芦荟苦素对黑素细胞与角质形成细胞混合培养模型中黑素合成的影响

目的：体外构建黑素细胞与角质形成细胞直接接触的混合培养模型，观察芦荟苦素、熊果苷及茶多酚对此模型中黑素细胞的影响。方法：以包皮组织作为细胞来源，分别培养角质形成细胞、黑素细胞；体外构建黑素细胞与角质形成细胞直接接触的混合培养模型，检测芦荟苦素、熊果苷及茶多酚作用于此模型后对黑素细胞酪氨酸酶活性以及黑素合成的影响。结果：黑素细胞与角质形成细胞混合培养5天后出现浅灰色，继续培养，颜色逐渐加深。三种退色剂对酪氨酸酶活性及黑素的合成均呈浓度依赖性抑制。结论：成功构建了黑素细胞与角质形成细胞直接接触的混合培养模型，三种退色剂均对黑素细胞酪氨酸酶活性及黑素合成产生浓度依赖性抑制，以茶多酚作用最强，芦荟苦素次之。     

构建含黑色素细胞组织工程表皮的实验研究

目的将黑色素细胞和表皮角质形成细胞，接种于壳聚糖-明胶薄膜材料上，构建含黑色素细胞的组织工程表皮。方法从小儿包皮分离黑色素细胞和表皮角质形成细胞，分别对黑色素细胞和表皮角质形成细胞进行鉴定，并进行不同代次细胞生长动力学检测。将黑色素细胞和表皮角质形成细胞接种于壳聚糖-明胶薄膜上，进一步检测两种细胞在材料上的生长情况。结果分离培养黑色素细胞和表皮角质形成细胞，经鉴定两者分别表达S-100和角蛋白。接种于壳聚糖-明胶薄膜上的黑色素细胞和表皮角质形成细胞能成功构建组织工程化表皮替代物。两种细胞在材料上生长状态良好，分布均匀，并表达各自特异性标志物。结论以壳聚糖-明胶薄膜材料作为支架材料，成体黑色素细胞和表皮角质形成细胞为种子细胞，能有效地构建含黑色素细胞的组织工程表皮。     

应用胶原凝胶构建组织工程化皮肤的研究

目的探讨以胶原凝胶为支架材料构建组织工程化皮肤的可行性。方法体外分离、培养人皮肤表皮细胞和成纤维细胞；利用自制的胶原蛋白制成胶原凝胶作为组织工程支架材料；在成功构建人工真皮的基础上种植表皮细胞，构建复合人工皮肤；采用HE染色与免疫组织化学的方法对复合人工皮肤进行组织学检测。结果HE染色可见，构建的复合人工皮肤具有表皮和真皮双层结构；免疫组织化学染色显示，Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性，在形态结构上与正常皮肤相似。结论培养的人表皮细胞和成纤维细胞种植于胶原凝胶支架上，气一液界面培养可构建出具有类似正常皮肤结构的组织工程化皮肤。     

人表皮干细胞的体外分离培养和鉴定

目的:探讨人表皮干细胞的体外快速分离培养及鉴定方法。方法:中性蛋白酶和胰蛋白酶两步法从手术切除的人包皮组织中分离表皮层和真皮层,并获得表皮单细胞悬液,采用Ⅳ型胶原铺板选择性粘附、分离和角质形成细胞无血清培养基(K-SFM)培养表皮干细胞。倒置显微镜下观察培养细胞的生长状况,检测细胞克隆形成率,免疫组化染色观察表皮干细胞标志物β1整合素和角蛋白19(K19)的表达;以角质形成细胞作为对照。结果:组织学观察显示,培养24h后细胞呈克隆状生长;所分离、培养细胞的克隆形成率高于对照角质形成细胞组;免疫组化染色显示,培养细胞β1整合素及Kl9均呈阳性表达。结论:运用Ⅳ型胶原粘附结合K-SFM培养可以实现人表皮干细胞的体外快速分离和培养。     

角质形成细胞在脱细胞异种真皮上培养的实验研究

目的 在脱细胞异种(猪)真皮上培养角质形成细胞探讨体外复合皮的构建。方法 取出生24h内的SD大鼠全厚皮肤，采用低温酶消化法和密度梯度离心法分离获得纯角质形成细胞；以不用任何滋养层的角质形成细胞培养作为空白对照组，脱细胞异种真皮作为支架的角质形成细胞培养为实验组，原代培养后行气——液面培养。形态学，常规组织学HE染色观察，免疫组化染色(SABC法)检测复合皮肤中的Pancyrtokeratin和层粘连蛋白(Laminin)。结果 HE染色显示有4层以上的角质形成细胞和基底膜层形成，并有轻度角质化；免疫组化染色显示：Pancytokeratin( )，提示在脱细胞猪真皮上生长的为角质形成细胞；Laminin( )，提示培养的角质形成细胞产生了新的基底膜。结论 体外培养的角质形成细胞能在脱细胞异种真皮上良好生长、存活，并有基底膜形成，在体外成功构建了具有表皮和真皮的复合皮，可以作为一种新的组织工程化皮肤。     

