Enzyme digestion to isolate and culture human scalp dermal papilla cells: a more efficient method

In this study, we show a more efficient method for isolation and cultivation of dermal papilla cells from hair follicles of human scalp skin. The dermal partments of low hair follicles were pulled out from cutaneous fat and the bulb epithelium was teased out from the fibrous sheath with attached dermal papilla by applying gentle pressure with the tip of an occal forceps. When these fibrous sheathes were entirely digested into isolated cells by collagenase D but the dermal papillae were justly to be digested, collagenase D was discarded and the dermal papillae were isolated completely out from the resuspension solution by repeated low-speed centrifugation and transferred to another dish for free-floating culture. This procedure markedly simples the steps of isolated dermal papilla operation and relieves the laborious tension. Furthermore, dermal papillae could be isolated on a large-scale and remained intact. After collagenase digestion, the dermal papillae showed very high adherent rate and quicker growth than that of microdissection, which suggests that the definition factor of dermal papilla cell migration was relaxed and some structure had been activated or exposed. The cells exhibited a multi-layer forming property and spread-out growth style. They showed positive with alcian blue, with toluidine blue O for different gradient pH and PAS, which was similar to the staining results of in situ dermal papilla. It suggests that the culture papilla cells still synthesize and excrete neutral and acid mucopolysaccharides. Our results demonstrate that the papilla cells in culture condition still remain the ability to synthesize the specific extracellular matrix components of in situ dermal papilla, which supports the concept that the dermal papilla cell, a highly specialized fibroblast, especially is involved in hair growth regulation. This work was supported by National Natural Science Foundation of China (No:30070701).     

毛囊混合细胞诱导毛囊重建的研究

目的分离培养人毛囊外根鞘细胞株(ORSC)和人毛囊毛乳头(DPC),在体内和体外诱导毛囊形成。方法利用酶消化法和组织块法获得第三代DPC和ORSC,将上述两种细胞混合后接种于海藻酸钠3D细胞支架,构建毛囊的体外三维模型,同时将该三维模型移植入SD大鼠皮下,8周后移植部位取材,行HE染色、免疫组化和电镜观察毛囊形成情况。结果 SD大鼠移植部位取材,HE染色可见细胞聚集成团状的毛囊样结构,CK14,CK15和波形蛋白染色阳性,电镜下可见支架中有散在细胞分布。结论 SD大鼠体内三维模型培养,证明在该模型中诱导出了具有向毛囊分化倾向的毛囊样结构,为以后成功重建毛囊做出了有益探索。     

人腋窝毛乳头细胞的分离与培养

【摘要】 目的 探讨高效快速分离培养人腋窝毛乳头细胞的方法。方法 收集2015年10月至2016年5月陆军军医大学第一附属医院皮肤科腋臭术后含毛皮肤标本,分别用改良二步酶消化法、一步酶消化法和显微解剖法分离腋窝毛乳头,比较3种方法操作过程差异以及毛乳头分离效率、贴壁率、细胞迁出时间、总操作时间、实际操作时间的差异,并鉴定培养的腋窝毛乳头细胞。结果 与一步酶消化法和显微解剖法相比,改良二步酶消化法操作更简单,分离效率可达30%以上,1周后毛乳头贴壁率高达96%,细胞迁出时间缩短3 ~ 4 d,总耗时长于一步酶消化法和显微解剖法,但实际操作时间短于一步酶消化法和显微解剖法,且污染率低于显微解剖法。培养的腋窝毛乳头细胞早期可呈凝集性生长,6代以后的细胞呈非凝集性生长。免疫荧光结果示腋窝毛乳头细胞层黏连蛋白和Ⅳ型胶原阳性。结论 改良二步酶消化法是一种简单﹑高效﹑快速分离人腋窝毛乳头细胞的方法,少量标本即可培养出腋窝毛乳头细胞。     

Soluble factors from human hair papilla cells and dermal fibroblasts dramatically increase the clonal growth of outer root sheath cells

Depending on environmental influences, follicular outer root sheath (ORS) cells in vivo can differentiate either towards interfollicular keratinocytes or, as demonstrated in the rat vibrissa, hair matrix cells. Crucial regulators of both their proliferation and differentiation are the mesenchymal cells of the respective tissues. The interactions of human ORS cells with human hair papilla cells (HPC) or human dermal fibroblasts (HDF) were studied using a two-chamber model separating the two cell types either by a microporous membrane or additionally by a medium layer. The results of ³H-thymidine incorporation studies indicated that ORS cell growth was markedly enhanced in co-culture with either HPC or HDF, the highest stimulatory effect resulting when ORS cells were in close association with the mesenchymal cells. No correlation was found between ORS cell proliferation and IL-6 production in the co-culture system, thus pointing to the secretion by HPC and HDF of growth-promoting soluble factors that are different form IL-6 as well as from EGF, bFGF and insulin present in the culture medium.     

