自体头皮与脱细胞异体真皮复合移植修复巨大色素痣北大核心CSCD

目的 探讨自体头皮与脱细胞异体真皮复合移植方法修复儿童巨大色素痣的效果.方法 采用自体刃厚头皮与脱细胞异体真皮复合移植的方法修复儿童巨大色素痣,并以自体薄中厚皮片移植方法作对照,观察移植皮片成活率、创面愈合时间及瘢痕增生状况.结果 供皮区实验组创面愈合时间为（5.31±1.45）d,对照组为（11.63±1.69）d,两组比较差异有统计学意义（P＜0.05）,实验组瘢痕评分为1.62±0.38,对照组为6.38±0.58,两组比较差异有统计学意义（P＜0.05）;受皮区实验组皮肤移植成活率为（94.44±2.56）％,对照组为（95.13±3.13）％,移植皮片成活率于两组间差异无统计学意义,实验组瘢痕评分为5.38±0.62,对照组瘢痕评分为8.40±0.41,两组差异有统计学意义（P＜0.05）.结论 自体头皮与脱细胞异体真皮复合移植修复儿童巨大色素痣,对供区损伤小,移植区形态及功能良好,值得应用.     

抗P_(200)血清对隐性遗传性营养不良性大疱性表皮松解症皮肤的免疫荧光研究

目的 确定P_(200)蛋白质抗原的性质。方法 收集了10例抗P_(200)类天疱疮血清,对6例隐性遗传性营养不良性大疱性表皮松解症(RDEB)皮肤切片进行了间接免疫荧光研究。结果 发现10例抗P_(200)类天疱疮血清均与5例RDEB皮肤基底膜带(BMZ)反应,而获得性大疱表皮松解症(EBA)血清对这些皮肤为阴性。另外,在1例RDEB,EBA血清既与BMZ反应又与Ⅶ型胶原沉积部位的胞浆反应,而抗P_(200)类天疱疮血清无此反应。结论 结果提示200 kDa抗原不是Ⅶ型胶原成份,而是一种特异的自身抗原。     

回复镶嵌现象与隐性营养不良型大疱性表皮松解症治疗的研究进展

隐性营养不良型大疱性表皮松解症是一种遗传性水疱病,由编码Ⅶ型胶原的基因突变引起,对患者生活质量有很大影响,目前治疗多为对症处理,迫切需要更好的治疗方法.近年研究发现,隐性营养不良型大疱性表皮松解症患者存在回复突变所致的片状外观正常皮肤.这种现象称为回复镶嵌现象,也称为天然基因治疗.这一发现开启了回复细胞治疗的可能性,即回复的角质形成细胞体外培养移植到受损皮肤.假如结合患者特异性诱导性多能干细胞方法,可以有机会培养出大片健康皮肤移植物.另外,回复体来源的诱导性多能干细胞还可以分化为造血细胞和间质干细胞,骨髓移植后归巢到水疱区域,即“从皮肤到血细胞,再修复皮肤”.     

皮肤创面覆盖物的研究进展

近年来 ,皮肤移植已从开始的自体皮肤移植和同种异体移植发展到生物皮肤替代物移植。本文综述了已商品化应用于临床的角质形成细胞移植物、角质形成细胞与异体真皮、无细胞真皮基质、合成的和组织工程的真皮基质组成的复合皮及其优缺点。     

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。     

皮肤创面覆盖物的研究进展

近年来，皮肤移植已从开始的自体皮肤移植和同种虱体移植发展到生物皮肤替代物移植，本文综述了已商品化应用于临床的角质形成细胞移植物，角质形成细胞与异体真皮，无细胞真皮基质，合成的和组织工程的真皮基质组成的复合民缺点。     

获得性大疱性表皮松解症合并症的研究进展

获得性大疱性表皮松解症是一种自身免疫性大疱性皮肤病,其发病与真皮-表皮连接的锚丝主要成分Ⅶ型胶原的自身免疫有关.获得性大疱性表皮松解症病变可累及皮肤和黏膜,主要表现多样的表皮下水疱,愈后可留有粟丘疹和萎缩性瘢痕.近年来,越来越多的研究证实,该病可合并其他皮肤病以及其他系统性疾病,包括消化系统疾病、自身免疫性疾病、内分泌系统疾病、感染性疾病、血液系统疾病以及肿瘤性疾病.部分合并疾病仅为个例报道,而另一些则有相关机制的研究.     

