瘢痕疙瘩组织中内皮素—1及碱性成纤维细胞生长因子mRNA的 …

目的 了解内皮素－１和碱性成纤维细胞生长因子（ｂＦＧＦ）在瘢痕疙瘩组织的表达情况。方法 组织活检标本分为３组，瘢痕疙瘩、萎缩性瘢痕和正常皮肤。用地高辛标记的ｃＤＮＡ探针，采取冰冻切片原位杂交的方法检测内皮素－１和ｂＦＧＦｍＲＮＡ的表达。结果 瘢痕疙瘩真皮组织内内皮素－１ｍＲＮＡ的表达明显强于萎缩性瘢痕和对照组。阳性染色主要位于真皮浅层血管和部分真皮成纤维细胞。瘢痕疙瘩组６／８例真皮成纤维细胞内皮素     

瘢痕疙瘩组织中ICAM-1、VEGF、c-fos表达的免疫组化检测

采取免疫组化SP方法检测了细胞间粘附分子 1(ICAM 1)、血管内皮细胞生长因子 (VEGF)、以及原癌基因 (c fos)在瘢痕疙瘩、普通瘢痕、正常皮肤组织中的表达。结果表明ICAM 1、VEGF、c fos在瘢痕疙瘩中表达皆增强。ICAM 1主要表达在真皮浅层血管、浸润的炎细胞、成纤维细胞 ;VEGF、c fos主要表达在表皮、真皮血管、皮肤附属器 ;部分瘢痕疙瘩标本成纤维细胞c fos染色阳性 ,提示瘢痕疙瘩组织中血管内皮细胞处于一种激活状态 ,瘢痕疙瘩组织血管内皮细胞和成纤维细胞增殖异常。     

人真皮网状层成纤维细胞在瘢痕疙瘩皮损组织中的表达与分布

【摘要】 目的 探讨人真皮乳头层成纤维细胞（Fp）、网状层成纤维细胞（Fr）和肌成纤维细胞（MFB）在瘢痕疙瘩皮损组织中的表达与分布。方法 2019年5 - 12月在武汉大学人民医院皮肤科门诊确诊的15例瘢痕疙瘩患者,男8例,女7例,年龄20 ~ 50岁,取皮损组织,以15例年龄匹配的女性乳房整形术正常皮肤组织为对照。采用双重免疫荧光染色法检测成纤维细胞活化蛋白（FAP）、CD90和α平滑肌肌动蛋白（α-SMA）在瘢痕疙瘩和正常皮肤组织中的分布。从3例正常皮肤和3例瘢痕疙瘩组织中分离成纤维细胞原代培养,采用10 ng/ml 转化生长因子β1（TGF-β1）体外处理两组细胞0 ~ 48 h,观察细胞表型的变化,荧光定量RT-PCR和Western印迹检测FAP、CD90和α-SMA mRNA和蛋白表达。两组间差异比较采用t检验。结果 免疫荧光结果显示,正常皮肤组织中,FAP+/CD90-细胞主要分布在真皮浅层,FAP-/CD90+细胞集中在真皮深层,CD90+细胞几乎不表达α-SMA;瘢痕疙瘩组织深层可见大量FAP+和CD90+细胞,大量CD90+细胞同时表达α-SMA。双重免疫荧光染色显示,正常皮肤成纤维细胞几乎不表达α-SMA,瘢痕疙瘩成纤维细胞表达α-SMA;TGF-β1处理24 h时,正常成纤维细胞和瘢痕疙瘩成纤维细胞α-SMA+细胞荧光强度（21.058 ± 0.709、27.112 ± 0.097）均高于未处理组（11.312 ± 0.636、21.306 ± 0.464）,t值为22.430、13.370,P < 0.05。RT-PCR和Western印迹显示,TGF-β1处理48 h时,瘢痕疙瘩成纤维细胞FAP、CD90、α-SMA mRNA相对表达水平（92.610 ± 3.667、1.366 ± 0.105、3.240 ± 0.141）与蛋白表达水平（0.652 ± 0.073、1.046 ± 0.119、0.946 ± 0.117）均高于处理前（均P < 0.05）。结论 瘢痕疙瘩组织真皮深层的CD90+（Fr）细胞异常增生,提示针对真皮深层异常增殖活跃的FAP-/CD90+（Fr）细胞群进行定向干预可能提高瘢痕疙瘩治疗疗效。     

