毛囊干细胞与组织工程研究进展

毛囊干细胞研究取得突破性进展的标志是由孙同天、Lavker和Cotsarelis等提出了隆突激活假说,明确了其定位的部位,是少数几种成体干细胞首先被明确定位的干细胞之一,并逐步得到了验证。毛囊干细胞不仅可以分化为毛囊上皮细胞,而且可以分化为皮脂腺细胞、表皮角质形成细胞,其分化过程是干细胞分化为短暂倍增细胞,最后分化为终末分化细胞。近年来进一步证明了毛囊隆突部也是多种干细胞的居住地,包括黑素干细胞,和毛囊干细胞存在同步化激活的机制。Wnt/β-catenin是毛囊发育、再生循环的基本信号通路,使毛囊在再生过程出现着色一致的毛发。诱导和重建毛囊形成是组织工程皮肤最主要的目标之一,毛乳头细胞的凝集性生长特性是诱导毛囊形成的条件,因此,维持毛乳头细胞凝集性生长特性主要有细胞团块法、低黏附性培养板(或高分子材料膜)法、悬滴培养法和胶原凝胶成球法,以实现体外重建毛乳头的目的。毛囊组织工程在体外还不能真正诱导形成,现有的毛囊再生或新生的成功方法还是须移植到动物体内,才能形成完整毛囊并产生毛发。主要方法有移植小室法、混合游离细胞注射法和皮瓣法。诱导毛囊形成比较成功的细胞是啮齿类动物来源的胚胎或新生鼠毛囊细胞和人胚胎毛囊细胞,成年人头皮毛囊来源细胞目前仍未能诱导出毛囊新生或再生。     

毛乳头细胞与毛发上皮细胞相互作用的研究

目的探讨人毛乳头细胞和毛发上皮细胞间的相互作用,了解毛发生长周期的调控,寻找体内外毛囊重建的途径。方法人毛乳头细胞和毛发上皮细胞分室和共同培养于 DMEM培养基中,在不同时相计数两种细胞,并观察细胞的生长方式。结果毛乳头细胞和毛发上皮细胞的生长能相互促进,毛发上皮细胞能诱导培养的毛乳头细胞形成毛乳头样结构。结论毛囊真－表皮间的作用是双向的,它们共同控制着毛囊的生长和发育。     

培养毛乳头细胞生物学特性和毛囊重建的研究

目的 观察毛乳头细胞在体内外诱导毛囊再生和支持毛囊生长情况。方法 采用免疫组化、原位杂交、毛囊器官型培养和裸鼠移植技术，观察不同传代培养的毛乳头细胞碱性成纤维细胞生长因子，内皮素和干细胞因子的表达变化情况。结果 低传代培养的毛乳头细胞的内皮素和干细胞因子表达较强，传代6代后减弱。用低传代培养的毛乳头细胞与毛囊上皮细胞在毛囊器官型培养模型中可见毛囊样结构形成，移植到裸鼠后可见较为完整的毛囊形成。用低传代培养的毛乳头细胞与毛囊上皮细胞按一定比例混合后直接注射到裸鼠皮下，也可见毛囊样结构形成。发现毛乳头细胞诱导毛囊再生的能力与其表达内皮素和干细胞因子的强弱相关。结论 低传代培养的毛乳头细胞在体内外均具有诱导毛囊再生的能力，并且与其表达内皮素和干细胞因子的强弱相关。     

裸鼠重建毛囊的组织学研究

目的 观察毛乳头细胞诱导毛囊再生民政部和毛囊重建情况。方法 采用器官型培养技术和裸鼠移植技术,将培养的毛囊毛乳头细胞、真皮鞘细胞和头皮（或包皮）真皮成纤维细胞分别制成胶原凝胶,再接种于毛囊上皮上段、下段和球部细胞,进行体外增习移植试验,结果 在毛囊毛乳头细胞与毛囊各段上皮细胞的器官型培养中均可见毛囊样结构形成;毛囊真皮鞘细胞凝胶上培养的毛球部细胞也重建出毛囊样结构。移植到裸鼠皮下后则可见较为完整的     

