生存素在瘢痕疙瘩组织中的表达及其意义

目的 探讨生存素在瘢痕疙瘩组织中的表达及在瘢痕疙瘩发病中的作用。方法 采用免疫组化SABC法，检测25例瘢痕疙瘩（病例组）及15例正常皮肤（对照组）组织内生存素的表达情况。结果 25例瘢痕疙瘩组织中生存素表达阳性20例，阳性率80.0%，表达部位主要在成纤维细胞和血管内皮细胞内，正常皮肤组织内均表达阴性，两组比较差异有统计学意义（χ2 ＝ 24.00，P < 0.01）。瘢痕疙瘩临床分级低度、中度、重度生存素表达阳性率分别为57.14%（4/7）、81.81%（9/11）、100%（7/7），差异无统计学意义（P ＝ 0.133）；瘢痕疙瘩复发组与未治疗组生存素表达阳性率分别为90.91%（10/11）、71.43%（10/14），差异亦无统计学意义（P ＝ 0.341）。结论 生存素可能参与瘢痕疙瘩的发病。     

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。     

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。     

晚期糖基化终末产物及其受体在瘢痕疙瘩中的表达

目的 研究晚期糖基化终末产物（AGE）及其受体（AGER）在瘢痕疙瘩中的表达。方法 瘢痕疙瘩、增生性瘢痕和正常人群血清、皮肤组织标本各20份,以荧光分光光度计检测三组人群血清中AGE含量,分别采用免疫组化法、Western印迹分析检测三组人群皮肤组织标本中AGE和AGER表达情况。结果 瘢痕疙瘩组血清中AGE含量为（0.713 ± 0.098） AU/ml,增生性瘢痕组为（0.699 ± 0.077） AU/ml,明显高于正常人群组（0.179 ± 0.056） AU/ml,三组差异有统计学意义（F = 283.82,P < 0.01）。免疫组化显示,AGE、AGER在瘢痕疙瘩、增生性瘢痕皮肤组织标本中表达阳性,正常人群组织表达则为阴性。Western印迹检测显示,瘢痕疙瘩、增生性瘢痕组织中AGE和AGER蛋白表达均明显高于正常人群,差异有统计学意义（F值分别为18.04、42.80,P < 0.05）,而瘢痕疙瘩和增生性瘢痕组之间差异无统计学意义（P > 0.05）。结论 AGE和AGER在瘢痕疙瘩中表达升高,在其发病过程中可能发挥一定的促进作用。     

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.     

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。     

积雪苷软膏对BALB/C裸小鼠瘢痕疙瘩模型成纤维细胞及胶原蛋白表达的影响

目的：明确外用积雪苷软膏对瘢痕疙瘩裸鼠模型的成纤维细胞和胶原蛋白表达的影响。方法：取手术切除的瘢痕疙瘩组织植入16只裸鼠肩胛及骶尾部，建立瘢痕疙瘩裸鼠模型。随机分为A组（外用对照软膏基质），B组（外用0.00125%积雪苷软膏），C组 （外用0.0025%积雪苷软膏）和D组（外用0.005%积雪苷软膏），每组4只。观察瘢痕疙瘩大体形态学及光镜下变化；免疫组化检测瘢痕疙瘩I型胶原蛋白表达的影响；透射电镜观察瘢痕疙瘩成纤维细胞结构的变化。结果：大体观察裸鼠瘢痕疙瘩经不同浓度积雪苷软膏作用后组织块体积均变小，光镜观察成纤维细胞数明显减少。A～D组I型胶原蛋白的表达率分别为79.17%、70.83%、37.50％、33.33%，四组比较，差异具有统计学意义（Fisher精确概率检验法，P<0.05）。透射电镜观察B-D组瘢痕疙瘩成纤维细胞较A组明显减少，可见凋亡细胞，细胞核固缩，膜结构不清，有坏死的细胞碎片及裸核状态的成纤维细胞。结论：外用积雪苷软膏能减少瘢痕疙瘩成纤维细胞并下调I型胶原蛋白的表达。     

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。     

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     

非标记定量技术研究瘢痕疙瘩的蛋白质组学特点

目的 联合应用毛细管高效液相色谱与串联质谱技术,寻找瘢痕疙瘩组织表达的差异蛋白,探索其蛋白质组学特点。方法 选取非家族性瘢痕疙瘩患者病变组织为实验组,以正常皮肤组织为对照,分别提取两组蛋白质,经溶液内酶解获得肽段,应用液相色谱串联质谱鉴定和DeCyder MSTM软件进行非标记定量和蛋白质差异统计分析。结果 对比分析瘢痕疙瘩和正常皮肤组织,共鉴定到570个有显著性差异的肽段（P < 0.05）,对应1221个差异表达的蛋白质,其中差异表达超过1.5倍的蛋白质293个,124个在瘢痕疙瘩组织中表达上调,169个表达下调。结论 非家族性瘢痕疙瘩患者病变组织与正常皮肤组织之间存在蛋白质表达差异。     

