酮洛芬异丙酯在皮肤细胞中的代谢

目的：研究酮洛芬异丙酯在皮肤细胞中的代谢作用，为进一步研究利用酯类前体药物方法改善药物的经皮吸收特性提供实验依据。方法：将人包皮的第3代角质形成细胞或成纤维细胞超声破碎制成匀浆，加入不同量的酮洛芬异丙酯进行37℃温孵实验，通过高效液以谱法分别在不同时间测定细胞匀浆中酮洛芬异丙酯及酮洛芬的浓度。结果：酮洛芬异丙酯在表皮角质形成细胞和真皮成纤维细胞的匀浆中存在代谢现象，有前者的代谢能力明显大于后者。结论：酯类前体药物可在皮肤细胞中被代谢为活性母体药物本身。     

健康人皮肤间充质干细胞对HaCaT细胞增殖作用的研究

目的探讨健康人皮肤间充质干细胞(S-MSCs)对Ha Ca T细胞增殖的作用,为研究S-MSCs在病理状态下的作用提供理论依据。方法健康人皮肤20份,均取自我院整形和泌尿外科手术切除的多余新鲜皮肤组织。在体外无菌条件下进行真表皮分离、培养真皮MSCs,流式细胞术进行细胞鉴定,多向诱导分化及分化细胞的鉴定,将培养至第5代的S-MSCs与Ha Ca T细胞共培养,并与自然增殖组进行对照,采用实时动态细胞分析技术法进行Ha Ca T细胞增殖检测,培养72 h后用细胞计数法检测Ha Ca T细胞的数量。结果倒置相差显微镜下,健康人S-MSCs的细胞形态呈细长梭形,细胞表面抗原CD29、CD44、CD73、CD90、CD105呈高表达,而CD34、CD45及人类白细胞抗原(HLA)-DR表达阴性,并且可诱导分化为脂肪细胞、成骨细胞、成软骨细胞。Ha Ca T细胞自然增殖组细胞数为(2.74±0.36)×105,与健康人S-MSCs共培养组细胞数为(2.24±0.28)×105,差异有统计学意义(P<0.001)。结论健康人皮肤来源的MSCs可在体外抑制Ha Ca T细胞的增殖。     

组织工程皮肤种子细胞的研究进展

组织工程皮肤是应用生命科学和工程学的原理与方法,构建出的接近于人体组织结构的生物活性替代物。种子细胞的研究是组织工程皮肤产品研制中的关键部分。现就种子细胞相关研究的进展进行综述。     

组织工程皮肤替代物构建的实验研究

目的探索组织工程皮肤替代物的构建方法。方法采用传代培养的角质形成细胞和成纤维细胞作为种子细胞,I型胶原作为真皮基质,采用气—液界面方式进行培养,用HE染色观察培养物的结构。结果培养的组织工程皮肤替代物具有正常皮肤的形态结构,具有结构致密的真皮和分化良好的表皮。结论本方法可用于活性皮肤替代物的制备。     

酮洛芬异丙酯在离体裸小鼠和猴皮肤中的渗透和代谢

目的　研究酮洛芬异丙酯在离体裸小鼠和猴皮肤中的渗透和代谢 ,阐明皮肤酯酶代谢的主要活性部位。方法　采用外科手术和酶消化方法制备离体皮肤 ,用水平双室扩散池进行体外渗透实验 ,HPLC方法测定样品中的药物浓度。结果　经全皮和去角质层皮肤渗透的酮洛芬异丙酯被代谢成酮洛芬 ,接受室中酮洛芬的浓度随时间延长而成线性增加。在裸小鼠皮肤中 ,酮洛芬全皮渗透的稳态速率是酮洛芬异丙酯全皮渗透的 2 5倍 ,而渗透实验结束后酮洛芬异丙酯及代谢物酮洛芬在皮肤中残留量是酮洛芬渗透实验结束后残留量的 2 2 2倍 ;酮洛芬异丙酯经真皮渗透时 ,代谢物酮洛芬的稳态形成速率大大小于经全皮渗透时的稳态形成速率。在猴皮肤中 ,酮洛芬异丙酯经全皮渗透时代谢物酮洛芬的稳态形成速率是经去角质层皮肤的 0 7倍 ,而渗透实验结束后皮肤中酮洛芬和酮洛芬异丙酯的残留量是去角质层皮肤的 2 0倍 ;酮洛芬异丙酯经真皮渗透时 ,代谢物酮洛芬的稳态形成速率小于经全皮和去角质层皮肤渗透时的稳态形成速率 ,渗透结束后皮肤中酮洛芬的残留量也小。结论　酮洛芬酯前体药物在皮肤原位能被代谢成活性母体酮洛芬本身 ,且在皮肤中能长时间驻留 ,有助于提高和延长酮洛芬的局部疗效。表皮层是皮肤酯酶代谢的主要活性部位     

组织工程皮肤修复创面的研究进展

近20年来随着组织工程学的建立和形成，组织工程的研究正成为生命科学研究领域的焦点之一，而组织工程皮肤产品开发的成功，使人工皮肤的研究实现了质的飞跃，它是由活性细胞接种在支架材料上形成的组织工程化皮肤，有真皮层或同时具有表皮层和真皮层，因此是一种活性生物敷料。组织工程皮肤替代自体皮片移植是创面愈合治疗领域的一个重大进展。组织工程皮肤不仅具有正常皮肤的部分功能，而且具有良好的修复皮肤创伤的作用，有利于皮肤创面的愈合。本文综述了近20多年来的相关文献，介绍了一些组织工程皮肤产品临床应用情况及其优缺点。     

银杏叶提取物抑制HaCaT细胞氧化应激损伤作用的研究

目的 探讨银杏叶提取物对长波紫外线照射诱导的HaCaT细胞氧化应激性损伤的保护作用.方法采用MTT法测定细胞活力,酶生化法测定细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)活性和丙二醛(MDA)含量.结果银杏叶提取物能提高HaCaT细胞增生活力,降低细胞内丙二醛水平,增强抗氧化酶活性.结论银杏叶提取物...     

皮肤角质形成细胞的无血清培养方法

目的探索建立少量成年自体皮肤角质形成细胞体外无血清培养体系,为自体组织工程皮肤的构建与移植奠定物质基础。方法:无菌条件下,取2cm×2cm 兔耳皮肤组织块,Dispase 消化,分离真表皮,表皮以胰蛋白酶+EDTA 消化获得角质形成细胞,以含钙和不含钙的角质形成细胞培养液(K-SFM),按不同细胞密度接种于24孔板,观察细胞生长状况;免疫组化鉴定,MTT 法测定不同血清浓度对角质形成细胞增殖分化的影响。结果:在适当的 Ca~(2+)浓度下,少量自体角质形成细胞能够在无血清培养液 k-SFM 中培养扩增,最多可传4～6代。添加血清后可明显加速细胞分化。结论:无血清培养体系适用于少量成年自体角质形成细胞的体外连续培养扩增,培养的细胞可用于自体组织工程皮肤的构建。     

小型香猪复方壳多糖组织工程皮肤的构建及组织学特点

目的评价制备的小型香猪复方壳多糖组织工程皮肤在组织学方面的特性,为其修复皮肤缺损创面提供实验依据。方法采用传代培养的角质形成细胞和成纤维细胞作为种子细胞,I型胶原作为真皮基质。采用气—液界面方式进行培养,通过苏木精—伊红(HE)染色、比较小型香猪的体外构建组织工程皮肤与正常皮肤的组织学特征,包括表皮、真皮结构。结果制备的复方壳多糖组织工程皮肤表皮细胞增殖活跃,分层分化良好,厚约150μm,真皮层细胞生长正常,排列有序,与机体皮肤结构相似。结论复方壳多糖组织工程皮肤组织结构良好,符合新型皮肤替代物在治疗皮肤缺损时的组织学要求。     

人参皂苷Rg1和Rb1对UVB照射引起的HaCaT细胞损伤的保护作用

过量紫外线辐射是危害人类健康的环境因素之一，它对皮肤的损伤更为显著，可以引起皮肤损伤等一系列生物反应。日光中的紫外线辐射到地面后主要是中波紫外线（UVB，波长280～320nm）和少量的长波紫外线（UVA，波长320～390nm），UVB对DNA嘧啶二聚体的形成、诱发突变等方面的生物学效应远大于UVA。环境污染、臭氧层稀薄等因素导致人类受到辐射增多的危险。     

Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms,HaCaT keratinocytes and affects skin deposition

The use of silver nanoparticles (Ag NPs) within the healthcare sector and consumer products is rapidly increasing. There are now a range of diverse-shaped Ag NPs that are commercially available and many of the products containing nanosilver are topically applied to human skin. Currently, there is limited data on the extent to which the antimicrobial efficacy and cytotoxicity of Ag NPs is related to their shape and how the shape of the Ag NPs affects their distribution in both intact and burn wounded human skin after topical application. In this study, we related the relative Ag NP cytotoxicity to potential skin pathogens and HaCaT keratinocytes in vitro with the shape of the Ag NPs. We employed multiphoton fluorescence lifetime imaging to map the distribution of the native and unlabeled Ag NPs after topical application to both intact and burn wounded human skin using the localized surface plasmon resonance signal of the Ag NPs. Truncated plate shaped Ag NPs led to the highest cytotoxicity against both bacteria (IC₅₀ ranges from 31.25 to 125?μg/mL depending on the bacterial species) and HaCaT keratinocytes (IC₅₀ 78.65?μg/mL [95%CI 63.88, 96.83]) thus both with similar orders of magnitude. All Ag NPs were less cytotoxic than solutions of silver nitrate (IC₅₀ of 7.85?μg/mL [95%CI 1.49, 14.69]). Plate-shaped Ag NPs displayed the highest substantivity within the superficial layers of the stratum corneum when topically applied to intact skin and the highest deposition into the wound bed when applied to burned ex vivo human skin relative to other Ag NP shapes.     

酮洛芬巴布剂的研制及体外透皮研究

目的：研制酮洛芬巴布剂并研究其体外释药性能和经皮吸收特点。方法：以水溶性高分子材料为辅料制备酮洛芬巴布剂，用HPLC法测定酮洛芬的经皮吸收量，按《中华人民共和国药典》2005年版方法进行体外释放度测定，利用改良Franz扩散池研究巴布剂的经皮吸收特点。结果：酮洛芬巴布剂含膏量均匀，含量稳定，体外释药符合Weibull分布方程。巴布剂中的酮洛芬24h内以零级动力学经皮渗透，体外经皮释药方程为Q=10．196t-7．9547（r=0．9988），24h累积渗透量为244．70μg／cm^2结论：酮洛芬巴布剂为一种新型控释型经皮给药制剂。     

Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo

This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.     

Skin penetration and tissue permeation after topical administration of diclofenac

Objective: Topical delivery of drugs is an alternative to oral administration, often with similar efficacy but potentially a more favorable tolerability profile. However, topical formulations need to be able to penetrate the skin and permeate to the target areas in quantities sufficient to exert a therapeutic effect. Many factors can affect this process, including the physicochemical properties of the drug, the formulation used, and the site and mode of application. It is believed that measurement of drug concentrations at the sites of action may be an indicator of their likely efficacy. This review addresses these issues, with reference to topically administered diclofenac in osteoarthritis.

Methods: Articles relevant to this review were identified after a systematic search of Medline and Embase, using the key words “diclofenac”, "topical administration" and “osteoarthritis” in the search strategy.

Results: The sparse data available indicate that topical diclofenac can penetrate and permeate to deeper tissues, with a lower plasma to tissue ratio than oral diclofenac. The tissue diclofenac levels after topical delivery are sustained over time (at least several hours). However, there is not enough data to establish how diclofenac levels in the joint compare with IC₅₀ levels (50% of the maximum inhibition of prostaglandin synthesis) established following oral administration.

Conclusions: After topical application, diclofenac can penetrate the skin and permeate to deeper tissues, where it reaches a concentration that appears to be sufficient to exert a therapeutic effect. More robust methods are required for in vivo characterization to better estimate the clinical efficacy of topically applied drugs.     

中药挥发油作为透皮吸收促进剂的研究进展

近年来,透皮吸收制剂正受到人们的普遍关注,而其中存在的一个难题就是吸收率低,难以达到有效的治疗浓度。透皮吸收促进剂的应用可以有效地解决这一难题。中药挥发油作为透皮吸收促进剂不良反应小,有促渗、治疗双重作用,因此逐渐引起了广大医药工作者的注意。就中药挥发油作促透剂的研究现状做一综述。     

Hair follicles contribute significantly to penetration through human skin only at times soon after application as a solvent deposited solid in man

AIMS

The aim of this study was to define the underlying relative penetration of caffeine through hair follicles and through intact stratum corneum with time in vivo through pharmacokinetic modelling.

METHODS

Caffeine plasma concentration–time profiles after topical application into skin with or without hair follicle blocking were modelled using the Wagner–Nelson method or a compartmental model with first order absorption and elimination. Pharmacokinetic parameters describing absorption rate and extent of absorption through hair follicles or the stratum corneum were determined separately and compared with each other.

RESULTS

The obtained pharmacokinetic parameters from the two methods were similar. The absorption rate constant of caffeine for hair follicles was nearly 10 times higher than that for the stratum corneum and the percentage of absorption from hair follicles was more than half of that of the stratum corneum. In addition, the absorption from the stratum corneum showed an approximately 10 min delay while there was no delay for absorption from hair follicles. All caffeine absorbed by hair follicles occurs within 30 min of application and accounts for 10.5 to 33.8% of the total amount absorbed across the skin for all subjects, whereas absorption of caffeine through the stratum corneum can occur over several hours.

CONCLUSION

Hair follicles contribute significantly to percutaneous absorption of caffeine after topical application in man in vivo only at times soon after application.     

In vitro skin penetration of acetyl hexapeptide-8 from a cosmetic formulation

There is a concern that peptides in cosmetic creams marketed as anti-aging/anti-wrinkle may penetrate into the deep layers of the skin and potentially stimulate biological activity. Claims for one cosmetic peptide, acetyl hexapeptide-8 (Ac-EEMQRR-amide), suggest interference with neuromuscular signaling as its anti-wrinkle mechanism of action. Therefore, the skin penetration of commercially available Ac-EEMQRR-amide from a cosmetic formulation (oil-in-water (O/W) emulsion) was determined in hairless guinea pig (HGP) and human cadaver skin assembled into in vitro diffusion cells. An O/W emulsion containing 10% Ac-EEMQRR-amide was applied to skin at a dose of 2?mg/cm². After a 24-h exposure, the skin surface was washed to remove unabsorbed peptide. Skin disks were tape stripped to determine the amount of peptide in the stratum corneum. Removal of the stratum corneum layers was verified by confocal microscopy. The epidermis was heat separated from the dermis and each skin fraction was homogenized. Skin penetration of Ac-EEMQRR-amide was measured in skin layers by hydrophilic interaction liquid chromatography with tandem mass spectrometry using electrospray ionization (ESI) in the positive mode. Stable isotopically labeled hexapeptides were used as internal standards for the quantitation of native hexapeptides to correct for matrix effects associated with ESI. The results (percent of applied dose) showed that the majority of the Ac-EEMQRR-amide was washed from the surface of both HGP and human skin. Ac-EEMQRR-amide that penetrated skin remained mostly in the stratum corneum of HGP (0.54%) and human (0.22%) with the peptide levels decreasing as each layer was removed by tape stripping. Total Ac-EEMQRR-amide found in the epidermis of HGP and human skin was similar at 0.01%. No peptide was detected in the dermis or buffer collected underneath the skin for both human and HGP. There was no hexapeptide metabolite (H₂N-EEMQRR-amide) detected in any layers of HGP skin, human skin or buffer collected underneath the skin. This skin penetration data will be useful for evaluating the safety of cosmetic products containing small peptide cosmetic ingredients.     

溶剂和氮酮对噁丙嗪透皮速率的影响

目的：研究溶剂与氮酮对恶丙嗪渗透性的影响。方法：分别采用乙醇和丙二醇为溶剂,利用离体大鼠皮肤和Valia-Chien水平扩散池,测定恶丙嗪的经皮渗透速率。结果：氮酮在丙二醇溶液中具有明显的促渗作用。结论：恶丙嗪的体外渗透符合零级动力学过程。     

Human skin penetration of gold nanoparticles through intact and damaged skin

Abstract

Gold nanoparticles (AuNPs) are produced for many applications but there is a lack of available data on their skin absorption. Experiments were performed using the Franz diffusion cell method with intact and damaged human skin. A physiological solution was used as receiving phase and 0.5 mL (1st exp) and 1.5 mL (2nd exp) of a solution containing 100 mgL^-1 of AuNPs (15 and 45 μg cm^-2, respectively) was applied as donor phase to the outer surface of the skin for 24 h. Skin absorption was dose dependent. Mean gold content of 214.0 ± 43.7 ng cm^-2 and 187.7 ± 50.2 ng cm^-2 were found in the receiving solutions of cells where the AuNPs solution was applied in higher concentration on intact skin (8 Franz cells) and on damaged skin (8 Franz cells), respectively. Twenty-four hours gold flux permeation was 7.8 ± 2.0 ng cm^-2 h^-1 and 7.1 ± 2.5 ng cm^-2 h^-1 in intact and damaged skin, respectively, with a lag time less than 1 hour. Transmission Electron Microscope analysis on skin samples and chemical analysis using Inductively Coupled Plasma-Mass Spectrometry demonstrated the presence of AuNPs into epidermis and dermis. This study showed that AuNPs are able to penetrate the human skin in an in vitro diffusion cell system.