胎儿及成人皮肤创面愈合过程中PDGF和EGF表达的对比性研究

目的 探讨人胎儿和成人皮肤及其创面愈合过程中PDGF和EGF的表达及意义。方法 利用已建立的人胎儿无瘢痕愈合动物模型，获取相应标本，结合临床所取成人皮肤标本，采用免疫组化染色方法，观察PDGF，EGF的表达情况。结果 ①正常胎儿皮肤中未见明显的PDGF阳性染色；创伤后12h、1d的胎儿表皮及真皮浅层可见PDGF的弱阳性表达；创伤后3d、1周的胎儿皮肤中PDGF的表达呈阴性；正常成人皮肤可在成纤维细胞，巨噬细胞及毛细血管见到阳性表达；创伤后表达加强；②正常胎儿皮肤表皮全层和毛囊，皮脂腺及汗腺细胞可见EGF的阳性表达；创伤后的胎儿皮肤中EGF的表达未见到明显变化；正常成人皮肤可见表皮基底层有中度阳性表达，毛囊，汗腺细胞也可见到轻度表达，创伤后表达有所减弱。结论 生长因子在胎儿和成人皮肤创面愈合过程中的差异表达可能是胎儿无瘢痕愈合的重要原因之一。     

胎儿皮肤免疫细胞CD68、CD3的表达与无瘢痕愈合的关系

目的探讨人胎儿皮肤、正常成人皮肤及增生性瘢痕(HS)组织免疫细胞CD68、CD3与无瘢痕愈合的关系。方法选择笔者单位引产的16~33周胎龄的10例胎儿皮肤、7例正常成人皮肤以及18例HS标本,用免疫组织化学的方法分别检测其巨噬细胞、T淋巴细胞的表面标志CD68、CD3的表达。结果胎儿皮肤中CD68[(5±6)个/400倍视野]显著少于成人[(23±4)个/400倍视野,P<0.01],成人皮肤中CD68又显著少于HS[(38±16)个/400倍视野,P<0·01].随胎龄增加,CD68逐渐增多,在24~28周胎龄时迅速上升,28周以后上升缓慢。发育各期胎儿皮肤中均未见CD3;成人皮肤中可见少量CD3[(24±8)个/400倍视野],主要分布于表皮基底层;HS组织中CD3较多[(69±25)个/400倍视野],常聚集成片状,主要分布于真皮乳头层,在小血管周围呈袖套状分布,数量和染色强度大于成人皮肤(P<0.01).结论胎儿皮肤中CD68数量少可能与无瘢痕愈合存在一定的关系;同时胎儿皮肤无瘢痕愈合可能与胎儿皮肤中缺乏CD3有关。     

胎儿和成人皮肤及其创面愈合过程中碱性成纤维细胞生长因子的表达及意义

目的　探讨胎儿和成人皮肤及其创面愈合过程中碱性成纤维细胞生长因子 (b FGF)的表达及其意义。方法　将孕龄 2 0～ 2 4周胎儿皮肤移植至 BAL B/ C裸鼠背部皮下 ,皮片成活后制造创面 ,建立胎儿无瘢痕愈合动物模型 ,定期获取相应标本。对临床所取正常成人皮肤及创面愈合皮肤标本 ,采用免疫组织化学染色方法 ,观察 b FGF的表达情况。　结果　正常胎儿皮肤及创伤后胎儿皮肤中均未见明显的 b FGF阳性表达。正常成人皮肤中血管周围可见阳性表达 ;创伤后成人皮肤也可见阳性表达 ,尤其成纤维细胞和血管内皮细胞创伤后表达明显增强。高倍镜视野随机观察计数b FGF阳性表达细胞数 ,正常胎儿皮肤为 2 .1± 0 .1,创伤后 12小时 ,1、3天和 1周胎儿皮肤分别为 2 .2± 0 .1、2 .1± 0 .3、2 .1± 0 .3和 2 .0± 0 .1;正常成人皮肤为 2 3.2± 4 .2 ,创伤后成人皮肤为 4 0 .5± 3.6 ,胎儿正常皮肤和创伤皮肤 b FGF表达与正常成人皮肤和创伤后皮肤 b FGF表达比较 ,差异有统计学意义 (P<0 .0 1)。　结论　 b FGF的阴性表达可能是胎儿皮肤无瘢痕愈合的重要原因之一。     

