Duffy抗原趋化因子受体（DARC）在瘢痕疙瘩成因中的作用
摘要
一、研究背景及目的
瘢痕疙瘩是一类创伤异常愈合所致的疾病,主要以纤维组织异常增殖以及胶原纤维过量沉积为主要标志,通常在创伤后持续存在,不能自行消退,且常超过原创伤范围,侵犯周围正常的皮肤组织。瘢痕疙瘩不仅会导致持续的瘙痒以及疼痛等症状,还会导致功能异常甚至畸形,严重影响外观,给患者带来严重的心理及生理的困扰。因此,瘢痕疙瘩是整形外科面临的重大难题之一。但由于瘢痕疙瘩形成的病因尚不十分明确,对于瘢痕疙瘩的预防及治疗方案也尚难统一。所以,找寻瘢痕疙瘩的主要致病因子是诊断及治疗瘢痕疙瘩的关键。
目前针对瘢痕疙瘩的研究多集中在以下3个方面：(1)细胞外基质蛋白的沉积以及降解,(2)细胞因子和生长因子,(3)凋亡通路。
我们既往的研究已经对凋亡通路有了一定的探索,但仍然未能找到瘢痕疙瘩形成的关键原因。鉴于各种细胞因子与瘢痕疙瘩形成关系密切,因此我们针对细胞因子中的趋化因子及其受体进行了新的探索。趋化因子是一类与炎症介质密切相关的小分子蛋白质家族,与多种器官的炎症反应,肿瘤转移还有组织纤维化密切相关。趋化因子可促进炎症反应,血管形成,协助白细胞从血液循环进入组织,在炎症反应中,趋化因子可为细胞提供相互联系,趋化因子信号转导是炎症细胞从血管中迁移粘附血管壁的关键因素。
关于趋化因子与瘢痕疙瘩的研究已经展开。MGSA/GRO α以及其受体CXCR2仅在瘢痕疙瘩组织中检测到表达,而增生瘢痕和正常上皮组织中则没有检测到其表达。并且其免疫组化的染色阳性率与该区域炎症反应的程度呈正相关。此外还有报道关于MCP-1及其受体CCR2过度活跃与各种纤维化的病理过程密切相关,并且在多种纤维化疾病中起着关键作用。而趋化因子需与其相应受体结合而发挥作用。
DARC(Duffy antigen receptor for chemokines; Dufy antigen/receptor for chemokines, DARC)即达菲抗原受体或称达菲抗原/趋化因子受体,是一个重要的趋化因子诱骗受体,该受体首先是在血友病患者中发现。它是红细胞膜上的糖蛋白,能够结合CC族和CXC族趋化因子的杂性趋化因子受体。作为典型的趋化因子诱骗受体,DARC只与具有促进血管生成作用的趋化因子结合,由于缺乏位于第2个胞内环上的与G蛋白偶联受体相似的7次跨膜糖蛋白,与配体结合后并不引起细胞信号转导和细胞代谢的改变。有研究发现除了在红细胞上有表达外,DARC同样也在内皮细胞、淋巴结、淋巴管上皮细胞、血管静脉和肺、肾上皮细胞中同样有表达。DARC在炎症反应中发挥着重要的作用,主要通过对其受体的调节,调控白细胞的归巢,影响炎症反应,炎症反应可以上调DARC的表达,而DARC的表达也可以对炎症反应进行调控。DARC在不同时期发挥的不同作用主要包括：1)趋化因子释放入血；2)血液中趋化因子的改变；3)血浆中趋化因子的转录后调控；4)趋化因子相关的DARC开／关比率；5)依赖DARC的转运机制。
DARC的表达还与许多肿瘤呈负相关,如前列腺癌、非小细胞肺癌、乳腺癌以及食管癌中都发现DARC高表达患者其预后优于低表达者。尽管瘢痕疙瘩是一种良性的上皮纤维异常增殖性肿瘤,没有恶性征象,但瘢痕疙瘩的某些特征,包括持续性生长,超出原有创伤范围,切除后容易复发等,又与恶性肿瘤有一定相似性。至今尚未有关于DARC与瘢痕疙瘩相关性,以及其在瘢痕疙瘩形成过程中所占的地位的报道,鉴于DARC对趋化因子调节的重要地位和其在炎症反应,以及肿瘤发生发展中所起的作用,瘢痕疙瘩的部分恶性肿瘤征象等理由,我们猜想DARC的表达可能与瘢痕疙瘩的形成有着一定的相关性。若能弄清DARC在瘢痕疙瘩形成中的作用,势必为瘢痕疙瘩的治疗和预防打开新的思路。
为了研究DARC在瘢痕疙瘩和正常皮肤中的表达情况及其在瘢痕疙瘩形成中的作用与机制,我们首先对不同的人瘢痕疙瘩和正常皮肤进行了检测,以确定它们表达DARC的情况。并且分离培养了部分瘢痕疙瘩患者和正常皮肤组织中的成纤维细胞,对其中DARC的mRNA含量及蛋白含量进行分析。此外还对其部分受体(?)IL-8,MCP-1以及与瘢痕疙瘩胶原沉积异常相关的MMP-2在瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞中的表达进行了研究,并且观察在DARC表达改变的情况下这些受体及蛋白的表达情况,还对DARC是否影响瘢痕疙瘩成纤维细胞的增殖和细胞周期,及其迁移的影响进行了研究。本研究旨在为发现趋化因子受体在瘢痕疙瘩成纤维细胞中的表达及其对成纤维细胞生长、迁移的影响,并深入研究其机制,对瘢痕疙瘩的成因及从根本上治疗瘢痕疙瘩提供理论依据。
二、材料与方法
材料：
收集2007年1月至2012年广东省中医院和南方医院病理确诊瘢痕疙瘩标本30例,以及正常对照皮肤30例。本研究事先得到南方医院相关审核部门的同意,所有研究对象签署知情同意书。指定2名对瘢痕疙瘩诊断和治疗具有丰富临床经验的整形外科医师,根据瘢痕疙瘩诊断标准,对所有研究对象进行全面的体格检查、详细的病史询问和准确的临床诊断,填写个人临床资料登记表,并严格遵照赫尔辛基宣言的规定采集他们的组织。
方法：
2.1瘢痕疙瘩中DARC表达的检测
将组织用石蜡包埋后切成石蜡切片,经过免疫组化染色,检测组织中DARC的表达情况。检测新鲜瘢痕疙瘩及正常皮肤中DARCmRNA及蛋白含量。
2.2细胞原代培养
将3例瘢痕疙瘩和3例正常皮肤组织进行体外细胞培养,培养出相应的成纤维细胞。将传代小于15代的成纤维细胞用于以下实验。
2.3运用siRNA干扰DARC表达
取处于对数生长期的瘢痕疙瘩成纤维细胞接种于六孔板中,待其长至板底的60%-80%时依据转染试剂所提供步骤,各孔分别用细胞培养液+转染溶液；control siRNA+转染溶液,以及DARC siRNA+转染溶剂进行转染,转染后24h及48h分别检测DARC mRNA及其蛋白的表达,并进行其余相关实验。
2.4细胞增殖影响
将瘢痕疙瘩成纤维细胞按5000个/孔的浓度将处于对数期的细胞种至96孔板,基础培养基100m1,每孔设3个对照；分别在12h,24h,48h,72h对细胞增殖进行检测；在每孔细胞中加入10μCCK-8溶液,细胞培养箱内继续孵育1.5h；MD5多功能酶标仪在450nm测定吸光度。
2.5细胞周期检测
将细胞计数1×105/瓶的NF(正常皮肤成纤维细胞组),KFC(瘢痕疙瘩成纤维细胞空白对照组),KFP(瘢痕疙瘩成纤维细胞阴性对照组),KFS(瘢痕疙瘩成纤维细胞干扰组)细胞在转染后24h进行细胞周期检测我们通过流式细胞仪对细胞周期的变化进行检测。
2.6细胞迁移的改变
我们通过划痕实验和transwell实验对正常皮肤成纤维细胞以及转染后的瘢痕疙瘩成纤维细胞进行检测,观察其迁移能力和增殖能力改变的情况。
2.7细胞上清液中蛋白的检测
我们通过ELASA检测正常皮肤成纤维细胞以及转染后的瘢痕疙瘩成纤维细胞中IL-8, MCP-1和MMP2蛋白的表达及变化情况。
三、结果
3.1DARC为胞膜和细胞浆着色,在正常皮肤及瘢痕疙瘩组织的阳性染色组织中,均可见部分染色的成纤维细胞的细胞浆及细胞膜。对比30例瘢痕疙瘩和正常皮肤组织中成纤维细胞DARC表达情况,发现瘢痕疙瘩成纤维细胞的DARC高表达率高于正常皮肤组织。
3.2通过进一步对3例瘢痕疙瘩组织和3例对照正常皮肤组织中DARC RNA以及蛋白的检测,同样发现瘢痕疙瘩组织中DRAC含量在mRNA水平与蛋白水平均高于正常皮肤。在对体外培养的3组瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞进行DARC RNA及蛋白检测后,发现其DARC表达与组织中的表达规律基本一致。
3.3转染后的细胞生长曲线测定比较,发现转染DARC siRNA对细胞增殖并无明显影响,并且瘢痕疙瘩与正常细胞成纤维细胞生长增殖无显著差异。流式细胞仪检测发现DARC siRNA转染对细胞周期无明显影响,此外瘢痕疙瘩成纤维细胞的迁移能力弱于正常皮肤来源的成纤维细胞,而干扰DARC后瘢痕疙瘩成纤维细胞的迁移能力有了一定提高,但仍稍低于正常皮肤成纤维细胞。
3.4DARC表达减低对IL-8, MCP-1, MMP-2mRNA的表达无明显影响。DARC表达减少可以增加MCP-1蛋白的分泌。我们研究发现DARC的表达高低对IL-8以及MCP-1mRNA的表达无明显影响。而当对DARC进行干扰后,我们发现与对照组相比,MCP-1在细胞上清中的含量增加,但仍稍低于正常皮肤来源的成纤维细胞。而DARC干扰对细胞上清液中IL-8以及MMP2蛋白的含量无明显影响。
四、讨论
我们的研究结果显示,虽然有一定的恶性肿瘤表型,但与恶性肿瘤不同的是,DARC在瘢痕疙瘩组织中无论在蛋白水平还是在转录水平均高于正常皮肤组织。说明瘢痕疙瘩的形成过程与恶性肿瘤细胞不同,不是各种致瘤因素所致,可能与伤口的异常愈合相关。创伤愈合需要复杂的细胞间相互作用,以及其调控因子的调节。组织创伤修复机制的变化受制于皮肤创伤的愈合。瘢痕疙瘩中常出现细胞迁移,增殖,炎症反应,细胞外基质蛋白与细胞因子的合成及分泌异常。
炎症反应是创伤愈合过程的组成部分,一方面可以作为抗感染的免疫屏障,另一方面可以导致纤维增生,闭合创面。趋化因子在创伤愈合过程中的炎症反应和组织修复中都发挥着重要的作用。而皮肤由于感染等外在因素导致的慢性炎症反应常常是瘢痕疙瘩形成的重要诱因。
趋化因子对白细胞聚集的有效调控的关键在于对其运动的趋化调节,即在准确的时间到达准确的位置,并且对其的功能在一定的时间和范围进行有效控制。趋化因子是通过与其受体的相互作用对白细胞产生趋化和激活作用,从而发挥其在炎症反应中的重要功效。瘢痕疙瘩中DARC表达改变,对成纤维细胞的的生长增殖无明显影响。而瘢痕疙瘩成纤维细胞体外培养伤口愈合能力弱于正常皮肤,这也是瘢痕疙瘩伤口愈合异常的表现。在对DARC进行干扰后,成纤维细胞的迁移能力有一定提高。说明DARC可能通过对其相应的配体的结合,而影响成纤维细胞的迁移,导致伤口延迟愈合。
在对DARC相应配体及MMP-2的检测中,我们发现瘢痕疙瘩成纤维细胞中IL-8, MCP-1, MMP-2的mRNA含量高于正常皮肤成纤维细胞,但仅有MCP-1在细胞上清中的表达低于正常皮肤成纤维细胞,并且MCP-1的表达受DARC的调控。在组织创伤后的炎症反应中,趋化因子的主要作用是诱导炎症因子在合适的试剂到达合适的位置,为了达到这样的效果,趋化因子的调控必须精确。炎症反应区域趋化因子及其受体的表达上调对于局部炎症细胞的快速聚集有重要意义。我们认为在瘢痕疙瘩成纤维细胞中,DARC可能通过与MCP-1的结合,引起MCP-1的分泌减少。导致局部炎症反应不平衡,形成慢性炎症反应。而DARC并不在基因水平上影响其配体的表达,DARC对其配体的下调作用是一种翻译后的调控因素,是发生在蛋白质水平的,而这种作用并不影响细胞内的信号通路,不影响细胞的增殖等生物学行为,是一种趋化因子的翻译后调控。
本实验首次对诱骗受体DARC在瘢痕疙瘩中的表达,及其对瘢痕疙瘩成纤维细胞生长、迁移的影响进行了研究,并初步探讨了相关机制,为瘢痕疙瘩的病因研究打开了新的思路,也为进一步研究趋化因子及其受体在瘢痕疙瘩中形成中的作用奠定了一定的实验基础。
五、结论
5.1.与恶性肿瘤细胞DARC表达相反,DARC在瘢痕疙瘩组织成纤维细胞中表达高于正常皮肤组织的成纤维细胞；
5.2.DARC并不对瘢痕疙瘩成纤维细胞的生长增殖产生影响,但我们的研究发现DARC可能会抑制瘢痕疙瘩成纤维细胞的迁移,造成创口愈合异常,对其进行干扰后瘢痕疙瘩成纤维细胞的迁移能力有一定提高。
5.3.DARC可能通过某种转录后调控的方式,抑制MCP-1的分泌,使成纤维细胞MCP-1分泌减少,由于DARC表达异常,导致其相关趋化因子调控异常,造成炎症反应不平衡,形成持续的炎症反应,导致组织损伤和慢性炎症形成,是瘢痕疙瘩形成的可能因素。
1. Background and Objective
Keloids result from alterations in the normal process of wound healing, which is characterized with excessive growths of fibrous tissue and accumulation of unmature collagen, it frequently persists at the site of injury, don't regress all the time; often recurs after excision, and always overgrows the boundaries of the original wound manifested by invasion of clinically normal skin. Keloid not only could results in the malformation and dysfunction which impact the outlook, but also usually occur pruritus and ache symptoms, which cause much physical and psychological anguish on patients with keloids. Therefore, keloid has been regarded as one of the intractable problem to plastic surgery. Due to the unknown etiology of the causes for keloid formation, there is still no specific diagnostic method and valid therapeutic measure in clinic up to now. It is thus clear that to identify etiological factors for keloid will be the key to diagnose exactly and treat effectually it.