二聚糖对瘢痕疙瘩成纤维细胞迁移的影响

目的探讨二聚糖(biglycan, BGN)在瘢痕疙瘩中的表达及其对瘢痕疙瘩成纤维细胞迁移的影响。方法应用免疫组织化学及Western blot检测BGN在瘢痕疙瘩组织及瘢痕疙瘩成纤维细胞中的蛋白表达。采用siRNA的方法对BGN基因进行敲减,利用体外细胞划痕实验方法观察瘢痕疙瘩成纤维细胞的移行能力。结果免疫组织化学与Western blot结果显示,与瘢痕疙瘩旁正常皮肤组织比较,BGN在瘢痕疙瘩组织中表达增高,与正常皮肤成纤维细胞比较,BGN在瘢痕疙瘩成纤维细胞内和外液中表达增高。敲减BGN基因可显著抑制瘢痕疙瘩成纤维细胞的移行能力。结论敲减BGN有明显的抗瘢痕疙瘩成纤维细胞迁移作用。     

己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子-β1表达的影响北大核心CSCD

目的探讨己酮可可碱对瘢痕疙瘩成纤维细胞增殖、胶原合成及转化生长因子（TGF）-β1表达的影响。方法取人瘢痕疙瘩及正常皮肤组织培养的第5～8代成纤维细胞,在含有0．1—3g／L己酮可可碱的环境中培养。应用噻唑蓝（M1Tr）法检测成纤维细胞增殖,双抗体夹心-ELISA法测定TGF—β1表达,RT—PCR检测I、Ⅲ型前胶原mRNA表达。结果0．1～2g／L己酮可可碱能明显抑制瘢痕疙瘩和正常皮肤成纤维细胞的增殖,呈明显的量效一时效关系,抑制作用在浓度2g／L时达到最高。浓度为0．5～2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞TGF—B1表达,1或2g／L时己酮可可碱能降低瘢痕疙瘩和正常皮肤成纤维细胞I、Ⅲ型前胶原mRNA表达。结论己酮可可碱对瘢痕疙瘩和正常皮肤成纤维细胞的增殖、TGF—β1以及I、Ⅲ型前胶原表达均有明显的抑制作用。     

瘢痕疙瘩及其成纤维细胞p16基因甲基化状态和相关基因表达研究北大核心CSCD

目的 探讨瘢痕疙瘩中成纤维细胞p16基因甲基化在其发生发展中的作用.方法 分离、培养来自瘢痕疙瘩皮损和健康人皮肤原代成纤维细胞;免疫组化法检测瘢痕疙瘩皮损中p16表达情况;实时荧光定量PCR检测瘢痕疙瘩成纤维细胞中p16、DNA甲基转移酶mRNA表达;亚硫酸氢盐修饰后测序（BSP法）检测瘢痕疙瘩皮损及培养的原代成纤维细胞p16基因甲基化状态.结果 瘢痕疙瘩成纤维细胞中p16基因mRNA表达低于健康人皮肤成纤维细胞（相对表达量2-△△Ct分别为0.64±0.18和1.92±0.23,t=10.54,P＜0.05）.瘢痕疙瘩成纤维细胞三种DNA甲基转移酶（DNMT）基因mRNA表达水平（DNMT1、DNMT3A、DNMT3B分别为2.58±0.23、4.87±0.46、1.57±0.12）与健康人皮肤成纤维细胞（分别为1.13±0.21、2.38±0.32、0.57±0.16）相比均存在高表达,两组比较,t值分别为11.22、10.81、12.45,均P＜0.05.瘢痕疙瘩组织和瘢痕疙瘩原代成纤维细胞内p16启动子区甲基化程度分别为1.81％±0.46％和3.15％±0.94％,明显高于健康人皮肤组织（0.90％±0.35％,F=14.23,P＜0.01）和原代成纤维细胞（0.17％±0.29％,F=37.62,P＜0.01）.结论 瘢痕疙瘩成纤维细胞中p16基因甲基化及其低表达与瘢痕疙瘩的失控性生长可能相关,DNA甲基转移酶在其发病中可能起一定作用.     

