构建组织工程化毛囊的初步研究
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摘要
研究背景
毛发具有重要的生理及美容价值,目前治疗毛发缺损性疾病特别是雄激素性秃发最为有效的方法是自体游离毛发移植,但存在供区不足、供区瘢痕、移植密度低等问题。而毛囊组织工程的研究有望为临床各种类型不可逆性秃发的治疗提供一种比现行毛发移植术更加简便易行、微创且不受来源限制的全新治疗手段。
毛囊是由上皮成分(包括毛母质、内根鞘和外根鞘)和真皮成分(包括毛乳头、真皮鞘)组成的。其中毛乳头细胞是构成毛乳头的主要间质细胞,它们是一簇特化的成纤维细胞。所有的毛乳头细胞在外观上都呈成纤维细胞样,而且体外培养的毛乳头细胞有着聚集性生长的特性。毛乳头在毛囊的形态学发生及周期调控中起着重要的作用。真皮鞘是围绕毛囊最外层的真皮源性组织,在毛囊的再生过程中,可以观察到真皮鞘细胞向毛乳头细胞的转化。有研究认为真皮鞘细胞是毛乳头细胞的储备细胞,毛乳头部位的真皮鞘细胞与毛乳头具有相同的诱导能力。毛囊隆突区(Bulge)与外根鞘相连续,并有研究证实毛囊Bulge区聚集了大量具有潜在增殖能力的早期幼稚细胞。被认为是毛囊干细胞的所在。已有研究证实,毛囊的形态学发生及周期性生长是毛囊上皮成分和真皮成分之间双相调节作用的结果。
另有研究认为表皮干细胞在人体不同部位的皮肤分布差异,以包皮和阴囊皮肤表皮最多。并有日本学者将鼠毛囊真皮成分细胞和人包皮表皮细胞组合,进行裸鼠体内毛囊诱导重建获得成功,并有肉眼可见的毛发纤维长出。但尚无报道用人毛囊真皮成分细胞与人包皮表皮基底细胞组合进行此实验。
Ⅰ型胶原蛋白是目前组织工程研究中最常用的天然生物材料,由多种糖蛋白分子组成,它具有多方面的生物活性,不仅是组织支持物,而且是细胞外间质的重要成分。研究表明,胶原蛋白生物相容性好,降解速度可调,而且可参与组织修复,是一类优良的可引导组织再生的生物材料。国内外许多毛囊生物学研究均用鼠尾胶原作为支架进行毛囊真皮细胞成分和表皮细胞成分组合器官型培养。但尚未报道采用用固体Ⅰ型胶原蛋白海绵作为支架进行毛囊种子细胞培养。
DiI是一种亲脂性碳花青染料,易嵌入生物质膜内作侧向扩散运动,从而标记整个细胞膜,还可以通过活体细胞的胞饮作用进入胞浆,标记整个细胞浆。以往研究显示,DiI对活体细胞无毒性,对被标记细胞的存活、生长无影响。并有研究报道DiI可作为荧光示踪剂用于培养的神经细胞。随后又有DiI标记肝细胞、骨髓基质干细胞、脂肪干细胞等报道,但迄今未见有应用于标记毛囊细胞。
研究目的
1.建立高效、快速分离培养毛乳头细胞、真皮鞘细胞和毛囊上皮细胞,包皮表皮基底细胞的方法。
2.选择适合于细胞生长的支架及细胞标记方法,建立毛囊细胞器官型培养的模型。
3.探索有效的动物体内毛囊重建的方法。
研究方法
1.人毛囊真皮成分和表皮成分细胞的分离、培养和鉴定。将人头皮标本剪成小块,中性蛋白酶中37℃孵育2 h后镜下将带外根鞘的毛干从真皮鞘中拔出,组织块法培养外根鞘隆起(Bulge)细胞;从真皮-皮下组织交界处横断头皮,拔出含毛乳头的真皮鞘,胶原酶D 37℃孵育6～8 h,多次低速离心,毛乳头沉淀重悬后培养,收集的真皮鞘细胞上清液经高速离心后重悬培养;真皮鞘和毛乳头的分离也可采用显微解剖法,依参照文献中的操作进行。观察其在体外培养的形态学和生物学特点,比较两种分离方法毛乳头的贴壁率,培养的Bulge细胞和毛乳头、真皮鞘细胞分别用K19或α-平滑肌动蛋白免疫组化鉴定。
2.鼠触须毛乳头细胞和鼠触须毛囊上皮细胞的分离、培养。采用显微解剖法进行大鼠触须毛乳头细胞分离、培养,大鼠麻醉后修剪颊侧被毛,常规消毒后剪去全层皮肤,暴露其下方的触须毛囊,然后用镊子将其完整拔出,用显微解剖法分离毛乳头。将分离的毛乳头置入含15%FCS的DMEM培养基,以适当密度接种(每个35 mm培养皿含10个毛乳头),观察其在体外培养的形态学和生物学特点。鼠触须毛囊上皮细胞的分离、培养:参照文献中的操作进行,无菌条件下取7日龄Wistar大鼠触须部皮肤,镜下将毛囊从组织中分离出来,置入0.125%Dispase消化液中,4℃消化2 h后,镜下轻压毛球部即可将毛囊外根鞘及毛干完整地分离出来。切去带外根鞘的毛干中下段和上段,保留Bulge区毛囊。将分离的Bulge区毛囊置入无血清的K-SFM培养基中培养。观察其在体外培养的形态学和生物学特点。
3.包皮表皮基底细胞的分离、培养。采用Ⅳ型胶原快速粘附法培养包皮表皮细胞。包皮标本用含双抗的D-Hanks液反复冲洗,室温下浸泡30 min,剔除皮下组织,将包皮剪成1.0 cm×0.5 cm大小皮条,置0.5%的中性蛋白酶中,4℃消化12～15 h,PBS冲洗2次,将表皮层撕下,剪成1mm×1mm大小皮片,置0.25%胰酶中,37℃消化5 min左右,中止消化,过200目筛网,计数,离心,重悬,接种于预先铺有Ⅳ型胶原的培养瓶中,室温下静置10 min,换液,吸附未贴壁的角质细胞。观察其在体外培养的形态学和生物学特点。
4.细胞胶原凝胶制备。取传代培养数次(＜4代)的鼠毛囊毛乳头细胞和外根鞘细胞分别为6×10~5和3×10~5(真皮细胞与上皮细胞比例为2:1)梯度离心后加入0.5 ml胎牛血清。吸4 ml鼠尾胶原(质量浓度为1.0～1.5ml)于冰浴中的青霉素小瓶中,加入0.5 ml浓缩10倍的DMEM培养基,1N NaOH调Ph,立即加入含鼠毛囊毛乳头细胞和外根鞘细胞的胎牛血清细胞悬液,快速混合,制成细胞胶原凝胶,然后吸到35 mm培养皿中。静置于37℃、体积分数为0.05的CO_2孵箱培养。
5.用DiI标记人毛囊毛乳头细胞和外根鞘细胞,包皮表皮基底细胞。将收集细胞用PBS清洗离心2次,加入无血清的DMEM制成细胞悬液,以1×10~9/L密度加入5μL DiI应用液,37℃下孵育25 min,1500 r/min离心5 min,PBS清洗离心2次,加入10%DMEM培养基中,荧光显微镜观察细胞显色情况,置于37℃、体积分数为0.05的CO_2孵箱培养,观察24,48,72 h细胞荧光强度及形态变化。
6.细胞—支架复合物的形成。将Ⅰ型胶原蛋白海绵支架材料裁制成10 mm×10mm×10 mm大小,利用紫外线照射及酒精浸泡消毒,培养液平衡后待用。DiI标记的人毛乳头细胞和外根鞘细胞、包皮表皮基底细胞消化后制成细胞悬液,分两组(毛乳头细胞+外根鞘细胞组、毛乳头细胞+包皮表皮基底细胞组,真皮成分细胞和表皮成分细胞数比例为2:1),细胞密度为2×10~5/ml,表面种植1 ml细胞悬液,静置4 h后,加入含20%胎牛血清的DMEM培养液,37℃、5%CO_2培养箱中培养。采用倒置相差显微镜和扫描电镜,观察细胞在支架上黏附、生长情况。
7.裸鼠体内诱导实验。将鼠毛囊细胞胶原凝胶在无菌条件下注射移植到裸鼠背部两侧的皮下。于4周切下移植部位皮肤,石蜡切片行H-E染色光学显微镜下观察;人细胞—支架复合物在无菌条件下注射移植到裸鼠背部两侧的皮下,分别于6、8、12周切下移植部位皮肤,冰冻切片和石蜡切片荧光显微镜下观察,并进行H-E染色光学显微镜下观察。
研究结果
1.结合运用显微法和酶消化法等基本分离技术,同时从头皮毛囊标本中分离培养毛囊三种细胞成分,获得了较纯化的外根鞘Bulge细胞,真皮鞘细胞和毛乳头细胞。酶消化法获得的毛乳头贴壁率高,可达到90%,毛乳头和真皮鞘细胞体外培养表现出明显的凝集性生长,α-SMA免疫组化染色呈现阳性。组织块法培养的头皮毛囊外根鞘Bulge细胞,可在K-SFM培养基中生长良好,呈铺路石样。角蛋白19免疫组化染色阳性。
2.采用显微解剖成功分离大鼠触须毛乳头细胞,细胞体外培养表现出明显的凝集性生长。采用酶消化法和显微解剖法成功分离到大鼠触须毛囊上皮细胞,可在K-SFM培养基中生长良好,呈铺路石样。
3.采用Ⅳ型胶原快速粘附法培养的包皮表皮基底细胞外培养过程中呈典型的克隆性生长,生长周期长。
4.刚接种于胶原凝胶的细胞呈圆形,在胶原凝胶内均匀分布。24 h内细胞伸展,粘附,2 d后部分细胞已伸展变形,以三角形和梭形为主。
5.DiI标记的人毛乳头细胞、外根鞘细胞和包皮表皮基底细胞荧光显微镜下观察,见全部细胞标记后均显红色荧光,标记的细胞呈梭形或圆形,胞浆丰富,保持了良好的正常形态,DiI标记阳性率为100%。DiI标记后早期细胞形态呈荧光环状,48 h后细胞中荧光颗粒增多,荧光增强,细胞核未染荧光。
6.细胞—支架复合物体外倒置显微镜下和扫描电镜观察,将毛乳头细胞、毛囊外根鞘细胞和包皮表皮基底细胞在材料上接种并在孵箱孵育2 h后,材料周边及孔板底部开始有细胞黏附,随着时间的延长,附着于材料上的细胞逐渐增多;培养2-3天后,细胞在材料上逐渐伸出突起,可见大量细胞附着于材料的孔隙及材料周边,胶原蛋白材料上皆有细胞附着,细胞呈梭形圆形或多角形,部分材料上的细胞呈团簇状生长,粘附于胶原海绵壁上或孔隙内,各细胞或细胞团之间通过基质相连或重叠。细胞种植3 d后,细胞呈扁平多角形或圆形,在材料表面伸展、黏附,种植1周后,形态为梭形或圆形,细胞数量明显增多,呈聚集性分布,细胞周围有大量基质。
7.裸鼠体内诱导实验。裸鼠背侧皮肤观察未见有肉眼可见的毛发纤维生成。石蜡切片光学显微镜下观察发现鼠细胞胶原凝胶诱导实验组和人细胞-Ⅰ型胶原蛋白支架复合物毛乳头细胞与毛囊上皮Bulge细胞组合、毛乳头细胞与包皮表皮基底细胞组合诱导实验中4周、8周、12周均可见新生毛囊样结构形成。空白支架对照组无毛囊样结构生成。石蜡切片荧光显微镜下观察DiI标记的人毛乳头细胞、外根鞘Bulge细胞组合和DiI标记的人毛乳头细胞、包皮表皮细胞组合实验组新生毛囊样结构显红色荧光。未用DiI标记的实验对照组新生毛囊样结构无红色荧光显示。
研究结论
1.结合运用显微解剖法和酶消化法等基本分离技术,避免了单独运用显微法和酶消化法的缺点,并在分离步骤和次序上进行改进,首次同时从头皮毛囊标本中分离培养毛囊三种细胞成分,最大限度的利用了标本量,毛囊毛乳头因为经过胶原酶的处理,其贴壁与融合时间与文献报道的两步酶法无明显差别。因毛囊各种细胞成分来源于同一标本,进行细胞组合诱导等毛囊试验时避免了毛囊各细胞因来源不同而在毛囊组织工程中组合应用可能面临的免疫原性问题。
2.经Ⅳ型胶原快速粘附法多次筛选的包皮表皮细胞外培养过程中呈典型的克隆性生长,生长周期长。说明用这种方法培养筛选的包皮表皮基底细胞可能含有较多的未分化表皮干细胞。
3.DiI标记的人毛乳头细胞、外根鞘细胞和包皮表皮基底细胞标记阳性率为100%。细胞在Ⅰ型胶原蛋白支架上形态为梭形或圆形,呈聚集性分布,细胞周围有大量基质,表明细胞在支架上粘附、伸展和生长良好。
4.裸鼠体内诱导实验组织切片观察DiI标记的人毛乳头细胞、外根鞘Bulge细胞组合和DiI标记的人毛乳头细胞、包皮表皮基底细胞组合实验组新生毛囊样结构显红色荧光。未用DiI标记的实验对照组新生毛囊样结构无红色荧光显示。证明了新生的毛囊是由细胞支架复合物诱导生成。
Background
Hair plays an important role in biological necessity and performs a variety of beauty function.At present,autograft of hair follicles is the most important treatment for hair loss,especially for male pattern baldness.But there are some problems for this treatment,such as formation of scar in donor site,scantiness of donor site,low density of transplantations,so hair follicle tissue engineering provides a new minimally invasive treatment not restricted by the source for repairing hair loss.