The molecular aberrances in keloids that correlate with the molecular mechanisms in normal wound healing can be categorized into three groups:(1) extracellular matrix proteins and their degradation,(2) cytokines and growth factors, and (3) apoptotic patheways.
We have aim to elucidate its possible mechanisms in apoptotic patheways, however, we haven't found the key etiology of the causes for keloid formation. Considering the relationship between cell factor and keloids, we decide to find the correlation between chemokines and the keloids. Chemokines and their receptors are growing family of inflammatory molecules that are associated with many tissue specific inflammatory event, cancer metastasis and fibrosis. Chemokines promote inflammation, angiogenesis, facilitate the passage of leukocytes from circulation into the tissue, and contribute to the regulation of epithelialization. They integrate inflammatory events and reparative processes that are important for modulating wound healing.
There have been many reports about the association of chemokin and the keloids. MGSA/GRO a, and its receptor, CXCR2, were found not observed in hypertrophic scars or normal dermis, but was present in some myofibroblasts in nodular areas of the keloid samples. And the staining positively correlated with the degree of inflammatory infiltrate in the lesions. The enhanced MCP-1and CCR2in the keloid tissues augments fibroblast proliferation has also been reported.
The Duffy antigen is an antigenic determinant of the Duffy blood group system and is the receptor exploited by the malaria parasite Plasmodium vivax for its entry into human red blood cells. The Duffy receptor is a typical decoy receptor referred to as Atypical Chemokine Receptors (ACRs), interceptors, or chemokine 'decoy' receptors, which selectively binds CXC and CC chemokines with high ffinity. Inflammation can further up-regulate DARC expression in postcapillary venules and veins, and induce DARC to appear in vascular segments. As well as red blood, DARC is also espressed on a subset of endothelial cells, lymph node, lymphatic endothelial cells, venules of the skin, and epithelium in lung and kidney collecting ducts. Thus, DARC on human rbc influences infectious disease and leukocyte homeostasis. The precise role of DARC at a given time will therefore be influenced by a host of variables, including(i) chemokine release into the blood,(ii) the diversity of the blood chemokine repertoire,(iii) post-translational modifications of plasma chemokines,(iv) the relative DARC on/off rates of these chemokines, and (v) the effectiveness of DARC-independent removal mechanisms.
In addition, the expression of DARC is a negative regulator of many cancers, such as prostate cancer, NSCLC, breast cancer, and laryngeal squamous cell carcinoma.
Although keloids behave as benign dermal fibroproliferative tumors lacking malignant potential, it shares many characteristics, including persistent growth and the tendency to recur after excision. So far, the role of DARC during keloid progression, especially the relationship between DARC and keloid has not been investigated. Based upon these findings, we proposed that the DARC expression might be associated with keloid.
To test this hypothesis we examined the expression of the DARC in keloid lesions as compared to normal skin, as well as the expression of the chemokines such as IL-8,,MCP-1in cultured fibroblasts from normal skin and keloid lesions. The effects of DARC on the growing and moving of fibroblasts from keloid was also investigated.
2. Materials and Methods
Actively growing keloid tissues (N=30) and normal skin (N=30) were collected from patients undergoing elective excision of keloids, abdominoplasty, skin grafting, and circumcision procedures. Tissue samples were obtained in accordance with procedures approved by the Institutional Review Board of the Nanfang Hospital, Southern Medical University in advance. The all of informed consent form (ICF) from the patients have been obtained. None of the keloids had received corticosteroid injection within a one-year period and all were removed from the truncal region..
2.1DARC expression detection
After processed the tissues through paraffin embedding and cut into slices, the immunohistochemical staining method was exerted to observe the expression of DARC. We also detected the mRNA and protein of DARC in keloids.
2.2Cell cultures
Keloid tissue obtained from patients undergoing keloid excision(N=3) and normal skin(N=3) obtained during surgery were collected.After dissected and cultred the fibroblasts in vitro, the cultured fibroblasts of less than15generations were used for the experiments.
2.3Transfection of siRNA plasmids
Duffy siRNA and control siRNA were transfected into target cells. After24hours and48hours of transfection, real-time PCR and western blot analyses were performed to assess the knockdown of the target gene.
2.4Cell proliferation
For determination of cell proliferation and viability, skin fibroblasts were cultured in a96-well microtiter plate. After reaching80%confluence, cultured fibroblasts were washed twice with PBS and incubated in DMEM containing10%FBS for a time period of12h,24h,48h and72h, respectively. The determination of cell proliferation was then performed with Cell Counting Kit-8(Dojindo) according to instructions provided by the manufacturer.
2.5cell cycle analysis
24h after the transfection, we use Flow Cytometry to detecte the change of cell cycle. NF(fibroblasts from normal skin), KFC (fibroblasts from keloid tissue), KFP (fibroblasts from keloid tissue transfected with control siRNA), KFS (fibroblasts from keloid tissue transfected with DARC siRNA).
2.6Shift assay
Shift experiments were conducted with a Matrigel invasion chamber. The bottom chamber contained10%FBS-supplemented medium as a chemoattractant. Keloid fibroblast cells were transfected with DARC siRNA and control siRNA respectively were plated in the upper chambers and incubated at37℃for48hours. The cell suspension was aspirated using a cotton swab. Then, the filters were fixed in10%formalin solution and stained with H&E. Cells that had invaded through the filter and reached the lower surface of the filter were counted under a light.
2.7Enzyme linked immunosorbent assay analysis
The protein level of human IL-8, MCP-1and MMP-2present in conditioned cell supernatants were determined using a sandwich enzyme linked immunosorbent assay (ELISA) kit.
3. Results
3.1Immunostaining for DARD in keloids and normal skin
To study the relation between DARC and keloids in the clinical setting, tissue slices of60patients'samples(30keloid tissues and30normal skin) were tested with DARC antibody. Our results revealed the expression of DARC in fibroblasts was higher in keloid tissue than that of normal skin.
3.2Expression of DARC in human fibroblasts
DARC expression in human fibroblasts (3normal and3keloid fibroblasts culltures) was determined utilizing reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. W e found that keloid fibroblasts expressed relative high DARC levels, while normal fibroblasts had a significant lower DARC expression.
3.3Knockdown of DARC using siRNA has no effect on proliferation, and chemokines gene expression
To investigate whether the change of DARC expression could modulate the proliferation of keloid fibroblasts in vitro, we assessed the growth of DARC siRNA and mock-transfected keloid fibroblasts. Knokdown of DARC had no effect on the proliferation of DARC siRNA-transfected keloid fibroblast when compared to mocktransfected and wild-type cells. As well as the DARC si has no effect on cell cycle. In addition, DARC expression had no effect on CXCL8(CXC chemokine ligand8, interleukin-8, IL-8), CCL2(CC chemokine ligand2, monocyte chemoattractant protein-1, MCP-1), MMP2(matrix metalloproteinase2) mRNA expression.
3.4Knockdown of DARC using siRNA enhance chemokines CCL2on protein level in vitro
The DARC functions as a promiscuous chemokine decoy receptor. Therefore, it is interesting to investigate the influence of high expression of DARC on the secretion of chemokines in this system. There were no significant differences of mRNA expression of CXCL8and CCL2in DARC, Mock-transfected and control fibroblasts. However, the CCL2level detected in conditioned media from the fibroblasts was markedly increased in the DARC siRNA-tansfected fibroblasts as compared to the control fibroblasts. In contrast, the DARC expression had no effect on CXCL8and MMP2levels in conditioned media from the fibroblasts
3.5Knockdown of DARC using siRNA enhance migrational property of the fibroblast
We then examined the migrational activities of DARC siRNA-transfected keloid fibroblasts using the Boyden Chamber. Knockdown of DARC increased the migration of keloid fibroblasts.
4. Discussion
Our research showed that although with some malignant character, the DARC expression in keloids still different from malignant tumor. DARC mRAN and protein were high expression in keloid tissues than in the normal skin. These results may indicate that the formation of keloids is different from malignant tumor. The keloids formation may result from abnormal wound healing, but not the tumorigenesis. Wound healing requires a complex series of reactions and interactions among cells and their mediators. The altered tissue repair mechanism appears to be restricted to dermal wound healing. Abnormalities in cell migration, proliferation, inflammation, synthesis and secretion of extracellular matrix proteins and cytokines, and remodeling of the wound matrix have all been described in keloids.
Inflammation is an important part of wound healing. One hand, it can build the Immunologic barrier to defect the infection. Another hand, it can cause the proliferation of fibers. Chemokines play an important role in inflammation and tissue repair. Infection leading to chronic inflammation in the skin could predispose to keloid formation.