生物工程设计的人类皮肤替代物研究进展

对于烧伤、急性创伤、某些疾病导致的皮肤缺损，临床上常采用自体皮片、皮瓣移植；然而，对于大面积皮肤缺损的患者，常存在自体皮源不足的问题。生物工程的出现找到了一种可取代自体、同种异体皮肤移植的新方法，即根据人体皮肤的组织学及功能特点构建的生物工程设计的人类皮肤替代物。现在，一些人工皮肤已应用于临床取得了较好的疗效，并进入商品化生产。综述了表皮替代物、真皮替代物、复合人工皮肤的研究进展。     

获得性大疱性表皮松解症伴宫颈鳞状细胞癌一例

患者女,54岁,因反复眼干涩2年,躯干四肢水疱伴口腔破溃2个月入院.皮肤科检查:鼻腔黏膜破溃结痂、充血.口腔大片浅溃疡,可见有水疱,表层水肿发白,边缘充血,下唇散在小溃疡,双峡颊黏膜水肿,溃疡与舌背相似.肩部、颈部及躯干见红斑周围或红斑上散在少许水疱,疱壁稍紧张,疱壁薄,尼氏征可疑阳性.该例以黏膜为突出表现,临床表现类似于瘢痕性类天疱疮,容易误诊,且皮疹和黏膜损害随着肿瘤的病情而转变.获得性大疱性表皮松解症与Ⅶ胶原有关,可能与恶性肿瘤及鳞状细胞癌有一定的相关性.     

生物工程设计的人类皮肤替代物研究进展

对于烧伤、急性创伤、某些疾病导致的皮肤缺损 ,临床上常采用自体皮片、皮瓣移植 ;然而 ,对于大面积皮肤缺损的患者 ,常存在自体皮源不足的问题。生物工程的出现找到了一种可取代自体、同种异体皮肤移植的新方法 ,即根据人体皮肤的组织学及功能特点构建的生物工程设计的人类皮肤替代物。现在 ,一些人工皮肤已应用于临床取得了较好的疗效 ,并进入商品化生产。综述了表皮替代物、真皮替代物、复合人工皮肤的研究进展。     

Grafting of venous leg ulcers. An intraindividual comparison between cultured skin equivalents and full-thickness skin punch grafts

Skin equivalents that consisted of a noncontracted collagen gel populated with allogeneic fibroblasts and covered with autologous cultured keratinocytes were used for grafting venous leg ulcers. The results were compared in the same patient with those obtained with a routinely used standard method of grafting with autologous full-thickness punch grafts. The skin equivalents and the punch grafts were grafted successfully in four of five patients. The median healing time of ulcers grafted with skin equivalents was 18 days whereas that of ulcers covered with punch grafts was 15 days. The cosmetic appearance of the skin equivalent-grafted ulcers was better than that of the punch-grafted ulcers.     

An allogeneic cultured dermal substitute suitable for treating intractable skin ulcers and large skin defects prior to autologous skin grafting: three case reports

Intractable skin ulcers that arise as secondary lesions from disease and full-thickness skin defects that result from skin tumor excision often need autologous skin grafting to close the wound. We developed an allogeneic cultured dermal substitute (CDS) to shorten the time needed to prepare a wound bed suitable for autologous skin grafting. The CDS was prepared by plating normal human fibroblasts on a spongy matrix consisting of hyaluronic acid and atelo-collagen. The allogeneic CDS was then placed on the rinsed wound surface. This procedure was repeated twice a week for up to five weeks, until the wounds were closed by autologous skin grafting. In all three cases, after CDS treatment for two to five weeks, the wound conditions became suitable for skin grafting; these conditions had not been improved by conventional topical treatments, including topical basic fibroblast growth factor (bFGF). Healthy granulation tissue developed rapidly, concomitant with wound size reduction. The present results indicate that CDS is an excellent biological wound dressing for improving wound conditions so that they are suitable for subsequent autologous skin grafting as well as for shortening the treatment duration for skin ulcers and full-thickness skin defects.     

