表皮干细胞及其信号通路的研究进展

1表皮干细胞特点及其在创伤愈合中的作用皮肤是人体最大的器官,表皮位于皮肤最外层,具有自我更新能力的组织(约每个月更新1次)。近年来,人们逐渐认识到存在于上皮组织的成体干细胞EScs与皮肤的发生、修复、改建以及与上皮源肿瘤、皮肤免疫性疾病有着密切     

感觉神经肽P物质特异性受体在表皮干细胞上的表达

近10年来，表皮干细胞（epidermal stem cells，ESCs）作为皮肤细胞发生、修复、改建的源泉，因其与上皮源肿瘤、皮肤变应性疾病的发生密切相关而成为研究的热点。体内实验结果显示，皮肤伤口中外源性感觉神经肽P物质可促进ESCs增殖并将其大量募集于创缘，加速修复过程。为进一步探讨P物质对ESCs生物学行为影响的机制，本实验分别从蛋白水平和mRNA水平观察体外培养的ESCs上P物质的两种特异性受体：神经激肽1受体（NK—1R）和NK-2R的表达情况。     

表皮干细胞生物学特性的研究进展

<正>皮肤作为保护人体的天然屏障,在避免机体水分大量丢失、维持机体体温恒定、抵抗微生物感染、减少机械损伤中均具有重要作用,并且具有终生动态保持自身结构状态稳定的能力。皮肤结构由表及里依次为表皮、真皮、皮下组织及其附属器官。表皮干细胞(epidermal stem cells,ESCs)是来源于胚胎外胚层的特异性干细胞,能分化为皮肤各层组织,从而使表皮始终处于持续不断的增殖、分化、凋亡状态,保证表     

表皮干细胞表面标志物的研究进展

表皮干细胞（epidermal stem cells,ESCs）是来源于胚胎外胚层的皮肤组织的专能干细胞,可以分化成各种表皮细胞,使表皮始终处于持续的增殖、分化、脱落、消亡状态,大约每月更新一次,并且增殖与消亡的速度保持大致平衡,从而保证了皮肤形态和功能的完整性。     

人表皮干细胞分离培养和纯化鉴定的进展

皮肤体现出很强的再生能力,是由于表皮基底层中的表皮干细胞(epidermal stem cells,ESCs)终身不断自我更新、持续分化以取代终末分化细胞,从而在维持组织结构的更新、细胞内环境的稳定及在创伤后创面修复过程中起至关重要的作用。随着分子生物学、细胞生物学、组织工程学及生     

microRNAs对调控胚胎干细胞自我更新与分化的影响

胚胎干细胞（embryonic stem cell,ESCs）是指存在于胚泡内细胞群中、具有高度自我更新和多向分化潜能的细胞。ESCs可以在体外无限扩增并保持未分化状态,具有分化为胚胎或成体的各种细胞类型的潜能。因此,研究ESCs增殖与分化机制,可以更好地了解其生物学特性,对实现ESCs的定向分化和组织工程产品的研制具有推动作用。尽管目前ESCs自我更新与分化过程中的分子机制是研究热点,但距离全面厘清此问题仍相去甚远。     

含小鼠表皮干细胞的真皮替代物体内移植追踪实验

目的追踪种植于真皮替代物中的小鼠表皮干细胞（epidermal stem cells，ESCs）移植到同基因受者体内后的转归。方法在体外诱导胚胎干细胞（embryonic stem cells，ES细胞）分化为ESCs，采用荧光染料Hoechst 33342染色后，种植到含有成纤维细胞的胶原一明胶海绵真皮替代物内，移植到与ES细胞同基因的129／J小鼠皮下，采用HE染色、免疫组化和Van Gieson染色等观察这些细胞的存活和分化情况。结果移植后至少3周内仍可以在荧光显微镜下清晰观察到ESCs。这些细胞能够存活并分化形成毛囊样、腺管样结构，形成与天然真皮相似的结构。在这些结构中有CD29（整合素口，业单位）和细胞角蛋白18等表皮干细胞特异性标志物的表达。在移植后至少10周内未观察到明显排斥反应或严重毒副反府。结论ESCs能够在所构建的真皮支架内存活并分化形成毛囊样和腺管样结构，提示这些来源于ES细胞的ESCs可以作为种子细胞，用于研究制备含有皮肤附属器的真皮替代物。     