The efficient regulation of leucocyte recruitment by chemokines requires their appropriate localization in functional micro-anatomical domains, as well as setting limits to their effects in space and time. Binding to the receptor is the key step for chemokines mediating leukocyte migration into the wound bed during wound healing. The change of DARC in keloid fibroblasts have nothing to do with the proliferation of fibroblasts. However, wound closure rates were slower in injured keloid fibroblasts than in normal fibroblasts. With DARC interruption, the migration of keloid fibroblasts was improved. This result may indicate that, by binding to the chemokines, DARC can reduce the maigration of keloid fibroblasts and cause the abnormal wound healing.
The mRNA of IL-8, MCP-1, MMP-2were higher in keloid fibroblasts than in the fibroblasts from normal skin. But only MCP-1protein expression was lower in the supernatant of keloid fibroblasts. In regulated tissue repair, inflammation resolves quickly and tissue remodelling follows. A balance between extracellular matrix synthesis and degradation is carefully maintained. We considered that, in keolid fibroblasts, DARC may bind to the MCP-1and reduce its secretion. These factors might result in delayed and prolonged activation of injury-induced inflammation. The decreasing of MCP-1protein level but not mRNA expression in the keloid fibroblasts may also due to the DARC, a powerful chemokine controller in post-translational but not translational stage.
Our research exploded the expression of DARC in keloids for the first time, and detected the role of DARC in the proliferation and migration of keloid fibroblasts. We also discussed the mechanism of DARC in keloid formation. These results may open up a new thought in keloid research, and establish the foundation on studying the relationship between chemokine and the keloids.
5.Conclusion
5.1Different from cancer cells, DARC mRAN and protein were high expression in keloid fibroblast than in the fibroblast from normal skin. The inflammation plays an important role in the formation of keloid.
5.2DARC si has no effect on the proliferation of keloid fibroblasts, but can improve the migration of keloid fibroblasts. We considered the high expression of DARC in keloid fibroblasts may reduce the migration of fibroblasts and cause the abnormity of wound healing.
5.3DARC can inhibit the expression of MCP-1protein, but not lead to degradation. This behavior may cause the inbalance of inflammation, and may lead to the persistence of an inflammatory response and may result in tissue damage and chormic inflammation. This may be an etiology of the causes for keloid formation.
瘢痕疙瘩是一类创伤异常愈合所致的疾病,主要以纤维组织异常增殖以及胶原纤维过量沉积为主要标志,通常在创伤后持续存在,不能自行消退,且常超过原创伤范围,侵犯周围正常的皮肤组织。瘢痕疙瘩不仅会导致持续的瘙痒以及疼痛等症状,还会导致功能异常甚至畸形,严重影响外观,给患者带来严重的心理及生理的困扰。因此,瘢痕疙瘩是整形外科面临的重大难题之一。但由于瘢痕疙瘩形成的病因尚不十分明确,对于瘢痕疙瘩的预防及治疗方案也尚难统一。所以,找寻瘢痕疙瘩的主要致病因子是诊断及治疗瘢痕疙瘩的关键。
目前针对瘢痕疙瘩的研究多集中在以下3个方面：(1)细胞外基质蛋白的沉积以及降解,(2)细胞因子和生长因子,(3)凋亡通路。
我们既往的研究已经对凋亡通路有了一定的探索,但仍然未能找到瘢痕疙瘩形成的关键原因。鉴于各种细胞因子与瘢痕疙瘩形成关系密切,因此我们针对细胞因子中的趋化因子及其受体进行了新的探索。趋化因子是一类与炎症介质密切相关的小分子蛋白质家族,与多种器官的炎症反应,肿瘤转移还有组织纤维化密切相关。趋化因子可促进炎症反应,血管形成,协助白细胞从血液循环进入组织,在炎症反应中,趋化因子可为细胞提供相互联系,趋化因子信号转导是炎症细胞从血管中迁移粘附血管壁的关键因素。
关于趋化因子与瘢痕疙瘩的研究已经展开。MGSA/GRO α以及其受体CXCR2仅在瘢痕疙瘩组织中检测到表达,而增生瘢痕和正常上皮组织中则没有检测到其表达。并且其免疫组化的染色阳性率与该区域炎症反应的程度呈正相关。此外还有报道关于MCP-1及其受体CCR2过度活跃与各种纤维化的病理过程密切相关,并且在多种纤维化疾病中起着关键作用。而趋化因子需与其相应受体结合而发挥作用。
DARC(Duffy antigen receptor for chemokines; Dufy antigen/receptor for chemokines, DARC)即达菲抗原受体或称达菲抗原/趋化因子受体,是一个重要的趋化因子诱骗受体,该受体首先是在血友病患者中发现。它是红细胞膜上的糖蛋白,能够结合CC族和CXC族趋化因子的杂性趋化因子受体。作为典型的趋化因子诱骗受体,DARC只与具有促进血管生成作用的趋化因子结合,由于缺乏位于第2个胞内环上的与G蛋白偶联受体相似的7次跨膜糖蛋白,与配体结合后并不引起细胞信号转导和细胞代谢的改变。有研究发现除了在红细胞上有表达外,DARC同样也在内皮细胞、淋巴结、淋巴管上皮细胞、血管静脉和肺、肾上皮细胞中同样有表达。DARC在炎症反应中发挥着重要的作用,主要通过对其受体的调节,调控白细胞的归巢,影响炎症反应,炎症反应可以上调DARC的表达,而DARC的表达也可以对炎症反应进行调控。DARC在不同时期发挥的不同作用主要包括：1)趋化因子释放入血；2)血液中趋化因子的改变；3)血浆中趋化因子的转录后调控；4)趋化因子相关的DARC开／关比率；5)依赖DARC的转运机制。
DARC的表达还与许多肿瘤呈负相关,如前列腺癌、非小细胞肺癌、乳腺癌以及食管癌中都发现DARC高表达患者其预后优于低表达者。尽管瘢痕疙瘩是一种良性的上皮纤维异常增殖性肿瘤,没有恶性征象,但瘢痕疙瘩的某些特征,包括持续性生长,超出原有创伤范围,切除后容易复发等,又与恶性肿瘤有一定相似性。至今尚未有关于DARC与瘢痕疙瘩相关性,以及其在瘢痕疙瘩形成过程中所占的地位的报道,鉴于DARC对趋化因子调节的重要地位和其在炎症反应,以及肿瘤发生发展中所起的作用,瘢痕疙瘩的部分恶性肿瘤征象等理由,我们猜想DARC的表达可能与瘢痕疙瘩的形成有着一定的相关性。若能弄清DARC在瘢痕疙瘩形成中的作用,势必为瘢痕疙瘩的治疗和预防打开新的思路。
为了研究DARC在瘢痕疙瘩和正常皮肤中的表达情况及其在瘢痕疙瘩形成中的作用与机制,我们首先对不同的人瘢痕疙瘩和正常皮肤进行了检测,以确定它们表达DARC的情况。并且分离培养了部分瘢痕疙瘩患者和正常皮肤组织中的成纤维细胞,对其中DARC的mRNA含量及蛋白含量进行分析。此外还对其部分受体(?)IL-8,MCP-1以及与瘢痕疙瘩胶原沉积异常相关的MMP-2在瘢痕疙瘩成纤维细胞与正常皮肤成纤维细胞中的表达进行了研究,并且观察在DARC表达改变的情况下这些受体及蛋白的表达情况,还对DARC是否影响瘢痕疙瘩成纤维细胞的增殖和细胞周期,及其迁移的影响进行了研究。本研究旨在为发现趋化因子受体在瘢痕疙瘩成纤维细胞中的表达及其对成纤维细胞生长、迁移的影响,并深入研究其机制,对瘢痕疙瘩的成因及从根本上治疗瘢痕疙瘩提供理论依据。
二、材料与方法
材料：
收集2007年1月至2012年广东省中医院和南方医院病理确诊瘢痕疙瘩标本30例,以及正常对照皮肤30例。本研究事先得到南方医院相关审核部门的同意,所有研究对象签署知情同意书。指定2名对瘢痕疙瘩诊断和治疗具有丰富临床经验的整形外科医师,根据瘢痕疙瘩诊断标准,对所有研究对象进行全面的体格检查、详细的病史询问和准确的临床诊断,填写个人临床资料登记表,并严格遵照赫尔辛基宣言的规定采集他们的组织。
方法：
2.1瘢痕疙瘩中DARC表达的检测
将组织用石蜡包埋后切成石蜡切片,经过免疫组化染色,检测组织中DARC的表达情况。检测新鲜瘢痕疙瘩及正常皮肤中DARCmRNA及蛋白含量。
2.2细胞原代培养
将3例瘢痕疙瘩和3例正常皮肤组织进行体外细胞培养,培养出相应的成纤维细胞。将传代小于15代的成纤维细胞用于以下实验。
2.3运用siRNA干扰DARC表达
取处于对数生长期的瘢痕疙瘩成纤维细胞接种于六孔板中,待其长至板底的60%-80%时依据转染试剂所提供步骤,各孔分别用细胞培养液+转染溶液；control siRNA+转染溶液,以及DARC siRNA+转染溶剂进行转染,转染后24h及48h分别检测DARC mRNA及其蛋白的表达,并进行其余相关实验。
2.4细胞增殖影响
将瘢痕疙瘩成纤维细胞按5000个/孔的浓度将处于对数期的细胞种至96孔板,基础培养基100m1,每孔设3个对照；分别在12h,24h,48h,72h对细胞增殖进行检测；在每孔细胞中加入10μCCK-8溶液,细胞培养箱内继续孵育1.5h；MD5多功能酶标仪在450nm测定吸光度。
2.5细胞周期检测
将细胞计数1×105/瓶的NF(正常皮肤成纤维细胞组),KFC(瘢痕疙瘩成纤维细胞空白对照组),KFP(瘢痕疙瘩成纤维细胞阴性对照组),KFS(瘢痕疙瘩成纤维细胞干扰组)细胞在转染后24h进行细胞周期检测我们通过流式细胞仪对细胞周期的变化进行检测。
2.6细胞迁移的改变
我们通过划痕实验和transwell实验对正常皮肤成纤维细胞以及转染后的瘢痕疙瘩成纤维细胞进行检测,观察其迁移能力和增殖能力改变的情况。
2.7细胞上清液中蛋白的检测
我们通过ELASA检测正常皮肤成纤维细胞以及转染后的瘢痕疙瘩成纤维细胞中IL-8, MCP-1和MMP2蛋白的表达及变化情况。
三、结果
3.1DARC为胞膜和细胞浆着色,在正常皮肤及瘢痕疙瘩组织的阳性染色组织中,均可见部分染色的成纤维细胞的细胞浆及细胞膜。对比30例瘢痕疙瘩和正常皮肤组织中成纤维细胞DARC表达情况,发现瘢痕疙瘩成纤维细胞的DARC高表达率高于正常皮肤组织。
3.2通过进一步对3例瘢痕疙瘩组织和3例对照正常皮肤组织中DARC RNA以及蛋白的检测,同样发现瘢痕疙瘩组织中DRAC含量在mRNA水平与蛋白水平均高于正常皮肤。在对体外培养的3组瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞进行DARC RNA及蛋白检测后,发现其DARC表达与组织中的表达规律基本一致。
3.3转染后的细胞生长曲线测定比较,发现转染DARC siRNA对细胞增殖并无明显影响,并且瘢痕疙瘩与正常细胞成纤维细胞生长增殖无显著差异。流式细胞仪检测发现DARC siRNA转染对细胞周期无明显影响,此外瘢痕疙瘩成纤维细胞的迁移能力弱于正常皮肤来源的成纤维细胞,而干扰DARC后瘢痕疙瘩成纤维细胞的迁移能力有了一定提高,但仍稍低于正常皮肤成纤维细胞。
3.4DARC表达减低对IL-8, MCP-1, MMP-2mRNA的表达无明显影响。DARC表达减少可以增加MCP-1蛋白的分泌。我们研究发现DARC的表达高低对IL-8以及MCP-1mRNA的表达无明显影响。而当对DARC进行干扰后,我们发现与对照组相比,MCP-1在细胞上清中的含量增加,但仍稍低于正常皮肤来源的成纤维细胞。而DARC干扰对细胞上清液中IL-8以及MMP2蛋白的含量无明显影响。
四、讨论
我们的研究结果显示,虽然有一定的恶性肿瘤表型,但与恶性肿瘤不同的是,DARC在瘢痕疙瘩组织中无论在蛋白水平还是在转录水平均高于正常皮肤组织。说明瘢痕疙瘩的形成过程与恶性肿瘤细胞不同,不是各种致瘤因素所致,可能与伤口的异常愈合相关。创伤愈合需要复杂的细胞间相互作用,以及其调控因子的调节。组织创伤修复机制的变化受制于皮肤创伤的愈合。瘢痕疙瘩中常出现细胞迁移,增殖,炎症反应,细胞外基质蛋白与细胞因子的合成及分泌异常。
炎症反应是创伤愈合过程的组成部分,一方面可以作为抗感染的免疫屏障,另一方面可以导致纤维增生,闭合创面。趋化因子在创伤愈合过程中的炎症反应和组织修复中都发挥着重要的作用。而皮肤由于感染等外在因素导致的慢性炎症反应常常是瘢痕疙瘩形成的重要诱因。
趋化因子对白细胞聚集的有效调控的关键在于对其运动的趋化调节,即在准确的时间到达准确的位置,并且对其的功能在一定的时间和范围进行有效控制。趋化因子是通过与其受体的相互作用对白细胞产生趋化和激活作用,从而发挥其在炎症反应中的重要功效。瘢痕疙瘩中DARC表达改变,对成纤维细胞的的生长增殖无明显影响。而瘢痕疙瘩成纤维细胞体外培养伤口愈合能力弱于正常皮肤,这也是瘢痕疙瘩伤口愈合异常的表现。在对DARC进行干扰后,成纤维细胞的迁移能力有一定提高。说明DARC可能通过对其相应的配体的结合,而影响成纤维细胞的迁移,导致伤口延迟愈合。
在对DARC相应配体及MMP-2的检测中,我们发现瘢痕疙瘩成纤维细胞中IL-8, MCP-1, MMP-2的mRNA含量高于正常皮肤成纤维细胞,但仅有MCP-1在细胞上清中的表达低于正常皮肤成纤维细胞,并且MCP-1的表达受DARC的调控。在组织创伤后的炎症反应中,趋化因子的主要作用是诱导炎症因子在合适的试剂到达合适的位置,为了达到这样的效果,趋化因子的调控必须精确。炎症反应区域趋化因子及其受体的表达上调对于局部炎症细胞的快速聚集有重要意义。我们认为在瘢痕疙瘩成纤维细胞中,DARC可能通过与MCP-1的结合,引起MCP-1的分泌减少。导致局部炎症反应不平衡,形成慢性炎症反应。而DARC并不在基因水平上影响其配体的表达,DARC对其配体的下调作用是一种翻译后的调控因素,是发生在蛋白质水平的,而这种作用并不影响细胞内的信号通路,不影响细胞的增殖等生物学行为,是一种趋化因子的翻译后调控。
本实验首次对诱骗受体DARC在瘢痕疙瘩中的表达,及其对瘢痕疙瘩成纤维细胞生长、迁移的影响进行了研究,并初步探讨了相关机制,为瘢痕疙瘩的病因研究打开了新的思路,也为进一步研究趋化因子及其受体在瘢痕疙瘩中形成中的作用奠定了一定的实验基础。
五、结论
5.1.与恶性肿瘤细胞DARC表达相反,DARC在瘢痕疙瘩组织成纤维细胞中表达高于正常皮肤组织的成纤维细胞；
5.2.DARC并不对瘢痕疙瘩成纤维细胞的生长增殖产生影响,但我们的研究发现DARC可能会抑制瘢痕疙瘩成纤维细胞的迁移,造成创口愈合异常,对其进行干扰后瘢痕疙瘩成纤维细胞的迁移能力有一定提高。
5.3.DARC可能通过某种转录后调控的方式,抑制MCP-1的分泌,使成纤维细胞MCP-1分泌减少,由于DARC表达异常,导致其相关趋化因子调控异常,造成炎症反应不平衡,形成持续的炎症反应,导致组织损伤和慢性炎症形成,是瘢痕疙瘩形成的可能因素。
1. Background and Objective
Keloids result from alterations in the normal process of wound healing, which is characterized with excessive growths of fibrous tissue and accumulation of unmature collagen, it frequently persists at the site of injury, don't regress all the time; often recurs after excision, and always overgrows the boundaries of the original wound manifested by invasion of clinically normal skin. Keloid not only could results in the malformation and dysfunction which impact the outlook, but also usually occur pruritus and ache symptoms, which cause much physical and psychological anguish on patients with keloids. Therefore, keloid has been regarded as one of the intractable problem to plastic surgery. Due to the unknown etiology of the causes for keloid formation, there is still no specific diagnostic method and valid therapeutic measure in clinic up to now. It is thus clear that to identify etiological factors for keloid will be the key to diagnose exactly and treat effectually it.
