人工毛乳头异种移植诱导大鼠足垫毛囊形成

目的观察人头皮毛乳头细胞微囊(人工毛乳头)异种移植诱导大鼠足垫毛囊形成的能力。方法以海藻酸钠-多聚赖氨酸-海藻酸钠(alginate-polylysine-alginate,APA)微囊包裹分离培养的毛乳头细胞;对体外培养1、4周的毛乳头细胞微囊及无APA的微囊对照组行组织学观察;取培养4周的毛乳头细胞微囊移植至大鼠足垫皮下,6周后取材行组织学检查。结果毛乳头细胞微囊体外培养1周后,毛乳头细胞周围出现细胞外基质;4周后,囊中形成“类毛乳头样结构”;人头皮毛乳头细胞微囊移植至大鼠足垫6周后,移植部位及其周围皮下有大量毛囊及皮脂腺结构形成。结论人工毛乳头诱导并参与了无毛区域新生毛囊及皮脂腺的组织构成。     

人工毛乳头异种移植诱导大鼠足垫毛囊形成

目的观察人头皮毛乳头细胞微囊（人工毛乳头）异种移植诱导大鼠足垫毛囊形成的能力。方法以海藻酸钠-多聚赖氨酸-海藻酸钠（alginate- polylysine - alginate, APA）微囊包裹分离培养的毛乳头细胞；对体外培养1、4周的毛乳头细胞微囊及无APA的微囊对照组行组织学观察；取培养4周的毛乳头细胞微囊移植至大鼠足垫皮下，6周后取材行组织学检查。结果毛乳头细胞微囊体外培养1周后，毛乳头细胞周围出现细胞外基质；4周后，囊中形成“类毛乳头样结构”；人头皮毛乳头细胞微囊移植至大鼠足垫6周后，移植部位及其周围皮下有大量毛囊及皮脂腺结构形成。结论人工毛乳头诱导并参与了无毛区域新生毛囊及皮脂腺的组织构成。     

微囊化人头皮毛乳头细胞移植诱导毛发形成

目的探索微囊化人头皮毛乳头细胞（以下简称毛乳头细胞）对裸鼠背部皮肤毛囊形成的诱导作用。方法胶原酶消化法体外分离、培养毛乳头细胞，再将毛乳头细胞以海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine—alginate，APA）微囊包裹，以胶原凝胶作为载体，植入裸鼠背部皮下；移植空囊、游离毛乳头细胞各作为对照。观察移植部位毛发生长情况，利用组织学方法观测所形成的毛囊结构。结果微囊化毛乳头细胞皮下移植4周后，裸鼠背部移植区有白色、浓密、分布均匀的毛发长出。局部组织切片见大量发育完整的毛囊结构；而空囊及游离毛乳头细胞移植均未能诱导出上述现象。结论聚集性生长的微囊化毛乳头细胞能够保持诱导皮肤毛囊形成的作用，且这种作用无种属特异性。     

人工毛乳头最佳直径的选择

目的观察葡聚糖-荧光素在APA微囊中的扩散方式，对比其在不同直径APA微囊中的扩散速度，确定最佳的人工毛乳头直径；对比最佳直径人工毛乳头的微囊膜与毛乳头基膜的通透性。以进一步评价微囊膜的通透性能。方法在共聚焦显微镜下，分别观察分子量10、40、70ku的葡聚糖-荧光素在APA微囊中的扩散速度和扩散方式，对比、分析相同时间、不同直径的APA微囊中葡聚糖-荧光素的强度，结合囊内细胞数量及成囊所需电压等因素，确定最佳的人工毛乳头直径；比较最佳直径人工毛乳头的微囊膜与新鲜分离的毛乳头基膜的通透性。结果相同分子量、相同时间内荧光强度的比较：小囊组〉中囊组〉大囊组，大囊组、小囊组间的差异具有极显著的统计学意义（P〈0．01）；大囊组与中囊组间的差异具有显著的统计学意义（P〈0．05）；直径400μm微囊中的荧光强度高于新鲜分离毛乳头中的荧光强度。结论直径约400肿人工毛乳头的微囊膜能保证营养物质、代谢产物和信号分子的自由进出，其内部结构适合毛乳头细胞聚集性生长的特性，且其制作过程对细胞的影响较小。     