结缔组织生长因子在瘢痕疙瘩中表达的研究

目的探讨结缔组织生长因子(CTGF)在瘢痕疙瘩发病中的作用。方法应用半定量逆转录聚合酶链反应技术检测30例瘢痕疙瘩患者皮损及对应邻近未受累皮肤中CTGFmRNA的表达,并以15例正常人皮肤组织作为对照。同时应用SP免疫组化技术对5例瘢痕疙瘩组织和5例正常人皮肤标本进行了检测。结果CTGFmRNA在瘢痕疙瘩及其边缘正常皮肤中的表达均明显高于正常人对照,差异有显著性(P<0.01)。瘢痕疙瘩CTGF的表达高低与病程无相关性(P>0.05)。免疫组化研究证实CTGF在瘢痕疙瘩中呈强表达,而在正常人皮肤中无表达。在瘢痕疙瘩组织边缘,CTGF表达呈现由强至弱的过渡现象。结论CTGF在瘢痕疙瘩中持续高度表达,提示其与瘢痕疙瘩的慢性纤维化有关。CTGF可能成为临床治疗瘢痕疙瘩的一个有力靶位。     

端粒酶在瘢痕疙瘩及其周围外观正常皮肤中的表达

目的 探讨端粒酶的活性与瘢痕疙瘩发病的关系。方法 采用链霉亲和素-过氧化物酶(SP)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例正常人对照组标本的成纤维细胞中端粒酶的活性进行检测,用SPSS统计软件进行相应统计学分析。结果 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,21.1%的成纤维细胞中端粒酶活性检出阳性;在正常人皮肤标本中,7.9%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深度明显浅于前两组。各组间两两比较,差异均有显著性(P<0.05)。结论 端粒酶的活性增高在瘢痕疙瘩的发病机制中有重要作用。     

Clinical genetics of familial keloids.

BACKGROUND: Keloids are proliferative fibrous growths that result from an excessive tissue response to skin trauma. Most keloids occur sporadically, but some cases are familial. However, the genetics of keloid formation have only rarely been documented, and the mode of inheritance is not known. OBJECTIVE: To elucidate the clinical genetic characteristics of keloid wound-healing disorder. OBSERVATIONS: We studied the clinical and genetic characteristics of 14 pedigrees with familial keloids. The ethnicity of these families is mostly African American (n = 10), but also white (n = 1), Japanese (n = 2), and African Caribbean (n = 1). The pedigrees account for 341 family members, of whom 96 displayed keloids. Of the affected family members, 36 are male and 60 are female. The age of onset varies from early childhood to late adulthood. There is variable expression of keloids within the same families: some affected members have only minor earlobe keloids, whereas others have very severe keloids affecting large areas of the body. In the described pedigrees, 7 individuals are obligate unaffected carriers, revealing nonpenetrance in about 6.8% of keloid gene carriers. Syndromes associated with keloids, namely Rubinstein-Taybi and Goeminne syndrome, were not found in these families. Additionally, linkage to the gene loci of these syndromes and X-chromosomal linkage were excluded. CONCLUSIONS: The pattern of inheritance observed in these families is consistent with an autosomal dominant mode with incomplete clinical penetrance and variable expression. This is the most comprehensive collection of keloid families described to date, and it allows for the first time the elucidation of the clinical genetic characteristics of the familial form of this wound-healing disorder.     

Upregulation of the NNP-1 (novel nuclear protein-1, D21S2056E) gene in keloid tissue determined by cDNA microarray and in situ hybridization

BACKGROUND: A keloid results from excessive collagen deposition, the cause of which remains elusive. A thorough understanding of the pathophysiology of keloid tissue can help determine the most appropriate treatment strategy. OBJECTIVES: To assess the differences in gene expression between keloids and adjacent normal skin in order to define the genes involved in keloid formation. METHODS: Three Korean patients with keloids underwent excision of the keloid and adjacent normal skin, which was used as the control. We investigated expression patterns of genes in the keloids and the normal skin using cDNA microarray and in situ hybridization techniques. RESULTS: Nine genes in the keloid tissue were consistently upregulated over the 2.0 ratio compared with the normal control from the cDNA microarray composed of 3063 clones: collagen type I alpha1 (NM_000088), DNA segment on chromosome 21 (unique) 2056 expressed sequence (D21S2056E, NNP-1, NM_003683), suppressor of Ty 5 homologue (NM_003169), phosphoglycerate dehydrogenase (NM_032692), adenosine triphosphate synthase beta (NM_001686), serine (or cysteine) proteinase inhibitor, clade H (heat shock protein 47, NM_001235), LIV-1 protein, oestrogen regulated (LIV-1, NM_012319), interleukin-11 receptor alpha (IL11RA, NM_004512) and carbonyl reductase 3 (CBR3, NM_001236). From the in situ hybridization study, the staining signals in the keloid tissue hybridized with anti sense probes of NNP-1 mRNA were stronger than signals in normal controls. Further, endothelial epithelium, but not the epidermis, expressed the signal equally in both keloid and normal control tissue. CONCLUSIONS: We identified nine upregulated genes in keloid tissue using cDNA microarray. Of the nine, the NNP-1 gene was confirmed by topological information using the in situ hybridization technique. We conclude that these nine genes, especially NNP-1, probably contribute either directly or indirectly to keloid formation.     