干细胞构建组织工程皮肤的研究进展

组织工程学是近年来细胞生物学、工程材料学和临床医学交叉发展起来的一门新兴学科。组织工程学最基本的思路是在体外将分离的种子细胞接种到具有一定空间结构的载体支架上,通过细胞与细胞之间的相互作用形成具有一定结构功能的组织或器官。从组织工程学的观点来看,人工皮肤主要有三类：①表皮替代物,即培养表皮片;②真皮替代物,即胶原凝胶、胶原海绵合成膜、     

以胶原海绵为载体培养的人表皮细胞移植

目的：将体外培养的人表皮细胞接种到胶原海绵上,构建一种表皮替代物,移植到鼠创面后研究观察皮肤的再生,方法：将手术切下的包皮经中性蛋白酶分离表皮,真皮,再经胰蛋白酶消化制成表皮细胞悬液,移入培养瓶中培养,将处于对数生长期的人表皮细胞直接接种到培养皿中的胶原海绵 ,再加上表皮细胞液培养3天后,移植到裸鼠全层皮肤缺损的创面上,以单纯无接种细胞的胶原海绵作为对照,并进行组织学,免疫组织化学及电子显微镜观察。结果：这种表皮替代物移植到创面后,表皮细胞继续增殖分化,形成一层新生表皮,与对照组相比,创面闭合早且收缩程度小,表皮成熟早且分层较多,基底膜形成较早,表皮下胶原纤维较少。结论：体外培养的表皮细胞能在胶原海绵上生长,移植到创面上后,该细胞可自动移行到创面并形成多层表皮结构,抑制创面收缩,可用于修复皮肤缺损。     

人胎儿表皮干细胞的体外分离培养及基因转染

目的：探讨人胎儿表皮干细胞体外分离培养的方法以及作为体外基因转染靶细胞的可行性。方法：利用Ⅳ型胶原快速贴附法分离人胎儿表皮干细胞，以人胎儿成纤维细胞条件培养液配制表皮干细胞培养基，通过角蛋白19（K19）和整合素β1免疫组化染色、细胞周期分析及克隆形成率测定，对培养细胞进行鉴定。采用脂质体介导法，以含血管内皮细胞生长因子165（VEFG165）基因片段的真核表达载体pcDNA3.1(pcDNA3.1/VEGF165)转染培养细胞；采用病毒载体介导法，以含报告基因绿色荧光蛋白（GFP）的重组腺相关病毒载体（raav/GFP)转染培养细胞。应用免疫组化染色及荧光显微镜观察检测转染效果。结果：人胎儿表皮干细胞呈明显克隆性生长、克隆形成率高，G1期细胞比例明显高于普通基底层角质细胞，K19和整合素β1免疫组化染色呈强阳性。pcDNA3.1/VEGF165转染的表皮干细胞VEGF165免疫组化染色阳性，raav/GFP转染的表皮干细胞呈现强荧光。结论：利用Ⅳ型胶原快速贴附法及人胎儿成纤维细胞条件培养基，可初步实现人胎儿表皮干细胞的分离培养。以质体为介导或以腺相关病毒为载体进行人胎儿表皮干细胞的体外基因转染是可行的。     

抗角蛋白自身抗体在黑素细胞无血清纯化培养中的应用

目的：探索抗角蛋白自身抗体(AK auto Ab)在黑素细胞纯化培养中的应用，寻找黑素细胞无血清纯化培养的安全而简易的方法。方法：按常规方法制备表皮细胞悬液，应用角质形成细胞无血清培养基(KC-SFM)中加入AK auto hb(用于替代TPA)、bFGF和BPE培养细胞，48h后换液一次，以后则用仅含bFGF和BPE的KC—SFM换液培养。结果：AK auto Ab对角质形成细胞有选择性杀伤作用，用含AK auto hb、bFGF和BPE的KC—SFM进行黑素细胞培养时原代即可获得纯化的黑素细胞。结论：AK auto hb可取代TPA用于黑素细胞无血清纯培养，方法可靠、简单。因AK auto hb是正常人血清中提取的物质，用其培养的黑素细胞在临床移植应用中将更具安全性。     