Bax和Bcl-2在增生性瘢痕中表达及其对瘢痕形成的影响

目的观察Bax和Bcl-2在增生性瘢痕和正常皮肤组织内的表达特征及其对增生性瘢痕形成的影响.方法用免疫组化方法和常规病理技术确定这两种蛋白在8例增生性瘢痕和8例正常皮肤中的表达变化规律.结果在正常皮肤内,Bax主要存在于表皮基底层细胞和部分成纤维细胞的胞浆中;Bcl-2蛋白则分布于表皮基底层细胞的胞浆内.在增生性瘢痕中,Bax主要定位于表皮基底层细胞内,阳性细胞率明显低于正常皮肤水平(P<0.05);含有Bcl-2的阳性细胞主要为表皮细胞和部分成纤维细胞,阳性细胞率显著升高(P<0.05).结论增生性瘢痕的发生可能与Bcl-2蛋白过度表达,抑制细胞凋亡相关,而Bax表达降低,其介导的细胞凋亡受阻亦可能是增生性瘢痕形成的原因之一.     

microRNA-21调控TGF-β通路促进增生性瘢痕形成的机制研究

目的探讨micro RNA-21在增生性瘢痕(Hypertrophic scars,HS)形成中的作用及机制,为生物学防治提供新靶点。方法收集临床患者正常皮肤及HS标本,组织学检测、组织胶原定量检测;免疫组化、Real-time PCR检测正常细胞外基质Col 1 A1、Col 3 A1、Fibronectin(FN)及α-SMA的表达;Real-time PCR检测micro RNA-21 m RNA表达水平;培养HS成纤维细胞,构建micro RNA-21反义表达载体,并加入TGF-β诱导,Real-time PCR检测Col 1 A1、Col 3A1及micro RNA-21的表达。结果 HS中胶原的含量及细胞外基质表达明显高于正常皮肤;HS组织中micro RNA-21表达高于正常皮肤组织;TGF-β可增强HS成纤维细胞中micro RNA-21的表达,而瘢痕成纤维细胞转染micro RNA-21反义表达载体后可明显降低瘢痕相关基因的表达。结论皮肤创伤愈合后局部micro RNA-21表达升高,促进了细胞外基质的合成,而TGF-β进一步加强了micro RNA-21对瘢痕形成的促进作用,可能是导致HS形成的重要原因。     

p53基因在病理性瘢痕中的表达及意义

目的 探讨p53基因在病理性瘢痕中的表达及其发生机制,为临床防治病理性瘢痕提供分子生物学依据.方法 选取病理性瘢痕患者30例,将手术切除后弃用的瘢痕组织作为实验组,根据临床表现及病理性质分为瘢痕疙瘩A组(10例),增生性瘢痕增生期B组(10例),增生性瘢痕成熟期C组(10例).选取普通瘢痕患者及正常皮肤者各10例,分别设为对照D,E组.将每例标本行石蜡包埋后制成3张切片,1张行HE染色,2张行免疫组织化学染色.于显微镜下观察、拍照,并计算各组P53蛋白阳性细胞的表达率.结果 P53蛋白的表达:在A,B,C组中,主要表达于表皮基底层细胞和部分成纤维细胞的胞核中;在D组中,主要表达于少部分成纤维细胞的胞核中;在E组中,主要表达于表皮基底层细胞的胞核中.A,B,C组的阳性表达率分别为90%,80%,60%,D,E组的阳性表达率分别为30%,10%.实验组与对照组比较,其差异有统计学意义(P<0.01);在实验组中,A组与B组比较,差异有统计学意义(P<0.05),A组与C组比较,差异也有统计学意义(P<0.01),而B组与C组比较,差异无统计学意义(>0.05).在病理性瘢痕组织中,P53蛋白呈高表达;而在普通瘢痕及正常皮肤组织中,P53蛋白呈低表达.结论 p53基囚参与病理性瘢痕的形成,且在不同类型的瘢痕中所起到的作用不同.因此,抑制P53蛋白的表达,可能成为防治病理性瘢痕的一个有效途径.     

蛋白糖基化抑制剂对病理性瘢痕成纤维细胞Fas蛋白的表达与功能的影响

目的 研究N-糖链合成抑制剂衣霉素对病理性瘢痕成纤维细胞Fas蛋白的表达与诱导凋亡功能的影响.方法 瘢痕疙瘩及增生性瘢痕各5例,以健康皮肤为对照,免疫组织化学法检测组织中成纤维细胞Fas蛋白表达;组织块贴壁法培养成纤维细胞;Western Blot法及流式细胞术检测衣霉素处理及未处理各组成纤维细胞Fas蛋白水平的表达及凋亡率的变化.结果 病理性瘢痕及健康皮肤成纤维细胞胞质及胞膜中均可见Fas蛋白表达;增生性瘢痕、瘢痕疙瘩及健康皮肤成纤维细胞Fas蛋白糖基化水平依次降低,3组成纤维细胞在Fas单克隆抗体(Fas monoelonal antibody,FasMcAb)作用后凋亡率与Fas蛋白糖基化成正相关,衣霉素可明显降低病理性瘢痕成纤维细胞Fas蛋白糖基化水平,但对健康皮肤成纤维细胞Fas蛋白糖基化水平抑制作用不明显.结论 FasMcAb诱导病理性瘢痕成纤维细胞凋亡与成纤维细胞Fas蛋白糖基化水平成正相关,而衣霉素可显著降低成纤维细胞Fas蛋白糖基化水平.     