人工毛乳头最佳直径的选择

目的 观察葡聚糖-荧光素在APA微囊中的扩散方式,对比其在不同直径APA微囊中的扩散速度,确定最佳的人工毛乳头直径;对比最佳直径人工毛乳头的微囊膜与毛乳头基膜的通透性,以进一步评价微囊膜的通透性能. 方法 在共聚焦显微镜下,分别观察分子量10、40、70ku的葡聚糖-荧光素在APA微囊中的扩散速度和扩散方式,对比、分析相同时间、不同直径的APA微囊中葡聚糖-荧光素的强度,结合囊内细胞数量及成囊所需电压等因素,确定最佳的人工毛乳头直径;比较最佳直径人工毛乳头的微囊膜与新鲜分离的毛乳头基膜的通透性. 结果 相同分子量、相同时间内荧光强度的比较:小囊组>中囊组>大囊组,大囊组、小囊组间的差异具有极显著的统计学意义(P<0.01);大囊组与中囊组间的差异具有显著的统计学意义(P<0.05);直径400μm微囊中的荧光强度高于新鲜分离毛乳头中的荧光强度. 结论 直径约400μm人工毛乳头的微囊膜能保证营养物质、代谢产物和信号分子的自由进出,其内部结构适合毛乳头细胞聚集性生长的特性,且其制作过程对细胞的影响较小.     

微囊化人头皮毛乳头细胞体外培养及异种移植

目的探讨微囊化人头皮毛乳头细胞体外培养及异种移植的可行性,并对海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine-alginate,APA）微囊与海藻酸钠-BaCi2（barium-alginate,BA）微囊的物理、生物性能进行评价.方法采用“一步酶消化法”分离、培养人头皮毛乳头细胞,分别用APA微囊与BA微囊包裹.对两种微囊的生物相容性、机械强度、免疫隔离效果及微囊内细胞活性进行比较.结果APA微囊生物相容性优于BA微囊（P＜0.01）,但机械强度低于BA微囊（P＜0.01）;成囊后短期BA微囊内细胞活性高于APA微囊（P＜0.01）,但APA微囊内细胞活性增高较快（P＜0.05）.在微囊完整、表面无纤维化时,两种微囊均可起到良好的免疫隔离作用.结论微囊化人头皮毛乳头细胞可在体外及异种体内培养.综合评价APA微囊与BA微囊的利弊,在不同情况下选择不同的成囊方式是必要的.     

海藻酸钠-多聚赖氨酸-海藻酸钠微囊对骨髓间充质干细胞生长分化性状的影响

目的探讨运用海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊化免疫隔离技术对骨髓间充质干细胞(MSCs)生长分化的影响。方法采用脉冲式高压静电微囊制备仪制备出APA-MSCs微囊复合体,在成囊后第1天和第4天运用荧光素双醋酸酯/溴化乙锭(FDA/EB)染色确定MSCs成活率。采用离心破囊方法破囊,培养并观察破囊后MSCs形态和成骨作用等。结果微囊化后细胞在其内存活率约70%～80%;破囊后的细胞有活跃的增殖能力,经茜素红染色证实具有成骨潜力。结论MSCs在APA微囊内生长良好,保持干细胞特有生长分化,APA微囊可用于对MSCs进行免疫隔离。     

一种适用于大规模高效快速分离培养人头皮毛乳头细胞的方法

目的:寻求适合于毛囊组织工程的大规模高效快速分离培养人头皮毛乳头细胞的方法。方法:将含有毛囊中下部的皮下组织剪碎,加入胶原酶Ⅰ消化2h,悬液经400目筛网过滤,收集被筛网截留的毛乳头进行培养,计算其贴壁率,绘制细胞生长曲线,并与显微解剖法相比较。结果:用这种方法成功分离培养了人头皮毛乳头细胞。结论:消化过滤法是一种能够大规模高效、快速分离培养人头皮毛乳头细胞的方法。     

