表皮干细胞及其微环境研究进展

表皮干细胞是皮肤重要的干细胞之一,主要位于毛囊隆突部,表面具有多种标记物.表皮干细胞生长于特定微环境niche中,有多种细胞因子、信号通路参与了niche调节表皮干细胞归巢、自我更新和分化,参与皮肤创伤修复、皮肤肿瘤形成等多种功能.对表皮干细胞及其微环境niche的深入研究,对了解表皮发育、表皮肿瘤形成及组织工程有着重要的意义.     

人表皮干细胞的体外培养与MSCs对其诱导的初步研究

目的：探讨人表皮干细胞体外分离培养的理想方法，初步研究骨髓间充质干细胞（MSCs）上清对其生长的影响。方法：采用中性蛋白酶-胰蛋白酶两步法收获表皮细胞，Ⅳ型胶原快速粘附分选表皮干细胞，用无血清培养基进行体外培养，采用SABC—FITC进行培养细胞鉴定。结果：胎儿皮肤和人包皮皮肤均可成功分离到表皮干细胞集落，且增殖能力强，细胞表达Bl整合素和角蛋白CK19。MSCs上清形成的条件培养基的诱导下，收集的表皮细胞形成花环样克隆，并形成成纤维样细胞。结论：用酶消化法、胶原快速粘附和无血清培养法是较理想的体外分离培养表皮干细胞的方法，MSCs可以诱导表皮细胞的定向分化。     

表皮干细胞研究新进展

表皮干细胞对研究皮肤组织发育、疾病发生具有重要意义，对今后转基因技术和皮肤组织工程的开展也极具重要应用前景。而目前表皮干细胞研究尚处于初步阶段，本文就表皮干细胞定位、生物学标志及其分化调控机制的研究进展进行综述。     

表皮干细胞的研究进展

干细胞是一类具有高度增殖潜能和无限自我更新能力的细胞亚群。角质形成细胞干细胞主要存在于毛囊隆突部，也见于毛囊间表皮网嵴的底部，其特征为慢周期及高度的增殖潜能，它能表达某些整合素亚单位，并可与细胞外基质发生快速粘连，据此特性可将其分离纯化。角质形成细胞干细胞不仅在表皮及其附属器的组织发生学、银屑病的发病机理中具有重要作用，也是肿瘤发生及基因治疗的重要靶细胞。     

人表皮干细胞的体外培养

表皮干细胞(epidermal stem cells, ESCs)是表皮发生、分化和创面修复的摇篮,1其正常增殖分化是维持皮肤正常组织结构和细胞内环境稳定的基本要求,但由于ESC数量较少,只占基底细胞总数的1%～10%,2缺乏明确的特异性分子标志,体外培养很容易丢失其干细胞生物学特性,因而如何大量获得纯度较高的表皮干细胞将成为目前干细胞研究领域的热点.本研究利用酶消化法和IV胶原筛选法从整形手术的正常人头皮、包皮中进行表皮干细胞分离和培养,报道如下.     

分离用于组织工程的纯表皮干细胞群

表皮干细胞可长期保留核标记 ,为标记保留细胞 (ＬＲＣｓ)。表皮干细胞在组织中保持活力 ,缓慢增殖 ,维持着表皮组织的不断更新。表皮基底层细胞通过分裂、增殖和不断取代最外面角质层已分化的细胞 ,以维持表皮组织的完整性。表皮干细胞是自我更新的细胞群 ,每一次表皮干细胞分裂产生一个干细胞和一个一过性放大 (ＴＡ)细胞 ,后者的分裂次数有限 ,其子代细胞分化并最终脱落。表皮中只保留干细胞并不断地终生增殖 ,这意味着任何基因治疗必须直接针对干细胞的基因组 ,但分离纯表皮干细胞群非常困难。该文报告了一种分选分离系统所产生的纯角质…     

