毛囊混合细胞植入胶原/壳聚糖多孔支架诱导裸鼠毛发生长及支架内形成血管样结构

目的 探讨利用毛囊混合细胞植入胶原,壳聚糖多孔支架内重建毛囊的可行性与支架内血管形成状态.方法 用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接种至胶原,壳聚糖多孔支架,培养2周后,将含毛囊混合细胞胶原/壳聚糖多孔支架植入裸鼠皮下,肉眼观察裸鼠背部毛发形成情况.6周后取移植区皮肤组织经10%甲醛固定后行组织学观察(HE染色).结果 移植至裸鼠皮下5周后,裸鼠背部在含毛囊混合细胞的胶原,壳聚糖多孔支架移植区皮肤出现毛发,丰长,6周后取皮肤组织作HE染色发现有分化成熟的毛囊形成,且支架内有血管样结构形成.而空白胶原/壳聚糖多孔支架移植区皮肤未发现毛生长,组织检杳也未发现有毛囊形成,且只有在支架表浅部位有少黑血管样结构形成.结论 毛囊混合细胞植入胶原/壳聚糖多孔支架内可诱导裸鼠毛发的形成,促进支架血管样结构的形成.     

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.     

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     

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。     

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。     

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。     

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。     

人毛囊内无色素黑素细胞的原子力显微镜观察

目的用原子力显微镜观察人头发毛囊内无色素黑素细胞的表面结构。方法分离酶Ⅱ消化带毛囊的头皮，游离单个毛囊，以胰酶／EDTA消化之，获取细胞悬液，以添加黑素细胞生长物质的254培养基重悬细胞，常规培养。传第3代后接种到内置云母片的6孔培养板中，细胞贴附到云母片上后，固定．原子力显微镜常温常压下，触摸式扫描。结果毛囊无色素性黑素细胞多呈梭形，有2个突起，少数有3个树突。每个树突无明显的二级分枝，个别在树突侧缘有隆起。在一级树突的顶端可见丝状伪足。结论毛囊内无色素性黑素细胞形态不成熟，不能转运黑素颗粒到角质形成细胞，但它有黑素小体运动的物质基础——丝状伪足。     

不同中药色素对C57BL/6小鼠触须毛囊体外培养的研究

目的 研究不同中药色素对小鼠体外毛囊生长的影响和作用机制.方法 分离小鼠触须毛囊,采用不同中药色素分多个浓度分别培养毛囊.在倒置显微镜下观察毛囊组织的形态学变化并测定毛干的长度,2次/d拍照记录;噻唑蓝(MTT)比色法检查毛囊细胞活力.结果 ①与阴性对照组比较,3μg/mL及10μg/mL的姜黄素、30μg/mL的紫草...     

胎儿头皮毛囊的黑素细胞定位及精细结构观察

目的 探讨胎儿毛囊黑素细胞的定位及精细结构。方法 6个月胎儿因宫内发育畸形而引产、死亡后，取其带毛头皮，一部分常规包埋切片，分别用NKI/beteb、HMB-45、酪氨酸酶、酪氨酸酶相关蛋白1（TRP1）单抗染色。另一部分无菌处理后，0.1 g/L的胶原酶Ⅱ和胰酶消化获得毛囊细胞，培养并传代后，透射电镜和原子力显微镜观察。结果 胎儿头皮毛囊NKI/beteb阳性细胞位于外根鞘，而在毛球内许多细胞HMB-45、酪氨酸酶、TRP1单抗染色阳性。在毛囊细胞的体外培养中，除去成纤维细胞和角质形成细胞后，可见两种黑素细胞，一种数目极少，色素很多，传代后消失；另一种数目较多，开始无色素，但增殖很快。传第3代后，几乎所有细胞NKI/beteb染色阳性。扫描电镜和原子力显微镜下，多数细胞为双极梭形，偶尔有3个树突。细胞体呈圆形或卵圆形，极突上无明显的分支，其内有少数散在的黑素体。结论 胎儿头皮毛囊外根鞘的黑素细胞推测为成黑素细胞和（或）其子代细胞。在早期的体外培养中，细胞增殖很快，但形态及功能上不成熟。     

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.     