其他

部分创面外用抗菌药物与成纤维细胞生长因子2、表皮生长因子、重组人生长激素对成纤维细胞生物学特性影响的实验研究；皮肤圆锥体结构损伤致增生性瘢痕的病理学观察；正常人不同部位皮肤表皮干细胞分布规律的研究；微囊化人头皮毛乳头细胞诱导小鼠耳毛囊再生的研究；皮肤软组织扩张器在瘢痕整复应用中外露发生原因及预防……[编者按]     

一步酶消化法高效快速分离培养人头皮毛乳头细胞

目的：寻求高效、快速体外培养人头皮毛乳头细胞的方法。方法：将含有毛囊中下部的皮下脂肪剪碎，加入胶原酶I进行消化，以吸管机械吹打帮助毛乳头游离后进行培养；对其贴壁率、细胞迁出率、工作强度、污染机会与显微解剖法、显微解剖加酶消化法进行比较。结果：“一步酶消化法”能显著降低工作强度、减少污染机会，并保留了显微解剖加酶消化法促进毛乳头贴壁和细胞迁出的优点。结论：“一步酶消化法”是一种高效、快速分离培养人头皮毛乳头细胞的方法。     

Induction of hair follicle regeneration in rat ear by mi-croencapsulated human hair dermal papilla cells

Objective: To induce hair follicle regeneration in rat ear by microencapsulated dermal papillae (DP) cells.Methods: Intact dermal papillae were obtained from human scalp follicles which were digested with collagenase I. The human hair DP cells were encapsulated with alginate-polylysine-alginate (APA) by a high-voltage electric field droplet generator. The diameters of the DP cell microcapsules were optimized by regulating the voltage, the distance be-tween the needle head and the solution surface and the injection speed. Then DP cell microencapsulations were xenotransplanted into ears of 20 SD rats with a novel method. One rat was killed every week at the postoperative 2-12 weeks and the implantation sites were biopsied for histo-logical observation.Results: The DP cell microencapsulations were found in a group of round, smooth and transparent microcapsules under a phase-contrast microscope. The optimal combina-tion of parameters to obtain 0.4 mm DP cell microcapsules was voltage 7.0 kV, injection speed 55 mm/h, and distance 10mm. After 4-12 weeks, 18 of 20 DP cell microcapsule implan-tations had produced high-density hair. Histological obser-vation indicated that both large follicles and sebaceous gland structures were formed in the rat ear within 3-12 weeks.Conclusions: These findings show that the DP cell microencapsulation maintain the capacity for initiating the follicle regeneration and can be considered as a substitute for fresh isolated dermal papillae.     

Role of hair papilla cells on induction and regeneration processes of hair follicles

Hair dermal papilla cells are specialized mesenchymal cells that exist in the dermal papilla located at the bottom of hair follicles. These cells play pivotal roles in hair formation, growth, and cycling. Hair follicle formation is usually directed by an aggregation of dermal mesenchymal cells, the origin of dermal papilla cells, in the embryonic skin. We noticed that cultured dermal papilla cells also have hair-forming activity and do not lose the activity even after long-term cultivation, if they are cultured with conditioned medium from keratinocytes obtained from the sole or with a medium containing fibroblast growth factor. The secreted factors from keratinocytes and fibroblast growth factor are, therefore, important for maintaining the cellular properties of dermal papilla cells. Even if the hair bulb, including the hair matrix and the dermal papilla, has been removed from vibrissal follicles in vivo, the new hair matrix and papilla can regenerate from the rest of the follicle, and eventually a hair shaft regrows. It has been reported that hair bulb regeneration does not occur when the lower half of a hair follicle is removed. However, new hair bulbs were formed in the remaining upper halves of vibrissal follicles if the amputated follicles had been implanted under the kidney capsule. The formed bulbs were small and pelage-type, not large vibrissa-type. Histological studies showed that the new dermal papillae were derived from dermal sheath cells surrounding upper follicular epidermis, and the new hair matrices were produced from the follicular epidermis. Moreover, the upper halves of vibrissal follicles reformed large vibrissa-type bulbs when they were associated with dermal papillae or cultured papilla cells and implanted in the kidney. Thus, dermal papilla cells and probably dermal sheath cells have the ability to induce and form hair bulbs under preferred environmental conditions. Attempts to identify the genes and proteins associated with hair-forming activity of dermal papilla cells have been carried out. We and other groups successfully isolated the molecules that were specifically expressed in dermal papilla cells. The nature of the hair-producing factors could be understood through the studies of these molecules.     