曲尼司特对人正常皮肤和瘢痕疙瘩成纤维细胞部分细胞因子表达的影响

目的 ：研究曲尼司特对正常皮肤和瘢痕疙瘩成纤维细胞转化生长因子-β1（TGF-β1）、碱性成纤维细胞生长因子（bFGF）和白介素-6（IL-6）表达的影响。方法：在体外无血清培养的人正常皮肤成纤维细胞和瘢痕疙瘩成纤维细胞中,分别加入0、10、25、50和250μg／mL曲尼司特孵育24、72、96h,用双抗体夹心酶联免疫吸附试验（ABC—ELISA）法测定其上清液中TGF-β1、bFGF和IL-6的表达水平。结果：与对照组相比。25、50和250μg／mL曲尼司特能抑制瘢痕疙瘩成纤维细胞TGF-β1的表达;50μg／mL和250μg／mL曲尼司特能增加瘢痕疙瘩成纤维细胞bFGF的表达;10～250μg／mL曲尼司特可降低IL-6的表达,上述改变在一定时段差异有统计学意义（P〈0．05）。不同浓度曲尼司特对正常皮肤成纤维细胞的影响相似。结论：曲尼司特能降低瘢痕疙瘩成纤维细胞TGF-β1的产生。增加bFGF的合成,减少IL-6的表达,这或许可解释其在抑制异常瘢痕形成中的作用。     

皮肤肿瘤

20 0 0 4 0 31　瘢痕疙瘩组织中 ICAM- 1、VEGF、c- fos表达的免疫组化检测 /阎国富 (三军大新桥医院皮肤科 )…∥临床皮肤科杂志 .- 2 0 0 0 ,2 9( 3) .- 139～ 141采用免疫组化 SP方法检测了细胞间粘附分子 - 1( ICAM- 1)、血管内皮细胞生长因子 ( VEGF)及原癌基因 ( c- fos)在瘢痕疙瘩、普通瘢痕、正常皮肤组织中的表达。结果表明 ICAM- 1、VEGF、c- fos在瘢痕疙瘩中表达皆增强。ICAM- 1主要表达在真皮浅层血管、浸润的炎细胞、成纤维细胞 ;VEGF、c- fos主要表达在表皮、真皮血管、皮肤附属器 ;部分瘢痕疙瘩标本成纤维细胞…     

瘢痕疙瘩组织中ICAM—1,VEGF,c—fos表达的免疫组化检测

采用免疫组化ＳＰ方法检测了细胞间粘附分子－１,血管内皮细胞生长因子、以及原癌基因在瘢痕疙瘩,普通颜痕、正常皮肤组织中的表达。结果表明ＩＣＡＭ－１、ＶＥＧＦ、ｃ－ｆｏｓ在般痕疙瘩中表达皆增强。ＩＣＡＭ－１主要表达在真皮浅层血管,浸润的炎细胞,成纤维细胞;ＶＥＧＦ、ｃ－ｆｏｓ主要表达在表皮,真皮血管,皮肤附属器;部分瘢痕疙瘩标本成纤维细胞ｃ－ｆｏｓ染色阳性,提示瘢痕疙瘩组织中血管内皮细胞处于一激活状态     

皮肤肿瘤

20 0 4 2 4 44　端粒酶在瘢痕疙瘩及其周围外观正常皮肤中的表达 /王强 (中国医科院协和医大皮研所 )… //中华皮肤科杂志 .- 2 0 0 ,36 (10 ) .- 589～ 590采用链霉亲和素 -过氧化物酶免疫组化法 ,对 17例瘢痕疙瘩标本、10例瘢痕疙瘩周围外观正常皮肤标本及 9例正常人皮肤标本的成纤维细胞端粒酶的活性进行检测。结果显示在瘢痕疙瘩标本中 ,50 .5%的成纤维细胞中端粒酶活性检出阳性 ,且阳性细胞平均染色较深 ;在瘢痕疙瘩周围外观正常皮肤标本中 ,2 1.1%阳性 ;在正常人皮肤标本中 ,7.9%阳性 ,且阳性细胞平均染色深度明显浅于前两组。各组间…     

Expression of basic fibroblast growth factor and its receptor by fibroblast, macrophages and mast cells in hypertrophic scar.