血管紧张素转化酶抑制剂对表皮干细胞增殖、分化、迁移的影响

目的 观察血管紧张素转化酶(ACE)抑制剂卡托普利对表皮干细胞(ESCs)增殖、迁移、分化的影响,探讨ACE在维持ESCs生物学功能中的作用.方法 利用差速贴壁法获得10例儿童的ESCs,进行离体培养,检测ACE在ESCs的表达.用XTT法检测不同浓度(1 ×l0-5、1×10-6、l×10-7、1×10-8mol/L) ACEI卡托普利对ESCs增殖的影响;体外创伤模型观察1×10-6mol/L的卡托普利对ESCs 6、12、18、24h各时间段的迁移能力;流式细胞仪检测K10的表达观察1×10-6mol/L的卡托普利对ESCs分化的影响.结果 培养的细胞经β1-整合素和K19免疫荧光双重标记染色,83.55％细胞为双标记阳性细胞,即ESCs.免疫荧光染色和逆转录-聚合酶链反应(RT-PCR)结果显示ESCs表达ACE.流式细胞仪定量检测培养的细胞ACE阳性率为74.2％.1×10-6mol/L的卡托普利可明显抑制ESCs的增殖(P＜0.05),且在第5天达到峰值.1×10-6 mol/L的卡托普利可明显抑制细胞的迁移能力(P＜0.05)和克隆能力(P＜0.05).流式细胞仪检测结果表明,l×10-6 mol/L的卡托普利并不影响K10的表达(P＞0.05).结论 ACE通过影响ESCs的增殖,迁移从而影响皮肤的损伤修复和自我更新.     

菲立磁标记胚胎干细胞适宜示踪浓度的研究

目的 探讨适宜体内移植示踪研究的胚胎干细胞(ESCs)菲立磁标记浓度.方法 分别以0.05、0.10 g/L硫酸鱼精蛋白-菲立磁复合物标记胚胎干细胞,行电镜检测及MRI扫描了解菲立磁标记细胞体外生物学特征.结果 0.05 g/L浓度的硫酸鱼精蛋白-菲立磁复合物标记胚胎干细胞效率最高,达到91.5%以上,标记细胞数量为1×105时能有效引发MRI信号改变.结论 以0.05 g/L硫酸鱼精蛋白-菲立磁复合物浓度标记胚胎干细胞可以作为胚胎干细胞体内移植适宜的标记浓度.

Abstract：

Objective To investigate the feasible feridex concentration for labeling embryonic stem cells (ESCs) in tracking study in vivo.Methods ESCs were labeled with 0.05,0.10 g/L protamine sulfate-feridex complex respectively,to find out the biological character of ESCs labeled with feridex through electron microscopy and magnetic resonance imaging (MRI) examination.Results With 0.05 g/L protamine sulfate-feridex complex,the feridex labeling efficiency of ESCs was above 91.5%.The number of labeled cells which induced MRI signal changes in vitro was 1 × 105.Conclusion Labeled ESCs with 0.05 g/L protamine sulfate-feridex complex is the optimal choice for transplanted ESCs in vivo.     

SDF-1对创缘表皮干细胞定向趋化及促创面愈合效应的研究

目的：观察干细胞趋化因子SDF-1在创面愈合过程中调控表皮干细胞定向迁移促进创面愈合的过程。方法：制作大鼠背部全层皮肤缺损模型,随机分为三个实验组：①SDF-1组;②AMD3100（CXCR4受体的拮抗剂）组;③空白对照组。分别于致伤后1天、3天、5天、7天和12天照相计算伤后不同时间创面占初始创面的百分比并取材。运用HE染色和免疫组化技术观察创缘表皮干细胞定向迁移分化的特点,寻找其规律。结果：与其他两个处理组相比,SDF-1处理组愈合时间缩短,其创缘皮表皮基底层细胞分裂增殖旺盛,创缘表皮干细胞数目明显多于其他两组。结论：在创面愈合过程中,SDF-1可以调控表皮干细胞向创缘迁移,加速创面上皮化。     

Akermanite bioceramic enhances wound healing with accelerated reepithelialization by promoting proliferation,migration, and stemness of epidermal cells