A hair follicle is composed of epithelial cells including matrix,out root sheath, inner root sheath and dermal cells including dermal papilla,dermal sheath cells. Dermal papilla cells are a kind of fibroblast cells,which can aggregate during growing in mediums containing FCS.According to the different phages of hair cycle, the shape of the dermal papilla change periodically.Dermal papilla plays an important role in the morphogenesis of hair follicle and hair cycle.Dermal sheath around the hair fllicle of the outermost layer are the dermis derived tissue,dermal sheath cells can be observed transforming to dermal papilla cells in the hair follicle regeneration process,It has been demonstrated that dermal sheath cells are the reserves cells of dermal papilla cells and the dermal sheath cells neighbor dermal papilla have the same capacity induction as dermal papilla cells.The human hair follicle bulge connectted with outer root sheath is an important niche for keratinocyte stem cells.It is widely accepted that a highly coordinated series of bidirectional epithelial-mesenchymal interactions(EMIs) is essential to hair follicle morphogenesis.
Several lines of evidence have suggested that foreskin and scrotum epidermal is an important niche for keratinocyte stem cells.Although Japanese scholars has demonstrated that hair follicle would be reconstructed and hair fibers visible to the naked eye grow induced by mouse hair follicles dermis cells and human foreskin epidermal cells portfolio when transplanted into nude mice dorsal.There is no reported that human hair follicle cells and human dermis foreskin epidermal cells portfolio have been used for the experimental replication.
Type I collagen scaffold composed of various glycoprotein molecules has been the most widely used in tissue engineering experiment in the present.Type I collagen scaffold,which is a kind of important component mesenchymal outside of cells,has many biological activity as an organizational support.It has been demonstrated that collagen could be participated in tissue repair as a kind of excellent biological materials because of its cellular compatibility and adjustable speed degradation.Rat tail collagen has been widely used in hair follicle biology studies as a scaffold for hair follicle cells in the dermis and epidermis cells composition organotypic culture. However,there has been no report that solid typeⅠcollagen could be used as a kind of scaffold for hair follicle seed cells culture in hair follicle engineering.
DiI is a lipophilic carbon anthocyanin dye easily embedded within a biological membrane by lateral diffusion movement,so DiI can effectively label cytomembrane, and DiI can effectively label cytoplasm by living cells' pinocytosis.It has been demonstrated that DiI labeled cells have good morphology and DiI is no toxicity for living cells.There are some research reports show that DiI can be used as a fluorescent tracer for cultured nerve cells,then come DiI label of hepatocyte,bone marrow stromal stem cells and adipose derived stem cells,but so far there has been no labeled for cells derived from human hair follices.
Objective
1.To establish the method for isolating dermal papilla cells,dermal sheath cells, follicular epithelial cells and foreskin epidermal basal cells rapidly and efficiently.
2.To explore appropriate scaffold materials for hair follicle tissue engineering, to establish the model for hair follicle cells organotypic culture.
3.To explore the method of reconstructing hair follicles.
Methods
1.The isolation,culture and identification of dermis and epidermis cells derived from human hair follicle.Small scalp specimens were incubated in the presence of dispase at 37℃for 2 h,the hair shafts with ORS embedded in the dermal sheath (DS) were extracted under dissecting microscope,and the ORS tissue were inoculated onto Petri dish.The specimens were transected at the interface between the dermis and subcutaneous tissue.The portions of DS and DP(linked with and enclosed by DS) embedded in the adipose tissue were pulled out and incubated with collagenase at 37℃for 6-8 h,and the DP and DSCs were isolated by repeated low-speed centrifugation and cultured respectively on petri dishes.The cultured ORS bulge cells were identified by immunohistochemistry with K19 antibody and DPCs and DSCs by immunohistochemistry withα-actin antibody.
2.The separation and culture of dermal papilla cells and epithelial cells of rat vibrissa follicle.Dermal papilla cells of rate vibrissa hair follicles were isolated by microdissecting method,and cultured in DMED medium containing 15%FCS,the bioligical behavior in vitro was observed.Epithelial cells of rate vibrissa hair follicles were isolated by microdissecting and enzyme digesting method,and cultured in K-SFM medium,Dermal papillas were removed from the dermal sheathes under the microscope.The bioligical behavior in vitro was observed.
3.The separation and culture of epidermal basal cells from the foreskin. Epidermal cells from the human foreskin were isolated by typeⅣcollagen rapid adhesion method,and cultured in K-SFM medium,the bioligical behavior in vitro was observed.
