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1.
Visualising the penetration pathway of a lipophilic model dye into the hair follicle of fresh unfixed human skin would facilitate optimisation of drug formulations for local delivery to the pilosebaceous unit. A block of fresh human scalp skin was mechanically fixed in a newly designed combination of cutting device/on-line diffusion cell, manual cross-sectioned perpendicular to the skin surface and sealed to create the donor and acceptor compartment. The donor phase consisted of a saturated solution of Bodipy FL C(5) in a citric acid buffer solution. Images were obtained on-line by confocal laser scanning microscopy (CLSM) every 30 min for 16 h. For each time point and each skin region relative intensity values were calculated. The on-line visualisation showed a fast diffusion of the label into the gap of the hair follicle followed by a fluorescent staining in the gap itself. The data strongly indicate that the fluorescence in the cuticle originates mainly from the dye of the gap and not from the surrounding epidermis. The on-line visualisation provides a new and excellent tool to monitor simultaneous changes in distribution profiles in the various skin layers including the hair follicle. This information can be used to determine penetration pathways in the skin.  相似文献   

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De novo skin regeneration with human keratinocytes amplified in culture is a life‐saving procedure for patients with extensive skin loss and chronic wounds. It also provides a valuable platform for gene function and therapeutic assessments. Nevertheless, tissues generated in this manner lack hair follicles that are important for skin homeostasis, barrier function, and repair. In this study, we generated skin tissues with human keratinocytes combined with dermal papilla (DP) cells isolated from mouse whisker hair. For this, cultured keratinocytes and mouse DP (mDP) cells were mixed at 10:1 ratio and seeded onto devitalized human dermal matrix derived from surgically discarded human abdominoplasty skin. After 1 week in submerged culture, the cell/matrix composites were grafted onto the skin wound beds of immunocompromised NSG.SCID mice. Histological analysis of 6‐week‐old skin grafts showed that tissues generated with the addition of mDP cells contained Sox2‐positive dermal condensates and well‐differentiated folliculoid structures that express human keratinocyte markers. These results indicate that cultured mDP cells can induce hair follicle neogenesis in the de novo regenerated skin tissues. Our method offers a new experimental system for mechanistic studies of hair follicle morphogenesis and tissue regeneration and provides insights to solving an important clinical challenge in generation of fully functional skin with a limited source of donor cells.  相似文献   

4.
Cutaneous infection is the most common form of human anthrax, but little is known of Bacillus anthracis-epidermal interactions. To study the latter, we used experimental inoculations of B. anthracis Sterne spores onto mouse flank skin. In DBA/2 mice (a sensitive strain) 10(7) spores injected intradermally or applied under occlusive dressings to abraded skin produced ipsilateral inguinal edema and rapid death. Epicutaneous application to shaved-only skin produced edema and death in most animals, but at longer times. Mortality after inoculation onto abraded skin was less in C57BL/6 mice (a relatively resistant strain). Inoculations onto shaved-only skin immunized C57BL/6 mice, and they survived later intradermal spore injections. Histology revealed massive organism proliferation in remaining epidermis and hair follicles of inoculated abraded skin, but less growth in the dermis itself. Conversely, no foci could be located by microscopic examination after inoculation onto shaved-only skin. High-dose nonocclusive dressing inoculations onto unshaved skin in DBA/2 mice revealed small numbers of infective foci, all in hair follicles. These results suggest that epidermal damage may increase infection susceptibility to B. anthracis of hair follicle contents and remaining epidermal remnants; the findings also indicate that access may occur through hair follicles and the denuded dermis.  相似文献   

