毛囊混合细胞在胶原/壳聚糖多孔支架上重建毛囊样结构

目的探讨利用毛囊混合细胞植入胶原／壳聚糖多孔支架内体外重建毛囊的可行性。方法用滴加法或注射法将体外培养的C57BL／6J近交系乳鼠背部皮肤毛囊混合细胞以不同传代数、不同细胞密度接种至胶原／壳聚糖多孔支架，倒置显微镜下观察支架表面或浅层的细胞生长或毛囊形成情况。将支架经10％甲醛固定后行组织学观察（H-E染色）。另用共聚焦激光扫描显微镜观察支架内活细胞的生长以及毛囊样结构的形成。结果在一定传代次数和细胞密度下，在支架内可形成具有毛干的毛囊样结构。激光共聚焦扫描显微镜发现团块内细胞排列呈同心圆状，整个三维结构似一长颈花瓶，且该结构仅见于注射法接种细胞的支架内。结论毛囊混合细胞植入胶原／壳聚糖多孔支架内体外可形成具有毛干的毛囊样结构。     

毛囊混合细胞在海藻酸钠支架上诱导毛囊重建

目的 探讨原代培养的毛囊外根鞘细胞、毛乳头细胞和成纤维细胞,采用不同接种顺序在体外和体内诱导毛囊形成.方法 将培养的毛囊外根鞘细胞(ORSC)、毛乳头细胞(DPC)和丝裂霉素干预后的成纤维细胞按一定比例制成细胞悬液,并按不同的接种顺序接种于海藻酸钠3D细胞支架上,构建毛囊的体外三维模型并培养8周,行HE染色光镜观察毛囊形成的情况;同时将该三维模型移植入bal/bcl裸鼠皮下体内培养8周,移植部位取材后分别行HE染色、免疫组化和电镜观察毛囊形成的情况.结果 体外构建的毛囊三维模型未见到角化物质及毛囊样结构;裸鼠皮下移植物HE染色可见细胞聚集成团,有呈环状排列的毛囊样结构,CK14,CK15、β1整合素和波形蛋白染色阳性;电镜下模型中可见贴附在支架上的毛囊细胞和红细胞.先接种用丝裂霉素干预的成纤维细胞于海藻酸钠3D细胞支架上培养1周后再接种DPC∶ORSC (1∶5)细胞悬液,裸鼠体内移植物HE染色不仅可见明显的毛囊样结构形成,而且形成毛囊样结构的数目较多.结论 模型中DPC保持了诱导毛囊形成的能力,ORSC保持了毛囊干细胞的特点.并明确了诱导毛囊形成的最佳细胞组合、接种顺序和比例,为体外构建含有毛囊的人工皮肤奠定了一定基础.     

中波紫外线诱导小鼠皮肤光损伤中miRNA141表达的研究

【摘要】 目的 探讨小室移植法重建小鼠毛囊,观察细胞成分对毛囊再生的影响。 方法 取0 ～ 2 d的C57BL/6乳鼠背部皮肤,胰酶消化后分离表真皮,再分离出毛囊上皮细胞。实验分表皮细胞混合毛囊胚芽组、真皮细胞组、表皮细胞混合毛囊胚芽 + 真皮细胞组、毛囊上皮细胞 + 真皮细胞组。用小室移植法接种于裸鼠背部,并于移植后1、2、4、8周观察变化,HE染色观察毛囊组织学形态。 结果 小鼠毛囊细胞移植1周时,背部小室开始脱落,伤口结痂;2周时除表皮细胞组外,另3组均长出了短小的毛发,镜下可见毛囊样结构;4周及8周时除表皮细胞组外,均长出了正常的毛发,表皮细胞与真皮细胞混合组及毛囊上皮细胞与真皮细胞混合组毛发生长情况良好,优于单独的真皮细胞组。 结论 毛囊细胞小室移植后可形成新的毛囊,上皮细胞及真皮细胞在毛囊重建中具有重要的作用。 【关键词】 毛囊; 毛囊重建; 鼠科     