4.Cell collagen gels preparation,mouse hair follicle dermal papilla cells(6×10~5) and outer root sheath cells(3×10~5) subcultured from several(＜4 generation) were gradient centrifugated and added into 0.5ml fetal bovine serum.4 ml of collagen from mice tendon(1.0-1.5g/L) was taken to small penicilin ampoule in ice-bath and 0.5ml 10×DMEM culture medium was added into and was mixed immediately.0.1 ml 1N NaOH was added dropwise to it until it changed to a pink colour.The mixture was then inoculated with cells and put into 35-mm culture dishes in a 37℃incubator for gel formation to take place.Gels were maintained in the incubator until use.
5.Fluorescent reactive dye DiI labeled human dermal papilla cells and outer root sheath cells,epidermal cells from foreskin.Cells in 3- 4 generations were collected for the trial,washed with PBS and centrifugalized twice.Serum-free DMEM was added to make into cell suspension before 5μL DiI liquor at the density of 1×10~9/L was added,and then cells were incubated for 20 minutes at 37℃,1500 r/min centrifugalized for 5 minutes,washed with PBS and centrifugalized twice. DMEM medium containing 0.1 volume fraction FBS was added before coloration and morphologic change of cells were observed at hours 24,48 and 72 with fluorescent microscope.
6.Cells- scaffold complex formation.The morphology and function of the human dermal papilla cells and outer root sheath cells,epidermal cells from foreskin were observed by inverted phase contrast microscope,scanning electron microscope when cocultured typeⅠcollagen scaffold with cells in vitro.
7.Nude mice induced hair follicle reformation experimental.Mouse hair follicle cells in the collagen gel were transplanted in the subcutaneous tissue of dorsal part of the nude mice.After 4 weeks,The cultured tissue and local skin with cell transplantation were sectioned with routine paraffin,and stained by HE,and observed under optical microscope and fluorescence microscope.Cells- scaffold complex were transplanted in the subcutaneous tissue of dorsal part of the nude mice. Respectively,after 4,8,12 weeks,local skin with cell transplantation were sectioned with routine frozen sections and paraffin,and stained by HE,and observed under optical microscope and fluorescence microscope.
Results
1.Dermal papilla cells,dermal sheath cells and purified bulge-derived cells were isolated and cultured from the scalp hair follicles by using method of combination of microdissection and enzyme digestion,and other basic separation technology.Dermal papilla cells isolated by using enzyme digestion method. proliferated rapidly in vitro,the adhering rate was high and can reached 90%, Dermal papilla cells and dermal sheath cells grew in a aggregating way and were positive for immunohistochemical stain ofα-SMA.Bulge-derived cells cultured by tissue cultured method proliferated rapidly in vitro in the medium of K-SFM,like paving stones,and were positive for immunohistochemical stain of K-19.
2.Microdissection method succeeded in isolating dermal papilla cells of rat vibrissa hair follicless.Cells proliferated rapidly in vitro,they grew in a aggregating way.ORS cells isolated by combination of microdissection and enzyme digestion. Cells proliferated rapidly in vitro in the medium of K-SFM,like paving stones.
3.Epidermal basal cells from the foreskin isolated by using typeⅣcollagen attachment method grew in a typical cloning of growth way in vitro,and cells growth cycle is long.
4.Cells just inoculated in collagen gel are round shape,uniform distributed within the collagen gel.Afeter 24 h,cells extended,adhesion to scaffold.After 2 d, some cells extended and deformated in triangle-shap or fusiform-shape.
5.Fluorescent microscope showed all the cells was with red fluorescence after labeling and cells with plenty kytoplasm were fusiform shape or round shape, keeping good normal shape,DiI positive rate was 100%.In the early period the cells were fluorescent ring shape,and 48 hours later fluorescent granules increased and fluorescence enhanced.No fluorescence was found in cell nucleus.
6.Dermal papilla cells,bulge-derived cells and epidermal basal cells from foreskin inoculated in scaffold material and incubated for 2 h in sterile box.Cells began to attach on the scaffolds and gradually increased,after 2-3 days,cells in the material gradually extended processes,we can see that a large number of cells attached to the pore and the surrounding material,cells were polygonal, spindle-shape or round-shape,some cells attach to collagen sponge or pore walls, with cluster-like growth,after 3 d,cells were flat polygonal or round,extending in the material surface,after 1 week,cells were round or spindle shape,significantly increased,distributing in aggregated way,there was a large number of stromal surrounding the cells.
7.Nude mice induced experimental.There was no hair fiber formation in the dorsal skin of nude mice.paraffin sections were observed under an optical microscope.Follicle-like structures formation were seen in the cells collagen gel experimental group,cell - typeⅠcollagen scaffold protein complexes(dermal papilla cells and bulge-derived ceils ) experimental group and cell - typeⅠcollagen scaffold protein complexes(dermal papilla cells and foreskin epidermal cells ) experimental group in 4 week,8 week and 12 week.There was no follicle-like sturcture formation in blank control group.De novo hair follicle-like structures were with red fluorescence under fluorescence microscope in the experimental group of DiI labeled dermal papilla cells combined with bulge-derived cells and in the experimental group of DiI labeled dermal papilla cells combined with epidermal cells from foreskin.There was no red fluorescent display in unlabeled control group. Conclusion
1.Three cells derived from human hair follicle could be isolated and cultured from the same sample at the same time by using the method of combination of microdissection and enzyme digestion and other basic separation technology,and the sequencing of the isolated steps was improved,so the sample was to be maxmize used.Adhering rate and integration time of dermal papilla cells isolated by this method is no significant difference with that of dermal papilla cells isolated by enzyme digestion.The problems of immmunogenicity in experimental induction of hair follicle would be avoid because of all hair follicle cells derived from the same samples.