Reconstitution of structure and cell function in human skin grafts derived from cryopreserved allogeneic dermis and autologous cultured keratinocytes

Grafts of allogeneic dermis plus autologous epidermal cell cultures were used to replace extensively burned skin. Cryopreserved split-thickness cadaveric skin was grafted onto debrided burn wound, and autologous keratinocytes were cultured from uninjured donor sites. Several weeks later, allograft epidermis was abraded and replaced with the keratinocyte cultures. The final grafts were thus composites of autologous cultured epidermis and allogeneic dermis. In a case with 28 months follow-up, reconstitution of the dermal-epidermal (BMZ.1) and microvascular (BMZ.2) basement membrane zones was studied immunohistochemically and ultrastructurally. Immediately before grafting, thawed cryopreserved skin reacted with antibodies against laminin and type IV collagen in normal patterns. Twenty-nine days after grafting, BMZ.1 reacted weakly with both antibodies, and anticollagen type IV reactivity was absent from BMZ.2. Antilaminin reactivity of BMZ.2, however, was moderately intense, consistent with recent neovascularization. On day 29, the allograft epidermis was replaced with autologous keratinocyte cultures. Twenty-five days later (54 d after allografting), staining of both BMZs was intense with both antibodies. Ultrastructurally, at day 76 (47 d after culture placement) BMZ.1 revealed only small hemidesmosomes, few incipient anchoring fibrils, and a discontinuous lamina densa. BMZ.2, however, was fully reconstituted. By 124 d, both BMZs appeared normal. Observations in the dermis at 76 d included the presence of lymphocytes, organellar debris, and hyperactive collagen fibrillogenesis, all indicative of dermal remodelling. The microvasculature was well differentiated, but no elastic fibers or nerves were found. In the epidermis, melanocytes and evidence of melanosome transfer were seen at 5, 47, and 95 d after grafting of keratinocyte cultures. We conclude that the composite procedure reconstitutes skin with excellent textural and histologic qualities.     

皮肤组织蛋白酶D表达与晚期糖基化终末产物堆积的相关性研究

目的 研究组织蛋白酶D（CatD）与晚期糖基化终末产物（AGE）在不同年龄、不同曝光程度皮肤中的表达及其相关性,初步探讨CatD在光老化皮肤AGE降解和堆积中的作用。方法 15 ~ 20岁、35 ~ 40岁、55 ~ 60岁及75 ~ 80岁患者曝光和非曝光部位皮肤组织,共8组,每组6份。采用免疫组化和免疫荧光双染法分别检测CatD和AGE在各组皮肤中的表达。采用析因设计方差分析、Wilcoxon秩和检验和Kruskal?Wallis秩和检验分析CatD和AGE表达与年龄、曝光的关系,并用Pearson相关系数分析CatD和AGE两者表达的相关性。结果 免疫组化显示,CatD表达随年龄增加而明显下降,而AGE的沉积则随年龄增加而逐渐增多;曝光部位CatD表达较同年龄组非曝光部位明显降低,而AGE沉积比同年龄组非曝光部位明显升高。析因设计方差分析显示,曝光会降低CatD的表达（F = 58.70,P < 0.001）,但会增加AGE的表达（F = 158.18,P < 0.001）。年龄的增长亦会引起CatD表达降低（F = 79.49,P < 0.001）,但AGE表达随年龄的增长而增加（F = 106.06,P < 0.001）。除了15 ~ 20岁年龄组,其他年龄组曝光组和非曝光组间CatD（35 ~ 40岁组：0.020 ± 0.005比0.032 ± 0.005;55 ~ 60岁组：0.012 ± 0.004比0.026 ± 0.002;75 ~ 80岁组：0.002 ± 0.001比0.013 ± 0.004）和AGE平均吸光度值（35 ~ 40岁组：0.030 ± 0.008比0.010 ± 0.003 ;55 ~ 60岁组：0.066 ± 0.010比0.021 ± 0.004 ;75 ~ 80岁组：0.085 ± 0.015比0.035 ± 0.009 ）差异均有统计学意义（均P < 0.001）。在固定曝光因素各水平条件下,不同年龄组间CatD和AGE表达差异亦均有统计学意义（均P < 0.001）。免疫荧光双染结果与免疫组化结果相似。Pearson相关分析显示,曝光部位皮肤CatD表达与AGE沉积呈高度负相关（r = -0.915,P < 0.05）,在非曝光皮肤中两者呈中度负相关（r = -0.730,P < 0.05）。结论 随年龄增长皮肤组织中CatD表达水平下降,而AGE表达水平上升。非曝光部位皮肤CatD和AGE表达呈中度负相关,曝光部位皮肤CatD和AGE表达呈高度负相关,CatD很可能在光老化皮肤AGE降解和堆积中起重要作用。     