Shh及Gli-1蛋白在人增生性瘢痕和正常皮肤成纤维细胞中的表达及意义

目的研究Hedgehog信号通路中分子Shh及Gli-1蛋白在人增生性瘢痕和正常皮肤成纤维细胞中的表达,探讨此通路在增生性瘢痕形成中的作用和意义。方法应用免疫蛋白印记法(western blot)及免疫细胞化学检测15例人增生性瘢痕和正常皮肤成纤维细胞中Shh、Gli-1蛋白的表达情况。结果(1)Western blot检测结果显示,Shh和Gli-1蛋白在增生性瘢痕及正常皮肤成纤维细胞中均有表达,二者在增生性瘢痕成纤维细胞中的表达量(0.640±0.093和0.560±0.120)均比正常成纤维细胞的表达量(0.330±0.023和0.340±0.050)高,其差异具有统计学意义(P0.05);(2)免疫细胞化学染色结果显示,Shh及Gli-1蛋白在增生性瘢痕及正常皮肤成纤维细胞均有阳性表达,但在增生性瘢痕成纤维细胞组织中的表达明显高于止常皮肤成纤维细胞,其差异均有统计学意义(P0.05)。结论增生性瘢痕成纤维细胞中,Shh及Gli-1蛋白呈现高表达,提示Shh信号通路异常激活可能在增生性瘢痕发生过程中起到了重要作用。     

胎儿和成人皮肤组织中TGF-β1,2,3因子蛋白含量的变化及其与创面无瘢愈合的关系

目的观察转化生长因子β三种异构体(TGF-β1、2和3)和α-平滑肌肌动蛋白(α-ASMA)在胎儿和成人皮肤组织中的表达特征以及与胎儿创面无瘢愈合的关系.方法12例被测标本中包括不同胎龄的胎儿皮肤6例,其对应的成人皮肤组织6例.用免疫组化方法和常规病理技术确定这四种蛋白在胎儿和成人皮肤组织中的定位及表达量的变化规律.结果TGF-β1、2、3细胞因子的阳性信号主要见于表皮基底层细胞、汗腺和毛囊细胞的胞浆和胞外基质中,α-ASMA蛋白则定位于肌动成纤维细胞内.在胎儿生长发育过程中,TGF-β1、2、3和α-ASMA等蛋白在皮肤组织中的阳性细胞率逐渐升高,在成人皮肤组织中,四种蛋白的含量更加明显增大.在胎儿发育的不同时期,这四种蛋白在皮肤组织内的含量变化规律相近似.结论在成人皮肤组织内,TGF-β1和α-ASMA两种蛋白含量的升高可能与伤口愈合形成瘢痕相关,而胎儿皮肤中这两种蛋白含量的降低可能与胎儿创面无瘢愈合密切相关,而TGF-β2、3在其中所起的作用尚需进一步深入研究.     

增生性瘢痕组织角质形成细胞中神经丝蛋白表达的特征及生物学意义

目的观察瘢痕组织与成人正常皮肤上皮细胞中神经丝蛋白(NFP)及其表皮细胞生长因子受体(EGFR)表达变化,阐明神经再支配对增生性瘢痕形成的影响.方法标本取自烧伤瘢痕愈合后11～26月来我院进行修复手术的病人,正常对照选自同一病人的供皮区.利用单克隆抗体免疫组织化学染色技术,光镜下观察瘢痕组织及正常皮肤上皮细胞中NFP和EGFR阳性细胞表达情况.结果瘢痕组织与正常皮肤上皮中神经丝蛋白与表皮细胞生长因子受体的表达存在差异,随着瘢痕的成熟,神经丝抗体的表达逐渐增强,而瘢痕早期表皮细胞生长因子受体的表达较强,瘢痕后期的表达减弱.结论增生性瘢痕与正常的皮肤中NFP的变化与EGFR活性与创面愈合的结局密切相关.神经调节在瘢痕形成的过程中可能充当了重要的角色.     