2.Epidermal cells from foreskin rapid isolated by using typeⅣcollagen attachment method grew in a typical cloning of growth way in vitro,and cells growth cycle is long.It show that epidermal cells from foreskin isolated by this method may contain more undifferertiated epidermal stem cells.
3.DiI positive rate was 100%.Cells in typeⅠcollagen scaffold cells were polygonal,spindle-shape or round-shape,in a gathering distuibution.There was a large number of stromal surrounding the cells.It shew that cells were able to attach, grow and proliferate well on the scaffolds.
4.Nude mice induced experimental tissue slices were observed under an optical microscope.The de nove follicle-like structures shew red fluorescent induced by the experimental group of DiI labeled human dermal papilla cells and bulge-derived cells and by the group of DiI labeled human dermal papilla cells and epidermal cells from foreskin,while the de nove follicle-like structures induced by the control group of DiI unlabelled cells were no red fluorescent display.This phenomenon shew that reformation of the de nove follicle-like structures were induced by cytoskeletal complexes
毛发具有重要的生理及美容价值,目前治疗毛发缺损性疾病特别是雄激素性秃发最为有效的方法是自体游离毛发移植,但存在供区不足、供区瘢痕、移植密度低等问题。而毛囊组织工程的研究有望为临床各种类型不可逆性秃发的治疗提供一种比现行毛发移植术更加简便易行、微创且不受来源限制的全新治疗手段。
毛囊是由上皮成分(包括毛母质、内根鞘和外根鞘)和真皮成分(包括毛乳头、真皮鞘)组成的。其中毛乳头细胞是构成毛乳头的主要间质细胞,它们是一簇特化的成纤维细胞。所有的毛乳头细胞在外观上都呈成纤维细胞样,而且体外培养的毛乳头细胞有着聚集性生长的特性。毛乳头在毛囊的形态学发生及周期调控中起着重要的作用。真皮鞘是围绕毛囊最外层的真皮源性组织,在毛囊的再生过程中,可以观察到真皮鞘细胞向毛乳头细胞的转化。有研究认为真皮鞘细胞是毛乳头细胞的储备细胞,毛乳头部位的真皮鞘细胞与毛乳头具有相同的诱导能力。毛囊隆突区(Bulge)与外根鞘相连续,并有研究证实毛囊Bulge区聚集了大量具有潜在增殖能力的早期幼稚细胞。被认为是毛囊干细胞的所在。已有研究证实,毛囊的形态学发生及周期性生长是毛囊上皮成分和真皮成分之间双相调节作用的结果。
另有研究认为表皮干细胞在人体不同部位的皮肤分布差异,以包皮和阴囊皮肤表皮最多。并有日本学者将鼠毛囊真皮成分细胞和人包皮表皮细胞组合,进行裸鼠体内毛囊诱导重建获得成功,并有肉眼可见的毛发纤维长出。但尚无报道用人毛囊真皮成分细胞与人包皮表皮基底细胞组合进行此实验。
Ⅰ型胶原蛋白是目前组织工程研究中最常用的天然生物材料,由多种糖蛋白分子组成,它具有多方面的生物活性,不仅是组织支持物,而且是细胞外间质的重要成分。研究表明,胶原蛋白生物相容性好,降解速度可调,而且可参与组织修复,是一类优良的可引导组织再生的生物材料。国内外许多毛囊生物学研究均用鼠尾胶原作为支架进行毛囊真皮细胞成分和表皮细胞成分组合器官型培养。但尚未报道采用用固体Ⅰ型胶原蛋白海绵作为支架进行毛囊种子细胞培养。
DiI是一种亲脂性碳花青染料,易嵌入生物质膜内作侧向扩散运动,从而标记整个细胞膜,还可以通过活体细胞的胞饮作用进入胞浆,标记整个细胞浆。以往研究显示,DiI对活体细胞无毒性,对被标记细胞的存活、生长无影响。并有研究报道DiI可作为荧光示踪剂用于培养的神经细胞。随后又有DiI标记肝细胞、骨髓基质干细胞、脂肪干细胞等报道,但迄今未见有应用于标记毛囊细胞。
研究目的
1.建立高效、快速分离培养毛乳头细胞、真皮鞘细胞和毛囊上皮细胞,包皮表皮基底细胞的方法。
2.选择适合于细胞生长的支架及细胞标记方法,建立毛囊细胞器官型培养的模型。
3.探索有效的动物体内毛囊重建的方法。
研究方法
1.人毛囊真皮成分和表皮成分细胞的分离、培养和鉴定。将人头皮标本剪成小块,中性蛋白酶中37℃孵育2 h后镜下将带外根鞘的毛干从真皮鞘中拔出,组织块法培养外根鞘隆起(Bulge)细胞;从真皮-皮下组织交界处横断头皮,拔出含毛乳头的真皮鞘,胶原酶D 37℃孵育6～8 h,多次低速离心,毛乳头沉淀重悬后培养,收集的真皮鞘细胞上清液经高速离心后重悬培养;真皮鞘和毛乳头的分离也可采用显微解剖法,依参照文献中的操作进行。观察其在体外培养的形态学和生物学特点,比较两种分离方法毛乳头的贴壁率,培养的Bulge细胞和毛乳头、真皮鞘细胞分别用K19或α-平滑肌动蛋白免疫组化鉴定。
2.鼠触须毛乳头细胞和鼠触须毛囊上皮细胞的分离、培养。采用显微解剖法进行大鼠触须毛乳头细胞分离、培养,大鼠麻醉后修剪颊侧被毛,常规消毒后剪去全层皮肤,暴露其下方的触须毛囊,然后用镊子将其完整拔出,用显微解剖法分离毛乳头。将分离的毛乳头置入含15%FCS的DMEM培养基,以适当密度接种(每个35 mm培养皿含10个毛乳头),观察其在体外培养的形态学和生物学特点。鼠触须毛囊上皮细胞的分离、培养:参照文献中的操作进行,无菌条件下取7日龄Wistar大鼠触须部皮肤,镜下将毛囊从组织中分离出来,置入0.125%Dispase消化液中,4℃消化2 h后,镜下轻压毛球部即可将毛囊外根鞘及毛干完整地分离出来。切去带外根鞘的毛干中下段和上段,保留Bulge区毛囊。将分离的Bulge区毛囊置入无血清的K-SFM培养基中培养。观察其在体外培养的形态学和生物学特点。
3.包皮表皮基底细胞的分离、培养。采用Ⅳ型胶原快速粘附法培养包皮表皮细胞。包皮标本用含双抗的D-Hanks液反复冲洗,室温下浸泡30 min,剔除皮下组织,将包皮剪成1.0 cm×0.5 cm大小皮条,置0.5%的中性蛋白酶中,4℃消化12～15 h,PBS冲洗2次,将表皮层撕下,剪成1mm×1mm大小皮片,置0.25%胰酶中,37℃消化5 min左右,中止消化,过200目筛网,计数,离心,重悬,接种于预先铺有Ⅳ型胶原的培养瓶中,室温下静置10 min,换液,吸附未贴壁的角质细胞。观察其在体外培养的形态学和生物学特点。
4.细胞胶原凝胶制备。取传代培养数次(＜4代)的鼠毛囊毛乳头细胞和外根鞘细胞分别为6×10~5和3×10~5(真皮细胞与上皮细胞比例为2:1)梯度离心后加入0.5 ml胎牛血清。吸4 ml鼠尾胶原(质量浓度为1.0～1.5ml)于冰浴中的青霉素小瓶中,加入0.5 ml浓缩10倍的DMEM培养基,1N NaOH调Ph,立即加入含鼠毛囊毛乳头细胞和外根鞘细胞的胎牛血清细胞悬液,快速混合,制成细胞胶原凝胶,然后吸到35 mm培养皿中。静置于37℃、体积分数为0.05的CO_2孵箱培养。
5.用DiI标记人毛囊毛乳头细胞和外根鞘细胞,包皮表皮基底细胞。将收集细胞用PBS清洗离心2次,加入无血清的DMEM制成细胞悬液,以1×10~9/L密度加入5μL DiI应用液,37℃下孵育25 min,1500 r/min离心5 min,PBS清洗离心2次,加入10%DMEM培养基中,荧光显微镜观察细胞显色情况,置于37℃、体积分数为0.05的CO_2孵箱培养,观察24,48,72 h细胞荧光强度及形态变化。
6.细胞—支架复合物的形成。将Ⅰ型胶原蛋白海绵支架材料裁制成10 mm×10mm×10 mm大小,利用紫外线照射及酒精浸泡消毒,培养液平衡后待用。DiI标记的人毛乳头细胞和外根鞘细胞、包皮表皮基底细胞消化后制成细胞悬液,分两组(毛乳头细胞+外根鞘细胞组、毛乳头细胞+包皮表皮基底细胞组,真皮成分细胞和表皮成分细胞数比例为2:1),细胞密度为2×10~5/ml,表面种植1 ml细胞悬液,静置4 h后,加入含20%胎牛血清的DMEM培养液,37℃、5%CO_2培养箱中培养。采用倒置相差显微镜和扫描电镜,观察细胞在支架上黏附、生长情况。
7.裸鼠体内诱导实验。将鼠毛囊细胞胶原凝胶在无菌条件下注射移植到裸鼠背部两侧的皮下。于4周切下移植部位皮肤,石蜡切片行H-E染色光学显微镜下观察;人细胞—支架复合物在无菌条件下注射移植到裸鼠背部两侧的皮下,分别于6、8、12周切下移植部位皮肤,冰冻切片和石蜡切片荧光显微镜下观察,并进行H-E染色光学显微镜下观察。
研究结果
1.结合运用显微法和酶消化法等基本分离技术,同时从头皮毛囊标本中分离培养毛囊三种细胞成分,获得了较纯化的外根鞘Bulge细胞,真皮鞘细胞和毛乳头细胞。酶消化法获得的毛乳头贴壁率高,可达到90%,毛乳头和真皮鞘细胞体外培养表现出明显的凝集性生长,α-SMA免疫组化染色呈现阳性。组织块法培养的头皮毛囊外根鞘Bulge细胞,可在K-SFM培养基中生长良好,呈铺路石样。角蛋白19免疫组化染色阳性。
2.采用显微解剖成功分离大鼠触须毛乳头细胞,细胞体外培养表现出明显的凝集性生长。采用酶消化法和显微解剖法成功分离到大鼠触须毛囊上皮细胞,可在K-SFM培养基中生长良好,呈铺路石样。
3.采用Ⅳ型胶原快速粘附法培养的包皮表皮基底细胞外培养过程中呈典型的克隆性生长,生长周期长。
4.刚接种于胶原凝胶的细胞呈圆形,在胶原凝胶内均匀分布。24 h内细胞伸展,粘附,2 d后部分细胞已伸展变形,以三角形和梭形为主。
5.DiI标记的人毛乳头细胞、外根鞘细胞和包皮表皮基底细胞荧光显微镜下观察,见全部细胞标记后均显红色荧光,标记的细胞呈梭形或圆形,胞浆丰富,保持了良好的正常形态,DiI标记阳性率为100%。DiI标记后早期细胞形态呈荧光环状,48 h后细胞中荧光颗粒增多,荧光增强,细胞核未染荧光。
6.细胞—支架复合物体外倒置显微镜下和扫描电镜观察,将毛乳头细胞、毛囊外根鞘细胞和包皮表皮基底细胞在材料上接种并在孵箱孵育2 h后,材料周边及孔板底部开始有细胞黏附,随着时间的延长,附着于材料上的细胞逐渐增多;培养2-3天后,细胞在材料上逐渐伸出突起,可见大量细胞附着于材料的孔隙及材料周边,胶原蛋白材料上皆有细胞附着,细胞呈梭形圆形或多角形,部分材料上的细胞呈团簇状生长,粘附于胶原海绵壁上或孔隙内,各细胞或细胞团之间通过基质相连或重叠。细胞种植3 d后,细胞呈扁平多角形或圆形,在材料表面伸展、黏附,种植1周后,形态为梭形或圆形,细胞数量明显增多,呈聚集性分布,细胞周围有大量基质。
7.裸鼠体内诱导实验。裸鼠背侧皮肤观察未见有肉眼可见的毛发纤维生成。石蜡切片光学显微镜下观察发现鼠细胞胶原凝胶诱导实验组和人细胞-Ⅰ型胶原蛋白支架复合物毛乳头细胞与毛囊上皮Bulge细胞组合、毛乳头细胞与包皮表皮基底细胞组合诱导实验中4周、8周、12周均可见新生毛囊样结构形成。空白支架对照组无毛囊样结构生成。石蜡切片荧光显微镜下观察DiI标记的人毛乳头细胞、外根鞘Bulge细胞组合和DiI标记的人毛乳头细胞、包皮表皮细胞组合实验组新生毛囊样结构显红色荧光。未用DiI标记的实验对照组新生毛囊样结构无红色荧光显示。
研究结论
1.结合运用显微解剖法和酶消化法等基本分离技术,避免了单独运用显微法和酶消化法的缺点,并在分离步骤和次序上进行改进,首次同时从头皮毛囊标本中分离培养毛囊三种细胞成分,最大限度的利用了标本量,毛囊毛乳头因为经过胶原酶的处理,其贴壁与融合时间与文献报道的两步酶法无明显差别。因毛囊各种细胞成分来源于同一标本,进行细胞组合诱导等毛囊试验时避免了毛囊各细胞因来源不同而在毛囊组织工程中组合应用可能面临的免疫原性问题。
2.经Ⅳ型胶原快速粘附法多次筛选的包皮表皮细胞外培养过程中呈典型的克隆性生长,生长周期长。说明用这种方法培养筛选的包皮表皮基底细胞可能含有较多的未分化表皮干细胞。
3.DiI标记的人毛乳头细胞、外根鞘细胞和包皮表皮基底细胞标记阳性率为100%。细胞在Ⅰ型胶原蛋白支架上形态为梭形或圆形,呈聚集性分布,细胞周围有大量基质,表明细胞在支架上粘附、伸展和生长良好。
4.裸鼠体内诱导实验组织切片观察DiI标记的人毛乳头细胞、外根鞘Bulge细胞组合和DiI标记的人毛乳头细胞、包皮表皮基底细胞组合实验组新生毛囊样结构显红色荧光。未用DiI标记的实验对照组新生毛囊样结构无红色荧光显示。证明了新生的毛囊是由细胞支架复合物诱导生成。
Background
Hair plays an important role in biological necessity and performs a variety of beauty function.At present,autograft of hair follicles is the most important treatment for hair loss,especially for male pattern baldness.But there are some problems for this treatment,such as formation of scar in donor site,scantiness of donor site,low density of transplantations,so hair follicle tissue engineering provides a new minimally invasive treatment not restricted by the source for repairing hair loss.
