应用表皮干细胞与去表皮的真皮体外构建组织工程皮肤

目的 用人皮肤表皮干细胞 （ESCs）接种至同种去表皮的真皮上，体外构建组织工程皮肤。方法 用两步酶消化法分离获得角质形成细胞，并用Ⅳ型胶原快速粘附法筛选ESCs并培养；进行细胞形态学观察和用免疫组化法对ESCs进行鉴定；将ESCs接种至去表皮的真皮上浸没培养7 d后转成气-液面培养，4周后取培养的组织工程皮肤切片分别做HE染色和角蛋白的免疫组化染色。结果 培养的ESCs呈铺路石样、克隆状生长；角蛋白K19和β1-整合素的免疫组化染色阳性。培养5周的组织工程皮肤可见有3 ～ 6层细胞，并可见角质层，表皮角蛋白染色阳性。结论 应用人皮肤的ESCs与去表皮的真皮可以体外构建组织工程皮肤。     

构建生长因子缓释功能的复合型组织工程皮肤替代物

目的设计制备具有缓释表皮生长因子(EGF)功能的复合型组织工程皮肤替代物并进行相关性能检测。方法在组织工程脱细胞真皮的研究基础上,利用天然材料制备凝胶状类似表皮结构的生物膜作为覆盖,并加载表皮生长因子缓释系统,构建具有促表皮愈合功能的复合型组织工程皮肤替代物。并对此皮肤替代物的形态学、体外释放性能以及实际疗效做以考察。结果缓释EGF的复合型组织工程皮肤替代物具有良好的形态学性能,生物相容性好,EGF在体外的释放可达14d以上。动物实验表明实验组创面愈合率在术后各时间点均高于对照组(P<0.05);组织学观察显示愈合创面的表皮愈合程度明显优于对照组。结论具有EGF缓释功能的复合型组织工程皮肤替代物能有效促进猪全层皮肤缺损的创面愈合,可作为具有应用前景的覆盖材料进一步研究。     

组织工程皮肤自体及异体移植后表皮分化及重建的动物实验研究

目的研究猪组织工程皮肤移植后表皮层角蛋白分化标志的变化情况,为组织工程皮肤临床应用提供实验依据。方法按本科已建立的方法构建猪的组织工程皮肤,进行自体及同种异体移植实验,并于移植后1周、4周、8周取标本,行组织学检查和免疫组化染色,观察移植后组织工程皮肤表皮层角蛋白的表达、表皮细胞分化和重建情况。结果猪组织工程皮肤自体及同种异体移植后均存活,创面达到理想的愈合效果;组织学检查显示表皮分化良好,表皮层广谱角蛋白抗体免疫组化染色阳性;基底层以上细胞角蛋白K10抗体免疫组化染色阳性;基底层细胞角蛋白K5抗体免疫组化染色阳性。结论构建的猪组织工程皮肤自体及同种异体移植后,可获得理想的创面愈合效果,表皮分化和再构建良好,与正常皮肤相似。本实验为组织工程皮肤临床应用提供了有力的实验依据。     

长波紫外线诱导人组织工程皮肤的表皮基底层突变北大核心CSCD

白人中皮肤癌是最为常见的肿瘤.在动物模型中,中波紫外线（UVB）可诱导基因突变、免疫抑制,进而诱发皮肤癌.由于UVB曾一直被认为是最重要的光致癌因素,因此化妆品行业最初研发的防晒剂主要是针对UVB,忽略了对UVA的有效防护.     