Basic fibroblast growth factor (bFGF) is a potent mitogenic and chemotactic factor for endothelial cells and fibroblasts. To investigate the pathological role of bFGF in hypertrophic scar, we performed an immunohistochemical study on bFGF and bFGF receptor (bFGF-R) in hypertrophic scar (HS) including keloid, in comparison with normal scar (non-HS) and normal skin. To identify bFGF and bFGF-R positive cells, double immunostaining with antibody to mast cell (MC, tryptase) or tissue macrophage (CD68) was carried out. The expression of bFGF and bFGF-R in cultured fibroblasts from scars was also examined. In HS, many positive cells for bFGF or bFGF-R were observed between collagen bundles in addition to the positive area in normal skin. Although most of the positive cells for bFGF or bFGF-R were fibroblasts, the positive rates of bFGF in macrophages was also increased (p < 0.005). The positive rate of bFGF in MCs and the positive rates of bFGF-R in macrophages and MCs were not changed. No obvious difference was observed between non-HS and normal skin in the expression of bFGF and bFGF-R. Cultured fibroblasts from HS showed a strong nuclear staining of bFGF, but not from non-HS and normal skin. bFGF-R was equally expressed with a diffuse cytoplasmic pattern by fibroblasts from all sources. bFGF may play an important role in the pathological fibrotic process of HS in which fibroblasts are persistently activated. Cellular source of the abnormal bFGF in HS may be both fibroblasts themselves and macrophages.     

Studies of transforming growth factors beta 1-3 and their receptors I and II in fibroblast of keloids and hypertrophic scars

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterized by an abnormal deposition of extracellular matrix components, particularly collagen. There is uncertain evidence that transforming growth factor-beta (TGFss) is involved in keloid formation. Therefore we investigated the expression of TGFss1, 2 and 3 and their receptors in keloids, hypertrophic scars and normal skin. Dermal fibroblasts were obtained from punch biopsies of patients with keloids and hypertrophic scars and from normal skin of healthy individuals. Total RNA was isolated and the expression of TGFss1, 2 and 3 and of TGFss receptors I and II (TGFssRI and II) was analysed by real-time PCR using the Lightcycler technique. Our data demonstrate significantly lower TGFss2 mRNA expression in hypertrophic scar fibroblasts as compared with fibroblasts derived from keloids and normal skin (p<0.05). In contrast, TGFss3 mRNA expression was significantly lower in keloid fibroblasts in comparison with fibroblasts derived from hypertrophic scar and normal skin (p<0.01). TGFssRI mRNA expression was significantly decreased in hypertrophic scar fibroblasts (p<0.01) and TGFssRII mRNA expression was decreased in keloids compared with hypertrophic scar fibroblasts (p<0.001). The ratio of TGFssRI/TGFssRII expression was increased in keloids compared with hypertrophic scar and normal skin fibroblasts. As recently supposed, an increased TGFssRI/TGFssRII ratio could promote fibrosis. Therefore our data support a possible role of TGFssRI and TGFssRII in combination with a certain TGFss expression pattern as fibrosis-inducing factors in keloids.     