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。     

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。     

小鼠皮肤移植细胞有效重建毛发

目的研究将新鲜分离的新生小鼠皮肤细胞移植到免疫缺陷鼠上有效重建毛囊的新技术。方法新生C57小鼠取其皮肤,将表皮和真皮细胞分别分离并混合,做成高浓度的细胞悬液,移植到免疫缺陷鼠NU/NU,CB-17 SCID,NOD SCID上,观察皮肤再生情况。结果细胞移植2周之后,伤口处肉眼可见黑色皮肤形成,3周后长出大量毛发,8周后观察重建的毛发维持良好状态。没有移植细胞的对照观察到伤口愈合但没有毛发形成。组织学分析显示再生的皮肤含有成熟的毛囊和毛干并连有皮脂腺及真皮毛乳头,并且新形成的皮肤不仅有表皮层和真皮层,还有皮下脂肪层,表明移植的细胞可重建完整的皮肤。细胞移植到三种不同的免疫小鼠NU/NU,CB-17 SCID和NOD SCID上,毛囊重建的结果相似。结论创建了一套分离和移植皮肤细胞的技术,可在免疫缺陷鼠上高效地重建毛发。该技术可以用于毛囊再生的研究,并具有潜在的临床应用价值。     

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     

中药黄芪、女贞子、人参促毛发生长的在体研究

目的:以C57BL/6小鼠为动物模型,探讨中药黄芪、女贞子、人参混合煎剂促进C57BL/6小鼠毛发生长的可能机制。方法:中药组小鼠给予中药混合煎剂口饲,对照组给予等量生理盐水,观察小鼠背部皮肤颜色变化和毛发生长情况。同时在光镜下观察小鼠毛囊组织学变化及毛囊内细胞的凋亡的状况。结果:中药组小鼠饲饮中药煎剂后背部皮肤颜色由粉红色变为黑色比对照组提前了1d,背部皮肤颜色由黑色变为灰黑色比对照组延长了1.5d。组织学上,拔毛后第18天对照组小鼠毛囊处于退行中晚期,而中药组仍为生长Ⅵ期和退行早期毛囊。中药组小鼠毛囊内凋亡细胞数量比对照组减少(P<0.001)。结论:黄芪、女贞子、人参混合煎剂促毛发生长作用可能与抑制退行期毛囊内细胞凋亡,诱导和延长C57BL小鼠毛发的生长期有关。     

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。     

皮肤黏蛋白病

报告1例皮肤黏蛋白病.患者男,19岁.背部、右上肢结节半年余.皮肤科检查见背部有3枚直径2.5～9.5 cm的斑块,右上臂伸侧有1枚直径5 cm的皮肤结节,质地中等有弹性,边界欠清,无红肿及破溃,无压痛.皮损组织病理检查示:真皮胶原稀疏,血管、汗腺、毛附属器周围可见致密的单-核细胞浸润.阿新蓝染色示:真皮、皮下、毛囊周嗣均见染淡蓝色物质.直接免疫荧光示:免疫球蛋白IgM、补体C3基膜带呈线状沉积,免疫球蛋白IsG、IgA阴性.电镜检查示:胶原束间出现团状电子密度较高的颗粒状、纤维状物质沉积,未见血管病变.符合皮肤黏蛋白病.     

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.     