角质形成细胞与黑素细胞体外构建含黑素的组织工程皮肤

目的 探讨体外构建含黑素的组织工程皮肤.方法 采用两步酶消化法处理健康小儿包皮,获得表皮细胞悬液,分别用人角质形成细胞(KC)无血清培养基(SFM)及改良的M254黑素细胞培养基培养KC、黑素细胞(MC),并传至第3代.将第3代KC接种于培养瓶中,48 h后加入第3代MC(MC:KC为1:10)混合培养.制备人去表皮的真皮(DED),将培养第3代的MC、KC制成细胞悬液,按照1:10的比例接种于DED表面,采用液下培养和空气-液面培养相结合的方式进行培养,2周后取培养的组织工程皮肤分别做HE染色、角蛋白免疫组化染色及Masson-Fontana染色.结果 KC、MC混合培养于培养皿5 d后,于倒置显微镜下观察到KC呈铺路石状生长,其间散布MC,且树突延伸到KC细胞间.两种细胞混合接种于DED 15 d后,HE染色显示在DED上有3～6层表皮细胞,并可见角质层,角蛋白免疫组化染色阳性,银氨染色显示基底层见黑素着色.结论 MC、KC混合接种于培养皿可构建MC和KC接触生长的单细胞层,将MC、KC接种于DED可构建含有黑素成分的组织工程皮肤.

Abstract：

Objective To construct tissue-engineered skin containing melanin with mixed culture of human keratinocytes (KCs) and melanocytes (MCs) on de-epidermized dermis (DED) in vitro. Methods Single-cell suspension was obtained by digestion of isolated preputial epidermis with pancreatin. Keratinocyte serum-free medium (K-SFM) and modified M254 culture medium were used to culture KCs and MCs respectively. Third-passage KCs were seeded into cell culture flasks and cultured for 48 hours; then, third-passage MCs were seeded into the same cell culture flasks with the MC/KC ratio being 1: 10 followed by a 5-day coculture. The suspension of third-passage KCs and MCs with the MC/KC ratio of 10:1 were seeded onto the surface of a prepared DED and maintained at the air-liquid interface for 11 days following a 4-day submerged culture.Subsequently, the constructed tissue-engineered skin was examined with HE staining, immunohistochemical staining for keratin and Masson-Fontana staining. Results After coculture in flasks for 5 days, KCs exhibited a typical paving-stone appearance, MCs with projected dendrites were scattered in the extracellular space between KCs. HE staining revealed 3 to 6 layers of cells with the formation of stratum corneum after mixed culture on DED for 15 days. Keratin protein was positive throughout the artificial epidermis, and melanin pigments were located in the basal layer of the epidermis as Masson-Fontana staining showed. Conclusions The co-culture of MCs and KCs can form single-cell layers with the contact between MCs and KCs in flasks, and construct tissue-engineered skin with melanin component on DED in vitro.     

SD大鼠组织工程皮肤构建及移植的实验研究

目的：构建SD大鼠全层组织工程皮肤,观察其在修复SD大鼠创面中的作用。方法：将分离、培养的SD乳鼠角质形成细胞和成纤维细胞种植于以牛I型胶原所构建的胶原海绵真皮支架两侧,构建出组织工程皮肤。分别观察自体皮肤、油纱布、组织工程皮肤覆盖SD大鼠皮肤缺损的愈合情况。第7、10、15d取创面皮肤标本行HE染色,组织工程皮肤再行纤维连接蛋白（fibronectin,FN）免疫组化染色。结果：构建的SD大鼠全层组织工程皮肤,移植于大鼠创面后,无出血、感染、坏死等不良反应,愈后良好。FN的表达呈现先升高后降低的规律性变化。结论：动物实验模型表明,以胶原海绵为真皮支架的组织工程皮肤是一种良好的皮肤替代物,可用于修复动物全层皮肤缺损。     

不同类型胶原蛋白构建的组织工程皮肤收缩情况研究

目的研究不同类型及不同浓度胶原蛋白构建的组织工程皮肤随着培养时间的增加皮肤收缩的情况。方法分别采用不同浓度牛肌腱胶原蛋白和鼠尾胶原蛋白构建组织工程皮肤,测量组织工程皮肤的收缩度,采用Wilcoxon Signed Ranks Test和Friedman Test对结果进行分析。结果牛肌腱胶原蛋白构建的组织工程皮肤收缩度要小于鼠尾胶原蛋白构建的组织工程皮肤(P<0.01);不同浓度胶原蛋白构建的组织工程皮肤其收缩度也不相同(P<0.01)。组间比较差异有显著性(P<0.01)。结论牛肌腱胶原蛋白构建的组织工程皮肤在保持其形态稳定性上显著优于鼠尾胶原蛋白构建的组织工程皮肤。牛肌腱胶原构建的组织工程皮肤,胶原浓度越高其形态稳定性越好。     

组织工程全层皮肤的研究现状

组织工程全层皮肤在一定程度上克服了原有的皮肤供区不足、二次表皮移植、免疫排斥、传播疾病等问题,在临床应用中已经取得了一定的疗效,但移植成功率低,也不具备完整的皮肤结构.目前国内外学者研究的热点集中在构建含正常附属结构的组织工程全层皮肤上.组织工程全层皮肤是目前较为理想的皮肤替代物,具有远大的开发前景.     

人源性组织工程皮肤白念珠菌感染模型的构建

【摘要】 目的 利用人皮肤来源的细胞体外构建含色素组织工程皮肤白念珠菌感染模型。方法　利用人皮肤原代培养成纤维细胞、角质形成细胞、黑素细胞,成纤维细胞与胶原孵育3 d后得到组织工程真皮,在其表面接种角质形成细胞和黑素细胞培养12 d,获得组织工程皮肤,再在其表面滴加1个麦氏浓度的白念珠菌悬液5 μl,分别在6、12、24、48和72 h进行HE染色和PAS染色观察。结果　利用成纤维细胞、角质形成细胞、黑素细胞及胶原构建的组织工程皮肤结构良好,表皮分化层次分明。HE染色和PAS染色显示,感染白念珠菌后,随着时间的延长,白念珠菌对表皮结构的破坏逐渐加重,菌丝由浅表向真皮逐渐侵入。感染至72 h,表皮结构完全破坏,真皮内充满大量菌丝。结论　利用人皮肤来源的细胞,培养出的含色素组织工程皮肤层次结构分明;初步建立了人皮肤来源的组织工程皮肤念珠菌感染模型。 【关键词】 组织工程; 皮肤,人工; 念珠菌,白色; 模型,生物学     

复合骨髓间充质干细胞的组织工程皮肤治疗慢性难愈性溃疡探讨

目的探讨复合骨髓间充质干细胞(MSCs)的组织工程(TE)皮肤在修复皮肤缺损中的疗效,为临床治疗类似疾病提供新方法。方法利用组织工程学方法,以牛胶原为支架,自体MSCs为种子细胞,构建TE皮肤。对1例双下肢溃疡15年的患者进行移植治疗,观察疗效。结果移植后1周溃疡边缘变平,健康的肉芽组织形成,2周后皮岛扩大,边缘形成了较薄的上皮组织。4周后溃疡面积缩小40%,9周后溃疡面积缩小90%。结论构建的复合骨髓间充质干细胞的组织工程真皮移植后,提高了愈合速度,获得了较理想的创面愈合效果。为临床治疗类似溃疡型疾病提供了新的治疗思路。     