Differential effects of hexoses and sucrose, and platelet-derived growth factor isoforms on cyclooxygenase-1 and -2 mRNA expression in keloid, hypertrophic scar and granulation tissue fibroblasts

Abstract Cyclooxygenase (COX) is the key enzyme in the formation of prostaglandins in inflammation. In the present study the effects of biomedically relevant hexose sugars (glucose, fructose, galactose, mannose) and sucrose disaccharide on the expression of COX-1 and COX-2 genes were evaluated in granulation tissue fibroblasts, hypertrophic scar fibroblasts and keloid fibroblasts. The effects of three isoforms (AA, AB and BB) of PDGF on COX gene expression in granulation tissue fibroblasts were also examined. All cell lines expressed COX-1 mRNA, whilst fibroblasts derived from abnormal scars did not express COX-2 mRNA. COX-1 mRNA expression was decreased by sugars in granulation tissue fibroblasts and increased in hypertrophic scar fibroblasts. No major changes were seen in keloid fibroblasts. On the other hand, COX-2 mRNA expression in granulation tissue fibroblasts was decreased dramatically in the presence of fructose, mannose and sucrose and moderately in the presence of galactose. All isoforms of PDGF increased COX-1 and COX-2 mRNA expression in granulation tissue fibroblasts, the most marked increases being elicited by PDGF-BB. All fibroblast cell lines studied expressed the COX-1 gene while the COX-2 gene was not expressed by abnormal scar-derived fibroblasts. Further, granulation tissue fibroblasts seemed to behave differently under the influence of sugars compared to hypertrophic scar fibroblasts whilst keloid fibroblasts seemed to be relatively unaffected by sugars. In addition, the PDGF-BB isoform is a potent inducer of COX-2 gene expression in wound fibroblasts. These findings may be relevant to the development of abnormal scars and indicate the need for further studies. Received: 9 March 2000 / Revised: 28 July 2000 / Accepted: 23 November 2000     

Distinct patterns of collagen gene expression are seen in normal and keloid fibroblasts grown in three-dimensional culture

The purpose of this study was to examine collagen gene expression in various types of scar fibroblasts as well as normal fibroblasts in a novel three-dimensional culture system and to compare them with those in a monolayer culture system. Cells in three-dimensional culture formed multiple layers within the self-produced dense extracellular matrix and formed a dermis-like structure. In monolayer culture, both normal and scar fibroblasts continued to express high levels of mRNA for proα1(I) and proα1(III) collagens. However, in three-dimensional culture, the mRNA levels gradually declined in normal fibroblasts. In contrast, mRNA levels remained high in keloid and hypertrophic scar fibroblasts. Atrophic scar fibroblasts demonstrated similar changes to normal fibroblasts in three-dimensional culture. When we compared mRNA expression in fibroblasts from the centre and the edge of hypertrophic scar, cells from the centre showed a persistently decreased level of collagenase mRNA expression. These results suggest that the mRNA expression pattern of proα1(I) and proα1(III) collagens varies depending on the culture system. Fibroblasts from keloids and hypertrophic scar may have a defective system of down-regulation in extracellular matrix metabolism.     

Gravin在瘢痕疙瘩中的表达

目的 探讨Gravin在瘢痕疙瘩形成中的可能作用机制。方法 荧光定量PCR法检测并比较Gravin在瘢痕疙瘩和正常皮肤中的表达情况,免疫荧光双标法分析Gravin在正常皮肤和瘢痕疙瘩中的定位。 结果 荧光定量PCR结果显示：Gravin在瘢痕疙瘩中的表达量明显降低（0.0953 ± 0.0664）,与正常皮肤相比（0.4565 ± 0.1728）差异有统计学差异（P < 0.01）。免疫荧光双标结果显示：正常皮肤组织中,Gravin主要定位于成纤维细胞;在瘢痕疙瘩中,Gravin定位于巨噬细胞和成纤维细胞,主要位于巨噬细胞。结论 瘢痕疙瘩中Gravin表达量明显下降且主要定位于巨噬细胞。这种表达和定位的改变可能通过影响瘢痕疙瘩中成纤维细胞和炎症细胞的增殖及活化,参与瘢痕疙瘩的形成。     

Insufficient expression of the melanocortin‐1 receptor by human dermal fibroblasts contributes to excess collagen synthesis in keloid scars