表皮干细胞的研究进展

干细胞是一类具有高度增殖潜能和无限自我更新能力的细胞亚群。角质形成细胞干细胞主要存在于毛囊隆突部 ,也见于毛囊间表皮网嵴的底部 ,其特征为慢周期及高度的增殖潜能 ,它能表达某些整合素亚单位 ,并可与细胞外基质发生快速粘连 ,据此特性可将其分离纯化。角质形成细胞干细胞不仅在表皮及其附属器的组织发生学、银屑病的发病机理中具有重要作用 ,也是肿瘤发生及基因治疗的重要靶细胞     

蛋白激酶C抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的初步研究

目的探讨蛋白激酶C(PKC)抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的可能性。方法采用酶消化法结合Ⅳ型胶原快速贴壁法获得人原代表皮干细胞及角质形成细胞,倒置相差显微镜下观察细胞生长状况,免疫细胞化学染色法检测GF109203X诱导培养后角质形成细胞的表型及功能改变。同期分离培养的人在体表皮干细胞作为本次实验的阳性对照,原代角质形成细胞培养2 d后加等体积二甲基亚砜为阴性对照。结果表皮干细胞快速黏附,培养4 d后细胞呈圆形,形态规则,折光性强,明显克隆,β1整合素、CK19及CK14呈阳性表达,CK10阴性表达;已分化角质形成细胞不能快速黏附,培养4 d细胞呈类圆形,大小不一,折光性较差,无明显克隆,β1整合素、CK19及CK14呈阴性表达,CK10呈阳性表达。角质形成细胞经GF109203X诱导培养2 d后,实验组与阳性对照组细胞群β1整合素、CK19、CK14均呈阳性表达,但实验组较阳性对照组中细胞β1整合素、CK19、CK14阳性细胞数少,CK10均呈阴性表达;阴性对照组中细胞群β1整合素、CK19及CKl4呈阴性表达,CKl0呈阳性表达。结论 GF109203X能够诱导角质形成细胞去分化形成表皮干细胞。     

表皮恶性肿瘤

基底细胞癌基底细胞癌(BCC)是一种表皮肿瘤,光镜下是由基质及其围绕的细胞岛组成的,其细胞类似成熟的表皮和毛囊基底细胞。电镜下瘤细胞象一种未分化的外胚叶细胞,类似未分化的毛母质细胞和原始上皮胚芽基底细胞,这似可解释BCCs有向任何附件结构分化的能力。退化期毛囊未分化的上皮细胞可存在于真皮,这可能是BCC的起源。某些BCCs的确与毛囊外根鞘相连,但95％与表面表皮附着。线状基底细胞痣是一种先天性损害,可解释为起源于静止的未分化的多潜能细胞。基质的质与量不同。富有粘蛋白。如果     

表皮生长因子受体及相关皮肤病

在正常人表皮，具免疫活性的表皮生长因子受体主要分布在基底细胞层和棘细胞层下部，为表皮多种功能的基本调节剂。表皮生长因子受体与其配体结合，可调节角质形成细胞的增殖和分化。文中综述了表皮生长因子受体与以表皮过度增殖和／或异常分化为特征的皮肤病（如脂溢性角化病、银屑病、基底细胞癌和鳞癌等）的关系，为相关疾病的治疗提供新的思路。     

As epidermal stem cells age they do not substantially change their characteristics

In this study, we ask the basic question: do stem cells age? We demonstrated that epidermal stem cells isolated from neonatal mice had the capacity to form multiple cell lineages during development. Here we demonstrate the cell lineages are clonal, and that epidermal stem cells isolated from the footpad epithelium of old mice have similar capabilities. Using Hoechst dye exclusion and cell size, we isolated viable homogenous populations of epidermal stem and transit-amplifying (TA) cells from GFP-transgenic mice, and injected these cells into 3.5-d blastocysts. Only the stem-injected blastocysts produced mice with GFP+ cells in their tissues. Furthermore, aged and young stem cells showed similar gene and protein expression profiles that showed some differences from the TA cell profiles. These data suggest that there may be a fundamental difference between somatic stem and TA cells, and that an epidermal stem cell placed in a developmental environment can alter its fate determination no matter what its age.     