A hair follicle is composed of epithelial cells including matrix,out root sheath, inner root sheath and dermal cells including dermal papilla,dermal sheath cells. Dermal papilla cells are a kind of fibroblast cells,which can aggregate during growing in mediums containing FCS.According to the different phages of hair cycle, the shape of the dermal papilla change periodically.Dermal papilla plays an important role in the morphogenesis of hair follicle and hair cycle.Dermal sheath around the hair fllicle of the outermost layer are the dermis derived tissue,dermal sheath cells can be observed transforming to dermal papilla cells in the hair follicle regeneration process,It has been demonstrated that dermal sheath cells are the reserves cells of dermal papilla cells and the dermal sheath cells neighbor dermal papilla have the same capacity induction as dermal papilla cells.The human hair follicle bulge connectted with outer root sheath is an important niche for keratinocyte stem cells.It is widely accepted that a highly coordinated series of bidirectional epithelial-mesenchymal interactions(EMIs) is essential to hair follicle morphogenesis.
Several lines of evidence have suggested that foreskin and scrotum epidermal is an important niche for keratinocyte stem cells.Although Japanese scholars has demonstrated that hair follicle would be reconstructed and hair fibers visible to the naked eye grow induced by mouse hair follicles dermis cells and human foreskin epidermal cells portfolio when transplanted into nude mice dorsal.There is no reported that human hair follicle cells and human dermis foreskin epidermal cells portfolio have been used for the experimental replication.
Type I collagen scaffold composed of various glycoprotein molecules has been the most widely used in tissue engineering experiment in the present.Type I collagen scaffold,which is a kind of important component mesenchymal outside of cells,has many biological activity as an organizational support.It has been demonstrated that collagen could be participated in tissue repair as a kind of excellent biological materials because of its cellular compatibility and adjustable speed degradation.Rat tail collagen has been widely used in hair follicle biology studies as a scaffold for hair follicle cells in the dermis and epidermis cells composition organotypic culture. However,there has been no report that solid typeⅠcollagen could be used as a kind of scaffold for hair follicle seed cells culture in hair follicle engineering.
DiI is a lipophilic carbon anthocyanin dye easily embedded within a biological membrane by lateral diffusion movement,so DiI can effectively label cytomembrane, and DiI can effectively label cytoplasm by living cells' pinocytosis.It has been demonstrated that DiI labeled cells have good morphology and DiI is no toxicity for living cells.There are some research reports show that DiI can be used as a fluorescent tracer for cultured nerve cells,then come DiI label of hepatocyte,bone marrow stromal stem cells and adipose derived stem cells,but so far there has been no labeled for cells derived from human hair follices.
Objective
1.To establish the method for isolating dermal papilla cells,dermal sheath cells, follicular epithelial cells and foreskin epidermal basal cells rapidly and efficiently.
2.To explore appropriate scaffold materials for hair follicle tissue engineering, to establish the model for hair follicle cells organotypic culture.
3.To explore the method of reconstructing hair follicles.
Methods
1.The isolation,culture and identification of dermis and epidermis cells derived from human hair follicle.Small scalp specimens were incubated in the presence of dispase at 37℃for 2 h,the hair shafts with ORS embedded in the dermal sheath (DS) were extracted under dissecting microscope,and the ORS tissue were inoculated onto Petri dish.The specimens were transected at the interface between the dermis and subcutaneous tissue.The portions of DS and DP(linked with and enclosed by DS) embedded in the adipose tissue were pulled out and incubated with collagenase at 37℃for 6-8 h,and the DP and DSCs were isolated by repeated low-speed centrifugation and cultured respectively on petri dishes.The cultured ORS bulge cells were identified by immunohistochemistry with K19 antibody and DPCs and DSCs by immunohistochemistry withα-actin antibody.
2.The separation and culture of dermal papilla cells and epithelial cells of rat vibrissa follicle.Dermal papilla cells of rate vibrissa hair follicles were isolated by microdissecting method,and cultured in DMED medium containing 15%FCS,the bioligical behavior in vitro was observed.Epithelial cells of rate vibrissa hair follicles were isolated by microdissecting and enzyme digesting method,and cultured in K-SFM medium,Dermal papillas were removed from the dermal sheathes under the microscope.The bioligical behavior in vitro was observed.
3.The separation and culture of epidermal basal cells from the foreskin. Epidermal cells from the human foreskin were isolated by typeⅣcollagen rapid adhesion method,and cultured in K-SFM medium,the bioligical behavior in vitro was observed.
4.Cell collagen gels preparation,mouse hair follicle dermal papilla cells(6×10~5) and outer root sheath cells(3×10~5) subcultured from several(＜4 generation) were gradient centrifugated and added into 0.5ml fetal bovine serum.4 ml of collagen from mice tendon(1.0-1.5g/L) was taken to small penicilin ampoule in ice-bath and 0.5ml 10×DMEM culture medium was added into and was mixed immediately.0.1 ml 1N NaOH was added dropwise to it until it changed to a pink colour.The mixture was then inoculated with cells and put into 35-mm culture dishes in a 37℃incubator for gel formation to take place.Gels were maintained in the incubator until use.
5.Fluorescent reactive dye DiI labeled human dermal papilla cells and outer root sheath cells,epidermal cells from foreskin.Cells in 3- 4 generations were collected for the trial,washed with PBS and centrifugalized twice.Serum-free DMEM was added to make into cell suspension before 5μL DiI liquor at the density of 1×10~9/L was added,and then cells were incubated for 20 minutes at 37℃,1500 r/min centrifugalized for 5 minutes,washed with PBS and centrifugalized twice. DMEM medium containing 0.1 volume fraction FBS was added before coloration and morphologic change of cells were observed at hours 24,48 and 72 with fluorescent microscope.
6.Cells- scaffold complex formation.The morphology and function of the human dermal papilla cells and outer root sheath cells,epidermal cells from foreskin were observed by inverted phase contrast microscope,scanning electron microscope when cocultured typeⅠcollagen scaffold with cells in vitro.
7.Nude mice induced hair follicle reformation experimental.Mouse hair follicle cells in the collagen gel were transplanted in the subcutaneous tissue of dorsal part of the nude mice.After 4 weeks,The cultured tissue and local skin with cell transplantation were sectioned with routine paraffin,and stained by HE,and observed under optical microscope and fluorescence microscope.Cells- scaffold complex were transplanted in the subcutaneous tissue of dorsal part of the nude mice. Respectively,after 4,8,12 weeks,local skin with cell transplantation were sectioned with routine frozen sections and paraffin,and stained by HE,and observed under optical microscope and fluorescence microscope.
Results
1.Dermal papilla cells,dermal sheath cells and purified bulge-derived cells were isolated and cultured from the scalp hair follicles by using method of combination of microdissection and enzyme digestion,and other basic separation technology.Dermal papilla cells isolated by using enzyme digestion method. proliferated rapidly in vitro,the adhering rate was high and can reached 90%, Dermal papilla cells and dermal sheath cells grew in a aggregating way and were positive for immunohistochemical stain ofα-SMA.Bulge-derived cells cultured by tissue cultured method proliferated rapidly in vitro in the medium of K-SFM,like paving stones,and were positive for immunohistochemical stain of K-19.
2.Microdissection method succeeded in isolating dermal papilla cells of rat vibrissa hair follicless.Cells proliferated rapidly in vitro,they grew in a aggregating way.ORS cells isolated by combination of microdissection and enzyme digestion. Cells proliferated rapidly in vitro in the medium of K-SFM,like paving stones.
3.Epidermal basal cells from the foreskin isolated by using typeⅣcollagen attachment method grew in a typical cloning of growth way in vitro,and cells growth cycle is long.