纤连蛋白及其额外结构域A和B在瘢痕疙瘩中的表达

目的研究瘢痕疙瘩及其成纤维细胞中的纤连蛋白(FN)及额外结构域A(EDA)、额外结构域B(EDB)的表达,探讨其在瘢痕疙瘩中的作用。方法使用蛋白质印迹分析及免疫组织化学等方法检测瘢痕疙瘩组织内及成纤维细胞中FN、FN-EDA、FN-EDB的含量。结果蛋白质印迹分析及免疫组织化学染色方法均证明FN、FN-EDA、FN-EDB在瘢痕疙瘩组织中增高明显,且FN-EDA、FN-EDB表达位置与FN差异有统计学意义(P<0.05)。结论瘢痕疙瘩中过表达的FN、FN-EDA、FN-EDB在瘢痕疙瘩形成过程中可能具有不同的促进作用。     

Elevated prolidase activity in keloids: correlation with type I collagen turnover

BACKGROUND: Keloid pathogenesis involves an altered balance of extracellular matrix metabolism, mainly accumulation of type I collagen. This could be due to excessive synthesis or decreased degradation of matrix, or a combination of both processes. Prolidase, an imidodipeptide-cleaving cytosolic enzyme, plays an important role in the collagen catabolic process by recycling proline for collagen synthesis. Collagen accumulation in keloids is due to an imbalance in the steady state of collagen turnover. OBJECTIVES: To investigate prolidase activity and its role in the steady state of collagen turnover between normal skin and keloid tissue and their derived fibroblasts. METHODS: Ten sets of keloid and normal skin tissues and their derived fibroblasts were employed. Measurements were made of tissue prolidase activity, free proline level, and concentrations of the collagen synthesis product aminoterminal propeptide of type I procollagen (PINP) and the collagen degradative product carboxyterminal telopeptide of type I collagen (ICTP). Also, synthesis of collagens type I and III and matrix metalloproteinases 1 and 2 was investigated using Western blot analysis. RESULTS: Keloid tissues had a significant increase in prolidase activity, up to fourfold that in normal skin. The elevated prolidase activity was accompanied by an increase in tissue PINP and ICTP concentrations in keloid; in addition, the collagen turnover index (PINP/ICTP) was higher in keloids. CONCLUSIONS: The combination of elevated prolidase activity and associated higher collagen synthesis to degradation ratio in keloids suggests a possible metabolic process for the excessive accumulation of type I collagen in keloids.     

Role of vascular endothelial growth factor in keloids: a clinicopathologic study

Background  Despite their benign nature, keloids are usually associated with considerable cosmetic effects and may lead to functional problems. Recently, it has been reported that vascular endothelial growth factor (VEGF), a potent angiogenic factor, is overexpressed in keloid tissue and may have a potential role in its evolution.
Methods  Twenty patients with keloids were included in this study and classified into two groups according to the treatment received: intralesional triamcinolone acetonide 20 mg/mL (group 1) and cryotherapy spray technique (group 2). Treatment was continued until clearance or for a maximum of six sessions, and the follow-up period was 1 year. Skin biopsies were taken from patients before and after treatment to evaluate keloid pathology and from patients and 10 healthy controls to detect the immunohistochemical expression of VEGF.
Results  Histopathologic examination revealed a remarkable resolution of the nodular arrangement of collagen after therapy, particularly in group 1. A statistically significant difference in VEGF expression was found between patients before therapy and controls, and between patients before and after therapy in each group. There was no significant difference in the treatment outcome between intralesional steroids and cryotherapy. No significant correlation was observed between the clinical variables of keloids and both VEGF expression and clinical response to therapy.
Conclusion  VEGF seems to play an important role in the pathogenesis of keloids and may be a useful guide in the evaluation of keloid therapeutics. Modulation of its production may provide a valuable treatment for keloids.     

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.