Isolation and identification of stem cells from adult cashmere goat skin

Background  Continuously renewing epithelia are maintained by stem cells that slowly proliferate and remain in the tissues for life. It has been known for decades that mouse epithelial stem cells can be selected by adherence to specific integrins.
Methods  The adherence of cashmere goat epidermal cells to collagen type IV for 10 min was used to obtain enriched epidermal stem cells. The characteristics of the rapidly adherent epidermal cells were determined.
Results  The rapidly adherent epidermal cells exhibited the stem cell characteristics of immaturity, were quiescent, showed a high colony formation efficiency, and expressed candidate surface markers for epidermal stem cells (keratin 15, keratin 19, p63, CD34, and β1-integrin).
Conclusions  The rapidly adherent epidermal cells represented the epidermal stem cell population.     

p53蛋白在UVB诱导凋亡的富集表皮干细胞的角质形成细胞中的表达

目的研究p53蛋白在中波紫外线(ultraviolet lightB,UVB)诱导凋亡的富集表皮干细胞的角质形成细胞群中的表达情况。方法分离富集人表皮干细胞的角质形成细胞群和正常角质形成细胞群,使用UVB诱导两种细胞群凋亡,蛋白印迹法比较不同剂量UVB诱导前后两组细胞的p53蛋白表达的差异。结果两种细胞在不同剂量的UVB照射后p53蛋白表达均比照射前显著增加,在20mJ/cm2与40mJ/cm2照射剂量时,富集人表皮干细胞的角质形成细胞群p53蛋白表达高于正常角质形成的细胞群,差异有统计学意义(P<0.05)。结论富集人表皮干细胞的角质形成细胞p53蛋白表达比其它角质形成细胞对中波紫外线的照射易感。     

Development of a new mouse model (xeroderma pigmentosum a-deficient, stem cell factor-transgenic) of ultraviolet B-induced melanoma

It is well established that exposure to sunlight or ultraviolet radiation (UVR) is the major environmental risk factor for the development of skin neoplasms. To date, however, there have been few appropriate mouse models available for studying the role of UVR in melanoma carcinogenesis, mainly because of the murine lack of the epidermal melanocyte, which is a major source of origin of human melanoma. In this study, we established xeroderma pigmentosum group A gene-deficient, stem cell factor-transgenic mice, which are defective in the repair of damaged DNA and do have epidermal melanocytes. The mice were exposed to UVR three times a week for 10 wk. More than 30% of the irradiated mice developed tumors of melanocyte origin that metastasized to the lymph nodes. Histologically, proliferated cells exhibited lentigo maligna melanoma or nodular melanoma. Immunohistochemistry confirmed that the tumor cells were characteristic of melanoma. Non-irradiated mice did not develop skin tumors spontaneously. The newly generated model mouse might be useful for studying the photobiological aspects of human melanoma, because the mice developed melanoma from epidermal melanocytes only after UVR exposures.     

Epithelial stem cells in the skin: definition, markers, localization and functions

In recent years, cutaneous epithelial stem cells have attained a genuine celebrity status. They are considered the key resource for epidermal and skin appendage regeneration, and are proposed as a preferential target of cutaneous gene therapy. Follicular epithelial stem cells may also give rise to a large variety of epithelial tumors, and cutaneous epithelial stem cells likely are crucial targets for physical or chemical agents (including carcinogens) that damage the skin and its appendages. However, as this Controversies feature illustrates, few experts can agree on how exactly to define and identify these elusive cells, or on where precisely in the skin they are localized. Given their potential importance in skin biology, pathology and future dermatological therapy, it is, therefore, timely to carefully reconsider the basic questions: What exactly is a stem cell, and how can we reliably identify epithelial stem cells? How many different kinds are there, and how do they differ functionally? Where exactly in the skin epithelium is each of the putative stem cell subpopulations located, and can we selectively manipulate any of them?     

