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。     

改良一步酶消化法分离培养人头皮毛乳头细胞的实验研究

目的进一步简化人头皮毛乳头的分离方法,实现高纯度毛乳头细胞的体外扩增。方法将含有毛球部的头皮皮下组织剪成肉泥状,胶原酶Ⅰ(2mg/ml)37℃消化2~3h后,加入10倍体积的DMEM,以吸管反复吹打消化液使毛乳头全部游离出来,在显微镜下用微量移液器逐个收集表面无杂质黏附的毛乳头。结果“改良一步酶消化法”分离毛乳头所需的操作时间进一步缩短,劳动强度降低;所得毛乳头纯度高,贴壁率达到99%,其细胞迁出加速。结论“改良一步酶消化法”是一种高效、快速分离培养人头皮毛乳头细胞的方法。     

改良一步酶消化法分离培养人头皮毛乳头细胞的实验研究

目的进一步简化人头皮毛乳头的分离方法,实现高纯度毛乳头细胞的体外扩增.方法将含有毛球部的头皮皮下组织剪成肉泥状,胶原酶Ⅰ(2 mg/ml) 37 ℃消化2～3 h后,加入10倍体积的DMEM,以吸管反复吹打消化液使毛乳头全部游离出来,在显微镜下用微量移液器逐个收集表面无杂质黏附的毛乳头.结果 "改良一步酶消化法" 分离毛乳头所需的操作时间进一步缩短,劳动强度降低;所得毛乳头纯度高,贴壁率达到99%,其细胞迁出加速.结论 "改良一步酶消化法"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。     

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。     

高效的毛乳头细胞分离培养方法

目的 寻求一种洁净且无须显微镜下操作的毛乳头分离方法 ,培养高纯度的毛乳头细胞.方法 中性蛋白酶消化头皮组织,将含有毛囊的皮下脂肪层剪下切碎,并用Ⅰ型胶原酶消化,之后通过多次离心的方法 获得毛乳头;对培养出的毛乳头细胞进行形态学观察,并检测毛乳头细胞的标志物alfa-SMA和碱性磷酸酶的表达情况.结果 通过新型的两步酶消化法可获得洁净的毛乳头,培养出的毛乳头细胞标志物检测阳性.结论 新型的两步酶消化法是一种高效低劳动强度的毛乳头分离方法.     

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.     

毛乳头细胞培养基血清浓度的探索

目的 研究不同血清浓度的培养基对毛乳头细胞生长速度及细胞状态的影响.方法 改良一步酶消化法分离培养人头皮毛乳头细胞,分别以无血清培养基及10%、15%等不同浓度小牛血清培养基,培养第3代毛乳头细胞,倒置相差显微镜下观察细胞生长状态,细胞消化后,计数并绘制生长曲线.结果 在无血清培养基培养条件下,毛乳头细胞传代后贴壁率较低.贴壁后细胞未见明显增殖;传代后前8 d,15%和20%血清培养基培养的毛乳头细胞生长速度,明显快于无血清培养基组和10%血清培养基组(P<0.05);传代第10天后,15%和20%血清培养基中毛乳头细胞生长速度差异无显著的统计学意义.结论 细胞生长速度与血清浓度密切相关.     

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.     

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.     

Viability of Isolated Single Hair Follicles Preserved at 4°C

Background. Hair follicle preservation for the purpose of delayed application would help us to transplant hair follicles more efficiently.
Methods. Isolated single hair follicles were preserved at 4°C in four different solutions. Viability of preserved follicles was judged by organ culture and cell culture. In addition, a small number of hair follicles were transplanted into athymic mice.
Results. By cell culture, both dermal papilla and outer root sheath cells could be cultivated after 7 days of preservation. Hair follicles preserved for 48 hours showed a significant increase of hair shafts in organ culture. Those preserved for 7 days regrew well when transplanted into athymic mice.
Conclusion. Preservation of hair follicles at 4°C could be one option to prepare many follicular units at one time for transplantation.     