Reepithelialization is an important step of wound healing, which is mainly completed by proliferation and migration of epidermal cells. Akermanite is a Ca‐, Mg‐, and Si‐containing bioceramic. This study evaluated the effects of Akermanite on wound healing and investigated the mechanisms. Using scald burn mice models, we demonstrated that local Akermanite treatment significantly accelerated wound healing by increasing reepithelialization and the stemness of epidermal cells. Epidermal cells were cultured in medium containing Akermanite extracts to explore the cellular mechanism of reepithelialization. Akermanite promoted the cell proliferation and migration, maintaining more cells in the S and G₂/M phases of the cell cycle. An additional study showed that Akermanite enhanced the expressions of integrinβ1, Lgr4, Lgr5, and Lgr6, which are specific molecular markers of epidermal stem cells, accompanied by the activation of the Wnt/β‐catenin pathway. These results suggested that Akermanite accelerated reepithelialization by increasing the proliferation, migration, and stemness of epidermal cells in a manner related to the Wnt/β‐catenin pathway, which might contribute, at least partially, to accelerated wound healing by Akermanite therapy.     

Epithelial stem cell islands in the regenerated epidermis

Objective: The effects of growth factors on wound healing have been studied extensively, however,their molecular and genetic mechanisms that regulate epidermal regeneration are not fully understood. In this study,we explore the cell reversion characteristics and epithelial stem cell distribution in human regenerated epidermis treated with recombinant human epidermal growth factor (rhEGF). Methods:Tissue biospies from 8 regenerated skins treated with rhEGF were used to evaluate the cell reversion and stem cell distribution in epidermis . The expression of β1 integrin, keratin 19 (K19), keratin 14 (K14) and keratin 10 (K10) in skins was detected with SP immunohistochemical methods. Another 8 biopsies from the regenerated epidermis treated without rhEGF, fetus, children and adults were used as the controls. Results:Immunohistochemical stain for β1 integrin and keratin 19 showed that there were some new stem cell islands in the epidermis treated with rhEGF. These cells were small, containing low RNA content and exhibiting positive expression with β1 integrin and K19 stain. They were isolated, bearing no anatomic relation with the epithelial stem cells in the basal layer. The serial identification experiments indicated that there treated without rhEGF. All of these results supported that these β1 integrin and K19 positive stain cells were the stem cells. Conclusions: The results indicated that these stem cell islands were the specific and individual cell structures in rhEGF treated wounds and rhEGF is the main factor in inducing the stem cell island formation. These results offer a direct evidence for epidermal cell reversion from the differentiated cells to undifferentiated stem cells in vivo and may be useful in the rational use of this growth factor to promote wound healing in clinic.     

Early cellular changes of human mesenchymal stem cells and their interaction with other cells

Cell-to-cell interactions between human mesenchymal stem cells and potential adjacent cells such as endothelial cells, dermal fibroblasts, and epidermal keratinocytes was investigated. A modified dual Boyden chamber assay using 8-microm pores revealed a more powerful chemotactic cell migration of human mesenchymal stem cells toward human epidermal keratinocytes than other cells, such as umbilical artery endothelial cells and dermal fibroblasts, during 16 hours of incubation (336.2+/-52.33, 36.0+/-11.20, and 62.7+/-18.16, cells/field, respectively, p<0.01; comparison between endothelial cells and keratinocytes, and fibroblasts and keratinocytes). Scanning electron microscopy showed human mesenchymal stem cell migration through the pores, with endothelial cells, fibroblasts, or keratinocytes in the lower chambers. Mesenchymal stem cell ultrastructural changes occurred, including a larger euchromatin nucleus, when the cells were placed in medium containing 10 percent fetal bovine serum, whereas basic fibroblast growth factor maintained the immature cell morphology for 4 days. Monolayer coculture also showed human mesenchymal stem cell changes in ultrastructural morphology in the vicinity of the epidermal keratinocytes. These data suggest that human mesenchymal stem cells may interact with human epidermal keratinocytes to accelerate wound healing and coverage.     