分段毛囊移植治疗毛发部位白癜风

【摘要】 目的 探讨分段毛囊移植治疗特殊部位白癜风的疗效。方法 2016年12月至2018年12月在杭州市第三人民医院皮肤科纳入10例19 ~ 28岁稳定期白癜风患者（男4例，女6例），共26处白斑合并白发，其中18处为头皮，8处为眉部。采用毛囊单位提取法从枕部或耳后区域获取毛囊，并均分为上下两部分。患者每处白斑合并白发皮损分为2部分，上下两部分毛囊分别移植于白斑的不同区域。术后每4周随访1次，观察复色及毛发生长情况。结果 12周时，移植毛囊上部26处白斑中痊愈3处，显效5处，好转12处，无效6处，有效率30.8%（8/26），且均未见毛发生长；移植毛囊下部的26处皮损中痊愈5例，显效7例，好转11例，无效3例，有效率46.2%（12/26），8处皮损出现毛发生长。移植毛囊上部组与移植毛囊下部组有效率差异无统计学意义（χ2 = 1.30，P = 0.25）。结论 分段毛囊移植术适用于头皮和眉部白癜风合并白发的治疗。     

Skin equivalent assay: An optimized method for testing for hair growth reconstitution capacity of epidermal and dermal cells

Hair follicle reconstitution requires highly organized epithelial‐mesenchymal interactions. Skin equivalents containing the epidermal and dermal cells with hair reconstitution capacity can reproduce these processes, but have not been established. This study was conducted to develop a hair follicle‐producing three‐dimensional (3D) skin equivalent assay using neonate mouse epidermal and dermal cells. A skin equivalent comprised of mouse dermal cells (MDCs) embedded in type I collagen and overlaid with mouse epidermal cells (MECs) was used. MDCs were mixed with type I collagen and cultured for 7 days. One day after adding MECs on top, the composites were grafted onto nude mice. MDCs cultured on a two‐dimensional (2D) plate for 7 days and mixed with MECs as a negative control, and freshly isolated MDCs and MECs mixture (chamber assay) as a positive control were also grafted. Six weeks after grafting, regenerated hair follicles were analysed. Our 3D skin equivalent culture assay reproducibly regenerated hair follicles, while MDCs precultured in the 2D model with MECs did not. Compared to the chamber assay, which produced randomly oriented hair follicles, nearly all regenerated hair follicles in our assay extruded through the skin and numerous regenerated hair follicles were higher than those in the chamber assay. Several representative genes associated with hair induction showed higher expression in our assay than in the 2D model. When Wnt3a was added, the number of regenerated hairs increased. Organized hair follicle regeneration was accomplished using our assay. This approach can be applied to assess a test agent with hair growth‐promoting effects.     

不同黑素细胞群在有色毛囊重建中作用的研究

目的观察不同种属小鼠的毛囊细胞能否嵌合重建有色毛囊, 探讨不同黑素细胞群在小鼠有色毛囊重建中的作用。方法选取C57BL/6J、BALB/C胎鼠或乳鼠皮肤, 分离出表皮细胞群、毛囊上皮细胞群和真皮细胞群, 培养、纯化从表皮细胞群获得的表皮黑素细胞。实验包含三部分, ①乳鼠C57BL/6J毛囊重建实验:分为表皮细胞+毛囊上皮细胞组、真皮细胞组;②嵌合毛囊重建实验:分为乳鼠C57BL/6J真皮细胞组、乳鼠BALB/C真皮细胞组、乳鼠BALB/C真皮+乳鼠C57BL/6J真皮细胞组、胎鼠BALB/C真皮细胞+胎鼠C57BL/6J真皮细胞组;③有色毛囊重建实验:分为乳鼠BALB/C真皮细胞+乳鼠C57BL/6J表皮细胞组、乳鼠BALB/C真皮细胞+乳鼠C57BL/6J毛囊上皮细胞组、乳鼠BALB/C真皮细胞+培养的C57BL/6J表皮黑素细胞组。采用小室移植法将不同细胞接种于裸鼠的背部, 每组4只。于移植后4周和8周, 通过大体观察、组织学及免疫荧光评估毛囊重建情况。结果移植后4周和8周, 乳鼠C57BL/6J毛囊重建实验中(本部分实验2组共8只BALB/C裸鼠, 7只存活, 1只因创面感染死亡...     