4.Cells just inoculated in collagen gel are round shape,uniform distributed within the collagen gel.Afeter 24 h,cells extended,adhesion to scaffold.After 2 d, some cells extended and deformated in triangle-shap or fusiform-shape.
5.Fluorescent microscope showed all the cells was with red fluorescence after labeling and cells with plenty kytoplasm were fusiform shape or round shape, keeping good normal shape,DiI positive rate was 100%.In the early period the cells were fluorescent ring shape,and 48 hours later fluorescent granules increased and fluorescence enhanced.No fluorescence was found in cell nucleus.
6.Dermal papilla cells,bulge-derived cells and epidermal basal cells from foreskin inoculated in scaffold material and incubated for 2 h in sterile box.Cells began to attach on the scaffolds and gradually increased,after 2-3 days,cells in the material gradually extended processes,we can see that a large number of cells attached to the pore and the surrounding material,cells were polygonal, spindle-shape or round-shape,some cells attach to collagen sponge or pore walls, with cluster-like growth,after 3 d,cells were flat polygonal or round,extending in the material surface,after 1 week,cells were round or spindle shape,significantly increased,distributing in aggregated way,there was a large number of stromal surrounding the cells.
7.Nude mice induced experimental.There was no hair fiber formation in the dorsal skin of nude mice.paraffin sections were observed under an optical microscope.Follicle-like structures formation were seen in the cells collagen gel experimental group,cell - typeⅠcollagen scaffold protein complexes(dermal papilla cells and bulge-derived ceils ) experimental group and cell - typeⅠcollagen scaffold protein complexes(dermal papilla cells and foreskin epidermal cells ) experimental group in 4 week,8 week and 12 week.There was no follicle-like sturcture formation in blank control group.De novo hair follicle-like structures were with red fluorescence under fluorescence microscope in the experimental group of DiI labeled dermal papilla cells combined with bulge-derived cells and in the experimental group of DiI labeled dermal papilla cells combined with epidermal cells from foreskin.There was no red fluorescent display in unlabeled control group. Conclusion
1.Three cells derived from human hair follicle could be isolated and cultured from the same sample at the same time by using the method of combination of microdissection and enzyme digestion and other basic separation technology,and the sequencing of the isolated steps was improved,so the sample was to be maxmize used.Adhering rate and integration time of dermal papilla cells isolated by this method is no significant difference with that of dermal papilla cells isolated by enzyme digestion.The problems of immmunogenicity in experimental induction of hair follicle would be avoid because of all hair follicle cells derived from the same samples.
2.Epidermal cells from foreskin rapid isolated by using typeⅣcollagen attachment method grew in a typical cloning of growth way in vitro,and cells growth cycle is long.It show that epidermal cells from foreskin isolated by this method may contain more undifferertiated epidermal stem cells.
3.DiI positive rate was 100%.Cells in typeⅠcollagen scaffold cells were polygonal,spindle-shape or round-shape,in a gathering distuibution.There was a large number of stromal surrounding the cells.It shew that cells were able to attach, grow and proliferate well on the scaffolds.
4.Nude mice induced experimental tissue slices were observed under an optical microscope.The de nove follicle-like structures shew red fluorescent induced by the experimental group of DiI labeled human dermal papilla cells and bulge-derived cells and by the group of DiI labeled human dermal papilla cells and epidermal cells from foreskin,while the de nove follicle-like structures induced by the control group of DiI unlabelled cells were no red fluorescent display.This phenomenon shew that reformation of the de nove follicle-like structures were induced by cytoskeletal complexes
引文
[1]程波,刘荣卿,伍津津等.改良毛乳头细胞培养法[J].中国皮肤科杂志,2001,34(5):387-388
[2]Magerl M,Kauser S,Paus R,et al.Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles[J].Experi Dermat,2002,11(4):381-385
[3]符刚,高强国,杨恬等.无血清K-SFM培养条件下大鼠毛囊Bulge细胞生物学特性的研究[J].第三军医大学学报,2005,27(15):1538-1540
[4]曾元临,辛国华,邱泽亮等.应用Ⅳ型胶原黏附法进行人表皮干细胞的体外快速分离与鉴定[J].中国组织工程研究与临床康复,2007,11(20):3861-3863
[5]Stenn K S,Paus R.Controls of hair follicle cycling[J].Physiol-Rev,2001,81(1):449-94
[6]Messenger G.The control of hair growth:An overview[J].J Invest Dermatol,1993,101(1 Supp 1):4s-9s.
[7]Reynolds AJ,Lawrence C,Cserhalmi-Friendman PB,et al.Trans-gender induction of hair follicles.[J].Nature.1999.402(1):33-34
[8]唐建兵,陈君,杨光成等.角蛋白在头皮中的分布及其在干细胞研究中的意义[J].中国美容医学,2005,14(4):392-393.
[9]Oshima H,Rochat A,Kedzia C,et al.Morphogenesis and renewal of hair follicles from adult multipotent stem cells[J].Cell,2001,104(2):233-45.
[10]Manabu Ohyama,Terunuma,Characterization and isolation of stem cell-enriched human hair follicle bulge cells.J Clin Invest.2006,116:249-260
[11]唐建兵,陈璧,汤朝武,等.人毛囊外根鞘细胞的培养[J].中华烧伤杂志 2003,19(1):47-49.
[12]张艺,杨恬,王韵,等.成人毛囊隆起细胞生长特性的初步观察[J].解剖学报,2005,36(3):269-71.
[13]Messenger AG.The culture of dermal papilla cells from human hair follicles.[J]Br J dermatol 1984,110:685-9.
[14]伍津津,刘荣卿,叶庆佾,等.毛乳头细胞的高效快捷培养方法[J].第三军医大学学报,1998,20(3):208-10.
[15]朱堂友,郝飞,伍津津,等.构建人头皮毛乳头细胞和真皮鞘细胞快捷高效分离培养的方法[J].中国临床康复,2005,9(18):72-3.
[1]Gharzi A,Reynolds AJ,Jahoda CA.Plasticity of hair follicle dermal cells in wound healing and induction.Exp Dermatol.2003 Apr;12(2):126-36.
[2]张云松,高建华,鲁峰,等.Ⅰ型胶原支架材料与人脂肪干细胞体外生物相容性研究[J].南方医科大学学报,2007,27(2):223-225.
[3]Noah EM,Chen JS,Jiao XY,et al.Impact of sterilization on the porous design and cekk behavior in collagen sponges prepared for tissue engineering[J].Biomaterials,2002,23:2855-61
[4]Berglund JD,MohseniMM,Nerem RM,Nerem RM,et al.A biological hybrid model for collagen 2 bassed tissue engineered vascular constructs[J]Biomaterials,2003,24:1241-54.
[5]王玉丽.胶原在生物医学中的应用[J].国外医学:药学分册,2002.29(2):113.
[6]吕中法,蔡绥勃,伍津津,等.培养毛乳头细胞生物学特性和毛囊重建的研究[J].中华皮肤科杂志,2006,39(2):64-67.
[7]韩明子,邹亚男,赵宪琪,等.Dil荧光示踪剂在小鼠骨髓细胞肝内移植中的研究[J].中华器官移植杂志.2003,24(4):251.
[8]Honig MG,Hume RI.Fluorescent carbocyanine dyes allow living neurons of identified origin to be s tudied in long-term cultures.J Cell Biol 1986; 103(1):171-187
[9] Soriano HE, Lewis D, Legner M, et al. The use of Dil-marked hepatocytes to demons trate orthotopic, intrahepatic engraftment following hepatocellular transplantation. Transplantation 1992; 54 (4) :717-723.
[1]Jahoda C A B,Home,K.A.R.F.Induction of hair growth by implantation of cultured dermal papilla cells[J].Nature,1984,311:560-562.
[2]Jahoda CA.Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds:vibrissa-type fibres are specified.Development,1992,115:110321109.
[3]Jahoda C A B,Reynolds A J.Dermal-epidermal interactions-follicle-derived cell populations in the study of hair growth mechanisms[J].J Invest Derm atol,1993,101(3):33-37
[4]Watson S A 1,Pisansarakit P,Moore G P M.Sheep vibrissa dermal papilla induce hair follicle formation in heterotypic equivalents[J].Br J Derm atol,1994,131(7):827-831
[5]Reynolds AJ,Jahoda CA.Hair follicle reconstruction in vitro[J].Dermatol Sci,1994,9(7):S84-97
[6]Reynolds AJ,Lawrence,C,Cserhalmi-Friedman,Trans-gender induction of hair follicles.[J].Nature,1999,402:33-34.
[7]Gharzi A,Reynolds A J,Jahoda CAB.Plasticity of hair follicle dermal cells in R wound healing and induction.[J],Experimental Dermatology 2003,Volume 12 Page 126.
[8]Ehama R,Ishimatsu-Tsuji Y,Iriyama S.Hair follicle regeneration using grafted rodent and human cells.[J]Invest Dermatol.2007,127(9):2106-15.
[9]Osada A,Iwabuchi T,Kishimoto J.Long-term culture of mouse vibrissal dermal papilla cells and de novo hair follicle induction.[J].Tissue Eng.2007,13(5):975-82.
[10]伍津津,刘荣卿.毛乳头细胞诱导毛囊形成的研究[J].中国修复重建外科志,2003,17(2):108-111.