The molecular aberrances in keloids that correlate with the molecular mechanisms in normal wound healing can be categorized into three groups:(1) extracellular matrix proteins and their degradation,(2) cytokines and growth factors, and (3) apoptotic patheways.
We have aim to elucidate its possible mechanisms in apoptotic patheways, however, we haven't found the key etiology of the causes for keloid formation. Considering the relationship between cell factor and keloids, we decide to find the correlation between chemokines and the keloids. Chemokines and their receptors are growing family of inflammatory molecules that are associated with many tissue specific inflammatory event, cancer metastasis and fibrosis. Chemokines promote inflammation, angiogenesis, facilitate the passage of leukocytes from circulation into the tissue, and contribute to the regulation of epithelialization. They integrate inflammatory events and reparative processes that are important for modulating wound healing.
There have been many reports about the association of chemokin and the keloids. MGSA/GRO a, and its receptor, CXCR2, were found not observed in hypertrophic scars or normal dermis, but was present in some myofibroblasts in nodular areas of the keloid samples. And the staining positively correlated with the degree of inflammatory infiltrate in the lesions. The enhanced MCP-1and CCR2in the keloid tissues augments fibroblast proliferation has also been reported.
The Duffy antigen is an antigenic determinant of the Duffy blood group system and is the receptor exploited by the malaria parasite Plasmodium vivax for its entry into human red blood cells. The Duffy receptor is a typical decoy receptor referred to as Atypical Chemokine Receptors (ACRs), interceptors, or chemokine 'decoy' receptors, which selectively binds CXC and CC chemokines with high ffinity. Inflammation can further up-regulate DARC expression in postcapillary venules and veins, and induce DARC to appear in vascular segments. As well as red blood, DARC is also espressed on a subset of endothelial cells, lymph node, lymphatic endothelial cells, venules of the skin, and epithelium in lung and kidney collecting ducts. Thus, DARC on human rbc influences infectious disease and leukocyte homeostasis. The precise role of DARC at a given time will therefore be influenced by a host of variables, including(i) chemokine release into the blood,(ii) the diversity of the blood chemokine repertoire,(iii) post-translational modifications of plasma chemokines,(iv) the relative DARC on/off rates of these chemokines, and (v) the effectiveness of DARC-independent removal mechanisms.
In addition, the expression of DARC is a negative regulator of many cancers, such as prostate cancer, NSCLC, breast cancer, and laryngeal squamous cell carcinoma.
Although keloids behave as benign dermal fibroproliferative tumors lacking malignant potential, it shares many characteristics, including persistent growth and the tendency to recur after excision. So far, the role of DARC during keloid progression, especially the relationship between DARC and keloid has not been investigated. Based upon these findings, we proposed that the DARC expression might be associated with keloid.
To test this hypothesis we examined the expression of the DARC in keloid lesions as compared to normal skin, as well as the expression of the chemokines such as IL-8,,MCP-1in cultured fibroblasts from normal skin and keloid lesions. The effects of DARC on the growing and moving of fibroblasts from keloid was also investigated.
2. Materials and Methods
Actively growing keloid tissues (N=30) and normal skin (N=30) were collected from patients undergoing elective excision of keloids, abdominoplasty, skin grafting, and circumcision procedures. Tissue samples were obtained in accordance with procedures approved by the Institutional Review Board of the Nanfang Hospital, Southern Medical University in advance. The all of informed consent form (ICF) from the patients have been obtained. None of the keloids had received corticosteroid injection within a one-year period and all were removed from the truncal region..
2.1DARC expression detection
After processed the tissues through paraffin embedding and cut into slices, the immunohistochemical staining method was exerted to observe the expression of DARC. We also detected the mRNA and protein of DARC in keloids.
2.2Cell cultures
Keloid tissue obtained from patients undergoing keloid excision(N=3) and normal skin(N=3) obtained during surgery were collected.After dissected and cultred the fibroblasts in vitro, the cultured fibroblasts of less than15generations were used for the experiments.
2.3Transfection of siRNA plasmids
Duffy siRNA and control siRNA were transfected into target cells. After24hours and48hours of transfection, real-time PCR and western blot analyses were performed to assess the knockdown of the target gene.
2.4Cell proliferation
For determination of cell proliferation and viability, skin fibroblasts were cultured in a96-well microtiter plate. After reaching80%confluence, cultured fibroblasts were washed twice with PBS and incubated in DMEM containing10%FBS for a time period of12h,24h,48h and72h, respectively. The determination of cell proliferation was then performed with Cell Counting Kit-8(Dojindo) according to instructions provided by the manufacturer.
2.5cell cycle analysis
24h after the transfection, we use Flow Cytometry to detecte the change of cell cycle. NF(fibroblasts from normal skin), KFC (fibroblasts from keloid tissue), KFP (fibroblasts from keloid tissue transfected with control siRNA), KFS (fibroblasts from keloid tissue transfected with DARC siRNA).
2.6Shift assay
Shift experiments were conducted with a Matrigel invasion chamber. The bottom chamber contained10%FBS-supplemented medium as a chemoattractant. Keloid fibroblast cells were transfected with DARC siRNA and control siRNA respectively were plated in the upper chambers and incubated at37℃for48hours. The cell suspension was aspirated using a cotton swab. Then, the filters were fixed in10%formalin solution and stained with H&E. Cells that had invaded through the filter and reached the lower surface of the filter were counted under a light.
2.7Enzyme linked immunosorbent assay analysis
The protein level of human IL-8, MCP-1and MMP-2present in conditioned cell supernatants were determined using a sandwich enzyme linked immunosorbent assay (ELISA) kit.
3. Results
3.1Immunostaining for DARD in keloids and normal skin
To study the relation between DARC and keloids in the clinical setting, tissue slices of60patients'samples(30keloid tissues and30normal skin) were tested with DARC antibody. Our results revealed the expression of DARC in fibroblasts was higher in keloid tissue than that of normal skin.
3.2Expression of DARC in human fibroblasts
DARC expression in human fibroblasts (3normal and3keloid fibroblasts culltures) was determined utilizing reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. W e found that keloid fibroblasts expressed relative high DARC levels, while normal fibroblasts had a significant lower DARC expression.
3.3Knockdown of DARC using siRNA has no effect on proliferation, and chemokines gene expression
To investigate whether the change of DARC expression could modulate the proliferation of keloid fibroblasts in vitro, we assessed the growth of DARC siRNA and mock-transfected keloid fibroblasts. Knokdown of DARC had no effect on the proliferation of DARC siRNA-transfected keloid fibroblast when compared to mocktransfected and wild-type cells. As well as the DARC si has no effect on cell cycle. In addition, DARC expression had no effect on CXCL8(CXC chemokine ligand8, interleukin-8, IL-8), CCL2(CC chemokine ligand2, monocyte chemoattractant protein-1, MCP-1), MMP2(matrix metalloproteinase2) mRNA expression.