毛乳头细胞诱导毛囊形成的研究

目的 探讨培养的毛乳头细胞在体内外条件下诱导毛囊形成的可能性。方法 采用酶消化法获得毛乳头细胞、真皮鞘细胞、毛囊上、下段及球部细胞，进行毛囊组织工程重建，或用游离细胞混合移植于棵鼠，组织学观察毛囊形成情况。结果 毛囊间表皮细胞、毛囊上段上皮细胞、下段上皮细胞和球部细胞在间质细胞凝胶上均可形成双层结构的组织工程皮肤，在真皮鞘细胞胶原凝胶上毛囊的上、下段上皮细胞形成了毛囊结构，移植于棵鼠后8周毛乳头细胞胶原凝胶诱导毛囊上、下段细胞形成了毛囊。低代毛乳头细胞与毛囊上皮细胞混合移植形成了数量较多、结构典型的毛囊，并有肉眼可见的毛发纤维产生。结论 毛囊的真皮成分细胞即毛乳头细胞、真皮鞘细胞在体内、外均具有诱导毛囊形成的能力，通过与毛囊上皮细胞之间的相互作用，可诱导毛囊形成。     

GMP-compliant culture of human hair follicle cells for encapsulation and transplantation

Human hair follicle cells, both bulge and dermal papilla cells, were isolated and cultured in a GMP cell factory, in order to obtain an in vitro hair follicle source for encapsulation end transplantation in alopecia regenerative cell therapy. An in vitro model, constituted by organotypic cultures of human skin sample, was set up to simulate the dermal-epidermal interaction between bulge cells and dermal papilla cells, evaluating the possible new follicles formation and the regenerative potentiality of these hair follicle cells. Both the bulge and dermal papilla cells show an excellent cellular proliferation as well as an abundant extracellular matrix production. The immunofluorescence investigation revealed the positivity of both cell lines to CK15 and CD200, whereas both cell lines were negative to CD71 and Oct-4. The pool of cultured bulge and dermal papilla cells was injected into the deep dermis; at day 28 of culture, some organized areas with a higher cell density can be observed: the cells self-organize into papilla-like lengthened aggregates. In samples in which the follicular cells have been seeded on the dermis surface, an epidermis-like homogeneous monolayer on the dermis surface can be seen, therefore showing a potentiality of these cells for epidermis regeneration. These data show the efficacy of a cellular isolation and amplification approach to obtain an in vitro human hair follicle regenerative source on industrial scale in a GMP cell factory. The results also proved an intrinsic potentiality of follicular cells to in vitro recreate the epidermis for tissue engineering purposes. Thus, it is feasible to produce bioengineered hair follicles in a GMP cell factory, for encapsulation and transplantation in alopecic patients.     

用毛囊隆突细胞构建复合皮的实验观察

目的 以人毛囊隆突细胞为种子细胞体外构建组织工程复合皮，在体观察其功能性修复全层皮肤缺损的可行性。方法 胶原酶消化法体外分离培养人毛囊隆突细胞和毛乳头细胞，实验分为A、B两组。A组将毛囊隆突细胞与毛乳头细胞按1：2混合，接种于胶原包被的聚羟基乙酸纤维支架中；B组单纯接种相同数量的毛乳头细胞。而后覆盖角质形成细胞膜片，构成组织工程复合皮，移植于裸鼠全层皮肤缺损创面。观察创面愈合情况，分别于术后2、4、6周在光学显微镜下观察移植物组织学变化。结果 组织工程复合皮能够有效修复A、B两组裸鼠全层皮肤缺损。术后2周，A、B组创面均可见完整的表皮及真皮结构。术后4-6周，A组复合皮表皮层明显增厚并形成基膜的钉突，可见毛囊样结构；B组仅表皮层有所增厚但基膜平整，未见钉突和毛囊结构形成。结论 以聚羟基乙酸真皮基质为支架，用角质形成细胞、毛囊隆突细胞和毛乳头细胞共同构建的组织工程复合皮，可以有效修复裸鼠全层皮肤缺损。其中毛囊隆突细胞参与了创面解剖修复，同时可能引导组织结构和功能的修复。     