应用胶原凝胶构建组织工程化皮肤的研究

目的探讨以胶原凝胶为支架材料构建组织工程化皮肤的可行性。方法体外分离、培养人皮肤表皮细胞和成纤维细胞;利用自制的胶原蛋白制成胶原凝胶作为组织工程支架材料;在成功构建人工真皮的基础上种植表皮细胞,构建复合人工皮肤;采用HE染色与免疫组织化学的方法对复合人工皮肤进行组织学检测。结果HE染色可见,构建的复合人工皮肤具有表皮和真皮双层结构;免疫组织化学染色显示,Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性,在形态结构上与正常皮肤相似。结论培养的人表皮细胞和成纤维细胞种植于胶原凝胶支架上,气-液界面培养可构建出具有类似正常皮肤结构的组织工程化皮肤。     

人源性表皮高色素纯黑素细胞的培养及其生物学特性

目的:探讨如何获得人源性表皮高色素性纯黑素细胞,并鉴定其生物学特性。方法:以包皮组织作为细胞来源,消化法获得表皮细胞悬液,用两种不同的培养基培养细胞,参照文献并结合本实验室设计的方法对细胞进行纯化,Dopa染色和S-100染色鉴定细胞来源,酪氨酸酶活性、生长曲线及MTT法观察细胞生长状态。结果:获得的黑素细胞色素颗粒较多,未见成纤维细胞和角质形成细胞污染,生物学特性观察显示:高色素性黑素细胞具有贴壁时间长、高酪氨酸酶活性和生长增殖慢的特点。结论:获得了大量人源性表皮高色素性纯黑素细胞。     

Development of a tissue-engineered human oral mucosa equivalent based on an acellular allogeneic dermal matrix: A preliminary report of clinical application to burn wounds

Tissue-engineered skin equivalents composed of epidermal and dermal components have been widely investigated for coverage of full-thickness skin defects. We developed a tissue-engineered oral mucosa equivalent based on an acellular allogeneic dermal matrix and investigated its characteristics. We also tried and assessed its preliminary clinical application. Human oral mucosal keratinocytes were separated from a piece of oral mucosa and cultured in a chemically-defined medium. The keratinocytes were seeded on to the acellular allogeneic dermal matrix and cultured. Histologically, the mucosa equivalent had a well-stratified epithelial layer. Immunohistochemical study showed that it was similar to normal oral mucosa. We applied this equivalent in one case with an extensive burn wound. The equivalent was transplanted three weeks after the harvest of the patient's oral mucosa and about 30% of the graft finally survived. We conclude that this new oral mucosa equivalent could become a therapeutic option for the treatment of extensive burns.     

Development of a tissue-engineered human oral mucosa equivalent based on an acellular allogeneic dermal matrix: a preliminary report of clinical application to burn wounds.

Tissue-engineered skin equivalents composed of epidermal and dermal components have been widely investigated for coverage of full-thickness skin defects. We developed a tissue-engineered oral mucosa equivalent based on an acellular allogeneic dermal matrix and investigated its characteristics. We also tried and assessed its preliminary clinical application. Human oral mucosal keratinocytes were separated from a piece of oral mucosa and cultured in a chemically-defined medium. The keratinocytes were seeded on to the acellular allogeneic dermal matrix and cultured. Histologically, the mucosa equivalent had a well-stratified epithelial layer. Immunohistochemical study showed that it was similar to normal oral mucosa. We applied this equivalent in one case with an extensive burn wound. The equivalent was transplanted three weeks after the harvest of the patient's oral mucosa and about 30% of the graft finally survived. We conclude that this new oral mucosa equivalent could become a therapeutic option for the treatment of extensive burns.     

构建含黑色素细胞组织工程皮肤的研究

目的 探讨运用组织工程方法构建含有黑色素细胞的组织工程皮肤。方法 以包皮组织作为细胞来源，采用消化法获得角朊细胞、成纤维细胞和黑色素细胞，在自行设计的组织工程皮肤培养系统构建组织工程皮肤。行Dopa染色、透射电镜和S-100免疫组织化学检测构建的皮肤中黑色素细胞的分布和状态。结果 构建的组织工程皮肤结构完整，细胞状态良好，Dopa染色、透射电镜和S-100免疫组织化学检测均显示含有大量黑色素细胞并处于良好状态。结论 构建了含黑色素细胞的组织工程皮肤，可用于下一步的动物实验和临床实验。     