Cross-reactive tumor antigens in the skin of mice exposed to subcarcinogenic doses of ultraviolet radiation

This study suggests that cross-reactive tumor-associated antigens (TAA) are induced in the epidermis of mice exposed to subcarcinogenic doses of ultraviolet radiation (UVR). Foot-pad immunization of C3H mice with viable epidermal cells from syngeneic UVR-exposed mice induced cytotoxic cells in the draining lymph nodes (DLN). These cells were capable of lysing a battery of UVR-induced tumor targets in a short-term chromium release assay. In contrast, the DLN cells of mice immunized with epidermal cells from normal non-UVR-exposed mice did not mature into effector cells with antitumor activity. The spectrum of tumor recognition of cytotoxic cells induced by immunization with UVR-exposed epidermal cells was identical to that of cytotoxic T cells obtained from animals that were foot-pad immunized with UVR-induced tumor cells. Both cytotoxic cell populations were shown to lyse tumors of diverse origin, including syngeneic UVR- and methylcholanthrene-induced tumors, as well as allogeneic UVR-induced tumors. These cells displayed minimal lytic activity against YAC-1 lymphoma cells, peritoneal exudate cells, and concanavalin A-stimulated splenocytes. The results from this study demonstrate that antigens are expressed on UVR-exposed mouse epidermal cells prior to the emergence of skin tumors, and these antigens can induce cytotoxic cells with specificity for TAA. We conclude that the early antigenic changes observed in UVR-exposed epidermis and their effect on the host's immune system may influence the emergence and progression of UVR-induced skin cancers.     

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.     

Keratinocyte stem cell assays: an evolving science

Although the existence of epithelial stem cells in the skin has been known for some decades from cell kinetic studies performed in vivo, attempts to prospectively isolate these cells for further biological characterization have been made possible relatively recently facilitated by the availability of antibodies that detect cell surface markers on epidermal cells. Elegant gene marking studies in vivo have provided confirmation of the patterns of epithelial tissue replacement predicted by classical cell turnover studies. But, the identification of candidate epidermal stem cells ex vivo remains an area of great controversy, requiring the re-evaluation of current experimental approaches that rely of necessity on predicted epidermal stem cell behavior in culture. Here we review the diverse experimental approaches utilized to identify keratinocyte stem cells and their underlying assumptions. We conclude that hair follicles and interfollicular epidermis each have their own self-renewing stem cell populations, contributing to distinct regions of the epithelium during homeostasis, although this is perturbed during wound healing. The need for the development of more rigorous assays for stem cell activity is highlighted given our recent observations using current assays and the discovery of new surface markers that identify putative epidermal stem cells.     

Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors

Nestin, a marker of neural stem cells, is expressed in the stem cells of the mouse hair follicle. The nestin-expressing hair follicle stem cells give rise to the outer-root sheath. Nestin-expressing hair follicle stem cells that are negative for the keratinocyte marker keratin 15 (K15) can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Recent studies suggest that the epithelial stem cells are important in tumorigenesis. In this study, we immunohistochemically examined the expression of three hair follicle stem cell and progenitor cell markers, nestin, K15, and CD34, in normal human epidermis and hair follicles and in epidermal and follicular tumors, trichilemmoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). In normal human skin, the cells in the epidermal basal layer were positive for K15 and negative for nestin and CD34. The hair follicle cells below the sebaceous glands were also positive for nestin and K15 and negative for CD34. The outer-root sheath cells under this area could be divided into three parts: an upper part of the outer-root sheath cells that was partially positive for nestin and positive for K15 and negative for CD34; a middle part that was CD34-positive and K15-negative; and a lower part that was positive for K15 and negative for CD34. In the tumor tissues, nestin immunoreactivity was observed in trichilemmoma but not in BCC. Also, immunoreactivity for K15 was strong in BCC and weak in trichilemmoma, and SCC was negative for nestin and partially positive for K15. No CD34 immunoreactivity was observed in any of the cases. These results suggested that trichilemmoma originates in the nestin-positive/K15-positive/CD34-negative outer-root sheath cells below sebaceous glands, BCC tumor cells from the more mature nestin-negative/K15-positive/CD34-negative outer-root sheath cells, and SCC from the nestin-negative/K15-positive/CD34-negative keratinocytes of the basal cell layer in the epidermis.