人毛乳头细胞体外培养生长曲线观察

目的：观察人正常头皮毛囊真皮成分的细胞体外培养的生长繁殖曲线及表皮细胞生长因子对它们的影响。方法：采用胶原酶消化法培养毛乳头细胞作真皮鞘细胞，在4种培养基中进行传代培养，并在基础培养基、常规培养基＋表皮细胞生长因子中进行^3H－TdR掺入，测定细胞代谢情况。结果：毛乳头细胞和真皮鞘细胞、成纤维细胞的生长呈现三个时相，停滞期、对数生长期和缓慢生长期，表皮细胞生长因子对3种细胞的生长具有显著的促进作用，尤其是对真皮鞘细胞的作用最为显著。结论：常规培养基＋表皮细胞生长因子对3种间质细胞的促生长作用表明，在器官型培养中对毛囊上皮细胞和真皮成分的细胞也会有促生长作用，为毛囊器官型培养和人工皮肤研究采用此培养基提供了实验依据。     

毛乳头细胞在毛囊形态学发生和生长调控中的作用

毛乳头细胞是一群特异的间质细胞 ,位于毛囊底部。在毛囊的胚胎发育过程中 ,这些细胞起源于真皮成纤维细胞。通过细胞外基质的变化 ,毛乳头细胞在毛囊的形态学发生和成年毛囊的周期性生长过程中具有重要的作用     

毛乳头细胞在毛囊形态学发生和生长调控中的作用

毛乳头细胞是一群特异的间质细胞，位于毛囊底部。在毛囊的胚胎发育过程中，这些细胞起源于真皮成纤维细胞。通过细胞外基质的变化，毛乳头细胞在毛囊的形态学发生和成年毛囊的周期性生长过程中具有重要的作用。     

毛乳头诱导毛囊形成和毛发生长信号通路的研究进展

毛乳头是位于毛囊底部的特殊成纤维细胞团块,在毛囊的周期性生长中,毛乳头细胞与周围细胞相互作用而发挥重要功能,被认为是控制毛囊形成和毛发生长信号传导的中心.近年来的研究表明,许多信号通路,包括Wnt、骨形成蛋白、Shh、Notch、成纤维细胞生长因子等信号通路,在毛乳头控制毛囊形成和毛发生长的机制中起作用.可以通过这些信号通路,促进体外培养毛乳头细胞的增殖、加强体外培养毛乳头细胞诱导毛发生长的能力,从而建立一种毛发重构技术用于临床治疗.     

毛乳头分子特征研究进展

上皮细胞和间质细胞相互作用形成不同的皮肤附属器。毛囊周期更替使其成为研究器官再生机制的主要模型,其核心是毛乳头,毛囊上皮、表皮干细胞协同对毛乳头信号做出反应。最近的研究揭示了毛乳头内新的分子调控机制,发现毛乳头是脱发和毛发再生的治疗靶点,鉴定其中新的分子靶点将有助于脱发的治疗。该文对毛乳头中分子调控机制的研究进展作一综述。     

