of the unique kinetics of these materials in solution. Particles in general and nanoparticles specifically, diffuse, settle, and agglomerate in cell culture media as a function of systemic and particle properties: media density and viscosity and particle size, shape, charge and density, for example. Cellular dose then is also a function of these factors as they determine the rate of transport of nanoparticles to cells in culture. Here we develop and apply the principles of dosimetry in vitro and outline an approach for simulation of nanoparticle particokinetics in cell culture systems. We illustrate that where equal mass concentrations (mug/ml) imply equal doses for dissimilar materials, the corresponding particle number or surface area concentration doses differ by orders of magnitude. More importantly, when rates of diffusional and gravitational particle delivery are accounted for, trends and magnitude of the cellular dose as a function of particle size and density differ significantly from those implied by "concentration" doses. For example, 15-nm silver nanoparticles appear approximately 4000 times more potent than micron-sized cadmium oxide particles on a cm(2)/ml media basis, but are only approximately 50 times more potent when differences in delivery to adherent cells are considered. We conclude that simple surrogates of dose can cause significant misinterpretation of response and uptake data for nanoparticles in vitro. Incorporating particokinetics and principles of dosimetry would significantly improve the basis for nanoparticle toxicity assessment, increasing the predictive power and scalability of such assays.     

大鼠肺间质纤维化造模的2种方法比较研究

目的探讨气管和腹腔注射2种给药方式复制特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)大鼠模型的差异。方法240~260g雄性SD大鼠24只,按照随机数字表法分为气管对照组(QC组)、气管给药组(Q组)、腹腔对照组(FC组)和腹腔给药组(F组),每组6只。分别经气管一次性注射生理盐水0.2mL或博莱霉素(BLM)5mg·kg~(-1)·d~(-1);腹腔注射生理盐水0.2mL或博莱霉素15mg·kg~(-1)·d~(-1),连续10d。28d后处死。观察每组小鼠生存率、体质量、肺脏外观、病理改变及肺组织羟脯氨酸含量。结果 Q组生存率为66.7%;F组生存率为83.3%,二者比较差异有统计学意义(P<0.05)。2种方法给药组大鼠体质量均减轻,F组动物体质量在1~7d内下降,第8天起开始恢复;Q组动物体质量在前15d内持续下降,第16d起开始恢复。2种方法给药组的肺脏外观基本相同,没有显著差别。病理检查证实,Q组的胶原纤维沉积主要分布在气管周围,而F组的胶原沉积则分布在胸膜和肺间质。2种方法复制的模型组肺组织羟脯氨酸含量均显著高于对照组,差异有统计学意义(P<0.05);模型组间差异无统计学意义。结论气管给药与腹腔给药对于复制大鼠IPF无明显差别,但气管给药法更方便、经济和可靠。     

大鼠肝睾酮羟化酶体外诱导模型的建立

目的建立大鼠原代培养肝细胞微粒体睾酮羟化酶系列同工酶(CYP2A1,CYP2B1/2,CYP2C11,CYP3A1/2)的体外诱导模型。方法应用胶原酶原位灌流法分离大鼠肝细胞进行原代培养;用苯巴比妥钠(PB,1mmol·L-1)、地塞米松(Dex,10μmol·L-1)和β-萘酚黄酮(β-NF,50μmol·L-1)诱导培养肝细胞72 h,提取肝细胞微粒体,进行其肝CYP总量、肝微粒体蛋白含量和睾酮羟化酶比活性的测定。另外采用体内诱导方法,SD大鼠分别给予PB 80mg·kg-1,Dex50mg·kg-1和β-NF80mg·kg-1,ip,每天1次,连续5d,停药24h后制备肝微粒体进行上述指标的测定。结果在体外和体内实验中,PB,Dex和β-NF对肝微粒体蛋白含量、CYP总量和睾酮羟化酶同工酶活性均具有较高的诱导效应。PB对肝CYP总量在体外的诱导效应高于体内,对睾酮不同位置羟化作用的诱导效应体内外无显著性差异;Dex和β-NF对肝CYP总量及睾酮不同位置羟化作用的诱导效应体内外无显著性差异。结论大鼠肝睾酮羟化酶体外诱导模型可替代体内实验,用于药物代谢、新药安全性评价及其他外源性化合物代谢和毒性研究。     

Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells.

Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein and hepatocyte nuclear factor (HNF)3beta decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18, and HNF1alpha were gradually upregulated. In contrast, cocktail treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially exposed cells featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and inducible cytochrome P450-dependent activity, far more efficiently compared to the cocktail condition. In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat BMSC into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a "virtually unlimited" source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development.     

Non-occlusive topical exposure of human skin in vitro as model for cytotoxicity testing of irritant compounds

Testing of irritant compounds has traditionally been performed on animals and human volunteers. Animal testing should always be restricted and for skin irritancy mice and rabbits hold poor predictive value for irritant potential in humans. Irritant testing on human volunteers is restricted by the duration subjects can be exposed, and by the subjectivity of interpreting the visual signs of skin irritation. We propose an irritant testing system using viable human full thickness skin with the loss of cell viability in the exposed skin area as end point measurement. Skin was exposed to sodium dodecyl sulfate (SDS) at 20% concentration by non-occluded topical exposure to establish a positive control response and subsequent test compounds were statistically compared with the 20% SDS response. Cell viability and metabolism were measured with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The model presents correlation between increased concentration of SDS and decreased viability of cells in the exposed skin area (R²?=?0.76). We propose the model to be used for cytotoxicity testing of irritant compounds. With fully intact barrier function, the model comprises all cells present in the skin with quantifiable end point measurement.     

缩宫素诱导的小鼠离体痛经模型的实验方法研究

目的对缩宫素诱发小鼠离体子宫收缩的药理模型的实验条件进行选择和优化。方法使用不同雌激素增加小鼠子宫敏感性,获取小鼠离体子宫,用0·5~10U·L-1缩宫素诱导离体子宫收缩,观察改变各种处理条件后子宫收缩的变化,对4种离体子宫收缩的描述指标进行比较。结果缩宫素5U·L-1为最适剂量;腹腔注射3d10mg·kg-1己烯雌酚或苯甲酸雌二醇均可使子宫产生较高的兴奋性;随着体重上升,离体子宫对缩宫素敏感性略有下降,但各组间差异无显著性;给予缩宫素后5~30min内子宫收缩较为恒定;在各项描述指标中平均收缩张力最为灵敏可靠;250μg·L-1氯丙嗪、1mg·L-1的维拉帕米及100μg·L-1硝苯地平可完全抑制离体子宫的收缩。结论缩宫素诱发的离体小鼠子宫收缩模型快速、简便、灵敏,适合用于痛经治疗药物的筛选。     

Solid lipid nanoparticles for transdermal delivery of diclofenac sodium: preparation,characterization and in vitro studies

The aim of this study was to prepare diclofenac sodium (DNa) solid lipid nanoparticles (SLNs) by a modified emulsion/solvent evaporation method for transdermal delivery. Five independent processing parameters including the lipid matrix, emulsifiers, co-emulsifiers, water-dispersed phase and organic phase were assessed systematically to enhance the entrapment of DNa. The SLNs produced by optimal formulation were submicrometre size with low polydispersity index, the entrapment efficiency was about 89% and the drug loading was about 9.5%. Shape and surface morphology were determined by transmission electron microscopy, which revealed the fairly spherical and core-shell shapes of the SLNs. The in vitro release of SLNs showed a two-step release pattern: one initial burst release followed by a second slow-release phase. In the in vitro cutaneous permeation studies, value of flux obtained for DNa solution was higher than that of SLNs suspension. SLNs had also been shown to improve the dermal localization of DNa.