3.4Knockdown of DARC using siRNA enhance chemokines CCL2on protein level in vitro
The DARC functions as a promiscuous chemokine decoy receptor. Therefore, it is interesting to investigate the influence of high expression of DARC on the secretion of chemokines in this system. There were no significant differences of mRNA expression of CXCL8and CCL2in DARC, Mock-transfected and control fibroblasts. However, the CCL2level detected in conditioned media from the fibroblasts was markedly increased in the DARC siRNA-tansfected fibroblasts as compared to the control fibroblasts. In contrast, the DARC expression had no effect on CXCL8and MMP2levels in conditioned media from the fibroblasts
3.5Knockdown of DARC using siRNA enhance migrational property of the fibroblast
We then examined the migrational activities of DARC siRNA-transfected keloid fibroblasts using the Boyden Chamber. Knockdown of DARC increased the migration of keloid fibroblasts.
4. Discussion
Our research showed that although with some malignant character, the DARC expression in keloids still different from malignant tumor. DARC mRAN and protein were high expression in keloid tissues than in the normal skin. These results may indicate that the formation of keloids is different from malignant tumor. The keloids formation may result from abnormal wound healing, but not the tumorigenesis. Wound healing requires a complex series of reactions and interactions among cells and their mediators. The altered tissue repair mechanism appears to be restricted to dermal wound healing. Abnormalities in cell migration, proliferation, inflammation, synthesis and secretion of extracellular matrix proteins and cytokines, and remodeling of the wound matrix have all been described in keloids.
Inflammation is an important part of wound healing. One hand, it can build the Immunologic barrier to defect the infection. Another hand, it can cause the proliferation of fibers. Chemokines play an important role in inflammation and tissue repair. Infection leading to chronic inflammation in the skin could predispose to keloid formation.
The efficient regulation of leucocyte recruitment by chemokines requires their appropriate localization in functional micro-anatomical domains, as well as setting limits to their effects in space and time. Binding to the receptor is the key step for chemokines mediating leukocyte migration into the wound bed during wound healing. The change of DARC in keloid fibroblasts have nothing to do with the proliferation of fibroblasts. However, wound closure rates were slower in injured keloid fibroblasts than in normal fibroblasts. With DARC interruption, the migration of keloid fibroblasts was improved. This result may indicate that, by binding to the chemokines, DARC can reduce the maigration of keloid fibroblasts and cause the abnormal wound healing.
The mRNA of IL-8, MCP-1, MMP-2were higher in keloid fibroblasts than in the fibroblasts from normal skin. But only MCP-1protein expression was lower in the supernatant of keloid fibroblasts. In regulated tissue repair, inflammation resolves quickly and tissue remodelling follows. A balance between extracellular matrix synthesis and degradation is carefully maintained. We considered that, in keolid fibroblasts, DARC may bind to the MCP-1and reduce its secretion. These factors might result in delayed and prolonged activation of injury-induced inflammation. The decreasing of MCP-1protein level but not mRNA expression in the keloid fibroblasts may also due to the DARC, a powerful chemokine controller in post-translational but not translational stage.
Our research exploded the expression of DARC in keloids for the first time, and detected the role of DARC in the proliferation and migration of keloid fibroblasts. We also discussed the mechanism of DARC in keloid formation. These results may open up a new thought in keloid research, and establish the foundation on studying the relationship between chemokine and the keloids.
5.Conclusion
5.1Different from cancer cells, DARC mRAN and protein were high expression in keloid fibroblast than in the fibroblast from normal skin. The inflammation plays an important role in the formation of keloid.
5.2DARC si has no effect on the proliferation of keloid fibroblasts, but can improve the migration of keloid fibroblasts. We considered the high expression of DARC in keloid fibroblasts may reduce the migration of fibroblasts and cause the abnormity of wound healing.
5.3DARC can inhibit the expression of MCP-1protein, but not lead to degradation. This behavior may cause the inbalance of inflammation, and may lead to the persistence of an inflammatory response and may result in tissue damage and chormic inflammation. This may be an etiology of the causes for keloid formation.
引文
[1]Bettinger, David A, Dome R. The effect of TGF-β on keloid fibroblast proliferation and collagen synthesis. Plast Reconstr Surg,1996,98(6):827.
[2]Boyce DE, Ciampolini J, Ruge F, et al. Inflammatory cell subpopulations in keloid scars. Br J Plast Surg,2001,54(6):511-6.
[3]Marnems AG, Norris JE, Olsen BR. Clinical genetics of familial keloids. Arch Dermatol,2001,137(11):1429-34.
[4]Chen Y, Gao JH, Liu XJ. Characteristics ofoccurrence for Han Chinese familial keloids. Burns,2006,32(8):1052-9.
[5]Clark JA, Turner ML, Howard L. Description of familial keloids in five pedigrees: evidence for autosomal dominant inheritance and phenotypic heterogeneity. BMC Dermatol,2009,9(8):1471-1479.
[6]Marneros AG, Norris JE, Watanabe S. Genome scans provide evidence for keloid susceptibility loci on chromosomes 2q23 and 7p11. J Invest Dermatol,2004,122(5): 1126-32.
[7]Bettinger DA, Yager DR, Diegelmann RF. The effect of TGF-beta on keloid fibroblast proliferation and collagen synthesis. Plast ReconstrSurg,1996; 98(5): 827-33.
[8]Sate M. Upregulation of the Wnt/beta-catenin pathway induced by transforming growth factor-beta in hypertrophic scars and keloids. Acta Derm Venereol,2006; 86(4):300-7.
[9]Lu L, Sanlis AS, Liu WR. The temporal effects at antiTGF-beta 1,2, and 3 monoclonal antibody on wound healing and hypertrophic scar formation. J Am Coll Surg,2005; 201(3):391-7.
[10]Wu Y, Zhang Q, Ann D K, et al. Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERKI/2 in keloid fibroblasts. Am J Physiol Cell Physiol,2004; 286(4):905-12.
[11]Yoshimoto H, Ishihara H, Ohtsuru A, et al. Overexpression of insulin like growth factor-I(IGF-I)receptor and the invasiveness of cuhured keloid fibroblasts. Am J Pathol,1999; 154(3):883-9.
[12]Ishihara H, Yoshimoto, Fujioka M, et al. Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor. J Invest Dermatol,2000; 115(6):1065-71
[13]Ghazizadeh M, Tosa M, Shimizu H, et al. Functional implications of the IL-6 signaling pathway in keloid pathogenesis. J Invest Dermatol,2007; 127(1):98-105.
[14]Khoo YT, Ong CT, Mukhopadhyay A, et al. Upregulation of secretory connective tissue growth factor(CTGF)in keratinocyte-fibroblast coculture contributes to keloid pathogenesis. J Cell Physiol,2006; 208(2):336-43.
[15]Meshkinpour A, Ghasri P, Pope K, et al. Treatment of hypertrophic scars and keloids with a radiofmquency device:a study of collagen effects. Lasers Surg Med, 2005; 37 (5):343-9.
[16]Galus R, Antiszko M, Wlodarski P. Clinical applications of hyaluronic acid. Pol Merkur Lekarski,2006; 20(119):606-8.
[17]Meyer LJ, Russell SB, Russell JD, et al. Reduced hyaluronan in keloid tissue and cultured keloid fibroblasts. J Invest Dermatol,2000; 114(5):953-9.
[18]Harty M, NeffAW, King Mw, et al. Regeneration or scarring:animmunologic perspective. Dev Dyn,2003; 226(2):268-79.
[19]Mukherjee A, Mukherjee A, Saha KC. Delayed type hypersensitivity reaction to cutaneous antigen in keloid. Indian J Dermatol,1982; 27 (4):125-30.
[20]Ana Cristina Garcia-Ulloa, Oscar Arrieta,Tubal occlusion causing infertility due to an excessive inflammatory response in patients with predisposition for keloid formation.medical hypotheses,2005;65(5):908-14
[21]Lee JY, Yang CC, Chao SC. et al. Histopathological differential diagnosis of keloid and hypertrophic scar. Am J Dermatopathol,2004; 26(5):379-84.
[22]Segerer S, Jedlicka J, Wuthrich RP. Atypical chemokine receptors in renal inflammation. Nephron Exp Nephrol,2010; 115(4):89-95.
[23]Zlotnik A, Yoshie O, Nomiyama H. The chemokine and chemokine receptor superfamilies and their molecular evolution. Genome Biol,2006; 7(12):243.
[24]Nirodi CS, Devalarja R, Nanney LB,Chemokine and chemokine receptor espression in keloid and normal fibroblasts.Wuond Repair Regen,2000; 8(5):371-82.
[25]Liao WT, Yu HS, Arbiser JL, Enhanced MCP-1 release by keloid CD14+cells augments fibroblast proliferation:role of MCP-1 and Akt pathway in keloids. Experimental Dermatology,2010; 19(8):142-50.
[26]Mantovani A, Bonecchi R, Locati M. Tuning inflammation and immunity by chemokine sequestration:decoys and more. Nat Rev Immunol,2006; 6:907-18.
[27]Hansell CA, Simpson CV, Nibbs RJ. Chemokine sequestration by atypical chemokine receptors.Biochem Soc Trans,2006; 34:1009-13.
[28]Uu XH, Hadley TJ, Xu L. Up-regulation of Duffy antigen receptor expression in children with renal disease. Kidney Int,1999; 55:1491-1500.
[29]Chris AH, Catherine EH, Robert JB. DARC and D6:silent partners in chemokine regulation? Immunol Cell Biol,2011; 89(2):197-206.
[30]Gardner L,Wilson C,Patterson AM, et al. Temporal expression pattern of Duffy antigen in rheumatoid arthritis:up-regulation in early disease. Arthritis Rheum,2006; 54(6):2022-6.
[31]Woolley IJ, Hotmire KA, Sramkoski RM. Diferential expression of the Dufy an tigen receptor for chemokines according to RBC age and FY genotypo. Transfusion, 2000;40(8):949-53.
[32]Comerford I, Nibbs RJ. Post-translational control of chemokines:a role for decoy receptors? Immunol Lett,2005; 96(2):163-74.
[33]Lentsch AB. The Dufy antigen/receptor for chemokines(DARC)and prostate cancer:A role as clear as black and white. FASEB J,2002; 16(9):1093-5.
[34]Addison CL, Belperio JA, Burdick MD. Overexpression of the dufy antigen receptor for chemokines(DARC)by NSCLC tumor cells results in increased tumor necrosis. BMC Cancer,2004;4(1):28.
[35]Wang J. Ou ZL, Hou YF. Enhanced expression of Dufy an tigen receptor for chemokines by breast cancer cells attenuates growth and metastasis potential. Oncogene,2006; 25(54):7201-11.
[36]Sun G, Wang Y, Zhu Y. Risk Factors for Neck Nodal Metastasis in Papillary Thyroid Microcarcinoma:A Study of 1066 Patients. Acta Otolaryngol,2011;131(2): 197-203.
[37]Atiyeh BS, Costagliola M, Hayek SN.Keloid or hypertrophic scar:The controversy:Review of the literature. Ann Plast Surg,2005;54 (6):676-80.
[38]Rekha A. Keloids-A frustrating hurdle in wound healing. Int Wound J,2004; 1(2):145-8.
[39]Cole-Tobian JL, Cortes A, Baisor M. Age-acquired immunity to a Plasmodium vivax invasion ligand, the duffy binding protein. J Infect Dis,2002;186(4):531-9.
[40]Marnems AG, Norris JE, Olsen BR. Clinical genetics of familial keloids. Arch Dermatol,2001;137(11):1429-34.
[41]Luo S, Benathan M, Raffoul W. Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions. Plast Reconstr Surg, 2001; 107(1):87-96.