[11]吕中法,蔡绥勃,伍津津.培养毛乳头细胞生物学特性和毛囊重建的研究[J].中华皮肤科杂志,2006,39(2):64-67.
[12]李宇,林常敏.微囊法体外重建人头皮毛乳头诱导毛囊再生功能的评价[J].中国临床康复,2005,9(6):46-48.
[1]钟白玉,吕中法,刘荣卿,等.毛囊真皮鞘成纤维细胞的培养[J].第三军医大学报,1999,21(12):935-937.
[2]Katsuoka K,Schell H,Homstein OP,et al.Comparative morphological and growth kinetics studies of human hair bulb papilla cells and root sheath fibroblasts in vitro[J].Arch Dermtol Res,1986,279(1):20-25.
[3]Jahoda CA,Oliver R,Reynolds A J,et al.Human hair follicle regeneration following amputation and grafting into the nude mouse.Invest Der matol,1996,107(6):804-807.
[4]Chiu HC,Chang CH,Chen JS,et al.Human hair follicle dermal papilla cell,dermal sheath cell and interstitial dermal fibroblast characteristics.Formos.MedAssoc,1996,95(9):667-674.
[5]Gharzi A,Reynolds A J,Jahoda CAB.Plasticity of hair follicle dermal cells inwound healing and induction.Experimental Dermatology,2003,12:126-136
[6]Jahoda CA,Whitehouse J,Reynolds AJ,et al.Hair follicle dermal cells differentiate into adipogenic and osteogeniclineages.Exp Dermatol.2003,12(6):849-59.
[7]Robinson M,Reynolds AJ,Gharzi A,et al.In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture.J Invest Dermatol.2001 Sep;117(3):596-604.
[8]唐建兵,陈璧.人毛囊外根鞘细胞的培养[J].中华烧伤杂志2003;19(1):47-49
[9]Yang JS,Lavker RM,Sun TT.Upper human hair follicle contains a subpopulation of keratinocytes with superior in vitro proliferative potential[J].J Invest Dermatol,1993,101:652-659
[10]Limat A,Mauri D, Hunziker T,Successful treatment of chronic logulcers with epidermal equivalents generated from cultured autologous out root sheath cells[J].J Invest Dermatol, 1996,107:128-315.
[11] Limat A,Salomon D,Carraux P,et al.Human melanocytes grown in epidermal equivalents transfer their melamin to follicular outer root sheath keratinocytes[J].Arch Dermatol Res,19991291:325-332.
[12]Jeschke MG,Richeter W,Ruf SG.Cultured autologous outer root sheath cells:a new therapeutic alternative for chronic decubitus ulcers[J].Plast Reconstr Surg,2001,107(7):1803-1806.
[13] Limat A,Hunziker T.Use ofepidernal equivalents generated from follicular outer root sheath cells in vitro and for autologous grafting of chronic wounds[J].Cells Tissues Organs,2002,172(2):79-85.
[14] Cotsarelis G, Sun TT, Lavker RM. Label-retaining cells residue in the bulge area of pilosebaceous unit:implications for follicular stem cells,hair cycle and skin carcinogenesis.Cell,1990,61(2):1329-1337.
[15] Taylor G, Lshrer NlS,Jensen PJ,et al. Involvement of follicular stem cells informing not only the follicle but also the epidermis .Cell,2000,102(3):451-461.
[16] Mayumi Ito,Kenji Kizawa ,Kazuto Hamada ,et al.Hair follicle stem cells in the lower bulge form the secondary germ,a biochemically distinct but functionally equivalent progenitor cell population at the termination of catagen.Differentiation, 2004,72(8):548-557.
[17] Rebecca J Morris,Yaping Liu,Lee Marles,et al.Capturing and profiling adult hair follicle stem cells.Nature Biotechnology,2004,22(4):411-417.
[18]Cedric Blanpain, William E.Lowry, Andrea Geoghegan,et al. Self-renewal, multipotency,and the existence of two cell populations within an epithelial stem cell niche.Cell,2004,118(3):635-648.
[19]Sieber-Blum M,Grim M,Hu YF,Szeder V.Pluripotent neural crest stem cells in the adult hair follicle.Dev Dyn 2004;231(2):258-69.
[20]Sieber-Blum M,Grim M.The adult hair follicle:cradle for pluripotent neural crest stem cells.Birth Defects Res C Embryo Today 2004;72(2):162-72.
[21]Manabu Ohyama,Terunuma,Characterization and isolation of stem cell-enriched human hair follicle bulge cells.The Journal of Clinical Investigation.2006;116:249-260.
[22]STENN K S,PAUS R.Controls of hair follicle cycling[J].Physiological eviews,2001,81(1):449-494.
[23]Jahoda C A B,Reynolds A J.Dermal-epidermal interactions-follicle-derived cell populations in the study of hair growth mechanisms.J Invest Derm atol,1993;101:33.
[24]Jahoda CA.Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds:vibrissa2 type fibres are specified.Development,1992,115:110321109.
[25]Watson S A 1,Pisansarakit P,M oore G P M.Sheep vibrissa dermal papilla induce hair follicle formation in heterotypic equivalents.Br J Derm atol,1994;131:827.
[26]Reynolds AJ,Jahoda CA.Hair follicle reconstruction in vitro.J Dermatol Sci.1994.7:S84-97.
[27]伍津津,刘荣卿.毛乳头细胞诱导毛囊形成的研究[J].中国修复重建外科杂志;2003;17(2):108-111.
[28]刘荣卿,钟白玉.裸鼠重建毛囊的组织学研究[J].中华皮肤科杂志;2000;33(4):232-23.
[29]李宇,林常敏.微囊法体外重建人头皮毛乳头诱导毛囊再生功能的评价[J].中国临床康复2005,9(06):46-48.
[30]Gharzi A,Reynolds AJ,Jahoda CAB.Plasticity of hair follicle dermal cells in wound healing and induction.[J].Experimental Dermatology 2003,12:126.
[31]Limat A,Mauri D,Hunziker T,Successful treatment of chronic logulcers with epidermal equivalents generated from cultured autologous outer sheath cells,J Invest Dermatol,1996,107:128-315.
[32]Limat A,Salomon D,Carraux P,et al.Human melanocytes grown in epidermal equivalents transfer their melanin to follicular outer sheath keratinocytes[J].Arch Dermatol Res,1999,291:325-332.
[33]Gharzi A,Reynolds AJ,Jahoda CAB.Plasticity of hair follicle dermal cells in wound healing and induction.[J].Experimental Dermatology 2003,12:126.
[2]Magerl M,Kauser S,Paus R,et al.Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles[J].Experi Dermat,2002,11(4):381-385
[3]符刚,高强国,杨恬等.无血清K-SFM培养条件下大鼠毛囊Bulge细胞生物学特性的研究[J].第三军医大学学报,2005,27(15):1538-1540
[4]曾元临,辛国华,邱泽亮等.应用Ⅳ型胶原黏附法进行人表皮干细胞的体外快速分离与鉴定[J].中国组织工程研究与临床康复,2007,11(20):3861-3863
[5]Stenn K S,Paus R.Controls of hair follicle cycling[J].Physiol-Rev,2001,81(1):449-94
[6]Messenger G.The control of hair growth:An overview[J].J Invest Dermatol,1993,101(1 Supp 1):4s-9s.
[7]Reynolds AJ,Lawrence C,Cserhalmi-Friendman PB,et al.Trans-gender induction of hair follicles.[J].Nature.1999.402(1):33-34
[8]唐建兵,陈君,杨光成等.角蛋白在头皮中的分布及其在干细胞研究中的意义[J].中国美容医学,2005,14(4):392-393.
[9]Oshima H,Rochat A,Kedzia C,et al.Morphogenesis and renewal of hair follicles from adult multipotent stem cells[J].Cell,2001,104(2):233-45.
[10]Manabu Ohyama,Terunuma,Characterization and isolation of stem cell-enriched human hair follicle bulge cells.J Clin Invest.2006,116:249-260
[11]唐建兵,陈璧,汤朝武,等.人毛囊外根鞘细胞的培养[J].中华烧伤杂志 2003,19(1):47-49.
[12]张艺,杨恬,王韵,等.成人毛囊隆起细胞生长特性的初步观察[J].解剖学报,2005,36(3):269-71.
[13]Messenger AG.The culture of dermal papilla cells from human hair follicles.[J]Br J dermatol 1984,110:685-9.
[14]伍津津,刘荣卿,叶庆佾,等.毛乳头细胞的高效快捷培养方法[J].第三军医大学学报,1998,20(3):208-10.
[15]朱堂友,郝飞,伍津津,等.构建人头皮毛乳头细胞和真皮鞘细胞快捷高效分离培养的方法[J].中国临床康复,2005,9(18):72-3.
[1]Gharzi A,Reynolds AJ,Jahoda CA.Plasticity of hair follicle dermal cells in wound healing and induction.Exp Dermatol.2003 Apr;12(2):126-36.
[2]张云松,高建华,鲁峰,等.Ⅰ型胶原支架材料与人脂肪干细胞体外生物相容性研究[J].南方医科大学学报,2007,27(2):223-225.
[3]Noah EM,Chen JS,Jiao XY,et al.Impact of sterilization on the porous design and cekk behavior in collagen sponges prepared for tissue engineering[J].Biomaterials,2002,23:2855-61
[4]Berglund JD,MohseniMM,Nerem RM,Nerem RM,et al.A biological hybrid model for collagen 2 bassed tissue engineered vascular constructs[J]Biomaterials,2003,24:1241-54.