成纤维细胞生长因子受体在人正常皮肤和伤口处的免疫定位

成纤维细胞生长因子(FGF)是一个有22个成员的细胞因子家族，与细胞表面受体结合调节细胞生长、增殖、分化和基质合成并参与炎症反应，其作用与细胞分化及生化状态和理化环境有关。目前发现有4种成纤维细胞生长因子受体(FGFR)，作者采用免疫组化技术检测了4种受体在正常皮肤和伤口处的表达。结果：①正常皮肤中FGFR的表达：FGFR-1表达于表皮各层、皮肤附属器、竖毛肌、血管和真皮成纤维细胞；FGFR-2在表皮、毛囊、皮脂腺、小汗腺、血管和成纤维细胞有表达，而且在基底层呈强阳性；FGFR-3在基底层的上部和毛囊内层呈强阳性；FGFR-4主要见于血管平滑肌、竖毛肌和小汗腺；②头皮生长期毛囊FGFR的表达：FGFR-1在毛发基质细胞、内外根鞘和毛乳头细胞强表达；FGFR-2的表达定位与FGFR-1相似，并且在毛囊根部基质细胞呈强阳性而角化区较弱；FGFR-3在毛球至峡部的内毛根鞘内段强表达，也见于外毛根鞘峡部和已分化的皮脂腺细胞及皮脂腺导管；FGFR-4仅见于毛囊周结缔组织鞘；③伤口处FGFR的表达：新生表皮基底层细胞FGFR-1和3呈强表达，而FGFR-2和4的表达较弱；基底层上部的细胞FG．FR-3强表达，而FGFR-1和2表达弱；肉芽和瘢痕组织中成纤维细胞／肌纤维母细胞表面FGFR-1和3的表达较强，而2和4呈弱阳性，而浸润单核细胞表面FGFR-1和3表达较强。讨论：正常表皮中4种FGFR表达的变化，提示在角质形成细胞分化过程中4种受体的表达发生着改变。研究表明，表皮细胞特异性表达FGFR-3亚型Ⅲb，而间质细胞同时表达FGFR-3的两个亚型Ⅲb和Ⅲc。其中Ⅲb与aFGF和FGF-9结合，而Ⅲc与aFGF和FGF-4高亲和力结合，与bFGF结合力弱而不与FGF-5结合。bFGF位于表皮基底层而aFGF则主要存在于棘层。aFGF和FGFR-3在基底层上部的正在分化的角质形成细胞中共表达，提示aFGF可能通过自分泌作用参与角质形成细胞的分化过程。Finch等发现基质细胞表达对人角质形成细胞有强促分裂作用的角质形成细胞生长因子，并发现其受体表达于正常人表皮基底层及银屑病表皮基底层和基底层上方的角质形成细胞。本研究发现，基底层中FGFR-2的表达，可能是角质形成细胞生长因子受体在表皮基底层和外毛根鞘的定位。Gonzalez等发现FGFR-1及其mRNA主要表达于表皮生发层，由此推测bFGF可能通过FGFR-1对基底细胞有促分裂作用，而aFGF则促进棘细胞的有丝分裂。动物实验表明：FGF和FGFR的表达与毛发的生长周期和发育有关。作者发现4种FGFR在正常人皮肤生长期毛囊的表达模式不同，特别在毛球根部毛母质细胞中FGFR-2的表达呈强阳性，结合动物实验结果，本研究提示真皮乳头FGF-2活化毛母质细胞表达FGFR-2并刺激毛母质细胞增生和毛发生长；毛囊内FGFR-1的表达，提示FGFR-1可能在角质形成细胞向毛发分化的终末过程中起一定的作用。FGFR-3在生长期毛囊中内外毛根鞘和皮脂腺的终末分化细胞中表达而在毛基质细胞内不表达，这可能是由于分化类型的差异，角质形成细胞表达不同的FGFR。     

Hair follicle reformation induced by dermal papilla cells from human scalp skin

To investigate the possibility of hair follicle reformation induced by dermal papilla cells in vivo and in vitro. Dermal papilla cells, dermal sheath cells obtained from human scalp skin by enzyme digestion were mixed with collagen to form mesenchymal cell-populated collagen gels. Superior and inferior epithelial cells and bulb matrical cells were then cultured on these gels by organotypic culture to recombine bilayer artificial skins. Dermal papilla cells and outer root sheath keratinocytes were mingled together and transplanted under subcutaneous tissue of the dorsal skin of nude mice. The results of histologic examination was observed with HE stain. These recombinants by organotypic culture all reformed bilayer structure like nature skin. Hair follicle-like structure reformation was found in dermal sheath cell-populated collagen gel when combined with superior or inferior epithelial cells. Dermal papilla cells also induced superior and inferior epithelial cells to form hair follicle on nude mice. Low passage dermal papilla cells mixed with hair follicle epithelial cells reformed many typical hair follicle structures and produced hair fibres after transplantation on nude mice. The dermal part of hair follicle, such as dermal papilla cells and dermal sheath cells, has the ability to induce hair follicle formation by interaction with the epithelial cells of hair follicle.     