[42]Su Y, Arnold F, Cherry G. Proliferation and apoptosis in chronic wounds. Wound Rep Reg,1996; 28(4):141-7.
[43]Bayat A, McGrouther DA, Ferguson MW. Skin scarring. Br Med J,2003; 326(7380):88-92.
[44]Hadley TJ, Peiper SC. From Malaria to chemokine receptor:The emerging physiologic role of the duffy blood group antigen. Blood,1997;89(9):3077-91.
[45]Dustan HP. Does keloid pathogenesis hold the key to understanding black/white differences in hypertension severity? Hypertension,1995; 26(6):858-62.
[46]Keeley EC, Mehrad B, Strieter RM. CXC chemokines in cancer angiogenesis and metastases. Adv Cancer Res,2010; 106:91-111.
[47]Atiyeh B.S., Costagliola M., Hayek S.N. Keloid or hypertrophic scar:The controversy:Review of the literature. Ann Plast Surg,2005;54 (6):676-80.
[48]Rekha A. Keloids-A, frustrating hurdle in wound healing. Int Wound J, 2004; 1(2):145-48.
[49]Horuk R, Wang ZX, Peiper SC, et al. Identification and characterization of a promiscuous chemokine-binding protein in a human erythroleukemic cell line. J Biol Chem 1994,269(26):17730-17733.
[50]Ogawa R. keloids as a serious disease such as malignancy,plast reconstr surg.2008;122(3):993-4.
[51]Sidle DM, Kim H. keloids:prevention and management. Facial Plast Surg Clin North Am,2011; 19(3):505-15.
[52]Mustoe TA, Cooter RD, Gold MH.International clinical recommendations on scar management.Plast Reconstr Surg,2002; 110(2):560-71.
[53]Peiper SC, Wang ZX, Neote K. The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor. J Exp Med,1995;181(4):1311-7.
[54]Bao P, Kodra A, Tomic-Canic M. The role of vascular endothelial growth factor in wound healing. J Surg Res,2009; 153(2):347-58.
[55]Harrison SL, Vavken P, Murray MM.Erythrocytes inhibit ligament fibroblast proliferation in a collagen scaffold. J Orthop Res,2011; 29(9):1361-6.
[56]Patterson AM, Siddall H, Chamberlain G. Expression of the duffy antigen/receptor for chemokines (DARC) by the inflamed synovial endothelium. J Pathol,2002; 197:108-16.
[57]Pruenster M., Mudde L., Bombosi P. The Duffy antigen receptor for chemokines transports chemokines and supports their promigratory activity. Nat. Immunol,2009; 10(1):101-8.
[58]Dawson TC, Lentsch AB, Wang Z. Exaggerated response to endotoxin in mice lacking the Duffy antigen/receptor for chemokines (DARC). Peiper SC Blood,2000; 96(5):1681-4.
[59]Geleff S, Draganovici D, Jaksch P.The role of chemokine receptors in acute lung allograft rejection. Segerer S Eur Respir J,2010;35(1):167-75.
[60]Koch AE, Polverini PJ, Kunkel SL. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science,1992; 258(5089):1798-1801.
[61]Kelly M, Kolb M, Bonniaud P. Re-evaluation of fibrogenic cytokines in lung fibrosis. Curr Pharm Des,2003; 9(1):39-49.
[62]Gira AK, Brown LF, Washington CV, Cohen C. Keloids demonstrate high-level epidermal expression of vascular endothelial growth factor. Am Acad Dermatol,2004; 50(6):850-3.
[63]Hoch RC, Schraufstatter IU, Cochrane CG. in vivo, in vitro, and molecular aspects of interleukin-8 and the interleukin-8 receptor. J Lab Clin Med, 1996;128(2):134-5.
[64]Onq CT, Khoo YT, Tan EK. epthelial-mesenchymal interactions in keloid pathogenesis modulate vascular endothelial growth factor exression and secretion.J pathol,2007;211(1):95-108.
[65]Lim CP, Phan TT, Lim IJ, Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. Epub,2009;129(4):851-61.
[66]Lee JS, Frevert CW, Wurfel MM. Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo. J Immunol,2003; 170(10):5244-51.
[67]Friedman DW, Boyd CD,Mackenzie JW. Regulation of collagen gene expression in keloids and hypertrophic scar. J Surg Res,1993; 55(2):214-22.
[68]Ghahary A, Shen YJ, Nedelec B. Collagenase production is lower in post-burn hypertrophic scar fibroblast than in normal fibroblasts and is reduced by insulin-like-growth factor-Ⅰ. J Invest Dermatol,1996; 106(3):476-81.
[69]Kast RE. Halatsch ME. Matrix metalloproteinase-2 and-9 in glioblastoma:a trio of old drugs-captopril, disulfiram and nelfinavir-are inhibitors with potential as adjunctive treatments in glioblastoma. Arch Med Res,2012,43:243-47.
[70]Chakraborti S, Mandal M, Das S. Regulation of matrix metalloproteinases: an overview. Mole Cellr Biochem,2003;253:269-85.
[71]Louw L. The keloid phenomenal:progress toward a solution. Clinical Anat, 2007; 20(1):3-14.
[72]McCaw A, Ewald J, Werb Z. Matrix metalloproteinases and the regulation of tissue remodeling. Nat Rev Mol Cell Biol,2007;8(3):221-33.
[73]Bullen EC, Longaker MT, Updike DL. Tissue inhibitor of metalloproteinases..1 is decreased and activated gelatinases are increased in chronic wounds. J Invest Dermatol,1995; 104(2):236-40.
[74]Yager DR, Zhang LY, Liang HX, et al. Wound fluids fromhuman pressure ulcers contain elevated matrix metalloproteinases levels and activity compared to surgical wound fluids. J Invest Dermatol,1996; 107(5):743-48.
[75]Neely AN, Clendening CE, Gardner J.Gelatinase activity in keloids and hypertrophic scars. Wound repair region,1999; 7(3):166-71.
[76]Yeh FL, Shen HD,Tai HY. Decreased production of MCP-1 and MMP-2 by keloid-derived fibroblasts, Burns,2009,35(3):348-51.
[77]Tanriverdi AS, Menderes A, Oktay G. Matrix metalloproteinase-2 and -9 activities in human keloids, hypertrophic and atrophic SCarS:a pilot study. Cell Biochem Funct,2009,27(2):81-7.
[78]Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure, function, and biochemistry. Circ Res,2003;92(8): 827-39.
[79]Goede V, Brogelli L, Ziche M, et al. Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1. Int J Cancer,1999,82(5):765-70.
[80]Anders HJ, Sayyed SA, Vielhauer V. Questions about Chemokine and Chemokine Receptor Antagonism in Renal Inflammation, Nephron Exp Nephrol, 2010;114(2):e33-8.
[81]Xia Y, Frangogiannis NG MCP-1/CCL2 as a therapeutic target in myocardial infarction and ischemic cardiomyopathy. Inflamm Allergy Drug Targets,2007;6(2): 101-7.
[82]Tesch G H. MCP-1/CCL2:a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol,2008; 294(4):697-701.
[83]Gharaee KM, Denholm EM, Phan SH. Costimulation of fibroblast collagen and transforming growth factor betal gene expression by monocyte chemoattractant protein-1 via special receptors. J Biol Chem,1996;271(30):17779-84.
[84]Fivenson DP, Faria DT, Nickoloff BJ, et al. Chemokine and inflammatory cytokine changes during chronic wound healing. Wound Repair Regen,1997; 5(4):310-22.
[85]Low QE, Drugea IA, Duffner LA, et al. Wound healing in MIP-lalpha(-/-) and MCP-1 (-/-) mice. Am J Pathol,2001;159(2):457-63.
[86]Zymek P, Winkelmann K, Koerting A, et al Abou-Khamis T, et al. CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res,2005; 96(8):881-9.
[87]Yeh FL, Shen HD, Tai HY. Decreased production of MCP-1 and MMP-2 by keloid-derived fibroblasts. Burns,2009;35(3):348-51.
[88]Lim C P, Phan T T, Lim I J, et al. Cytokine profling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. J Invest Dermatol,2009; 129(4):851-61.
[89]Girardeau S, Mine S, Pageon H, et al. The Caucasian and African skin types differ morphologically and functionally in their dermal component. Exp Dermatol, 2009; 18(8):704-11.
[90]Darbonne WC, Rice GC, Mohler MA. Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin. J Clin Invest,1991;88(4):1362-69.
[91]Mangalmurti NS, Xiong z, HulverM. Loss of red cell chemokine scavenging promotes transfusion-related lung inflammation. Blood,2009;113(5):1158-66.
[92]Vielhauer V, Allam R, Lindenmeyer MT. Efficient renal recruitment of macrophages and T cells in mice lacking the Duffy antigen/receptor for chemokines. Am J Pathol,2009; 175(1):119-31.
[93]Fukuma N, Akimitsu N,Hamamoto H. A role of the Duffy antigen for the maintenance of plasma chemokine concentrations.Biochemical and Biophysical Research Communications,2003;303(1):137-9.
[94]Zarboek A, Sehmolke M, Bockhom SG, el al. The Dufry antigen receptor for chemokines in acute renal failure:A facilitator of renat chemokine presentation. Crit Care Med,2007; 35(9):2156-63.
[1]Marnems AG, Norris JE,Olsen BR, et al. Clinical genetics of familial keloids[J]. Arch Dermatol,2001,137(11):1429-34.
[2]Brown JJ, Ollier W, Thomson W, et al. Bayat A Positive association of HLA-DRB1*15 with Dupuytren's disease in Caucasians[J]. Tissue Antigens, 2008,72(2):166-70.
[3]Butler PD, Longaker MT, Yang GP. Current progress in keloid research and treatment[J]. J Am Coll Surg,2008,206(4):731-41.
[4]Bloom D, et al Heredity of keloids; review of the literature and report of a family with multiple keloids in five generations[J]. N Y State J Med,1956,56(4):511-519.
[5]Omo-Dare P, et al Genetic studies on keloid[J]. J Natl Med Assoc,1975, 67(6):428-32.
[6]宋文刚,宋光江,李桂倍,瘢痕疙瘩四家系遗传学分析[J].中国皮肤性病学 杂志,1992,6(2)：109-10.
[7]Chen Y, Gao JI-I, Liu XJ, et al.Characteristics ofoccurrence for Han Chinese familial keloids[J]. Burns,2006,32(8):1052-9.
[8]Clark JA, Turner ML, Howard L,et a 1.Description of familial keloids in five pedigrees:evidence for autosomal dominant inheritance and phenotypic heterogeneity [J]. BMC Dermatol,2009,9(8):1471-9.
[9]Marneros AG, Norris JE, Watanabe S, et al. Genome scans provide evidence for keloid susceptibility loci on chromosomes 2q23 and 7p11[J]. J Invest Dermatol, 2004,122(5):1126-32.
[10]陈阳,高建华,刘晓军,中国人群瘢痕疙瘩家系与染色体2q23和7p11的连锁分析[J].中国实用美容整形外科杂志,2006,17(3)：221-4.
[11]刘晓军,高建华,宋玫,瘢痕疙瘩候选基因与染色体7p11微卫星扫描及连锁分析研究[J].中国组织工程研究与临床康复,2008,12(28)：5453-6.
[12]刘晓军,高建华,宋玫,瘢痕疙瘩候选基因与染色体2q23微卫星扫描及连锁分析的多家系研究[J].中国组织工程研究与临床康复,2008,12(33)：6483-7.
[13]严欣,高建华,陈阳,一汉族瘢痕疙瘩家系的易感基因定位分析研究[J].中华整形外科杂志,2007,23(1)：32-5.
[14]陈阳,高建华,严欣,中国汉族一瘢痕疙瘩家系易感基因的定位研究[J].中华整形外科杂志,2007,23(2)：137-40.