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。     

Identification of hematopoietic cell populations from the dermal papillae of human hair follicles

Hematopoietic stem cell (HSC) activity has been identified from the hair follicles (HFs) in mice; however, it has not been identified in human HFs. We used immunohistochemistry and flow cytometry to identify cultured dermal papilla (DP) cells expressing CD45 to test for hematopoietic activity in colony-forming assays of granulocyte/macrophage hematopoietic progenitors (CFU-GM). Occasional CD45-positive cells were detected in cultured DP cells. After in vitro stimulation with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) for 7 days, about 1% of the cells were CD45-positive by flow cytometry analysis, an fifty-fold expansion in cell numbers. We further examined whether mesenchymal stem/progenitor cells reside in human dermal papillae. Cultured DP papilla cells incubated with monoclonal antibodies to remove the CD45 positive cells were induced into multilineage differentiation with the formation of CFU-GM. Our findings preliminarily indicate that human dermal papilla contain at least a CD45-positive hematopoietic cell population and a mesenchymal stem/progenitor cell population.     

氟对游离毛囊凋亡影响的实验研究

目的观察离体培养条件下氟对人头皮毛囊各部位凋亡的影响及硒对氟影响的拮抗作用。方法构建人头皮游离毛囊培养模型,在模型中,加入不同浓度的氟化钠和亚硒酸钠,筛选浓度后,分为不同组;制作冰冻切片,利用TUNEL法原位检测各组毛囊不同位置的凋亡数量并做统计分析;用透射电镜进行观察照相。结果原位凋亡染色发现,1mmol/L和10mmol/L的氟化钠能使游离培养5d的人头皮毛囊中外根鞘、真皮鞘和毛乳头,及毛球下部的凋亡细胞显著增多,0.01mmol/L的亚硒酸钠能拮抗1mmol/L氟化钠对外根鞘、毛球部的促凋亡作用(P<0.05)。结论不同浓度的氟化钠对游离培养的第5天的人头皮毛囊的凋亡具有不同作用,一定浓度的氟化钠能促进游离毛囊一定部位的细胞凋亡。0.01mmol/L的亚硒酸钠对一定浓度的氟化钠的促凋亡作用具有拮抗性。     

人毛乳头细胞条件培养液对斑秃患者的治疗作用

目的:观察人毛乳头细胞(DPC)条件培养液对斑秃患者的治疗作用,探讨DPC分泌促进毛囊生长的活性物质。方法:收集体外培养低传代DPC培养上清,制成冻干粉,用于治疗50例斑秃患者,并分别选择其中26例和10例作去炎松组和空白组自身对照。结果:DPC条件培养液组、去炎松组和空白组治愈率分别为74%、54%、10%,有效率分别为96%、73%、30%,DPC条件培养液组其治愈率和有效率均明显高于去炎松组和空白组(P<0.01)。结论:人DPC条件培养液有促进斑秃患者毛囊生长的作用。     

Male androgenetic alopecia

Male pattern hair loss is the most common cause of balding. The pathogenesis involves androgen, and in particular dihydrotestosterone, binding to androgen receptors in the dermal papilla of sensitive hair follicles. Hair follicle sensitivity is genetically determined and shows regional specificity. Androgen stimulation of scalp dermal papilla cells induces transforming growth factor beta (TGF-B) and results in cyclical miniaturization of the entire hair follicle. The resulting hair produced from that follicle is shorter and finer and provides less complete scalp coverage. In contrast androgen stimulation of beard dermal papilla cells produces insulin growth factor -2 (IGF-2) and results in cyclical enlargement of the entire hair follicle. The resulting hair produced from that follicle is longer and thicker and provides more complete facial skin coverage. Some degree of androgenetic alopecia is universal among ageing men, especially bitemporally, however less than half become bald in the Hippocratic sense. Although scalp hair coverage has little functional importance, it has cosmetic significance. Baldness changes the facial appearance of affected men. When that change is perceived as adverse it has the potential to produce emotional morbidity.