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     

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.     

Dynamic ultrastructural changes of the connective tissue sheath of human hair follicles during hair cycle

Summary Ultrastructural changes of the connective tissue sheath (CTS), including the hyaline membrane, of human hair follicles during the hair cycle, were studied in normal scalp skin specimens. In early anagen, the CTS was composed of a thin basal lamina and surrounding collagen tissue. The collagen tissue gradually thickened during the development of the hair and hair follicle. In mature anagen hair follicles, the collagen tissue was separated into three layers. The inner collagen layer, just outside the basal lamina, was thin and composed of collagen fibres running longitudinally parallel to the hair axis. The middle collagen layer was very thick with its collagen fibres running transversely against the hair axis and surrounding the inner hair tissue. Many fibroblasts were present among the collagen fibres in the middle layer, whereas the inner layer contained almost none. In the outer collagen layer, collagen fibres ran in various directions parallel to the outer surface of the outer root sheath cells. In late anagen, the basal lamina became very thick. In catagen, the basal lamine and the inner collagen layer became corrugated and showed oedematous change and degeneration. Surrounding fibroblasts showed active production of new collagen fibres, which seemed to fill the spaces left by the retraction of the hair follicle and hyaline membrane. These ultrastructural changes of the CTS show that there may be dynamic metabolic changes of the connective tissue around human hair follicles during the hair cycle.     

人毛囊干细胞的定位及增殖特性

目的 探讨人毛囊干细胞定位和增殖特性.方法 取人枕部头皮毛囊进行角蛋白19(K19)免疫组化染色,并测量阳性段距毛囊下端的距离;将包含毛球部上方阳性段的毛囊上皮组织分别置于含有和不含有间质细胞的凝胶表面气液界面培养,当出现增殖灶时,测量其距毛囊下端的距离,分析K19阳性部位与培养出现的增殖灶之间的关系,并通过透射电镜观察增殖灶的超微结构.结果 人枕部头皮毛囊有两个K19阳性区域,即隆突部和毛球部上方一段外根鞘,毛囊下段上皮组织只在含有间质细胞的凝胶表面培养时毛球部上方出现一增殖灶,统计学分析支持毛球部上方的K19阳性部位和毛囊下段上皮组织培养出现的增殖灶为同一部位.透射电镜显示增殖灶含有幼稚细胞、成熟角质形成细胞和凋亡细胞.结论 人头皮毛囊可能有两个干细胞库,即隆突部和球部上方外根鞘的一个局限区域,干细胞的增殖需要间质细胞(如毛乳头细胞)的存在,增殖的干细胞可能向形成新的干细胞、成熟角质形成细胞和凋亡细胞三个方向演变.     

何首乌、女贞子等中药煎剂对体外培养的猪毛囊毛发生长的影响

目的 探讨何首乌、女贞子等中药混合煎剂对体外培养的猪毛囊毛发生长的影响。方法 将离体培养的猪毛囊分为对照组(Williams E培养基)和中药组(Williams E培养基+中药煎剂),显微镜下观察各组毛囊毛发生长和毛球部形态变化,并用末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(TUNEL)检测各组毛囊中的凋亡细胞。结果 培养第7天,对照组毛囊毛发生长长度低于中药组(P<0.001);对照组毛囊TUNEL阳性细胞数明显高于中药组(P<0.001);毛球形态观察,对照组毛囊出现退行性变化,而中药组毛囊仍维持生长期毛囊形态。结论 何首乌、女贞子等中药煎剂能在一定程度上抑制猪毛囊细胞内凋亡,延缓生长期毛囊进入退行期。     

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.     

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.