5.
BACKGROUND: The use of 2',7'-dichlorofluorescein diacetate (DCFH), dihydrorhodamine 123 (DHR) and hydroethidine (HE) has been described for detecting respiratory burst activity by flow cytometry in polymorphonuclear neutrophil (PMN) suspension. However, their specificities for reactive oxygen species are not well defined. We investigated the reactivity of these probes for detecting superoxide anion (O(2)(* -)), hydrogen peroxide (H(2)O(2)) and/or nitric oxide (NO(z.rad;))-dependent mechanisms. METHODS: PMNs (10(6)/ml) were preincubated for 15 min at 37 degrees C with DCFH (5 micro mol/l), DHR (1 micro mol/l) or HE (10 micro mol/l). Cell suspensions were then split for each probe into five different aliquots containing either no effector or one effector: N-ethylmaleimide (NEM, 150 micro mol/l, NADPH oxidase inhibitor), sodium azide (NaN(3), 50 micro mol/l, peroxidase and catalase inhibitor), N-nitro-L-arginine methyl ester (L-NAME, 1.5 micro mol/l, NO(z.rad;) synthase inhibitor) or H(2)O(2) (30%). At the same time, PMNs were stimulated with phorbol myristate acetate (PMA, 10 micro mol/l) for 10 min at 37 degrees C. Analyses were carried out on a Beckman-Coulter Epics XL equipped with an argon laser (488 nm). Green fluorescences from DCFH and DHR were measured in the FL1 channel and HE fluorescence was analyzed in the FL2 channel. RESULTS: NaN(3) decreased the fluorescence of PMNs incubated with DCFH, indicating that it needs a peroxidase activity to react with H(2)O(2). L-NAME reduced the oxidation of DCFH, showing that it reacts with reactive nitrogen species. DHR was specifically responsive to H(2)O(2) accumulation. HE seemed to be preferentially oxidized by O(2)(* -). CONCLUSIONS: Hence the choice of the probe to be used depends on the reactive species of interest.  相似文献   

6.
毛发移植是治疗脱发的重要手段,但受到供区毛囊数量有限的制约,提高毛囊存活率是毛发移植术成功的关键.而影响毛发移植存活率的关键在于毛囊的离体阶段.本文通过查阅国内外文献,综述毛囊保存液的发展、作用机制及研究进展,讨论其对毛发移植术中毛囊存活率的影响.目前临床可用的组织保存液有三种:生理盐水、细胞培养液、低温组织保存液,它们各有利弊.优良的毛囊保存液应具备抗缺氧、保护毛囊干细胞、促进毛囊新生血管等作用.通过了解毛囊保存液对毛囊的影响和作用机制,优化保存液方案,克服当前毛囊移植的技术壁垒,促进毛发再生医学的革新.  相似文献   

7.
黄珊珊  王青  李利 《中国临床康复》2008,(34):6723-6726
应用计算机检索Pubmed数据库1990-2008年、中文期刊全文数据库1998.2008年与皮肤干细胞定位相关的文献,44篇文献资料分析结果显示,毛囊bulge区细胞具有高度增殖潜能,为标记滞留细胞,具有多向分化潜能,不表达分化细胞特异性标志,表达干细胞标志,为目前公认的表皮干细胞的定居处。毛囊真皮鞘包含具有可塑性的干细胞,认为是真皮干细胞的定居处。毛囊真皮乳头内含有具一定增殖分化能力的前体细胞,与真皮鞘相互作用,能够诱导毛囊形成,目前观点认为,真皮乳头内前体细胞移行自真皮鞘干细胞,为其活化的子代细胞。毛囊间皮肤与无毛区皮肤是否存在皮肤干细胞尚存争论,部分实验表明,毛囊间表皮基底层亦有表皮干细胞的存在。  相似文献   

8.
Cooling the scalp during administration of chemotherapy can prevent hair loss. It reduces both skin blood flow and hair follicle temperature, thus affecting drug supply and drug effect in the hair follicle. The extent to which these mechanisms contribute to the hair preservative effect of scalp cooling remains unknown. The purpose of this study was to establish a relationship between local scalp skin temperature and cutaneous blood flow during scalp cooling. We measured skin temperature and cutaneous perfusion during a cooling and re-warming experiment. Experiments on a single subject showed that the measurements were reproducible and that the response was identical for the two positions that were measured. Inter-subject variability was investigated on nine subjects. We found that for the first 10 degrees C of cooling, perfusion of the scalp skin decreases to below 40%. Perfusion can be further reduced to below 30% by a few degrees more cooling, but a plateau is reached after that. We found that a generally accepted relation in thermal physiology between temperature and perfusion (i.e. Q(10) relation) does not describe the data well, but we found an alternative relation that describes the average behavior significantly better.  相似文献   

9.
The hair follicle bulge area is an abundant, easily accessible source of actively growing pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, is also expressed in follicle stem cells and their immediate, differentiated progeny. Green fluorescent protein (GFP), whose expression is driven by the nestin regulatory element in transgenic mice, serves to mark hair follicle stem cells. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34, but negative for keratinocyte marker keratin 15, suggesting their relatively undifferentiated state. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. In vivo studies show that nestin-driven GFP hair follicle stem cells can differentiate into blood vessels and neural tissue after transplantation to the subcutis of nude mice. Hair follicle stem cells implanted into the gap region of a severed sciatic or tibial nerve greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells, which are known to support neuron regrowth. The transplanted mice regain the ability to walk normally. Thus, hair follicle stem cells provide an effective, accessible, autologous source of stem cells for treatment of peripheral nerve injury.  相似文献   