人毛囊隆突细胞向皮脂腺细胞诱导分化的研究

目的研究人毛囊隆突细胞向皮脂腺细胞诱导分化的可行性。方法体外分离培养人毛囊隆突细胞，以3-甲基-1-异丁基黄嘌呤、地塞米松和牛胰岛素为诱导剂，体外观察细胞形态的变化，诱导剂作用2周后行油红染色和抗上皮膜抗原免疫细胞化学染色。以不加诱导剂组细胞作为对照。结果毛囊隆突细胞经诱导剂作用1周后，细胞体积明显增大，连接松散，出现特征性“扇贝”形细胞；2周后，细胞体积进一步增大，胞质内出现少量颗粒和液滴；3周后细胞达到融合，排列紊乱，部分细胞伸展，形态变为椭圆，纺锤或不规则形；4周后，伸展的细胞相互融合，包裹形成不规则的网格状。而未加诱导剂组细胞随培养时间延长，细胞融合，在克隆中心出现细胞的堆积，分层。诱导剂作用2周后油红染色及上皮膜抗原染色呈阳性。结论在适宜的微环境刺激下，人毛囊隆突细胞具有向皮脂腺细胞分化的潜能。     

Hair-bearing scalp reconstruction using a dermal regeneration template and micrograft hair transplantation

The scalp has traditionally presented a challenge to the reconstructive surgeon. Although a variety of techniques exists for providing stable coverage to the cranium that has become exposed secondary to trauma, infection, or surgical ablation, these techniques tend to require multiple operations and a significant time commitment on the part of the patient and the surgeon. Unfortunately, most if not all of these techniques are unable to recreate the appearance of natural-appearing hair on the reconstructed scalp. We present our unique experience utilizing a tissue-engineered dermal regeneration template for reconstruction of the traumatized scalp. Once vascularized and skin grafted, the neodermis received approximately 1600 microfollicular hair grafts, which resulted in a pleasing esthetic outcome. In this case, a 50-year-old man suffered a significant degloving of the posterior portion of the scalp. The area of exposed periosteum/galea was covered acutely with Integra dermal regeneration template, followed by skin grafting 2 weeks later. Since the patient refused tissue expansion reconstruction, a trial of hair micrografting (100 grafts) into the Integra was performed, with good results. Two subsequent sessions of 800 grafts placed also had good results. This report represents the first use of sequential hair micrografting into Integra dermal regeneration template to reconstruct hair-bearing scalp. Application of a dermal regeneration template with a thin split-thickness skin graft followed by sequential hair micrografting is a viable alternative for hair-bearing scalp reconstruction in cases where traditional methods (eg, tissue expansion, microvascular free tissue transfer) are unavailable or undesirable.     

创伤性毛囊新生:成年哺乳动物毛囊再生的新视角

目的综述近年来创伤性毛囊新生(wound-induced hair follicle neogenesis,WIHN)细胞来源及相关信号通路的研究进展。方法广泛查阅近年与WIHN相关的文献,从细胞来源、分子机制等方面进行总结讨论。结果WIHN是一种罕见的成年哺乳动物毛囊再生现象,新生毛囊细胞来源丰富,既有创面周围来源的毛囊干细胞,也有上皮来源的干细胞。WIHN分子机制复杂,是一种多种信号通路调控的再生过程,与免疫系统密切相关,免疫细胞及其产生的各种免疫因子为这一过程提供了适宜的条件。结论WIHN的细胞来源及分子机制等方面仍有许多未解决的问题,加强机制研究将加深对成年哺乳动物毛囊再生的了解,为皮肤功能性愈合提供新思路。