[5]王玉丽.胶原在生物医学中的应用[J].国外医学:药学分册,2002.29(2):113.
[6]吕中法,蔡绥勃,伍津津,等.培养毛乳头细胞生物学特性和毛囊重建的研究[J].中华皮肤科杂志,2006,39(2):64-67.
[7]韩明子,邹亚男,赵宪琪,等.Dil荧光示踪剂在小鼠骨髓细胞肝内移植中的研究[J].中华器官移植杂志.2003,24(4):251.
[8]Honig MG,Hume RI.Fluorescent carbocyanine dyes allow living neurons of identified origin to be s tudied in long-term cultures.J Cell Biol 1986; 103(1):171-187
[9] Soriano HE, Lewis D, Legner M, et al. The use of Dil-marked hepatocytes to demons trate orthotopic, intrahepatic engraftment following hepatocellular transplantation. Transplantation 1992; 54 (4) :717-723.
[1]Jahoda C A B,Home,K.A.R.F.Induction of hair growth by implantation of cultured dermal papilla cells[J].Nature,1984,311:560-562.
[2]Jahoda CA.Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds:vibrissa-type fibres are specified.Development,1992,115:110321109.
[3]Jahoda C A B,Reynolds A J.Dermal-epidermal interactions-follicle-derived cell populations in the study of hair growth mechanisms[J].J Invest Derm atol,1993,101(3):33-37
[4]Watson S A 1,Pisansarakit P,Moore G P M.Sheep vibrissa dermal papilla induce hair follicle formation in heterotypic equivalents[J].Br J Derm atol,1994,131(7):827-831
[5]Reynolds AJ,Jahoda CA.Hair follicle reconstruction in vitro[J].Dermatol Sci,1994,9(7):S84-97
[6]Reynolds AJ,Lawrence,C,Cserhalmi-Friedman,Trans-gender induction of hair follicles.[J].Nature,1999,402:33-34.
[7]Gharzi A,Reynolds A J,Jahoda CAB.Plasticity of hair follicle dermal cells in R wound healing and induction.[J],Experimental Dermatology 2003,Volume 12 Page 126.
[8]Ehama R,Ishimatsu-Tsuji Y,Iriyama S.Hair follicle regeneration using grafted rodent and human cells.[J]Invest Dermatol.2007,127(9):2106-15.
[9]Osada A,Iwabuchi T,Kishimoto J.Long-term culture of mouse vibrissal dermal papilla cells and de novo hair follicle induction.[J].Tissue Eng.2007,13(5):975-82.
[10]伍津津,刘荣卿.毛乳头细胞诱导毛囊形成的研究[J].中国修复重建外科志,2003,17(2):108-111.
[11]吕中法,蔡绥勃,伍津津.培养毛乳头细胞生物学特性和毛囊重建的研究[J].中华皮肤科杂志,2006,39(2):64-67.
[12]李宇,林常敏.微囊法体外重建人头皮毛乳头诱导毛囊再生功能的评价[J].中国临床康复,2005,9(6):46-48.
[1]钟白玉,吕中法,刘荣卿,等.毛囊真皮鞘成纤维细胞的培养[J].第三军医大学报,1999,21(12):935-937.
[2]Katsuoka K,Schell H,Homstein OP,et al.Comparative morphological and growth kinetics studies of human hair bulb papilla cells and root sheath fibroblasts in vitro[J].Arch Dermtol Res,1986,279(1):20-25.
[3]Jahoda CA,Oliver R,Reynolds A J,et al.Human hair follicle regeneration following amputation and grafting into the nude mouse.Invest Der matol,1996,107(6):804-807.
[4]Chiu HC,Chang CH,Chen JS,et al.Human hair follicle dermal papilla cell,dermal sheath cell and interstitial dermal fibroblast characteristics.Formos.MedAssoc,1996,95(9):667-674.
[5]Gharzi A,Reynolds A J,Jahoda CAB.Plasticity of hair follicle dermal cells inwound healing and induction.Experimental Dermatology,2003,12:126-136
[6]Jahoda CA,Whitehouse J,Reynolds AJ,et al.Hair follicle dermal cells differentiate into adipogenic and osteogeniclineages.Exp Dermatol.2003,12(6):849-59.
[7]Robinson M,Reynolds AJ,Gharzi A,et al.In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture.J Invest Dermatol.2001 Sep;117(3):596-604.
[8]唐建兵,陈璧.人毛囊外根鞘细胞的培养[J].中华烧伤杂志2003;19(1):47-49
[9]Yang JS,Lavker RM,Sun TT.Upper human hair follicle contains a subpopulation of keratinocytes with superior in vitro proliferative potential[J].J Invest Dermatol,1993,101:652-659
[10]Limat A,Mauri D, Hunziker T,Successful treatment of chronic logulcers with epidermal equivalents generated from cultured autologous out root sheath cells[J].J Invest Dermatol, 1996,107:128-315.
[11] Limat A,Salomon D,Carraux P,et al.Human melanocytes grown in epidermal equivalents transfer their melamin to follicular outer root sheath keratinocytes[J].Arch Dermatol Res,19991291:325-332.
[12]Jeschke MG,Richeter W,Ruf SG.Cultured autologous outer root sheath cells:a new therapeutic alternative for chronic decubitus ulcers[J].Plast Reconstr Surg,2001,107(7):1803-1806.
[13] Limat A,Hunziker T.Use ofepidernal equivalents generated from follicular outer root sheath cells in vitro and for autologous grafting of chronic wounds[J].Cells Tissues Organs,2002,172(2):79-85.
[14] Cotsarelis G, Sun TT, Lavker RM. Label-retaining cells residue in the bulge area of pilosebaceous unit:implications for follicular stem cells,hair cycle and skin carcinogenesis.Cell,1990,61(2):1329-1337.
[15] Taylor G, Lshrer NlS,Jensen PJ,et al. Involvement of follicular stem cells informing not only the follicle but also the epidermis .Cell,2000,102(3):451-461.
[16] Mayumi Ito,Kenji Kizawa ,Kazuto Hamada ,et al.Hair follicle stem cells in the lower bulge form the secondary germ,a biochemically distinct but functionally equivalent progenitor cell population at the termination of catagen.Differentiation, 2004,72(8):548-557.
[17] Rebecca J Morris,Yaping Liu,Lee Marles,et al.Capturing and profiling adult hair follicle stem cells.Nature Biotechnology,2004,22(4):411-417.
[18]Cedric Blanpain, William E.Lowry, Andrea Geoghegan,et al. Self-renewal, multipotency,and the existence of two cell populations within an epithelial stem cell niche.Cell,2004,118(3):635-648.
[19]Sieber-Blum M,Grim M,Hu YF,Szeder V.Pluripotent neural crest stem cells in the adult hair follicle.Dev Dyn 2004;231(2):258-69.
[20]Sieber-Blum M,Grim M.The adult hair follicle:cradle for pluripotent neural crest stem cells.Birth Defects Res C Embryo Today 2004;72(2):162-72.
[21]Manabu Ohyama,Terunuma,Characterization and isolation of stem cell-enriched human hair follicle bulge cells.The Journal of Clinical Investigation.2006;116:249-260.
[22]STENN K S,PAUS R.Controls of hair follicle cycling[J].Physiological eviews,2001,81(1):449-494.
[23]Jahoda C A B,Reynolds A J.Dermal-epidermal interactions-follicle-derived cell populations in the study of hair growth mechanisms.J Invest Derm atol,1993;101:33.
[24]Jahoda CA.Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds:vibrissa2 type fibres are specified.Development,1992,115:110321109.
[25]Watson S A 1,Pisansarakit P,M oore G P M.Sheep vibrissa dermal papilla induce hair follicle formation in heterotypic equivalents.Br J Derm atol,1994;131:827.
[26]Reynolds AJ,Jahoda CA.Hair follicle reconstruction in vitro.J Dermatol Sci.1994.7:S84-97.
[27]伍津津,刘荣卿.毛乳头细胞诱导毛囊形成的研究[J].中国修复重建外科杂志;2003;17(2):108-111.
[28]刘荣卿,钟白玉.裸鼠重建毛囊的组织学研究[J].中华皮肤科杂志;2000;33(4):232-23.
[29]李宇,林常敏.微囊法体外重建人头皮毛乳头诱导毛囊再生功能的评价[J].中国临床康复2005,9(06):46-48.
[30]Gharzi A,Reynolds AJ,Jahoda CAB.Plasticity of hair follicle dermal cells in wound healing and induction.[J].Experimental Dermatology 2003,12:126.
[31]Limat A,Mauri D,Hunziker T,Successful treatment of chronic logulcers with epidermal equivalents generated from cultured autologous outer sheath cells,J Invest Dermatol,1996,107:128-315.
[32]Limat A,Salomon D,Carraux P,et al.Human melanocytes grown in epidermal equivalents transfer their melanin to follicular outer sheath keratinocytes[J].Arch Dermatol Res,1999,291:325-332.
[33]Gharzi A,Reynolds AJ,Jahoda CAB.Plasticity of hair follicle dermal cells in wound healing and induction.[J].Experimental Dermatology 2003,12:126.