Exogenous transforming growth factor-beta amplifies its own expression and induces scar formation in a model of human fetal skin repair.

OBJECTIVE: Fetal skin wounds heal without scarring. To determine the role of TGF-beta 1 in fetal wound healing, mRNA expression of TGF-beta 1 was analyzed in human fetal and adult skin wounds. METHODS: Human fetal skin transplanted to a subcutaneous location on an adult athymic mouse that was subsequently wounded heals without scar, whereas human adult skin heals with scar formation in that location. In situ hybridization for TGF-beta 1 mRNA expression and species-specific immunohistochemistry for fibroblasts, macrophages, and neutrophils were performed in human adult wounds, fetal wounds, and fetal wounds treated with a TGF-beta 1 slow release disk. RESULTS: Transforming growth factor-beta 1 mRNA expression was induced by wounding adult skin. No TGF-beta 1 mRNA upregulation was detected in human fetal skin after wounding. However, when exogenous TGF-beta 1 was added to human fetal skin, induction of TGF-beta 1 mRNA expression in human fetal fibroblasts occurred, an adult-like inflammatory response was detected, and the skin healed with scar formation. CONCLUSIONS: Transforming growth factor-beta 1 is an important modulator in scar formation. Anti-TGF-beta 1 strategies may promote scarless healing in adult wounds.     

Wound healing in a fetal,adult, and scar tissue model: A comparative study

Early gestation fetal wounds heal without scar formation. Understanding the mechanism of this scarless healing may lead to new therapeutic strategies for improving adult wound healing. The aims of this study were to develop a human fetal wound model in which fetal healing can be studied and to compare this model with a human adult and scar tissue model. A burn wound (10 × 2 mm) was made in human ex vivo fetal, adult, and scar tissue under controlled and standardized conditions. Subsequently, the skin samples were cultured for 7, 14, and 21 days. Cells in the skin samples maintained their viability during the 21‐day culture period. Already after 7 days, a significantly higher median percentage of wound closure was achieved in the fetal skin model vs. the adult and scar tissue model (74% vs. 28 and 29%, respectively, p<0.05). After 21 days of culture, only fetal wounds were completely reepithelialized. Fibroblasts migrated into the wounded dermis of all three wound models during culture, but more fibroblasts were present earlier in the wound area of the fetal skin model. The fast reepithelialization and prompt presence of many fibroblasts in the fetal model suggest that rapid healing might play a role in scarless healing.     

Adult skin wounds in the fetal environment heal with scar formation.

OBJECTIVE: This study investigated the influence of the fetal environment on the healing characteristics of adult skin. SUMMARY BACKGROUND DATA: The remarkable ability of the fetus to heal without scarring is poorly understood. The unique qualities of fetal wound healing may be caused by the fetal environment, the fetal tissues, or a combination of both. There are numerous differences between the prenatal and postnatal environments that may play a role in the unique fetal response to injury. METHODS: Full-thickness adult sheep skin was transplanted onto the backs of 60-day-gestation fetal lambs (term, 145 days of gestation). The adult skin grafts were thus perfused by fetal blood and bathed in amniotic fluid. Previous work has demonstrated that, before midgestation, fetal lambs do not reject allogenic skin grafts. Forty days later (100 days of gestation), incisional wounds were made on both the adult skin graft and the adjacent fetal skin. The wounds were harvested 14 days postwounding and analyzed by both light microscopy and immunohistochemical testing using antibodies to collagen types I, III, and VI. RESULTS: The wounds in the adult skin grafts healed with scar formation. This observation contrasts strongly with the scarless healing of the incisional fetal skin wounds. CONCLUSIONS: This study suggests that scarless fetal skin healing properties are intrinsic to fetal skin and are not primarily the result of the fetal environment.     

Wound healing in oral mucosa results in reduced scar formation as compared with skin: Evidence from the red Duroc pig model and humans

Scar formation is a common, unwanted result of wound healing in skin, but the mechanisms that regulate it are still largely unknown. Interestingly, wound healing in the oral mucosa proceeds faster than in skin and clinical observations have suggested that mucosal wounds rarely scar. To test this concept, we created identical experimental wounds in the oral mucosa and skin in red Duroc pigs and compared wound healing and scar development over time. We also compared the pig oral mucosal wound healing to similar experimental wounds created in human subjects. The findings showed significantly reduced scar formation at both clinical and histological level in the pig oral mucosa as compared with skin 49 days after wounding. Additionally, the skin scars contained a significantly increased number of type I procollagen immunopositive cells and an increased fibronectin content, while the oral mucosal wounds demonstrated a prolonged accumulation of tenascin-C. Furthermore, the pig oral mucosal wounds showed similar molecular composition and clinical and histological scar scores to human oral mucosal wounds. Thus, the reduced scar formation in the pig oral mucosa provides a model to study the biological processes that regulate scarless wound healing to find novel approaches to prevent scar formation in skin.     

Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair.

OBJECTIVE: This review updates the surgeon about the cellular, matrix, and growth factor components of scarless fetal wound repair. SUMMARY BACKGROUND DATA: Fetal skin wound healing is characterized by the absence of scar tissue formation. This unique repair process is not dependent on the sterile, aqueous intrauterine environment. The differences between fetal and adult skin wound healing appear to reflect processes intrinsic to fetal tissue, such as the unique fetal fibroblasts, a more rapid and ordered deposition and turnover of tissue components, and, particularly, a markedly reduced inflammatory infiltrate and cytokine profile. Scarless fetal wounds are relatively deficient in the inflammatory cytokine, transforming growth factor beta (TGF-beta). In contrast, the fibrosis characteristic of adult wound repair may be associated with TGF-beta excess. Recent experimental studies suggest that specific anti-TGF-beta therapeutic strategies can ameliorate scar formation in adult wound repair and fibrotic diseases. Inhibitors of TGF-beta may be important future drugs to control scar. CONCLUSIONS: Based on the scarless fetal wound repair model, a number of ways in which the matrix and cellular response of the healing adult wound might be manipulated to reduce scarring are reviewed.     

新型增生性瘢痕裸鼠动物模型的建立

目的　建立一种稳定的增生性瘢痕 (HS)动物模型 ,为进一步研究HS的发病机制提供帮助。　方法　在 10 0只裸鼠背部分别作 2 .0cm× 1.5cm全层皮肤缺损创面 ,移植人全厚皮肤 ;皮片存活后用加热的铜棒造成深Ⅱ度烫伤 ,观察创面愈合后的瘢痕增生情况。　结果　皮片完全存活的 86只裸鼠中 ,6 7只有明显、持续的瘢痕增生 ,占 78%。增生瘢痕的外观和组织学特点与人体增生性瘢痕相似。瘢痕增生的平均厚度为 0 .34cm,最厚 0 .6 0cm,增生时间为 6 3～ 2 17d,平均 12 8d。结论　该模型瘢痕增生明显、增生持续时间长 ,可用于观察创面愈合至瘢痕形成的全过程 ,是目前较为稳定的增生性瘢痕动物模型。     

Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation

Wound healing in the mid-gestation fetus is scarless with minimal inflammation and a unique extracellular matrix. We have previously documented the relative lack of inflammatory cytokines in this environment. We demonstrate that interleukin (IL)-10 is highly expressed in mid-gestation human fetal skin but is absent in postnatal human skin. We hypothesize that overexpression of IL-10 in postnatal skin may replicate a permissive environment for scarless healing. To study the mechanism underlying this process we performed immunohistochemistry for IL-10 in human mid-gestation fetal and postnatal skin. We also determined if adenoviral-mediated overexpression of IL-10 could allow for scarless wound healing in a murine incisional wound model. Wounds were analyzed at 1–90 days postwounding for effects on scar formation, inflammatory response, and biomechanical properties. Ad-IL-10 reconstitutes a permissive environment for scarless healing as shown by reconstitution of a normal dermal reticular collagen pattern and distribution of dermal elements. Compared with controls, Ad-IL-10 treated wounds showed reduced inflammatory response and no difference in biomechanical parameters. Therefore, overexpression of IL-10 in postnatal wounds results in a permissive environment for scarless wound repair, possibly by replicating a fetal wound environment.     

Hydrogen peroxide disrupts scarless fetal wound repair

Cutaneous wound healing is a complex process that leads to the formation of a permanent scar in adult skin. In contrast, early gestation fetal skin undergoes scarless repair. Normally, the repair process in the skin begins with an acute inflammatory response. However, one of the most important aspects of scarless fetal wound repair appears to be a lack of inflammation, suggesting that inflammation promotes scar formation in the skin. While it is well accepted that inflammation causes scar formation in the fetus, it is not known what specific factors produced during inflammation are responsible for these effects. Oxidants released by activated inflammatory cells have the potential to be involved, although this has never been examined. The present studies, using a murine fetal wound repair model, show that hydrogen peroxide interferes with scarless healing, possibly through the induction of transforming growth factor-beta1. Hydrogen peroxide also increased the proliferation of fetal fibroblasts, which could contribute to an increase in the fibrosis seen with hydrogen peroxide. Defining the factors produced during the inflammatory response that contribute to scar formation could be important for the development of new therapies designed to minimize scarring.