[15]刘晓军,高建华,中国汉族瘢痕疙瘩家系易感基因位点的定位分析研究[J].中国美容整形外科杂志,2008,19(3)：179-82.
[16]刘晓军,高建华,中国汉族瘢痕疙瘩家系易感基因位点的定位分析[J].中国组织工程研究与临床康复,2008,12(37)：7241-4.
[17]刘晓军,高建华,瘢痕疙瘩家系17号染色体易感基因位点的定位分析术[J].广东医学,2008,29(5)：740-2.
[18]Shih B, Garside E, McGrouther DA, et al. Epub 2009 Dec 11 Molecular dissection of abnormal wound healing processes resulting in keloid disease [J]. Wound Repair Regen,2010,18(2):139-53.
[19]Satish L, Babu M, Tran KT, et al. Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways [J]. Wound Repair Regen,2004,12(2):183-92.
[20]段红杰,高建华,瘢痕疙瘩成纤维细胞p53基因突变检测[J].中国临床康复,2002,6(22)：3358-9.
[21]刘永波,高建华,段红杰,瘢痕疙瘩p53基因突变高发区基因结构的研究[J].中华整形外科杂志,2003,9(4)：258-60.
[22]蒋游晖,刘旺,李友良,瘢痕疙瘩组织p53基因突变的实验研究[J].中国美容医学,2003,11(6)：568-70.
[23]刘晓军,高建华,瘢痕疙瘩家系p53基因高发突变区基因突变检测的实验研究[J].中国美容整形外科杂志,2007,18(2)：108-11.
[24]卓阳,高建华,罗深秋,瘢痕疙瘩发病风险与p53基因第72位密码子多态性的关系[J].中华整形外科杂志,2005,21(3)：201-3.
[25]刘永波,高建华,瘢痕疙瘩临床表型与p53基因72位编码子基因多态性的相关性分析[J].中国修复重建外科杂志,2008,22(12)：1433-6.
[26]严笠,吕晓岩,王春梅,p53基因第72密码子多态性与部分中国人瘢痕疙瘩关系的研究[J].中华整形外科杂志,2007,23(5)：428-30.
[27]鲁峰,高建华,携带Fas基因重组腺病毒治疗瘢痕疙瘩的体外研究[J].中国修复重建外科杂志,2005,19(1)：35-8.
[28]刘永波,高建华,刘晓军,Fas基因和P53基因在瘢痕疙瘩成纤维细胞中的结构异常与其功能的关系[J].中国临床康复,2005,9(18)：106-108.
[29]刘晓军,高建华,瘢痕疙瘩家系Fas基因死亡域突变的实验研究[J].中国修复重建外科杂志,2007,21(7)：698-701.
[30]Laurentaci G. Dioguardi D. HLA antigens in keloids and hypertrophic seals [J], Arch Dermat,1977,113(12):1726-9.
[31]Brown JJ, Ollier WE, Thomson W, Bay at A Positive association of HLA-DRB1*15 with keloid disease in Caucasians[J]. Int J Immunogenet,2008, 35(4-5):303-7.
[32]孙东杰,陈东明,HLA-DR, HLA-DQ分子在瘢痕疙瘩患者外周血单个核细胞中的变化[J].中国美容医学,2008,17(3)：370-3.
[33]Lu WS, Wang JF, Yang S, Association of HLA-DQA1 and DQB1 alleles with keloids in Chinese Hans[J]. J Dermatol Sci,2008,52(2):108-17.
[34]Lu WS, Zuo XB, Wang ZX.Association of HLA haplotype with keloids in Chinese Hans[J]. burns,2011,37(5):794-9.
[35]Van Gersdorff G, Susztak K, Rezvan i F, et al. Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta[J]. J Biol Chem,2000,275(15):11320-6.
[36]Bay at A, Bock O, Mrowietz U, Genetic susceptibility to keloid disease: transforming growth factor beta receptor gene polymorphisms are not associated with keloid disease[J]. Exp Dermatol,2004,13(2):120-4.
[37]Bayat A, McGrouther DA, Ferguson MW, Skin scarring[J]. BMJ,2003, 326(7380):88-92.
[38]Brown JJ, Ollier W, Arscott G, Genetic susceptibility to keloid scarring:SMAD gene SNP frequencies in Afro-Caribbeans[J]. Exp Dermatol,2008,17(7):610-613.
[39]宋玫、高建华,瘢痕疙瘩家系外周血Smad2基因的突变检测[J].中国美容整形外科杂志,2009,20(3)：179-82.
[40]Archer HL, Gupta S, Enoch S, Distinct phenotype associated with a cryptic subtle omeric deletion of 19p13.3-pter[J].Am J Med Genet A,2005,136(1):38-44.
[41]Nakashima M, Chung S, Takahashi A, A genome-wide association study identifies four susceptibility loci for keloid in the Japanese population[J].2010, 42(9):768-71.
[1]Lee JY,Yang CC,Chao SC,Wong TW:histopathological differential diagnosis of keloid and hypertrophic scar. The American journal of dermatopathology 2004,26 (5):379-84.
[2]Ana Cristina Garcia-Ulloa, Oscar Arrieta,Tubal occlusion causing infertility due to an excessive inflammatory response in patients with predisposition for keloid formation.medical hypotheses 2005,65(5):908-14.
[3]Segerer S, Jedlicka J, Wtithrich RP. Atypical chemokine receptors in renal inflammation. Nephron Exp Nephrol 2010; 115(4):89-95
[4]Zlotnik A, Yoshie O, Nomiyama H. The chemokine and chemokine receptor superfamilies and their molecular evolution. Genome Biol,2006,7(12):243.
[5]Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure, function, and biochemistry. Circ Res 2003;92(8):827-39
[6]Goede V, Brogelli L, Ziche M, Augustin HG Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1. Int J Cancer 1999;82(5): 765-70.
[7]Xia Y, Frangogiannis N G. MCP-1/CCL2 as a therapeutic target in myocardial infarction and ischemic cardiomyopathy. Inflamm Allergy Drug Targets 2007:6(2): 101-7.
[8]Tesch G H. MCP-1/CCL2:a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol 2008: 294(4):697-701.
[9]Marra F. Renaming cytokines:MCP-1, major chemokine in pancreatitis. Gut 2005: 54(12):1679-81.
[10]Gharaee-Kermani M, Denholm E M, Phan S H. Costimulation of fibroblast collagen and transforming growth factor betal gene expression by monocyte chemoattractant protein-1 via special receptors. J Biol Chem 1996:271(30): 17779-84.
[11]Fivenson DP, Faria DT, Nickoloff BJ, Poverini PJ, Kunkel S, Burdick M, et al. Chemokine and inflammatory cytokine changes during chronic wound healing. Wound Repair Regen 1997;5(4):310-22.
[12]Low QE, Drugea IA, Duffner LA, Quinn DG, Cook DN, Rollins BJ, et al. Wound healing in MIP-lalpha(-/-) and MCP-1 (-/-) mice. Am J Pathol 2001;159(2):457-63.
[13]Zymek P, Winkelmann K, Koerting A, Ren G, Abou-Khamis T, et al. CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res 2005;96(8):881-9.
[14]Yeh FL, Shen HD, Tai HY. Decreased production of MCP-1 and MMP-2 by keloid-derived fibroblasts. burns 2009;35(3):348-351.
[15]Lim C P, Phan T T, Lim I J, Cao X. Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. J Invest Dermatol 2009:129(4):851-61.
[16]Girardeau S, Mine S, Pageon H, Asselineau D. The Caucasian and African skin types differ morphologically and functionally in their dermal component. Exp Dermatol 2009:18(8):704-11.
[17]Liao WT, Yu HS, Arbiser JL, Enhanced MCP-1 release by keloid CD 14+ cells augments fibroblast proliferation:role of MCP-1 and Akt pathway in keloids. Experimental Dermatology 2010; 19(8):142-50.
[18]Shaftel SS, Carlson TJ, Olschowka JA, Kyrkanides S, Matousek SB, O'Banion MK. Chronic interleukin-lbeta expression in mouse brain leads to leukocyte infiltration and neutrophil-independent blood brain barrier permeability without overt neurodegeneration. J Neurosci.2007;27(35):9301-9.
[19]Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, Moldawer LL, Nathan CF, Lowry SF, Cerami A. Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J Exp Med. 1988;167(2):570-81.
[20]Nirodi CS, Devalaraja R, Nanney LB, Arrindell S, Russell S, Trupin J, Richmond A:Chemokine and chemokine receptor expression in keloid and normal fibroblasts. Wound Repair Regen 2000,8(5):371-82.
[21]Koch AE, Polverini PJ, Kunkel SL, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science,1992,258(5089):1798-801.
[22]Kelly M, Kolb M, Bonniaud P, et al. Re-evaluation of fibrogenic cytokines in lung fibrosis. Curr Pharm Des,2003,9(1):39-49.
[23]Gira AK, Brown LF, Washington CV, Cohen C, Arbiser JL. Keloids demonstrate high-level epidermal expression of vascular endothelial growth factor. Am Acad Dermatol 2004;50(6):850-3.
[24]Lim CP, Phan TT, Lim IJ, Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. Epub 2009; 129(4):851-61.
[25]Weber KS, Nelson PJ, Grone HJ, expression of CCR2 by endothelial cells: implications for MCP-1 mediated wound injury reparir and in vivo inflammatory activation of endothelium. Atterioscler thromb vasc boil 1999;19(9):2085-93.
[26]Liu L, Callahan MKJ Huang DJ et al. Chemokine receptor CXCR3:an unexpected enigma. Curr Top Dev BiolJ 2005,68:49-181.
[27]Cecelia C. Yates, Diana Whaley, Priya Kulasekeran*,Delayed and Deficient Dermal Maturation in Mice Lacking the CXCR3 ELR-Negative CXC Chemokine Receptor.The American Journal of Pathology 2007,171(2):484-95.
[28]Cecelia C. Yates,Priya Krishna,Diana Whaley, Lack of CXC Chemokine Receptor 3 Signaling Leads to Hypertrophic and Hypercellular Scarring.The American Journal of Pathology 2010;176(4):1588-91.
[2]Boyce DE, Ciampolini J, Ruge F, et al. Inflammatory cell subpopulations in keloid scars. Br J Plast Surg,2001,54(6):511-6.
[3]Marnems AG, Norris JE, Olsen BR. Clinical genetics of familial keloids. Arch Dermatol,2001,137(11):1429-34.
[4]Chen Y, Gao JH, Liu XJ. Characteristics ofoccurrence for Han Chinese familial keloids. Burns,2006,32(8):1052-9.
[5]Clark JA, Turner ML, Howard L. Description of familial keloids in five pedigrees: evidence for autosomal dominant inheritance and phenotypic heterogeneity. BMC Dermatol,2009,9(8):1471-1479.
[6]Marneros AG, Norris JE, Watanabe S. Genome scans provide evidence for keloid susceptibility loci on chromosomes 2q23 and 7p11. J Invest Dermatol,2004,122(5): 1126-32.
[7]Bettinger DA, Yager DR, Diegelmann RF. The effect of TGF-beta on keloid fibroblast proliferation and collagen synthesis. Plast ReconstrSurg,1996; 98(5): 827-33.
[8]Sate M. Upregulation of the Wnt/beta-catenin pathway induced by transforming growth factor-beta in hypertrophic scars and keloids. Acta Derm Venereol,2006; 86(4):300-7.
[9]Lu L, Sanlis AS, Liu WR. The temporal effects at antiTGF-beta 1,2, and 3 monoclonal antibody on wound healing and hypertrophic scar formation. J Am Coll Surg,2005; 201(3):391-7.
[10]Wu Y, Zhang Q, Ann D K, et al. Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERKI/2 in keloid fibroblasts. Am J Physiol Cell Physiol,2004; 286(4):905-12.