Evaluation of Permacol as a cultured skin equivalent

Skin loss following severe burn requires prompt wound closure to avoid such complications as fluid and electrolyte imbalance, infection, immune suppression, and pain. In clinical situations in which insufficient donor skin is available, the development of cultured skin equivalents (dermal matrices seeded with keratinocytes and fibroblasts) may provide a useful alternative. The aim of this study was to assess the suitability of a porcine-derived dermal collagen matrix (Permacol™) to function as a cultured skin equivalent in supporting the growth of keratinocytes in vitro and providing cover to full thickness wounds in the BALB C/nude mouse model. A histological comparison was against Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (Gly-EO Dermis) which has successfully been used as a cultured skin equivalent in previous studies. Both Gly-EO Dermis and to a lesser extent Permacol™ were able to support the growth of cultured keratinocytes following a 16-day period of cell culture, however, this study was only able to demonstrate the presence of an epidermal layer on Gly-EO dermis 2 weeks after grafting onto full-thickness wounds in the BALB C/nude mouse model.     

全厚皮片移植后表皮黑素细胞形态学变化

目的 研究黑素细胞（MC）形态与功能的关系及其对全厚皮片移植后色泽变化的可能影响。方法 黑白花豚鼠18只，随机分为9组，每组2只，分别于皮片移植术前（对照组）及术后1、2、3、7、14、21、28、90d取材。使用表皮铺片及多巴反应技术，显示不同时间点皮片表皮MC；然后借助电子计算机辅助图像分析系统对MC的形态学变化进行观察及量化分析。结果 皮片移植术后3d内，手术损伤及缺血缺氧导致MC突起脱落，MC总面积、突起面积及突起数目减少；术后2～3周时各项指标恢复至术前水平。实验末期，特征性的表现是MC突起粗而短。结论 全厚皮片内MC的形态学变化与其功能变化基本一致；与MC胞体形态及面积变化相比较，其突起的数目、面积变化能更敏感地反映细胞的功能状态；与移植前正常状态下MC细而长的突起相比，术后1～3个月MC短而粗的突起适应了皮片移植后期较小的表皮黑素单位（EMU）的需要，更有利于黑素在MC突起内的转运过程。     

Human De-epidermized Dermis as a Stem Cell Carrier

Recently several types of skin equivalents have been developed, consisting of differentiated keratinocytes cultured on various dermal substitutes. Different models of reconstructed human skin have been proposed, such as human and animal de-epidermized dermis, inert filters, collagen matrices, lyophilized collagen membranes populated with fibroblasts, and other models populated with melanocytes and/or Langerhans cells. These skin equivalents mimic native skin in vivo. They have provided information about dermal-epidermal interactions, cell-cell, and cell-matrix interactions; responses of dermal and epithelial cells to biological signals and pharmacological agents; as well as effects of drugs and growth factors on wound healing. Human allodermis from tissue banks has been used for clinical purposes, namely, as support for autologous keratinocyte cultures and as a potentially ideal scaffold for dermal replacement. This bioproduct is considered to be the most suitable clinical carrier for autologous fibroblasts and keratinocytes, as well as an useful experimental model to study angiogenesis and to stimulate vascularization in reconstructed human skin. Because it is human-derived, it is in our opinion the safest of all available types of skin equivalent. Having epidermal and dermal structures, it can be used in one-stage grafting procedures for wound closure.     

Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice

Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patient's self-image. To address these morbidities, human fibroblasts were inoculated on a collagen-glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 x 10(6)/cm2 combined with either 0, 1.1 x 10(2), 1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm2. The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L-Dopa-positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL-5. The skin substitutes with 1.1 x 10(4) melanocytes/cm2 were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 x 10(3) melanocytes/cm2 group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm2 group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 x 10(3) and 1.1 x 10(4) melanocytes/cm2. These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 x 10(4) melanocytes/cm2 to skin substitutes.     

Tissue engineered artificial skin composed of dermis and epidermis

Abstract: We made an artificial skin comprised of a stratified layer of keratinocytes and a dermal matrix with a type I collagen containing fibroblasts. In this work, we showed keratinocyte behavior under primary culture, gel contractions varying with concentration of collagen solution, and cell growth plots in the collagen gel. The optimum behavior of dermal equivalent could be obtained using 3.0 mg/ml collagen solution and attached gel culture. The attached gel culture had a jumping effect of growth factor on cell growth at the lag phase. To develop the artificial skin, 1× 10⁵ cells/cm² of keratinocytes were cultured on the dermal equivalent at air-liquid interface. Finally, to overcome the problem that artificial skin of collagen gel was torn easily during suturing of grafting, we prepared histocompatible collagen mesh and attached the mesh to the bottom of the gel. Cultured artificial skins were successfully grafted onto rats.