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.     

Modelling the hair follicle dermal papilla using spheroid cell cultures

Please cite this paper as: Modelling the hair follicle dermal papilla using spheroid cell cultures. Experimental Dermatology 2010; 19: 546–548. Abstract: Human dermal papilla (DP) cells grown in two‐dimensional (2D) culture have been studied extensively. However, key differences exist between DP cell activities in vivo and in vitro. Using a suspension method of cell culture to maintain DP cells, we created three‐dimensional (3D) dermal spheres morphologically akin to intact (anagen) DPs. Analysis of these spheres using immunocytochemistry demonstrates that they have expression profiles different from papilla cells cultured in 2D but with many similarities to intact DPs. This method of DP cell culture may provide us with a tool to elucidate our understanding of signalling within the DP as it relates to induction, maintenance or even inhibition of hair growth.     

毛乳头细胞体外诱导毛囊上皮细胞分化的实验研究

目的:探讨间质细胞对毛囊上皮细胞分化的调节作用,研究毛囊上皮细胞的分化特性。方法:分别用团块状的毛乳头细胞、皮肤成纤维细胞制成间质细胞胶原凝胶,表面接种毛囊上皮细胞,进行气-液界面培养。结果:毛囊上皮细胞有向毛乳头细胞移动集结的趋势;团块状的毛乳头细胞诱导毛囊上皮细胞形成球形结构;皮肤成纤维细胞诱导毛囊上皮细胞形成表皮样层化结构。结论:(1)毛囊上皮细胞具有双向分化特性,它既能分化形成毛囊,也能分化形成表皮结构;(2)毛乳头细胞对毛囊上皮细胞有趋化作用;(3)间质细胞的种类及分布在毛囊上皮细胞分化的调节中起着重要的作用。     

毛乳头细胞分泌性蛋白质组的制备

毛乳头是毛囊的真皮部分,凝集性生长为其特性之一,有诱导毛囊形成和再生的能力［1］。研究证实,凝集性生长的毛乳头细胞（dermal papillae cell,DPC）分泌大量蛋白样因子,形成网络,共同调节其他DPC、外根鞘细胞、内皮细胞和角质形成细胞的增殖［２－３］;这些分泌性蛋白质通过自分泌、旁分泌等途径发挥作用,全面系统研究分泌性蛋白有助于全面认识、分析和解释毛乳头细胞的凝集性生长特性……     

黄芩苷对人毛囊生长和毛乳头细胞分泌血管内皮生长因子的影响

目的 探讨黄芩苷对体外培养的人毛囊生长、毛乳头细胞增殖和分泌血管内皮生长因子(VEGF)的影响.方法 选择不同浓度黄芩苷作用于体外培养的人头皮毛囊8天,观察人毛囊生长及形态变化:作用于人毛乳头细胞72h,四甲基偶氮唑监(MTF)法测定细胞增殖活性,ELISA法检测细胞分泌的VEGF水平.结果 7.5.15,30,45μg/mL的黄芩苷对体外培养的人头皮毛囊有明显促生长作用;3.75,7.5,15,30μg/mL的黄芩苷对毛乳头细胞增殖无明显影响,但促进细胞分泌VEGF,与阴性对照组相比,差异有统计学意义.结论 黄芩苷促进体外培养的人头皮毛囊生长,部分可能是通过增加毛乳头细胞分泌VEGF促使毛发生长.     

血管生成素在人毛囊中的表达及其对毛发生长的影响

目的 探讨血管生成素在人毛囊中的表达及其意义。方法 分离完整的人生长期毛囊,提取总RNA,RT-PCR法检测血管生成素mRNA的表达,同时应用免疫组化法检测血管生成素蛋白在人毛囊中的表达。在此基础上,在体外培养的人毛囊中加入不同浓度的重组人血管生成素（0 ～ 200 ng/mL）,培养6 d后测量毛囊的生长长度。利用两步酶法分离培养人毛乳头细胞,加入不同浓度的重组人血管生成素（0 ～ 200 ng/mL）,48 h后,MTT法检测细胞增殖,流式细胞仪检测细胞周期。结果 RT-PCR显示人毛囊表达血管生成素mRNA,免疫组化法发现人毛囊的毛乳头和真皮鞘表达血管生成素蛋白。25 ～ 200 ng/mL重组人血管生成素呈浓度依赖性促进体外培养的人毛囊生长（P < 0.05）,而12.5 ～ 200 ng/mL重组人血管生成素能够明显促进体外培养的人毛乳头细胞增殖（P < 0.05）。流式细胞仪检测12.5 ～ 200 ng/mL重组人血管生成素能够显著增加S期细胞比率和细胞增殖指数（P < 0.05）。结论 血管生成素可能是一种促进毛发生长的因子。     

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.     

Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells

Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell‐based co‐cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF‐1, two well‐known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.     

雄激素性秃发患者毛乳头细胞基因表达谱分析

【摘要】 目的 探讨3D培养下,雄激素性秃发患者毛乳头细胞基因表达谱的差异。 方法 分离雄激素性秃发患者毛乳头细胞进行3D培养,实验组经双氢睾酮处理,提取总RNA进行扩增,Cy3标记,用NimbleGen人类全基因组表达谱芯片杂交,筛选差异表达基因进行GO分析及pathway分析,实时 PCR对结果进行验证。结果 全基因组表达谱分析显示,有622 个基因有差异性表达,上调基因数359个,下调基因数为263个。其中GO分析显示,抑制细胞增殖、促进凋亡的基因出现上调,如CHEK1及Tob1等。促进细胞增殖、参与表皮生长发育的分子表达下调,如BAMBI、EFNA3、Dlx3及UCGC等。实时 PCR验证结果与芯片结果的差异趋势一致。Pathway分析显示,调节细胞周期信号转导通路的分子富集在首位。 结论 雄激素性秃发发病受多种信号分子及信号转导通路调节,可能集中在调节细胞周期、细胞增殖及凋亡等方面。     

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。     

永生化人毛乳头细胞系体内诱导毛囊的形成

目的研究永生化人毛乳头细胞(DPC)系DPC-hTERT在体内诱导毛囊形成的能力。方法将第30代DPC-hTERT与刚分离的毛囊上皮细胞混合后直接注射到裸鼠背部皮下,观察毛囊形成情况。结果 DPC-hTERT与刚分离的毛囊上皮细胞混合后注射到裸鼠背部皮下后,可见毛囊样结构形成,且此结构表达毛囊特有的角蛋白。结论 DPC-hTERT具有正常DPC的功能,具备在体内诱导毛囊样结构的能力。     

腺苷对人毛囊生长的促进作用及对血管生成素表达的影响

目的：探讨腺苷对人毛囊生长的促进作用及对血管生成素表达的影响。方法分离完整的人头皮生长期毛囊,分成6组,分别加入0、0.1、1、10、100、1000μmol/L腺苷作用,每个浓度组再分成两个亚组,其中一个亚组加入0.8 mg/L抗人血管生成素抗体,另外一个亚组不加抗体,显微镜下测量6 d内毛囊的生长长度。利用两步酶法分离培养人毛乳头细胞,分组同上,作用48 h后,噻唑蓝法检测细胞增殖,流式细胞仪检测细胞周期,逆转录（RT）?PCR和ELISA分别检测人毛乳头细胞血管生成素mRNA和细胞上清液中血管生成素蛋白的表达。结果与不加腺苷的对照组相比,10~1000μmol/L腺苷能够明显促进体外培养的人毛囊生长（F=377.776,P<0.05）,其中以100μmol/L腺苷的促进作用最为显著（P<0.05）,加入抗人血管生成素抗体能够部分拮抗腺苷促进人毛囊生长的作用。与对照组相比,0.1~1000μmol/L腺苷能够明显促进体外培养的人毛乳头细胞增殖（F=230.067,P<0.05）,其中以10μmol/L和100μmol/L腺苷促进细胞增殖的作用最为显著（均P<0.05）,加入抗人血管生成素抗体能够部分拮抗腺苷促进细胞增殖的作用。流式细胞仪检测显示,与对照组相比,10~1000μmol/L腺苷能够显著下调毛乳头细胞G1期细胞比例（F=75.5,P<0.05）,并显著上调G2期（F=15.7,P<0.05）和S期（F=81.8,P<0.05）细胞比例。而且,与对照组相比,10μmol/L和100μmol/L腺苷能够明显促进毛乳头细胞表达血管生成素mRNA（F=942.630,P<0.05）和血管生成素蛋白（F=148.544,P<0.05）。结论腺苷在体外具有促进人毛囊生长和毛乳头细胞增殖的作用,其机制可能与上调血管生成素表达有关。