Cultivation of murine hair follicles as organoids in a collagen matrix

Techniques are described for the isolation and cultivation of functionally intact mouse hair follicles. Follicles were isolated by collagenase digestion of dermis from 5-day-old mice and purified by differential centrifugation and filtration. Purified follicles were cultured in a Type 1 collagen matrix using Medium 199 and 8% fetal calf serum as the basic nutrient. Viability of follicles was maintained in culture since the cultures incorporated thymidine into DNA and methionine into proteins for at least 7 days. Furthermore, follicles isolated from the collagen matrix after 7 days could reattach to a plastic culture substrate or be further cultivated in a fresh collagen matrix. Functional integrity of cultured follicles was maintained since some follicle-specific cytoskeletal proteins were synthesized in vitro, and follicles isolated from the collagen matrix after 7 days formed a haired skin when recombined with dermal fibroblasts and grafted to a skin site on nude mice. Only a minority of follicles appeared to produce a mature hair shaft in vitro by morphologic criteria, however, and synthesis of the total complement of hair proteins was not observed. Cholera toxin was a strong mitogen for cultured follicles, whereas epidermal growth factor was slightly mitogenic. Epidermal growth factor stimulated the release of a Type 1 collagenase by follicle cells, however. This model system provides an opportunity for the systematic analysis of factors required for the induction of hair growth and the underlying physiology of hair follicle development. This model should also be useful for studying the role of the hair follicle in skin carcinogenesis.     

Single follicular unit transplantation reconstructs arrector pili muscle and nerve connections and restores functional hair follicle piloerection

The autologous transplantation of hair follicles that have been separated into single follicular units is an accepted treatment for androgenetic alopecia. Recent studies demonstrate that the multiple stem cell populations and surrounding cutaneous tissues coordinately regulate the hair follicle functions and skin homeostasis. Therefore, the critical issues for consideration regarding functional hair restoration therapy are reproduction the correct connectivity and cooperation with host cutaneous tissues, including the arrector pili muscle (APM) and nerve system. We report successful establishment of mouse single follicular transplantation model and autonomous restoration of transplanted hair follicle piloerection in mouse skin. Transplanted hair follicles were responsive to the neurotransmitter acetylcholine and formed proper connections with surrounding host tissues such as APM and nerve fibers, which in turn connect with not only the hair follicle bulge region but also the APM. These results demonstrate that the piloerection ability of transplanted hair follicles can be estimated quantitatively. This study makes a substantial contribution towards the development of transplantation therapy that will facilitate future functional regeneration therapy for skin and skin appendages.     

Human hair growth ex vivo is correlated with in vivo hair growth: selective categorization of hair follicles for more reliable hair follicle organ culture

Of the numerous assays used to assess hair growth, hair follicle organ culture model is one of the most popular and powerful in vitro systems. Changes in hair growth are commonly employed as a measurement of follicular activity. Hair cycle stage of mouse vibrissa follicles in vivo is known to determine subsequent hair growth and follicle behavior in vitro and it is recommended that follicles be taken at precisely the same cyclic stage. This study was performed to evaluate whether categorization of human hair follicles by the growth in vivo could be used to select follicles of the defined anagen stage for more consistent culture. Occipital scalp samples were obtained from three subjects, 2 weeks later after hair bleaching. Hair growth and follicle length of isolated anagen VI follicles were measured under a videomicroscope. Follicles were categorized into four groups according to hair growth and some were cultured ex vivo for 6 days. Follicles showed considerable variations with respect to hair growth and follicle length; however, these two variables were relatively well correlated. Hair growth in culture was closely related with hair growth rate in vivo. Moreover, minoxidil uniquely demonstrated a significant increase of hair growth in categorized hair follicles assumed at a similar early anagen VI stage of hair cycle. Selection of follicles at a defined stage based on hair-growth rate would permit a more reliable outcome in human hair follicle organ culture.Oh Sang Kwon and Jun Kyu Oh contributed equally.