创面愈合过程中创缘表皮干细胞的异位

目的观察创缘表皮干细胞在全层皮肤创面愈合过程中的分布特征,初步探讨其在创面愈合过程中的作用.方法将已行BrdU活体标记的 20只Wistar大鼠背部制备4个为2.54 cm2的全层皮肤创面,分别于伤后3、7、14和21 d(n=5)行组织学检查,动态观察创面愈合情况,并行β1整合素、角蛋白19(K19)与BrdU免疫组织化学法检测表皮干细胞在创面愈合过程中的分布情况.结果创面愈合率为83.75%(61/80).所有创面肉芽组织于各时相点均未见β1整合素、K19阳性细胞出现,但于创缘表皮的棘层或颗粒层均出现散在的β1整合素、K19和BrdU阳性细胞.且越接近创面阳性细胞越密集,组织学上与基底层的阳性细胞无直接联系;其数量随创面的缩小逐渐增加,直至创面愈合.创面上皮化后,阳性细胞逐渐减少,并随愈合创面表皮脚的出现而消失.而感染创面的阳性细胞数量明显少于未感染创面.结论表皮干细胞能主动参与创面的修复,创缘的表皮干细胞异位的主要功能可能是促进创面再上皮化.     

A simple microfluidic strategy for cell migration assay in an in vitro wound‐healing model

In vitro scratch wound assays are commonly used strategies to measure cell repair rate, facilitating the study of cell migration, tissue reorganization, and cell division. This work presented a simple and novel microfluidic device that allowed a quantitative investigation of the cell migration and cell proliferation behaviors in an in vitro wound‐healing model, especially focused on the scratch assay. The microfluidic device is composed of four units, which include cell growth regions and cell‐free regions created by micropillars. Using this device, we evaluated the proliferation and migration process of human gastric epithelial cells in the presence of different concentrations of the epidermal growth factor, and investigated the migration behavior of mesenchymal stem cells toward tumor cells as well. This approach has the unique capability to create localized cell‐free regions in parallel, and facilitate quantitative research on cell migration in the wound‐healing process, providing a powerful platform for elucidating the mechanism of cell migration in regeneration medicine.     

Role of adipose‐derived stem cells in wound healing

Impaired wound healing remains a challenge to date and causes debilitating effects with tremendous suffering. Recent advances in tissue engineering approaches in the area of cell therapy have provided promising treatment options to meet the challenges of impaired skin wound healing such as diabetic foot ulcers. Over the last few years, stem cell therapy has emerged as a novel therapeutic approach for various diseases including wound repair and tissue regeneration. Several different types of stem cells have been studied in both preclinical and clinical settings such as bone marrow‐derived stem cells, adipose‐derived stem cells (ASCs), circulating angiogenic cells (e.g., endothelial progenitor cells), human dermal fibroblasts, and keratinocytes for wound healing. Adipose tissue is an abundant source of mesenchymal stem cells, which have shown an improved outcome in wound healing studies. ASCs are pluripotent stem cells with the ability to differentiate into different lineages and to secrete paracrine factors initiating tissue regeneration process. The abundant supply of fat tissue, ease of isolation, extensive proliferative capacities ex vivo, and their ability to secrete pro‐angiogenic growth factors make them an ideal cell type to use in therapies for the treatment of nonhealing wounds. In this review, we look at the pathogenesis of chronic wounds, role of stem cells in wound healing, and more specifically look at the role of ASCs, their mechanism of action and their safety profile in wound repair and tissue regeneration.     

Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats

Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.     

Enhanced wound-healing quality with bone marrow mesenchymal stem cells autografting after skin injury

Adult stem cells exist in various tissues and organs and have the potential to differentiate into different cell lineages, including bone, cartilage, fat, tendon, muscle, and epithelial cells of the gastrointestinal tract. Here, we report that the in vitro expanded and purified bone marrow mesenchymal stem cells (MSCs) might take on phenotypes with characteristics of vascular endothelial cells (7% on day 3 and 15% on day 1) or epidermal cells (3% on day 3 and 13% on day 1) after being cultured under different lineage-specific culture conditions. Also, in vivo grafting experiments showed that 5-bromodeoxyuridine-labeled MSCs could convert into the phenotypes of vascular endothelial cells (3.43, 3.46, and 2.94% on days 7, 14, and 28, respectively) in granulation tissues, sebaceous duct cells, and epidermal cells (0-1.49%) in regenerated skin, implying that these grafted MSCs might have transdifferentiated into the above three cell types. Animal autografting experiments with MSCs further confirmed that indices pertaining to wound healing quality, such as the speed of reepithelialization, the number of epidermal ridges and thickness of the regenerated epidermis, the morphology and the number and arrangement of microvasculature, fibroblasts and collagen, were much enhanced. Our results indicate that locally delivered bone marrow MSCs can enhance wound healing quality, and may generate de novo intact skin, resulting in perfect skin regeneration after full-thickness injury.