高脂饮食对小鼠皮肤创面修复影响的实验研究

目的 观察长期高脂饮食对小鼠皮肤创面愈合的影响并初步探讨其相关机制。 方法 10周龄C57BL/6J野生型小鼠16只,随机分为2组,每组8只,分别采用高脂及普通膳食喂养8周后,在小鼠背部建立全层皮肤缺损模型。每日对创面愈合状况进行观察,记录创面愈合率及上皮化水平。于术后第14天处死所有小鼠并切取创面组织进行组织学检测,比较两组创面新生表皮厚度、创面床内胶原沉积率、新生血管数目、细胞增殖及炎症细胞浸润水平。喂养前、喂养8周后及手术后第14天检测体重。术后第14天禁食12 h后检测外周血总胆固醇（TC）、三酰甘油（TG）水平。采用t检验进行统计学分析。 结果 高脂膳食组在高脂喂养8周后及手术后第14天平均体重［分别为（27.3 ± 0.7） g和（28.8 ± 0.7） g］显著高于普通膳食组［分别为（21.2 ± 0.6） g和（23.1 ± 1.1） g］,两组比较,t值分别为21.98和25.22,均P < 0.001。术后第14天,高脂膳食组TG及TC水平［分别为（1.35 ± 0.32） mmol/L和（4.21 ± 0.41） mmol/L］远高于普通膳食组［分别为（0.99 ± 0.28） mmol/L和（2.71 ± 0.31） mmol/L, 两组比较,t值分别为2.24和6.49,均P < 0.05］;高脂膳食组创面愈合时间明显慢于普通膳食组［分别为（13.5 ± 0.5） d和（12.6 ± 1.1） d,t = 1.99,P < 0.05］,创面新生表皮薄于普通膳食组［分别为（47.8 ± 13.8） μm 和（95.7 ± 13.7） μm,t = 5.68,P < 0.001］,CD31阳性血管数低于普通膳食组［分别为（8 ± 1）个和（13 ± 3）个,t = 4.1,P < 0.001］,ki-67阳性细胞数目低于普通膳食组［分别为（21 ± 4）个和（49 ± 10）个,t = 3.33,P < 0.001］,但巨噬细胞及肥大细胞浸润水平明显高于普通膳食组（均P < 0.05）,各组创面床内胶原沉积率差异无统计学意义（P > 0.05）。 结论 长期高脂饮食会影响小鼠创面的愈合,延缓皮肤创面修复。     

Wound healing of human skin transplanted onto the nude mouse after a superficial excisional injury: human dermal reconstruction is achieved in several steps by two different fibroblast subpopulations

Abstract It has been established that human skin grafted onto the nude mouse is able to regenerate after being subjected to a full-thickness wound. In the present work, we sought to determine the cells involved in the connective tissue repair process following superficial wounding. Two months after transplantation, superficial wounds were made at the center of the graft using mechanical dermabrasion. At various times thereafter, ranging from 2 days to 6 weeks, healing grafts were harvested and processed for immunohistological study with species-specific and cross-reacting antibodies directed against human or mouse antigens. The grafted human skin regenerated according to the following series of events. First, the human dermis underneath the scab became devoid of human fibroblasts while the surrounding human dermis preserved its own characteristics. The TUNEL reaction on early-phase healing wounds indicated that apoptosis occurred steadily within this area and could be the mechanism by which cells disappeared. Moreover, cell death was reduced when the wound was covered with an occlusive dressing. The human dermis beneath the wound was then invaded by mouse cells which deposited type I collagen on the human extracellular matrix and produced mouse granulation tissue at the surface above it. Human keratinocytes migrated over the mouse granulation tissue to reconstruct the epidermis. Eventually, the mouse granulation tissue was progressively invaded by human fibroblasts, which formed a human neodermis. The overall process appeared to depend upon several successive epithelial-mesenchymal interactions, which were not species-specific. This suggests that myofibroblasts arise from a specific subpopulation of fibroblasts, probably located at the interface between the dermis and adipose tissue, and that the granulation tissue is eventually remodeled by another population of fibroblasts present in the human dermis. Received: 31 March 1999 / Accepted after revision: 15 July 1999 / Accepted: 13 August 1999     

Diversity of desmosomal proteins in regenerating epidermis: immunohistochemical study using a human skin organ culture model