10.
The purpose of the present study was to determinate the significance of ion pairing on the topical permeation of retinoic acid (R.A) using microemulsions as delivery vehicles. Phenylalanine methyl ester, phenylalanine ethylester, histidine methyl ester, tryptophan methyl ester and valine methyl ester were used as counter ions. Results of diffusion studies through polydimethylsiloxane membrane (PDMS) indicate that retinoic acid permeation from ethanol-pH 6.4 buffer mixture significantly increased in the presence of counter ions. A linear relationship was found between apparent partition coefficients and permeation coefficients. The highest values were with valine methyl ester and phenylalanine ethyl ester. In order to develop alternative formulations for topical administration of R.A, microemulsions were evaluated as delivery vehicles. Oil-in-water (O/W) and water-in-oil (W/O) microemulsion formulations were prepared using water, isopropyl myristate, lecithin, caprylyl-capryl glucoside and ethanol or 1,2 hexanediol. Experiments with PDMS membranes showed decreasing permeabilities of R.A from microemulsions in the presence of counter ions. This was related to the increased lipophilicity and different vehicle membrane affinity of the ion pairs The ability of the systems to deliver R.A through the skin was evaluated in vitro using pig-skin. R.A permeabilities were much lower with microemulsions than with solution, while a large increase in R.A skin deposition was observed only from O/W microemulsions in the presence of counter ions. The depth of skin accumulation was below 100 microm after 24 h application. The results suggest that O/W microemulsions containing a counter ion can be used to optimise drug targeting without a concomitant increase in systemic absorption.  相似文献   

11.
Repair of large skin defects caused by burns, trauma, or tumor operations is a clinical challenge. Hair follicle stem cells (HFSCs) are involved in epithelialization of wounds, formation of new hair follicles and promote vascularization in the newly formed skin, and human acellular amniotic membrane (hAAM) is a promising scaffold for skin substitute. Here, we investigated the ability of rat HFSCs (rHFSCs) combined with an hAAM to repair full thickness skin defects in nude mice. The effect of the rHFSC‐hAAM composite on the repair of skin defects in nude mice was assessed by hematoxylin and eosin staining, immunohistochemistry, and EdU‐labeled cell tracking. Isolated and cultured rHFSCs had strong cloning and proliferation potentials. Immunofluorescence staining and flow cytometry assays showed that rHFSCs expressed high levels of integrin α6, CK15, p63, and Sox9. Cells cultured in hAAM showed flaky and cluster‐like morphology and were able to adhere and grow effectively. After transplantation, the rHFSC‐hAAM composite promoted wound healing in nude mice. Moreover, cells in the rHFSC‐hAAM composite were directly involved in hair follicle formation and angiogenesis of tissue around the hair follicle. These results provide an experimental and theoretical basis for the clinical application of HFSCs in repair of human skin defects and a new approach for skin tissue engineering.  相似文献   

12.
Application of reconstructed human Skin (RhS) is a promising approach for the treatment of extensive wounds and for drug efficacy and safety testing. However, incorporating appendages, such as hair follicles, into RhS still remains a challenge. The hair follicle plays a critical role in thermal regulation, dispersion of sweat and sebum, sensory and tactile functions, skin regeneration, and repigmentation. The aim of this study was to determine whether human neopapilla could be incorporated into RhS (differentiated epidermis on fibroblast and endothelial cell populated dermis) and whether the neopapillae maintain their inductive follicular properties in vitro. Neopapillae spheroids, constructed from expanded and self‐aggregating dermal papilla cells, synthesized extracellular matrix typically found in follicular papillae. Compared with dermal fibroblasts, neopapillae showed increased expression of multiple genes (Wnt5a, Wnt10b, and LEF1) known to regulate hair development and also increased secretion of CXCL1, which is a strong keratinocyte chemoattractant. When neopapillae were incorporated into the dermis of RhS, they stimulated epidermal down‐growth resulting in engulfment of the neopapillae sphere. Similar to the native hair follicle, the differentiated invaginating epidermis inner side was keratin 10 positive and the undifferentiated outer side keratin 10 negative. The outer side was keratin 15 positive confirming the undifferentiated nature of these keratinocytes aligning a newly formed collagen IV, laminin V positive basement membrane within the hydrogel. In conclusion, we describe a RhS model containing neopapillae with hair follicle‐inductive properties. Importantly, epidermal invagination occurred to engulf the neopapillae, thus demonstrating in vitro the first steps towards hair follicle morphogenesis in RhS.  相似文献   

13.
目的:研究人体毛囊单位修复裸鼠创面的效果以及人体毛囊单位在裸鼠创面中的生长情况.方法:40只裸鼠随机分为5组,空白对照组(A组,n=8)、毛囊单位移植组(B组,n=8)、微粒皮移植组(C组,n=8)、脱细胞真皮加毛囊单位移植组(D组,n=8)、限制创面收缩毛囊单位移植组(E组,n=8),分别进行相关移植操作结果:A、B、C、D组术后2周的创面愈合率分别为(51.50±1.07)%、(64.49±0.75)%、(58.66±1.56)%、(46.25±2.27)%.A、B、C、D组创面愈合时间分别为(24.00±1.31)d、(18.63±1.69)d、(21.75±1.28)d、(27.13±1.46)d.B、C两组在愈合率和愈合速度方面均优于A、D两组,差异有统计学意义(P<0.0001);而B组愈合率和愈合速度方面又由于C组,差异有统计学意义(P<0.0001).结论:人体毛囊单位移植可以促进裸鼠创面愈合.人体毛囊单位在限制裸鼠创面收缩情况下,无法发挥创面修复作用.  相似文献   

14.
Androgenetic alopecia (AGA), also known as common baldness, is characterized by a marked decrease in hair follicle size, which could be related to the loss of hair follicle stem or progenitor cells. To test this hypothesis, we analyzed bald and non-bald scalp from AGA individuals for the presence of hair follicle stem and progenitor cells. Cells expressing cytokeratin15 (KRT15), CD200, CD34, and integrin, α6 (ITGA6) were quantitated via flow cytometry. High levels of KRT15 expression correlated with stem cell properties of small cell size and quiescence. These KRT15(hi) stem cells were maintained in bald scalp samples. However, CD200(hi)ITGA6(hi) and CD34(hi) cell populations--which both possessed a progenitor phenotype, in that they localized closely to the stem cell-rich bulge area but were larger and more proliferative than the KRT15(hi) stem cells--were markedly diminished. In functional assays, analogous CD200(hi)Itga6(hi) cells from murine hair follicles were multipotent and generated new hair follicles in skin reconstitution assays. These findings support the notion that a defect in conversion of hair follicle stem cells to progenitor cells plays a role in the pathogenesis of AGA.  相似文献   

15.
In order to investigate the effect of liposomal drugs on skin delivery, it was postulated that the process of liposomalization might lead the drug to an overpredicted solubility state which has far-reaching implications for drug skin permeation and accumulation. In this regard, conventional (CL) and flexible liposomes (FL) were prepared by the lipid film hydration method and the particles were downsized by sonication using hydrocortisone (HC) as a poorly water soluble model drug. The solutions derived from the whole CL and FL suspensions eluted on a Sephadex G-50 column (SG-50) demonstrated that most part of HC not only resides solely in the water phase but also it might exist in an improved solubility state. The results of the in vitro study using rat abdominal skin and occlusive application indicated that HC penetrated and accumulated much better solely than when associated with CL or FL. In regard to the penetration of the non-entrapped HC associated to liposomes bilayer fragments, a very small amount of phospholipids in the non-liposomal part eluted on SG-50 was found that could not justify by itself the penetration of HC associated to liposome bilayer fragments. It was proposed that all the steps of the liposomes preparation process might contribute for the increased HC solubility state, but definitively the presence of phospholipids played a crucial role on improving the HC solubility in the absence of sodium cholate. In comparison with commercially available ointments, the non-entrapped HC solution derived from the whole CL suspension eluted on SG-50 showed a higher concentration of HC accumulated and more uniformly distributed as well in the epidermis and dermis compartments. In addition, the thermodynamic activity of the non-entrapped HC solutions maintaining a driving force of the drug across the skin barrier pointed out that the level of HC solubility achieved during liposome preparation has far-reaching implication for drug skin permeation and accumulation in the experimental conditions used. The findings also indicated that the non-entrapped drug solutions obtained on the process of liposomalization could be useful on transdermal drug delivery systems, particularly for improving the permeation and accumulation capacity of poorly soluble drugs.  相似文献   