Accelerated healing of pyoderma gangrenosum treated with bioengineered skin and concomitant immunosuppression

Pyoderma gangrenosum is a rare, destructive, neutrophilic dermatosis, the origin of which remains largely obscure. The ulcerative variant of this inflammatory disorder causes painful, necrotic, rapidly enlarging ulcers. Because of pathergy, many clinicians avoid managing these nonhealing ulcers with aggressive surgical debridement and autologous grafts. This article proposes that the application of an allogeneic cultured human skin equivalent (Graftskin) not only circumvents this problem, but also hastens re-epithelialization of the ulcer bed. An added benefit of the possible improvement of the cosmetic appearance of the final scar by preventing severe wound contracture is also postulated. We report a newly diagnosed case of ulcerative pyoderma gangrenosum; the use of bioengineered skin as an adjunct to concurrent immunosuppressive therapy with cyclosporine hastened the healing and diminished pain in a rapidly enlarging leg ulcer. Within 2 weeks, the ulcer was 30% to 40% healed, achieving 100% re-epithelialization within 6 weeks.     

特应性皮炎患者皮肤屏障与水闸蛋白1表达的相关性研究

目的 研究特应性皮炎（AD）患者皮肤屏障功能情况,并分析其与水闸蛋白1（claudin-1）表达的相关性。 方法 纳入AD患者和健康人各11例。应用皮肤经表皮失水率测定仪和皮肤高频超声检测仪测定受试者经表皮失水率、表皮厚度与表皮致密度,并用双抗体夹心ELISA法定量检测血清中脱落claudin-1表达量。应用单因素方差分析和t检验比较不同组别之间相关参数的差异;应用Pearson相关系数分析不同参数之间的相关性。 结果 AD患者皮疹部位经表皮失水率为（36.9 ± 34.2） g·m-2·h-1,非皮疹部位为（9.1 ± 6.0） g·m-2·h-1,均高于健康对照［（4.4 ± 3.1） g·m-2·h-1］;AD患者皮疹部位表皮厚度（0.23 ± 0.04） mm,显著高于非皮疹部位［（0.18 ± 0.03） mm］和健康对照［（0.18 ± 0.02） mm］。AD患者皮疹部位有其特征性表皮下低回声带。AD患者claudin-1表达量为（0.80 ± 0.88） ng/ml,显著低于健康人［（1.73 ± 1.85） ng/ml］;claudin-1与表皮厚度显著负相关（r = -0.61）,与经表皮失水率的倒数显著正相关（r = 0.44）。 结论 AD患者损伤的皮肤屏障功能与claudin-1表达相关,屏障功能状态可用经表皮失水率、经表皮失水率倒数和表皮厚度进行定量表述。     

Gene therapy: pursuing restoration of dermal adhesion in recessive dystrophic epidermolysis bullosa

The replacement of a defective gene with a fully functional copy is the goal of the most basic gene therapy. Recessive dystrophic epidermolysis bullosa (RDEB) is characterised by a lack of adhesion of the epidermis to the dermis. It is an ideal target for gene therapy as all variants of hereditary RDEB are caused by mutations in a single gene, COL7A1, coding for type VII collagen, a key component of anchoring fibrils that secure attachment of the epidermis to the dermis. RDEB is one of the most severe variants in the epidermolysis bullosa (EB) group of heritable skin diseases. Epidermolysis bullosa is defined by chronic fragility and blistering of the skin and mucous membranes due to mutations in the genes responsible for production of the basement membrane proteins. This condition has a high personal, medical and socio‐economic impact. People with RDEB require a broad spectrum of medications and specialised care. Due to this being a systemic condition, most research focus is in the area of gene therapy. Recently, preclinical works have begun to show promise. They focus on the virally mediated ex vivo correction of autologous epithelium. These corrected cells are then to be expanded and grafted onto the patient following the lead of the first successful gene therapy in dermatology being a grafting of corrected tissue for junctional EB treatment. Current progress, outstanding challenges and future directions in translating these approaches in clinics are reviewed in this article.     