Abstract We recently established a skin organ culture model for epithelial healing by creating a central defect in freshly excised human skin specimens and keeping them in culture for up to 7 days, either untreated or with transplantation of allogenic or autologous keratinocytes. In this study the molecular diversity of cell-cell junction proteins in the regenerating epidermis was analysed immunohistochemically using a broad spectrum of monoclonal antibodies against glycoproteins (cadherins) and plaque proteins of desmosomes. At all stages studied the entire set of desmosomal cadherins [desmogleins (Dsg) 1–3 and desmocollins (Dsc) 1–3] was detected, with Dsg3, Dsc2 and Dsc3 being the most prominent. In the disordered neoepithelium at day 3 (after transplantation) some desmosomal cadherins appeared in their respective stratum compartments. In regenerating epidermis on day 7, which exhibited a more ordered stratification and a compact horny layer, stratification-related patterns of desmosomal cadherins were more pronounced. However, some immaturity of the day-7 neoepidermis was reflected by relatively low levels of the maturation-associated Dsg1 and Dsc1 and a strong basal layer expression of Dsg2 which is sparse in normal epidermis. Desmosomal plaque proteins showed expression patterns similar to those in normal healthy epidermis. The adherens junction-related E-cadherin was also detected. Dendritic cells (melanocytes, Langerhans cells) were mainly present at the wound margins. In conclusion, this study demonstrated partial but not complete epidermal maturation and junction development during regeneration up to day 7. This model should also be useful in future studies to evaluate the effects of growth hormones to be used in therapeutic trials on chronic leg ulcers. Received: 15 June 1998 / Received after revision: 22 February 1999 / Accepted: 9 April 1999     

自体与异体细胞混合构建的活性皮肤替代物修复隐性营养不良型大疱性表皮松解症患者手部瘢痕挛缩一例

目的 观察以人羊膜为基质、自体与异体细胞混合构建的活性皮肤替代物（AM?LSE）修复大疱性表皮松解症患者手部瘢痕挛缩的疗效。方法 采集1例隐性营养不良型大疱性表皮松解症（RDEB）患者及其母亲的皮肤组织,分别进行表皮角质形成细胞和真皮成纤维细胞分离、培养。将自体与异体细胞混合后构建AM?LSE并进行组织学检查和Ⅶ型胶原免疫荧光检查。移植AM?LSE皮片于RDEB患者手部瘢痕挛缩松解术后的皮肤缺损区域。术后观察皮片的成活以及创面修复效果。结果 构建的AM?LSE具有真皮层、重层分化良好的表皮层和发育完好的基底膜。免疫荧光检查显示,Ⅶ型胶原沿基底膜线性分布。术后观察半年,移植的AM?LSE存活良好,未发现明显的排异反应,未发现受皮区再发水疱及破溃,瘢痕挛缩不显著,皮片色泽接近正常皮肤,质地柔软,患手可抓握,满足一般生活自理的要求。结论 自体与异体细胞混合构建的AM?LSE具有良好的组织学形态,移植于RDEB患者瘢痕切除后的创面,无明显排异,不易因摩擦起水疱破溃或发生瘢痕挛缩。     

光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤MIF表达的影响

目的:探讨光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤巨噬细胞移动抑制因子(MIF)表达的影响。方法:选取BALB/c裸鼠,皮下注射人宫颈癌Caski细胞,10d后将达到荷瘤标准的40只裸鼠采用随机数字表法分为四组,即A组、B组、C组和D组,各10只。A组作为阴性对照、B组给予单次光动力疗法、C组给予多次光动力疗法和D组给予顺铂腹腔注射。对比治疗前和治疗2周后肿瘤体积,治疗前后裸鼠体质量、瘤体积、瘤质量变化及抑瘤率,并分别采用western-blot和免疫组化法对各组裸鼠肿瘤组织MIF表达情况进行检测,行对比分析。结果:B组、C组和D组体质量、瘤体积和瘤质量变化和A组比较差异均有统计学意义(P<0.05),且C组和D组上述指标及抑瘤率和B组比较差异均有统计学意义(P<0.05),而C组和D组间各项指标差异均无统计学意义(P>0.05);电镜分析结果显示各组肿瘤胞浆可见明显空泡,团间有明显坏死灶,并且随着光动力疗法剂量增高,坏死区域增大越明显。B组、C组和D组MIF蛋白IOD值均较A组显著降低(P<0.05),且C组和D组MIF蛋白相对表达量和IOD值均较B组显著降低(P<0.05),而C组和D组MIF蛋白相对表达量和IOD值比较差异均无统计学意义(P>0.05)。结论:采用多次光动力疗法对人宫颈癌Caski细胞裸鼠移植瘤效果和顺铂相近,且可显著降低MIF表达水平。     

Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche

Abstract:  Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR.
According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while β1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. α6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs.
These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ .     