Plasticity of hair follicle dermal cells in wound healing and induction

The capacity of adult hair follicle dermal cells to participate in new follicle induction and regeneration, and to elicit responses from diverse epithelial partners, demonstrates a level of developmental promiscuity and influence far exceeding that of interfollicular fibroblasts. We have recently suggested that adult follicle dermal cells have extensive stem or progenitor cell activities, including an important role in skin dermal wound healing. Given that up to now tissue engineered skin equivalents have several deficiencies, including the absence of hair follicles, we investigated the capacity of follicle dermal cells to be incorporated into skin wounds; to form hair follicles in wound environments; and to create a hair follicle-derived skin equivalent. In our study, we implanted rat follicle dermal cells labelled with a vital dye into ear and body skin wounds. We found that they were incorporated into the new dermis in a manner similar to skin fibroblasts, but that lower follicle dermal sheath also assimilated into hair follicles. Using different combinations of follicle dermal cells and outer root sheath epithelial cells in punch biopsy wounds, we showed that new hair follicles were formed only with the inclusion of intact dermal papillae. Finally by combining follicle dermal sheath and outer root sheath cells in organotypic chambers, we created a skin equivalent with characteristic dermal and epidermal architecture and a normal basement membrane - the first skin to be produced entirely from hair follicle cells. These data support the hypothesis that follicle dermal cells may be important in wound healing and demonstrate their potential usefulness in human skin equivalents and skin substitutes. While we have made progress towards producing skin equivalents that contain follicles, we suggest that the failure of cultured dermal papilla cells to induce follicle formation in wounds illustrates the complex role the follicle dermis may play in skin. We believe that it demonstrates a genuine dichotomy of activity for follicle cells within skin.     

Review of hair follicle dermal cells

Hair follicle stem cells in the epithelial bulge are responsible for the continual regeneration of the hair follicle during cycling. The bulge cells reside in a niche composed of dermal cells. The dermal compartment of the hair follicle consists of the dermal papilla and dermal sheath. Interactions between hair follicle epithelial and dermal cells are necessary for hair follicle morphogenesis during development and in hair reconstitution assays. Dermal papilla and dermal sheath cells express specific markers and possess distinctive morphology and behavior in culture. These cells can induce hair follicle differentiation in epithelial cells and are required in hair reconstitution assays either in the form of intact tissue, dissociated freshly prepared cells or cultured cells. This review will focus on hair follicle dermal cells since most therapeutic efforts to date have concentrated on this aspect of the hair follicle, with the idea that enriching hair-inductive dermal cell populations and expanding their number by culture while maintaining their properties, will establish an efficient hair reconstitution assay that could eventually have therapeutic implications.     

分段毛囊上皮细胞增生能力比较

目的 阐明分段毛囊上皮细胞之间的生物学特性和毛囊干细胞的定位。方法 将毛囊上皮细胞分成上段,下段的球部,分别培养于人成纤维细胞滋养层上,然后传代,或在纤维细胞胶原凝胶上行器官型培养。结果 上段细胞传代次数最多,形成的克隆数最多,细胞生长活力最强;而球部细胞的生长能力阳差,下段细胞有少数能形成进行性生长克隆。     

Proliferation and differentiation of organoid hair follicle cells co-cultured with fat cells in collagen gel matrix culture

Using rat skin, we studied the influence of fat cells on the proliferation and differentiation of organoid hair follicle cells in a three-dimensional collagen gel matrix culture system. We cultured organoid hair follicles embedded in collagen gel under each of the following three conditions: cell-free collagen gel for control experiments (condition 1); co-culture with fat cells in close apposition (condition 2); and co-culture with fat cells in spatial separation (condition 3). Outgrowths of epithelial cells from the organoid hair follicles associated with perifollicular proliferation of fibroblasts were observed under conditions 1 and 3. Under condition 2, proliferation of both organoid hair follicle cells and fibroblasts was inhibited, but differentiation of the hair follicle cells appeared to be accelerated. Fat cells are considered to have an inhibitory effect on the proliferation of perifollicular fibroblasts, which might have resulted in the inhibition of hair follicle cell proliferation and also in the better maintenance of normal follicular structure and integrity, allowing for hair-type differentiation to proceed. A direct accelerating effect of fat cells on hair follicle differentiation may also have been responsible. In a physiological state (co-culture with keratinocytes on the collagen gel), similar results were observed under conditions 1 and 2. The different findings under conditions 2 and 3 may be due to either of two possibilities: either the concentration gradient of the soluble factors released from fat cells, acting on either the hair follicle cells or the perifollicular fibroblasts as an inhibitor of proliferation, caused the difference in the results, or direct contact between the organoid hair follicle cells and fat cells may have influenced the accelerating effect of fat cells on the differentiation of hair follicle cells.     

人毛乳头细胞增殖和胶原合成测定

目的：观察毛乳头细胞、真皮鞘细胞与成纤维细胞的增殖和胶原合成情况。方法：采用^3H-胸腺嘧啶摄入、^3H-脯氨酸摄入方法，观察体外培养的人毛乳头细胞、真皮鞘细胞、成纤维细胞的增殖活性和胶原合成情况。结果：^3H-胸腺嘧啶掺入结果表明，细胞繁殖具有明显的活跃高峰，3种细胞在基础培养基中第11天达掺入高峰，而在常规培养基中有两个掺入高峰，即第5天和第11天。^3H-脯氨酸摄入结果表明，细胞内摄入无明显差异，而递质中的胶原含量有明显差异，成纤维细胞显著高于毛乳头细胞和真皮鞘细胞，表明两者的功能有一定的差异。结论：常规培养基是这3种真皮细胞很好的培养基，能够明显促进它们的生长和繁殖，而毛乳头细胞和真皮鞘细胞分泌胶原的能力低于成纤维细胞，说明它们在合成胶原的功能方面有一定的差异。     

Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages

The adult hair follicle dermal papilla (DP) and dermal sheath (DS) cells are developmentally active cell populations with a proven role in adult hair follicle-cycling activity and unique inductive powers. In stem cell biology, the hair follicle epithelium has recently been the subject of a great deal of investigation, but up to now, the follicle dermis has been largely overlooked as a source of stem cells. Following the sporadic appearance of muscle, lipid and bone-type cells in discretely isolated follicle DP and DS cell primary cultures, we demonstrated that cultured papilla and sheath cell lines were capable of being directed to lipid and bone differentiation. Subsequently, for the first time, we produced clonal DP and DS lines that had extended proliferative capabilities. Dye exclusion has been reported to be an identifying feature of stem cells; therefore, clonal papilla and sheath lines with differing capacity to exclude rhodamine 123 were cultured in medium known to induce adipocyte and osteocyte differentiation. Both DS- and DP-derived clones showed the capacity to make lipid and to produce calcified material; however, different clones had varied behaviour and there was no obvious correlation between their stem cell capabilities and dye exclusion or selected gene expression markers. As a highly accessible source, capable of being discretely isolated, the follicle has important potentially as a stem cell source for tissue engineering and cell therapy purposes. It will also be interesting to compare follicle dermal stem cell properties with the broader stem cell capabilities discovered in skin dermis and investigate whether, as we believe, the follicle is a key dermal stem cell niche. Finally, the discovery of stem cells in the dermis may have implications for certain pathologies in which abnormal differentiation occurs in the skin.     