The longevity of a bilayered skin substitute after application to venous ulcers

BACKGROUND: A bilayered skin substitute composed of allogeneic keratinocytes and fibroblasts in a collagen gel has been approved by the US Food and Drug Administration for the treatment of venous and diabetic ulcers. Its mechanism of action has not been fully determined. OBJECTIVE: To determine the longevity of allogeneic fibroblasts and keratinocytes in a bilayered skin substitute in patients with venous leg ulcers. METHODS: Ten patients with venous leg ulcers were treated with a bilayered skin substitute on day 0, days 3 to 5, and weeks 1 through 3. Biopsy specimens of the grafted wound were taken. We used polymerase chain reaction analysis to determine whether allogeneic DNA was present in the biopsy specimens. RESULTS: We detected allogeneic DNA in 2 of 8 specimens at 1 month after initial grafting. Neither of the 2 patients showed persistence of allogeneic DNA at 2 months after initial grafting. CONCLUSIONS: Allogeneic cells from a bilayered skin substitute do not appear to survive permanently after grafting for treatment of venous leg ulcers. Other mechanisms of action might include cytokine release, structural support, or provision of a moist wound environment.     

Correction of dog dystrophic epidermolysis bullosa by transplantation of genetically modified epidermal autografts

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering condition caused by mutations in the gene coding for collagen type VII. Genetically engineered RDEB dog keratinocytes were used to generate autologous epidermal sheets subsequently grafted on two RDEB dogs carrying a homozygous missense mutation in the col7a1 gene and expressing baseline amounts of the aberrant protein. Transplanted cells regenerated a differentiated and vascularized auto-renewing epidermis progressively repopulated by dendritic cells and melanocytes. No adverse immune reaction was detected in either dog. In dog 1, the grafted epidermis firmly adhered to the dermis throughout the 24-month follow-up, which correlated with efficient transduction (100%) of highly clonogenic epithelial cells and sustained transgene expression. In dog 2, less efficient (65%) transduction of primary keratinocytes resulted in a loss of the transplanted epidermis and graft blistering 5 months after transplantation. These data provide the proof of principle for ex vivo gene therapy of RDEB patients with missense mutations in collagen type VII by engraftment of the reconstructed epidermis, and demonstrate that highly efficient transduction of epidermal stem cells is crucial for successful gene therapy of inherited skin diseases in which correction of the genetic defect confers no major selective advantage in cell culture.     

Immunology of skin transplantation

Untreated viable allogeneic skin is highly immunogenic. Epidermal Langerhans migrate after transplantation out of the donor skin into the lymph node of the recipient where they can activate T cells capable to mediate rejection. Allogeneic skin is used as a temporary coverage of burn wounds, often in combination with autologous skin grafts. Several methods to pretreat the allogeneic skin have been used to delay the rejection process. Processing of allogeneic skin in 85% glycerol results in a non-viable skin with a well-preserved structure. Experiments in a full thickness porcine wound model showed that rejection of glycerol treated allogeneic skin grafts was up to six days delayed. Viable, untreated allogeneic skin grafts were rejected predominantly by CD8 positive T cells whereas in the glycerol treated grafts the influx of host cells was lower and the majority of the cells were macrophages. The outgrowth of the autologous skin grafts underneath glycerol treated allogeneic skin was three days earlier completed when compared to grafts in combination with untreated allogeneic skin. Thus, by processing the allogeneic skin into 85% glycerol, the direct route to induce graft rejection is blocked since the Langerhans cells are non-viable. The glycerol-preserved skin grafts are finally rejected via an indirect route mediated by macrophages; this process is less disturbing for the outgrowth of autologous cells.