16.
Hair follicle stem cells sustain growth and cycling of the hair follicle and are located in the permanent portion of the follicle known as the bulge. In this issue of the JCI, Ohyama et al. report the characterization of global gene expression patterns of human hair follicle stem cells after their isolation using sophisticated laser capture techniques to microdissect out bulge cells. They discovered a panel of cell surface markers useful for isolating living hair follicle stem cells, a finding with potential therapeutic implications since isolated stem cells in mice can generate new hair follicles when transplanted to other mice. The findings of Ohyama et al. validate the use of the mouse for studying hair follicle biology but also underscore critical differences between mouse and human stem cell markers. In particular, CD34, which delineates hair follicle stem cells in the mouse, is not expressed by human hair follicle stem cells, while CD200 is expressed by stem cells in both species. Ultimately, this information will assist efforts to develop cell-based and cell-targeted treatments for skin disease.  相似文献   

17.
The skin represents an excellent site for vaccine inoculation due to its natural role as a first line of contact with foreign pathogens and the high local frequency of antigen presenting cells. To facilitate skin-directed immunization, a new technique has been developed (termed microporation) whereby a vaporization process is used to remove tiny areas of the stratum corneum creating microscopic pores that allow access to the underlying viable epidermis. Reporter gene expression was 100-fold increased following application of an adenovirus vector to microporated skin when compared to intact skin. Furthermore, 10-100-fold greater cellular and humoral immune responses were observed following topical administration of an adenovirus vaccine to microporated skin versus intact skin. Hairless mice responded to the microporated adenovirus vaccine equivalently to mice with normal hair follicle distribution demonstrating the activity of the microporated vaccine was not related to follicle count. In a tumor challenge model using a surrogate antigen, microporation increased vaccine efficacy by approximately 100-fold compared to intact skin. Finally, microporation enabled delivery of an adenovirus vaccine carrying a relevant melanoma antigen resulting in the development of auto-immune vitiligo and tumor protection. Thus, the microporation technology has proven to be a reliable and easy method to enable skin-directed vaccination.  相似文献   

18.
背景毛囊在伤口愈合,肿瘤发生等过程中起主导作用,由于其在活体内影响因素较多,故难以研究其生物学作用机制.目的利用培养的毛乳头细胞观察体内外条件下诱导毛囊形成的可能.设计非随机非对照的实验研究.地点和对象实验在第三军医大学大坪医院野战外科研究所完成.对象为正常人头皮中获取毛乳头细胞、真皮鞘细胞、毛囊上、下段及球部细胞.干预将毛乳头细胞、真皮鞘细胞分别与毛囊上、下段上皮细胞进行体外三维培养重建,用游离细胞混合移植于裸鼠,组织学观察毛囊形成情况.主要观察指标毛囊毛乳头细胞、真皮鞘细胞对分段毛囊上皮细胞的诱导分化作用.结果毛囊间表皮细胞、毛囊上段上皮细胞、下段上皮细胞和球部细胞在间质细胞凝胶上均可形成双层结构的组织工程皮肤,在真皮鞘细胞胶原凝胶上毛囊的上、下段上皮细胞形成了毛囊结构,移植于裸鼠后毛乳头细胞胶原凝胶诱导毛囊上、下段细胞形成了毛囊.低代毛乳头细胞与毛囊上皮细胞混合移植形成了数量较多、结构典型的毛囊,并有肉眼可见的毛发纤维产生.结论毛囊的真皮成分细胞即毛乳头细胞、真皮鞘细胞在体内、外均具有诱导毛囊形成的能力,通过与毛囊上皮细胞之间的相互作用,诱导毛囊形成.  相似文献   

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26-Hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and other C27-steroids was demonstrated in cultured skin fibroblasts from healthy individuals. Activities in skin fibroblasts were approximately 5-10% of those previously found in human liver homogenates, and were inhibited by CO. The apparent Km was lowest for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (1.3 mumol/liter) and highest for 5-cholestene-3 beta, 7 alpha-diol (12 mumol/liter). The rate of 26-hydroxylation was highest with 7 alpha-hydroxy-4-cholesten-3-one. These characteristics are similar to those of hepatic mitochondrial C27-steroid 26-hydroxylase. In skin fibroblasts from three patients with cerebrotendinous xanthomatosis (CTX), 26-hydroxylation of C27-steroids proceeded at a rate of only 0.2-2.5% of healthy controls. No accumulation of endogenous 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol could be demonstrated in these cells, and the lowered formation of radioactive, 26-hydroxylated products could not be explained by dilution of the labeled exogenous substrate. The present results add strong evidence to the concept that the primary metabolic defect in CTX is a deficiency of C27-steroid 26-hydroxylase.  相似文献   

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