Activation of the α‐melanocyte‐stimulating hormone (αMSH)/melanocortin‐1 receptor (MC1R) signalling pathway exerts antagonistic actions on cutaneous inflammatory and fibrogenic responses in addition to promoting pigment production. Herein, the expression of MC1R by keloid‐derived fibroblasts and keloid scar tissue was investigated using a range of techniques. MC1R mRNA expression levels in five different keloid fibroblast cell lines were significantly reduced to less than half compared with five normal fibroblast cell lines (P < 0.05). Immunohistological analysis of tissue samples indicated that MCR1 immunoreactivity in both epidermal and dermal compartments of five keloid tissue samples was dramatically decreased compared with normal skin (P < 0.05). Insufficient expression of MC1R on human dermal fibroblasts might abolish the αMSH‐mediated suppression of collagen production and myofibroblast transformation elicited by the profibrotic cytokine‐transforming growth factor‐β1. Restoration of reduced MC1R by dermal fibroblasts may lead to novel scar‐reducing therapeutic approaches for treating this refractory fibrotic disease.     

Conditioned medium from keloid keratinocyte/keloid fibroblast coculture induces contraction of fibroblast-populated collagen lattices

BACKGROUND: Keloid scars represent a pathological response to cutaneous injury. Overproliferation of fibroblasts and overproduction of collagen characterize these abnormal scars. The pathology of these scars remains poorly understood. The role of epithelial-mesenchymal interactions in keloid pathogenesis and scar contracture has recently been explored. OBJECTIVES: To test our hypothesis that epithelial-mesenchymal interactions play a major role in modulating keloid scar contracture. METHODS: A coculture model was employed wherein keloid and normal keratinocytes were cocultured with keloid or normal fibroblasts, and the conditioned media from day 5 cocultures were collected to study the effect of the paracrine secretions on contraction of an in vitro fibroblast-populated collagen lattice (FPCL) model. RESULTS: Keloid keratinocyte/keloid fibroblast coculture conditioned media brought about increased contraction of the collagen lattice compared with non-cocultured conditioned media. When keloid fibroblasts populated the collagen lattice, significantly increased lattice contraction was induced compared with lattices populated by normal fibroblasts. The addition of antitransforming growth factor (TGF)-beta neutralizing antibody to the conditioned media produced an attenuation of the contraction of the FPCLs. When keloid and normal fibroblasts were cultured on chamber slides and treated with conditioned media from coculture and non-coculture series, immunohistochemical analysis demonstrated an increased expression of alpha-smooth muscle actin (a marker for fibroblast differentiation into myofibroblasts) in fibroblasts exposed to conditioned media from coculture. CONCLUSIONS: These data indicate that epithelial-mesenchymal interactions are likely to play a major role in scar contracture and scar pathogenesis, and underscore the role of TGF-beta1 as a key player in keloid pathogenesis.     

Decorin‐expressing adenovirus decreases collagen synthesis and upregulates MMP expression in keloid fibroblasts and keloid spheroids

Decorin is a natural transforming growth factor‐β1 (TGF‐β1) antagonist. Reduced decorin synthesis is associated with dermal scarring, and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid‐derived fibroblasts (KFs) were transduced with decorin‐expressing adenovirus (dE1‐RGD/GFP/DCN), and we examined the therapeutic potential of decorin‐expressing Ad for treating pathologic skin fibrosis. Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1‐RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR) and secreted TGF‐β1 were assessed by Western blotting and ELISA. And type I and III collagen, and matrix metalloproteinase‐1 (MMP‐1) and matrix metalloproteinase‐3 (MMP‐3) mRNA levels were measured by real‐time RT‐PCR. Additionally, we immunohistochemically investigated the expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1‐RGD/GFP/DCN. Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1‐RGD/GFP/DCN, secreted TGF‐β1 and EGFR protein expressions were decreased in TGF‐β1‐treated HDFs and KFs. Also, type I and III collagen mRNA levels were decreased, and the expression of MMP‐1 and MMP‐3 mRNA was strongly upregulated. In addition, the expression of type I and III collagen, fibronectin and elastin was significantly reduced in dE1‐RGD/GFP/DCN‐transduced keloid spheroids. These results support the utility of decorin‐expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids.