抑制性消减杂交筛选生长期毛乳头细胞差异表达基因

目的 应用抑制性消减杂交构建生长期毛囊毛乳头细胞(DPC)差异表达cDNA消减杂交文库,从中克隆鉴定出差异表达基因。方法 分别从生长期DPC及休止期DPC提取总RNA;采用SMARTcDNA合成技术合成cDNA,进行2次消减杂交及2次抑制性PCR;将产物与T/A载体连接构建生长期毛囊DPC的cDNA消减文库,通过反向RNA印迹杂交验证阳性克隆,测序并登录基因库寻找同源性基因。结果 成功构建了毛囊生长期DPC消减文库,并获得35个阳性克隆,其中功能已知基因22个,功能未知基因13个。结论 抑制性消减杂交技术是一种高效的筛选差异基因的方法,并可用于小量临床标本研究,本实验所发现的生长期DPC差异表达基因对今后研究毛囊生长调控可能具有重要意义。     

毛发疾病诊断技术和方法北大核心CSCD

毛发是机体健康的外表重要标志之一,涉及毛发的疾病直接影响心理健康和生活质量。毛发分布的密度和毛干直径是决定毛发是否能够正常地覆盖头皮的重要因素。毛囊是生成毛发的器官,由来源于不同胚层的多种细胞组成,具有生长期、退行期和休止期的周期性循环,毛干的产生和延长仅限于生长期。从毛囊的长轴来看,自表皮至皮下组织,毛囊分为永久和次级毛囊即暂时性毛囊,分水岭在立毛肌的附着点附近。次级毛囊经历生长期、退行期和休止期的周期性改变,它的最下端是毛囊的球囊部,是毛干形成的部位,由真皮结缔组织形成的毛乳头供给营养。     

毛发疾病诊断技术和方法

毛发是机体健康的外表重要标志之一,涉及毛发的疾病直接影响心理健康和生活质量.毛发分布的密度和毛干直径是决定毛发是否能够正常地覆盖头皮的重要因素.毛囊是生成毛发的器官,由来源于不同胚层的多种细胞组成,具有生长期、退行期和休止期的周期性循环,毛干的产生和延长仅限于生长期.从毛囊的长轴来看,自表皮至皮下组织,毛囊分为永久和次级毛囊即暂时性毛囊,分水岭在立毛肌的附着点附近.次级毛囊经历生长期、退行期和休止期的周期性改变,它的最下端是毛囊的球囊部,是毛干形成的部位,由真皮结缔组织形成的毛乳头供给营养.     

真皮干细胞在毛囊周期中的作用

毛囊由表皮(上皮)及真皮(间充质)组成,它们之间的相互作用在毛囊的形态发生及生长中发挥重要作用,二者之间相互作用是毛囊成功重建的关键因素.在毛发形成过程中,真皮细胞是诱导者,上皮细胞是应答者.真皮鞘和毛乳头内存在毛囊真皮干细胞,属于成体干细胞,具有慢周期、未分化、自我更新和体外增殖能力强的特点.真皮鞘中的真皮干细胞较长寿,可以历经几个毛囊周期,重建真皮鞘.在毛囊周期的生长期,真皮鞘中的干细胞产生新细胞提供给毛乳头;在退行期,真皮干细胞子代移出毛乳头或死亡.毛囊真皮细胞对于损伤和疾病之后的毛囊重建及修复具有重要意义.     

女贞子等中药对小鼠毛囊生长周期中生长因子mRNA表达的影响

目的 探讨女贞子等中药促毛囊生长的机制。方法 我们应用RT-PCR技术和凝胶密度扫描分析技术,对正常生长周期中毛囊肝细胞生长因子(HGF)和血管内皮细胞生长因子(VEGF) mRNA表达水平,以及女贞子等中药对体外培养小鼠触须毛囊HGF和VEGFmRNA表达水平的影响进行了深入的研究。结果 我们的研究发现正常毛囊生长周期中,生长期毛囊有明显的HGF和VEGF mRNA表达,而退行/休止期毛囊则很难检测出上述因子mRNA的表达。在毛囊生长发育的过程中,HGF和VEGF mRNA表达也逐渐减弱。与女贞子混合培养的毛囊,在培养的第6天仍有相当量的HGF和VEGF mRNA表达,而相应的对照组则未能测到这些因子的表达。而与齐敦果酸培养的毛囊在培养的第6天仅有HGFmRNA表达,而无VEGF mRNA的表达。结论 女贞子及齐敦果酸可促进体外培养的毛囊对HGF和(或)VEGFmRNA的表达。从而从分子生物学水平揭示了上述药物促进毛囊生长的可能机制。     

拉坦前列素对体外培养的猪毛囊生长的影响

目的探讨拉坦前列素对体外培养的猪毛囊生物学特性的影响。方法采用猪毛囊体外培养模型,记录体外培养的猪毛囊生长速度和毛球部形态学改变,评价拉坦前列素对毛发生长的作用。结果与阴性对照组比较,拉坦前列素质量浓度达到0.8ng/mL时即能使培养的猪毛囊生长速度加快(P<0.05),并持续生长达8d,且毛囊毛球部仍然呈现生长期样形态,而对照组则表现为退行期/休止期样外观。结论拉坦前列素对体外培养的猪毛囊生长具有明显的促进作用。能够延长毛囊的生长期。     

HMB-45抗原在毛囊黑素细胞中的表达

用单克隆抗体ＨＭＢ－４５，以ＡＰＡＡＰ法对２０份头皮活检标本的９８个毛囊组织进行染色。其中２０个毛囊为生长早期，６３个为成熟生长期，７个为退行期和８个是休止期。结果，５２个成熟生长期及２０个生长早期毛囊的黑素细胞与单克隆抗体ＨＭＢ－４５显著结合。所有退行期、休止期及工１１个成熟生长期毛囊未被染色。结果提示；ＨＭＢ－４５在毛囊中的表达随毛发生过周期而变化，且与黑素细胞功能活性状态有关。     

HSPC016基因在凝集性生长人毛乳头细胞的表达

目的 确定造血干细胞相关基因HSPC016的表达与毛乳头细胞凝集性生长的关系。方法 对凝集性生长的人毛乳头细胞、非凝集性生长的人毛乳头细胞和人真皮成纤维细胞分别进行细胞内mRNA原位杂交;用RT-PCR从以上3组细胞总RNA扩增HSPC016基因。结果 原位杂交发现HSPC016基因在凝集性生长的人毛乳头细胞表达,而在非凝集性生长的人毛乳头细胞和人真皮成纤维细胞均不表达;RT-PCR可以从凝集性生长的人毛乳头细胞RNA中扩增到约200bp目的片段,而不能从非凝集性生长人毛乳头细胞和人真皮成纤维细胞中扩增到相应目的片段。结论 HSPC016基因在凝集性生长的人毛乳头细胞特异性表达,可能与毛乳头细胞的分化和功能状态有关。     

脱发的治疗

毛发的生长和脱落呈现周期性。生长周期分为三期：即生长期(anagen)、退行期(catagen)及休止期(telogen)。正常头发约90％～95％的：毛囊在生长期，1％进入退行期，5％～10％为休止期，至休止期末则头发脱落，毛囊则进入下一生长周期。生长期持续的时间决定毛发的长度。头发生长期约2～6年，退行期2～3周，休止期2～3个月。生长期与休止期毛囊比例决定了毛     

HSPC011基因在毛乳头细胞的表达及其cDNA克隆

目的 确定HSPC011基因在毛乳头细胞的表达,并克隆其cDNA.方法 原位杂交技术证实HSPC011基因在毛乳头细胞表达;RT-PCR扩增目的基因并构建重组真核表达质粒pCI-neo/HSPCO11.结果 细胞内mRNA原位杂交证实HSPC011基因在凝集性生长的毛乳头细胞表达,而在非凝集性生长的毛乳头细胞和人真皮成纤维细胞不表达;RT-PCR从毛乳头细胞RNA中扩增到约430bp目的基因片段;构建了重组质粒pCI-neo/HSPCO11,酶切分析和测序结果均表明目的片段与HSPC011基因相应序列完全一致.结论 HSPC011基因在毛乳头细胞表达,其表达可能与毛乳头细胞的分化和功能状态有关.     

Expression of basement membrane proteins and interstitial collagens in dermal papillae of human hair follicles

The expression of basement membrane molecules and interstitial collagens in human hair follicle mesenchyme was studied by immunohistochemical staining of tissue sections and of cells cultured from dermal papillae. Type I and type III collagens were found in the dermal sheath and in the dermal papilla throughout the hair cycle. Laminin and type IV collagen were expressed at the outer root sheath basement membrane and in the extracellular matrix of the dermal papilla of anagen and catagen follicles. In telogen follicles, where the volume of the dermal papilla extracellular matrix is much reduced, outline staining of dermal papilla cells for laminin and type IV collagen was still apparent. Staining for bullous pemphigoid antigen was also seen at the outer root sheath basement membrane extending to the lower tip of the hair bulb. In anagen follicles, there was no staining for bullous pemphigoid antigen at the interface between hair bulb epithelium and the dermal papilla and no staining within the dermal papilla. However, linear staining for bullous pemphigoid antigen became continuous around hair follicle epithelium during catagen and telogen. Cells cultured from human dermal papillae also stained for interstitial collagens, type IV collagen and laminin. However, similar results were obtained when cultured dermal fibroblasts were stained with the same antibodies. The expression of basement membrane proteins in human dermal papillae resembles that seen in follicles from other mammalian species and suggests that this is relevant to dermal papilla function. Cultured dermal papilla cells express a similar pattern of interstitial collagens and basement membrane proteins to those seen in tissue sections but this finding is not specific to dermal papilla cells.     

Comprehensive analysis of FGF and FGFR expression in skin: FGF18 is highly expressed in hair follicles and capable of inducing anagen from telogen stage hair follicles

We quantified the mRNA expression of all 22 fibroblast growth factor family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair growth cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, 2, 5, 7, 10, 13, and 22), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair growth cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and 22 at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal keratinocytes and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair growth was observed. Our findings suggest that FGF18 is important for the regulation of hair growth and the maintenance of skin in adult mice.     

Hair cycle-specific expression of versican in human hair follicles

BACKGROUND: Versican, a large chondroitin sulfate proteoglycan molecule, is implicated in the induction of hair morphogenesis, the initiation of hair regeneration, and the maintenance of hair growth in mouse species. In contrast, in human hair follicles, the distribution and the roles of versican remains obscure. OBJECTIVES: To elucidate the implication of versican in normal human hair growth. METHODS: Versican expression was examined by in situ hybridization (mRNA) and immunohistochemistry (protein). RESULTS: The results clearly showed specific versican gene expression in the dermal papilla of anagen, which apparently decreased in the dermal papilla of catagen hair follicles. No specific signal was detectable in telogen hair follicles. Consistent with ISH results, versican immunoreactivity was extended over the dermal papilla of anagen hair follicles, and again, this staining diminished in the catagen phase of human hair follicles. Interestingly, versican proteins were deposited outside K15-positive epithelial cells in the bulge throughout the hair cycle. Versican immunoreactivity in the dermal papilla was almost lost in vellus-like hair follicles affected by male pattern baldness. CONCLUSION: Specific expression of versican in the anagen hair follicles suggests its importance to maintain the normal growing phase of human as well as mouse.     

Distribution of proteoglycans during the hair growth cycle in human skin.

The involvement of proteoglycans in hair growth has been recognized through the observation of increased hair growth in diseases such as the mucopolysaccharidoses and pre-tibial myxedema, which involve an increase in skin proteoglycan content. In an attempt to understand this, we have examined the distribution of chondroitin 6 sulphate (C6S), unsulphated chondroitin (COS), dermatan sulphate (DS), and heparan sulphate proteoglycans (HSPG) in frozen tissue sections of normal scalp by immunostaining. Results show that during anagen, the thick connective tissue sheath around the follicle strains strongly for C6S, COS, and DS. COS is uniquely associated with this region and is not found beneath the epidermis or infundibular epithelium. HSPG is, however, localized in the basement membrane zone adjacent to the outer root sheath. In addition, all of these proteoglycans are localized in the dermal papilla. In mid-catagen, we observed significant loss of C6S and COS staining from both the dermal papilla and the connective tissue sheath, but no decrease in staining for HSPG. In late catagen, very little staining of C6S and COS was observed. In early anagen, we observed that C6S was again present in the connective tissue sheath and dermal papilla; however, COS staining appeared to be weaker and less closely associated with the follicle. HSPG staining was observed in early anagen in a pattern very similar to that found for other basement membrane components. Results for DS were not obtained for catagen or early anagen. These results provide further evidence that hair growth is associated with the presence of chondroitin proteoglycans in the follicle environment and that the cessation of growth is associated with their removal. Further studies are underway to characterize the relationship between hair growth and proteoglycans.     

Protease-activated receptor-1 (thrombin receptor) is expressed in mesenchymal portions of human hair follicle

Protease nexin-1, a serine protease inhibitor, is expressed specifically in the dermal papilla (DP) of anagen hair follicles and is suggested to be one of the modulators of the cyclic growth of hair follicles. Accumulating evidence has shown that protease nexin-1 plays its biologic role by inhibiting thrombin action in various systems other than the hair follicle. Thrombin has various physiologic functions including blood coagulation cascade, mostly via activation of protease-activated receptors (PAR). In this study, we investigated the expression of PAR mRNA using RT-PCR in dissected human hair follicles. We showed that PAR-1 mRNA was expressed specifically in the mesenchymal portions, including DP and connective tissue sheath, of anagen hair follicles. Furthermore, immunoreactivity for PAR-1 was detected in the DP and lower portion of connective tissue sheath in the anagen and catagen phases and in the DP of telogen hair follicles. Because only a pharmacologic level (100 nM) of thrombin significantly stimulated cell proliferation and DNA synthesis of the cultured dermal papilla cells, thrombin does not seem to have a mitogenic effect on dermal papilla cells physiologically. These results raise the possibility that thrombin is involved in the cyclic hair growth through its receptor of PAR-1.     

The role of BMP signalling in the control of ID3 expression in the hair follicle

Both the production of the hair shaft in anagen and the initiation of a new hair cycle at telogen are the result of reciprocal interactions between the dermal papilla and the overlying epithelial cells. Secreted factors, such as those of the bone morphogenetic protein (BMP) family, play a crucial role in moderating these interactions. Analysis of hair follicles in different stages of the hair cycle showed that BMP signalling was only active during anagen and again during telogen. During catagen, no BMP signalling occurred in the dermal papilla. ID3, a gene expressed in the dermal papilla of both vibrissa and pelage follicles, is a BMP target, and as such, we found that ID3 was expressed from the earliest stages of morphogenesis. During the hair cycle, ID3 was only expressed in the dermal papilla at middle anagen and telogen. To test the significance of ID3 expression in the dermal papilla, we cultured dermal papilla cells and found that ID3 expression fell significantly after a single passage. ID3 expression was returned to in vivo levels in low- and high-passage cells by culturing to high confluence or by the addition of BMP4. These studies reinforce the requirement for active BMP signalling and cell-cell contacts in the dermal papilla during specific stages in the hair cycle.     

Hair follicle expression of 1,25-dihydroxyvitamin D3 receptors during the murine hair cycle

Because the hair follicle is a highly hormone-sensitive miniorgan, the role of hormones produced locally in the skin in the control of hair growth deserves systematic analysis. It has been shown previously that the potent steroid hormone 1,25-dihydroxyvitamin D₃ (1,25-D₃) modulates growth and differentiation of keratinocytes via binding to a high-affinity nuclear vitamin D receptor (VDR). In this study, we have examined the in situ expression of VDR during the murine hair cycle. VDR expression was detected immunohistochemically. To obtain defined stages of the murine hair cycle, hair growth was induced by depilation in C57 BL-6 mice. In addition to the recognized VDR expression of outer root sheath keratinocytes, we detected VDR immunoreactive cells in the dermal papilla, the mesenchymal key structure of the hair follicle. Furthermore, VDR immunoreactivity in the nuclei of outer root sheath keratinocytes and in dermal papilla cells was stronger during anagen IV-VI and catagen than during telogen and anagen I-III. This suggests hair cycle-associated changes in the expression of VDR, and points to a potential role for 1,25-D₃ in hair follicle biology. Selected follicular cell populations may display hair cycle-dependent sensitivity to 1,25-D₃ stimulation.     

Glycosaminoglycan synthesis by cultured human hair follicle dermal papilla cells: comparison with non-follicular dermal fibroblasts

The extracellular matrix of the hair follicle dermal papilla is rich in glycosaminoglycans, the expression of which varies during the hair growth cycle being maximal in anagen and becoming undetectable as the follicle enters telogen. These observations, together with other experimental and clinical evidence, suggest that glycosaminoglycans may be involved in regulating hair growth. To investigate the metabolism of glycosaminoglycans by the dermal papilla we have measured the incorporation of radiolabelled precursors into glycosaminoglycans released into extracellular matrix and culture medium by cultured human dermal papilla cells. We also studied glycosaminoglycan synthesis by cells cultured from the lower follicular connective tissue sheath and by non-follicular dermal fibroblasts. Compared with dermal fibroblasts, dermal papilla cells showed a three to fourfold higher level of incorporation of 35S-sulphate and 3H-glucosamine into extracellular matrix glycosaminoglycans. Dermal papilla cells also released more 3H-glucosamine-labelled glycosaminoglycan into culture medium than dermal fibroblasts but there was no difference in 35S-sulphate labelling. These findings indicate that dermal papilla cells maintain a high level of glycosaminoglycan synthesis in vitro. Specific enzyme/chemical degradation showed that dermal papilla cells and dermal fibroblasts synthesized the same glycosaminoglycan types. However, the results suggested that dermal papilla glycosaminoglycans are less sulphated than those synthesized by dermal fibroblasts and that a higher proportion of sulphated glycosaminoglycans is retained in an extracellular matrix. The synthesis of glycosaminoglycans by connective tissue sheath cells was similar to that of dermal papilla cells, supporting the view that the dermal papilla and connective tissue sheath share certain properties.