梅毒螺旋体膜蛋白Tpp47对血管内皮细胞黏附功能影响的实验研究

【摘要】 目的 探讨梅毒螺旋体膜蛋白Tpp47对血管内皮细胞的作用。 方法 利用基因工程技术重组合成的梅毒螺旋体膜蛋白Tpp47及脂多糖分别刺激人脐静脉内皮细胞（HUVEC）,ELISA检测上清中细胞间黏附分子1（ICAM-1）及E选择素的水平;荧光定量PCR检测HUVEC中ICAM-1及E选择素 mRNA的转录水平;MTT法检测HUVEC增殖水平;将重组蛋白Tpp47及脂多糖预处理的HUVEC与钙黄绿素AM标记的THP-1细胞共培养,荧光倒置显微镜下观察HUVEC与THP-1细胞的黏附情况。 结果 梅毒螺旋体膜重组蛋白Tpp47刺激HUVEC后,其分泌的黏附分子ICAM-1（1.28 ± 0.03）及E选择素（0.51 ± 0.01）水平显著提高,与空白对照组（0.90 ± 0.01与0.13 ± 0.03）比较,差异均有统计学意义（t值分别为18.28和18.19,均P ＜ 0.05）;将重组蛋白Tpp47刺激24 h后的HUVEC与THP-1细胞共培养,HUVEC与THP-1细胞的黏附率为56.1% ± 1.9%,较空白对照组（16.3% ± 2.1%）明显提高,差异有统计学意义（χ2 = 12.65,P ＜ 0.05）。MTT试验结果显示,重组蛋白Tpp47刺激HUVEC后,其增殖率（19.5% ± 1.7%）高于空白对照组（10.0% ± 3.1%）,差异有统计学意义（χ2 = 3.92,P ＜ 0.05）;较脂多糖低（41.2% ± 3.7%）,差异有统计学意义（χ2 = 10.42,P ＜ 0.05）。 结论 梅毒螺旋体膜重组蛋白Tpp47可体外上调HUVEC与THP-1的黏附能力及促进HUVEC增殖,可能在梅毒的发病中起一定作用。     

Ultra‐weak photon emission as a non‐invasive tool for monitoring of oxidative processes in the epidermal cells of human skin: comparative study on the dorsal and the palm side of the hand

Background/purpose: All living organisms emit spontaneous ultra‐weak photon emission as a result of cellular metabolic processes. Exposure of living organisms to exogenous factors results in oxidative processes and enhancement in ultra‐weak photon emission. Here, hydrogen peroxide (H₂O₂), as a strongly oxidizing molecule, was used to induce oxidative processes and enhance ultra‐weak photon emission in human hand skin. The presented work intends to compare both spontaneous and peroxide‐induced ultra‐weak photon emission from the epidermal cells on the dorsal and the palm side of the hand. Methods: A highly sensitive photomultiplier tube and a charge‐coupled device camera were used to detect ultra‐weak photon emission from human hand skin. Results: Spontaneous ultra‐weak photon emission from the epidermal cells on the dorsal side of the hand was 4 counts/s. Topical application of 500 mM H₂O₂ to the dorsal side of the hand caused enhancement in ultra‐weak photon emission to 40 counts/s. Interestingly, both spontaneous and peroxide‐induced ultra‐weak photon emission from the epidermal cells on the palm side of the hand were observed to increase twice their values, i.e. 8 and 80 counts/s, respectively. Similarly, the two‐dimensional image of ultra‐weak photon emission observed after topical application of H₂O₂ to human skin reveals that photon emission from the palm side exceeds the photon emission from the dorsal side of the hand. Conclusion: The results presented indicate that the ultra‐weak photon emission originating from the epidermal cells on the dorsal and the palm side of the hand is related to the histological structure of the human hand skin. Ultra‐weak photon emission is shown as a non‐destructive technique for monitoring of oxidative processes in the epidermal cells of the human hand skin and as a diagnostic tool for skin diseases.