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。     

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。     

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。     

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.     

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。     

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。     

人毛囊无色素性黑素细胞及其治疗白癜风的研究进展

毛囊无色素性黑素细胞可以作为皮肤黑素细胞的储库，给白癜风的治疗提供黑素细胞来源。人黑素细胞（melanocyte，MC）起源于胚胎神经嵴，在胚胎发育2～5周开始向表皮和毛囊移行。向表皮移行的黑素细胞最终定居在基底膜上，形成连续产生黑素的成熟黑素细胞；而向毛囊移行的黑素细胞则分为两种类型：一种是位于生长期毛囊毛母质和漏斗部具有黑素合成活性的黑素细胞；另一种是分布于生长期毛发外毛根鞘（Outer Root Sheath，ORS）中，无黑素合成活性、未活化的无色素性黑素细胞（amelanotic melanocyte，AMMC）。     

微囊化人头皮毛乳头细胞诱导小鼠耳毛囊再生的研究

目的 观察人头皮毛乳头细胞海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine-alginate，APA）微囊是否具备诱导小鼠耳部毛囊再生的功能；寻找理想的微囊直径。方法 以APA微囊包裹体外分离培养的人头皮毛乳头细胞；将毛乳头细胞微囊移植至小鼠耳部皮下，6周后局部取材行组织学检查；共聚焦显微镜下观察葡聚糖-荧光素在APA微囊中的扩散速度和扩散方式，并对比相同时间、不同直径的APA微囊中葡聚糖-荧光素的强度，综合分析确定最佳的微囊直径。结果 组织学检查显示：移植部位皮下有密集的同心圆状毛囊结构形成，其数量、大小、分化程度等与对照组明显不同。荧光素以同心圆状、逐层渗透的方式向APA微囊中扩散；相同时间内荧光强度比较：小囊组〉中囊组〉大囊组。结论 微囊化毛乳头细胞具备诱导毛囊再生的生理功能；微囊理想的直径是400μm。     

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.     

离体培养条件下氟与硒对毛囊影响的初步研究

目的:观察氟与硒对离体培养条件下的人头皮毛囊生长的影响。方法:在离体人头皮毛囊培养模型中,加入不同浓度的氟化钠和亚硒酸钠,目镜测微器测量毛囊长度的变化;连续观察毛囊形态学改变并做电镜切片检查。结果:1mmol/L和10mmol/L的氟化钠能不同程度的抑制离体毛囊的生长(P<0.05)。0.01mmol/L的亚硒酸钠对1mmol/L氟化钠毒性有拮抗作用(P<0.05)。0.1mmol/L氟化钠单独作用对毛囊生长无影响(P>0.05),0.1mmol/L氟化钠及0.01mmol/L亚硒酸钠共同作用对毛囊生长无影响(P>0.05)。电镜观察发现10mmol/L氟化钠作用的毛囊在培养5天时即可见较为严重的凋亡前改变及凋亡现象,10mmol/L氟化钠加0.01mmol/L亚硒酸钠组毛囊也可见到类似的改变,培养9天时还可见到细胞内有空泡,连接紊乱,桥粒连接破坏等现象。结论:不同浓度的氟化钠对游离的人头皮毛囊的生长有不同作用,一定浓度的亚硒酸钠能拮抗氟化钠对毛囊的毒性作用。     

Collagen expression by novel cell populations in the dermal wound environment

Numerous collagenous structures must be reconstituted following injury to the skin in order to return function to this tissue. The basement membrane zone, vascular basement membranes, and the dense connective tissue of the dermis are examples of structures that contain a number of different collagen types and that may need replacement following injury. In addition, a scar is deposited at the site of damage in order to substitute for elements lost in the trauma and for elements that cannot be successfully replaced. Clearly, cells resident within the different compartments of the skin are able to synthesize and deposit collagen to reform these multiple structures. However, accumulating experimental evidence suggests that in addition to these resident cells, blood-borne cells may be responsible for the deposition of a portion of the newly synthesized collagen. Studies from this laboratory point to the activated monocyte as a potential source of collagen in the wound environment. Given the dynamics of the process, the hypothesis is proposed that during normal wound healing, the activated monocyte is a source of collagen essential for the rapid formation of a provisional matrix conducive for the subsequent formation of granulation tissue. Collagen synthesis also occurs by expanded populations of resident cells, under the influence of inflammatory cell-derived mediators, which results in the major accumulation of collagen during normal wound repair. However, if a chronic inflammatory state is initiated, the activated monocytes may remain in sufficient numbers to deposit collagen leading to a pathological lesion.     