Co-culture of human hair follicles and dermal papillae in a collagen matrix

Human hair follicles, either alone or in combination with dermal papillae, were cultured in a collagen matrix. When plucked hair follicles were cultured alone, spike-like structures composed of outer root sheath cells started growing around the follicle and then radiated into the gel. When isolated dermal papillae were embedded close to the follicles, spikes started growing earlier and grew more rapidly than without the papillae. In cultures of excised follicles from which the dermal papilla had been removed, epithelial cells (possibly hair bulb cells) started growing out from the bulbous portion and then also formed spikes. In the presence of a papilla, the spikes elongated toward the papilla, finally reaching and surrounding it. These findings suggest that dermal papilla cells produce a factor(s) that enhances growth of follicular epithelial cells and also attracts those cells. In cultures of whole excised follicles, two major characteristic patterns of cellular growth were recognized. When the dermal papilla remained inside the bulb in contact with the hair bulb matrix, the hair matrix cells proliferated and differentiated in the normal manner, resulting in elongation of the hair shaft and follicle. But when the papilla was detached from the hair bulb matrix, epithelial cells proliferated from the bulbous portion and finally formed hair follicle-like structures. Thus, attachment of the dermal papilla to the hair bulb matrix in the bulbous portion appears to be necessary for growth of the hair and follicle in the normal manner. Our model may be useful for examining the interaction between follicular epithelial cells and dermal papillae and for studying the growth of hair and follicles in vitro.     

Parathyroid hormone-related peptide modulates signal pathways in skin and hair follicle cells

Parathyroid hormone-related protein (PTHrP) is secreted by skin epithelial cells and is thought to play an important role in the development and function of the hair follicle. It was hypothesized that PTHrP binds to receptors in dermal papilla cells and modulates intracellular signaling systems in these cells. We tested the effects of PTHrP on protein synthesis, protein kinase A (PKA) and protein kinase C (PKC) activities as well as tyrosine phosphorylation in rat vibrissa dermal papilla and capsular fibroblast cells. Cells were cultured in the presence or absence of the N-terminal peptide PTHrP1-34 for 48 h and detergent extracts prepared. Proteins were separated by electrophoresis. Phosphotyrosine and the PTH/PTHrP receptor immunoreactivity was identified by Western blot analysis. PKC and PKA activities in the cells were measured using colorimetric enzyme assays. Extracts of both dermal papilla cells and capsular fibroblasts displayed immunoreactivity to the PTH/PTHrP receptor. Electrophoresis showed that PTHrP treatment reduced the density of a 50-kDa protein in dermal papilla cells but not in capsular fibroblasts. Media conditioned by the cells showed similar changes, indicating that the PTHrP-modulated 50-kDa protein was secreted. Furthermore, 2-D gel electrophoresis indicated that the protein had a number of phosphorylation sites. Western analysis with antiphosphotyrosine antibodies confirmed a significant decrease in the intensity of a phosphorylated 50-kDa protein in papilla cells and papilla cell-conditioned medium. PKC and PKA activities of papilla cells were unaffected by PTHrP. However, activities of PKC were increased and PKA reduced in capsular fibroblasts following peptide treatment. These cell-specific effects showed that endogenous PTHrP may activate different intracellular pathways in mesenchymal cells of skin and elicit changes in levels of locally secreted proteins that specifically modulate normal follicular function.     

Interaction between dermal papilla cells and follicular epithelial cells in vitro: effect of androgen

Summary To investigate the mode of action of testosterone on human hair follicles, we studied the localization of androgen receptors in hair follicles and cultured dermal papilla cells from different body sites, using immunohistochemistry with a polyclona) antibody. Androgen receptors were detected in the dermal papilla cells of beard and axillary hair follicles, but not in those of occipital scalp hair follicles. Epithelial cells of the hair follicles were not stained by the antibody. Androgen receptors were also detected in the nuclei of cultured beard and axillary dermal papilla cells, but the dermal papilla cells from occipital scalp hair follicles showed little staining with this antibody. We also examined the effects of testosterone on the DNA synthesis and proliferation of cultured outer root sheath cells and dermal papilla cells. Testosterone did not have a proliferative effect on either type of cell when cultured alone. In contrast, testosterone significantly stimulated the proliferation of outer root sheath cells which were co-cultured with beard or axillary dermal papilla cells, without cell contact. No such effect was observed when dermal papilla cells from occipital scalp hair follicles were used in the cultures. This proliferative effect of testosterone was concentration-dependent, and was antagonized by cyproterone acetate. Our findings suggest that dermal papilla cells from the beard and axillary hair follicles produce androgen-dependent diffusible growth factors which stimulate follicular epithelial cells.