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.     

毛发诱导结合自体毛囊单位移植技术进行毛发定向重建的研究

目的 将异位诱导形成的毛发组织进行移植,实现毛发的定向重建.方法 制备新生C57鼠的皮肤细胞悬液注射于裸鼠体内,诱导形成皮下异位毛发组织,取出后在体视显微镜下分离成单个毛囊单位,移植至裸鼠体表,并进行大体观察和组织学检测.结果 按照预定密度移植后的毛发成活良好并呈周期性生长,组织学可见各时期相应的毛囊形态.结论 诱导形成的毛发组织可以作为移植物,通过毛囊单位移植的方式,实现毛发组织密度和方向的可控性重建.     

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.     

外用他克莫司小鼠-大鼠异种触须毛囊移植

目的 探讨建立小鼠-大鼠异种触须毛囊移植动物模型的可行性及局部辅助应用他克莫司的效果.方法 以C57BL/6J小鼠触须毛囊为供体、Wister大鼠为受体,进行异种毛囊移植.实验分两组:移植后局部外用他克莫司组(A组),移植后的空白对照组(B组).观察移植后毛囊的成活时间及形态学变化.结果 A组的毛囊最长成活15d,B组的毛囊最长成活10d.两组比较表明,他克莫司延长了异种移植毛囊的成活时间,但触须毛囊含血窦,易诱发免疫排斥反应.结论 本动物模型是一种研究异种毛囊移植和免疫赦免的良好动物模型.外用他克莫司可以延长异种移植触须毛囊的成活时间,但仅靠局部用药仍无法避免排斥反应的发生.     

男性健康人群和尖锐湿疣患者阴毛毛囊中人乳头瘤病毒的调查分析

目的:人乳头瘤病毒(human papillomavirus,HPV)在健康人群中广泛存在,但其在阴毛毛囊中的感染率目前尚不清楚,阴毛毛囊感染与尖锐湿疣的复发关系更不清楚,本课题旨在研究HPV在健康男性和尖锐湿疣患者阴毛毛囊中的感染情况,了解毛囊感染与尖锐湿疣复发的关系。方法:尖锐湿疣患者86例,年龄24~61岁,健康男性122例,年龄22~80岁,采用PCR技术对耻骨部阴毛毛囊中的HPV进行检测,并对两组资料进行分析。结果:男性尖锐湿疣患者的阴毛毛囊中HPV阳性率为32.55%(28/86),其中17例为HPV6(19.77%),7例为HPV11(8.14%),2例为其他亚型,混合感染2例(同时检测到HPV6和HPV11,以下同)。健康人群阴毛毛囊中的HPV检出率为17.21%(21/122),其中15例HPV6(12.30%),4例为HPV11(3.28%),1例其他亚型,1例为混合感染。结论:尖锐湿疣患者的阴毛毛囊中HPV感染率高于健康男性人群。尖锐湿疣患者与健康人群感染HPV的亚型基本相似,主要为HPV6和HPV11。     

HSPC011基因在人毛乳头细胞表达的生物学功能

目的 探讨HSPC011基因在人毛乳头细胞表达的生物学功能。方法 应用Nucleo-lector^TM基因转染技术，分别将质粒pCI-neo、绿色荧光蛋白质粒pEGFP-N1、真核表达重组质粒pCI-neo／HSPC011转染人毛乳头细胞和3T3细胞，了解HSPC011基因表达对这些细胞生长特性的影响。结果 通过观察绿色荧光蛋白在人毛乳头细胞和3T3细胞的表达，发现Nucleofector^TM基因转染效率分别约为70％和30％。HSPC011基因在人毛乳头细胞表达后，细胞形态幼稚而类似于3T3细胞，但其凝集性生长特性保持不变；HSPC011基因表达对毛乳头细胞的增殖活性无明显影响。HSPC011基因转染3T3细胞后，对3T3细胞的生长特性无任何影响。结论 初步体外实验表明，HSPC011基因在毛乳头细胞表达可能具有调节细胞分化的作用。