[11]Yoshimoto H, Ishihara H, Ohtsuru A, et al. Overexpression of insulin like growth factor-I(IGF-I)receptor and the invasiveness of cuhured keloid fibroblasts. Am J Pathol,1999; 154(3):883-9.
[12]Ishihara H, Yoshimoto, Fujioka M, et al. Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor. J Invest Dermatol,2000; 115(6):1065-71
[13]Ghazizadeh M, Tosa M, Shimizu H, et al. Functional implications of the IL-6 signaling pathway in keloid pathogenesis. J Invest Dermatol,2007; 127(1):98-105.
[14]Khoo YT, Ong CT, Mukhopadhyay A, et al. Upregulation of secretory connective tissue growth factor(CTGF)in keratinocyte-fibroblast coculture contributes to keloid pathogenesis. J Cell Physiol,2006; 208(2):336-43.
[15]Meshkinpour A, Ghasri P, Pope K, et al. Treatment of hypertrophic scars and keloids with a radiofmquency device:a study of collagen effects. Lasers Surg Med, 2005; 37 (5):343-9.
[16]Galus R, Antiszko M, Wlodarski P. Clinical applications of hyaluronic acid. Pol Merkur Lekarski,2006; 20(119):606-8.
[17]Meyer LJ, Russell SB, Russell JD, et al. Reduced hyaluronan in keloid tissue and cultured keloid fibroblasts. J Invest Dermatol,2000; 114(5):953-9.
[18]Harty M, NeffAW, King Mw, et al. Regeneration or scarring:animmunologic perspective. Dev Dyn,2003; 226(2):268-79.
[19]Mukherjee A, Mukherjee A, Saha KC. Delayed type hypersensitivity reaction to cutaneous antigen in keloid. Indian J Dermatol,1982; 27 (4):125-30.
[20]Ana Cristina Garcia-Ulloa, Oscar Arrieta,Tubal occlusion causing infertility due to an excessive inflammatory response in patients with predisposition for keloid formation.medical hypotheses,2005;65(5):908-14
[21]Lee JY, Yang CC, Chao SC. et al. Histopathological differential diagnosis of keloid and hypertrophic scar. Am J Dermatopathol,2004; 26(5):379-84.
[22]Segerer S, Jedlicka J, Wuthrich RP. Atypical chemokine receptors in renal inflammation. Nephron Exp Nephrol,2010; 115(4):89-95.
[23]Zlotnik A, Yoshie O, Nomiyama H. The chemokine and chemokine receptor superfamilies and their molecular evolution. Genome Biol,2006; 7(12):243.
[24]Nirodi CS, Devalarja R, Nanney LB,Chemokine and chemokine receptor espression in keloid and normal fibroblasts.Wuond Repair Regen,2000; 8(5):371-82.
[25]Liao WT, Yu HS, Arbiser JL, Enhanced MCP-1 release by keloid CD14+cells augments fibroblast proliferation:role of MCP-1 and Akt pathway in keloids. Experimental Dermatology,2010; 19(8):142-50.
[26]Mantovani A, Bonecchi R, Locati M. Tuning inflammation and immunity by chemokine sequestration:decoys and more. Nat Rev Immunol,2006; 6:907-18.
[27]Hansell CA, Simpson CV, Nibbs RJ. Chemokine sequestration by atypical chemokine receptors.Biochem Soc Trans,2006; 34:1009-13.
[28]Uu XH, Hadley TJ, Xu L. Up-regulation of Duffy antigen receptor expression in children with renal disease. Kidney Int,1999; 55:1491-1500.
[29]Chris AH, Catherine EH, Robert JB. DARC and D6:silent partners in chemokine regulation? Immunol Cell Biol,2011; 89(2):197-206.
[30]Gardner L,Wilson C,Patterson AM, et al. Temporal expression pattern of Duffy antigen in rheumatoid arthritis:up-regulation in early disease. Arthritis Rheum,2006; 54(6):2022-6.
[31]Woolley IJ, Hotmire KA, Sramkoski RM. Diferential expression of the Dufy an tigen receptor for chemokines according to RBC age and FY genotypo. Transfusion, 2000;40(8):949-53.
[32]Comerford I, Nibbs RJ. Post-translational control of chemokines:a role for decoy receptors? Immunol Lett,2005; 96(2):163-74.
[33]Lentsch AB. The Dufy antigen/receptor for chemokines(DARC)and prostate cancer:A role as clear as black and white. FASEB J,2002; 16(9):1093-5.
[34]Addison CL, Belperio JA, Burdick MD. Overexpression of the dufy antigen receptor for chemokines(DARC)by NSCLC tumor cells results in increased tumor necrosis. BMC Cancer,2004;4(1):28.
[35]Wang J. Ou ZL, Hou YF. Enhanced expression of Dufy an tigen receptor for chemokines by breast cancer cells attenuates growth and metastasis potential. Oncogene,2006; 25(54):7201-11.
[36]Sun G, Wang Y, Zhu Y. Risk Factors for Neck Nodal Metastasis in Papillary Thyroid Microcarcinoma:A Study of 1066 Patients. Acta Otolaryngol,2011;131(2): 197-203.
[37]Atiyeh BS, Costagliola M, Hayek SN.Keloid or hypertrophic scar:The controversy:Review of the literature. Ann Plast Surg,2005;54 (6):676-80.
[38]Rekha A. Keloids-A frustrating hurdle in wound healing. Int Wound J,2004; 1(2):145-8.
[39]Cole-Tobian JL, Cortes A, Baisor M. Age-acquired immunity to a Plasmodium vivax invasion ligand, the duffy binding protein. J Infect Dis,2002;186(4):531-9.
[40]Marnems AG, Norris JE, Olsen BR. Clinical genetics of familial keloids. Arch Dermatol,2001;137(11):1429-34.
[41]Luo S, Benathan M, Raffoul W. Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions. Plast Reconstr Surg, 2001; 107(1):87-96.
[42]Su Y, Arnold F, Cherry G. Proliferation and apoptosis in chronic wounds. Wound Rep Reg,1996; 28(4):141-7.
[43]Bayat A, McGrouther DA, Ferguson MW. Skin scarring. Br Med J,2003; 326(7380):88-92.
[44]Hadley TJ, Peiper SC. From Malaria to chemokine receptor:The emerging physiologic role of the duffy blood group antigen. Blood,1997;89(9):3077-91.
[45]Dustan HP. Does keloid pathogenesis hold the key to understanding black/white differences in hypertension severity? Hypertension,1995; 26(6):858-62.
[46]Keeley EC, Mehrad B, Strieter RM. CXC chemokines in cancer angiogenesis and metastases. Adv Cancer Res,2010; 106:91-111.
[47]Atiyeh B.S., Costagliola M., Hayek S.N. Keloid or hypertrophic scar:The controversy:Review of the literature. Ann Plast Surg,2005;54 (6):676-80.
[48]Rekha A. Keloids-A, frustrating hurdle in wound healing. Int Wound J, 2004; 1(2):145-48.
[49]Horuk R, Wang ZX, Peiper SC, et al. Identification and characterization of a promiscuous chemokine-binding protein in a human erythroleukemic cell line. J Biol Chem 1994,269(26):17730-17733.
[50]Ogawa R. keloids as a serious disease such as malignancy,plast reconstr surg.2008;122(3):993-4.
[51]Sidle DM, Kim H. keloids:prevention and management. Facial Plast Surg Clin North Am,2011; 19(3):505-15.
[52]Mustoe TA, Cooter RD, Gold MH.International clinical recommendations on scar management.Plast Reconstr Surg,2002; 110(2):560-71.
[53]Peiper SC, Wang ZX, Neote K. The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor. J Exp Med,1995;181(4):1311-7.
[54]Bao P, Kodra A, Tomic-Canic M. The role of vascular endothelial growth factor in wound healing. J Surg Res,2009; 153(2):347-58.
[55]Harrison SL, Vavken P, Murray MM.Erythrocytes inhibit ligament fibroblast proliferation in a collagen scaffold. J Orthop Res,2011; 29(9):1361-6.
[56]Patterson AM, Siddall H, Chamberlain G. Expression of the duffy antigen/receptor for chemokines (DARC) by the inflamed synovial endothelium. J Pathol,2002; 197:108-16.
[57]Pruenster M., Mudde L., Bombosi P. The Duffy antigen receptor for chemokines transports chemokines and supports their promigratory activity. Nat. Immunol,2009; 10(1):101-8.
[58]Dawson TC, Lentsch AB, Wang Z. Exaggerated response to endotoxin in mice lacking the Duffy antigen/receptor for chemokines (DARC). Peiper SC Blood,2000; 96(5):1681-4.
[59]Geleff S, Draganovici D, Jaksch P.The role of chemokine receptors in acute lung allograft rejection. Segerer S Eur Respir J,2010;35(1):167-75.
[60]Koch AE, Polverini PJ, Kunkel SL. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science,1992; 258(5089):1798-1801.
[61]Kelly M, Kolb M, Bonniaud P. Re-evaluation of fibrogenic cytokines in lung fibrosis. Curr Pharm Des,2003; 9(1):39-49.
[62]Gira AK, Brown LF, Washington CV, Cohen C. Keloids demonstrate high-level epidermal expression of vascular endothelial growth factor. Am Acad Dermatol,2004; 50(6):850-3.
[63]Hoch RC, Schraufstatter IU, Cochrane CG. in vivo, in vitro, and molecular aspects of interleukin-8 and the interleukin-8 receptor. J Lab Clin Med, 1996;128(2):134-5.
[64]Onq CT, Khoo YT, Tan EK. epthelial-mesenchymal interactions in keloid pathogenesis modulate vascular endothelial growth factor exression and secretion.J pathol,2007;211(1):95-108.
[65]Lim CP, Phan TT, Lim IJ, Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. Epub,2009;129(4):851-61.
[66]Lee JS, Frevert CW, Wurfel MM. Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo. J Immunol,2003; 170(10):5244-51.
[67]Friedman DW, Boyd CD,Mackenzie JW. Regulation of collagen gene expression in keloids and hypertrophic scar. J Surg Res,1993; 55(2):214-22.
[68]Ghahary A, Shen YJ, Nedelec B. Collagenase production is lower in post-burn hypertrophic scar fibroblast than in normal fibroblasts and is reduced by insulin-like-growth factor-Ⅰ. J Invest Dermatol,1996; 106(3):476-81.
[69]Kast RE. Halatsch ME. Matrix metalloproteinase-2 and-9 in glioblastoma:a trio of old drugs-captopril, disulfiram and nelfinavir-are inhibitors with potential as adjunctive treatments in glioblastoma. Arch Med Res,2012,43:243-47.
[70]Chakraborti S, Mandal M, Das S. Regulation of matrix metalloproteinases: an overview. Mole Cellr Biochem,2003;253:269-85.
[71]Louw L. The keloid phenomenal:progress toward a solution. Clinical Anat, 2007; 20(1):3-14.
[72]McCaw A, Ewald J, Werb Z. Matrix metalloproteinases and the regulation of tissue remodeling. Nat Rev Mol Cell Biol,2007;8(3):221-33.
[73]Bullen EC, Longaker MT, Updike DL. Tissue inhibitor of metalloproteinases..1 is decreased and activated gelatinases are increased in chronic wounds. J Invest Dermatol,1995; 104(2):236-40.
[74]Yager DR, Zhang LY, Liang HX, et al. Wound fluids fromhuman pressure ulcers contain elevated matrix metalloproteinases levels and activity compared to surgical wound fluids. J Invest Dermatol,1996; 107(5):743-48.
[75]Neely AN, Clendening CE, Gardner J.Gelatinase activity in keloids and hypertrophic scars. Wound repair region,1999; 7(3):166-71.
[76]Yeh FL, Shen HD,Tai HY. Decreased production of MCP-1 and MMP-2 by keloid-derived fibroblasts, Burns,2009,35(3):348-51.
[77]Tanriverdi AS, Menderes A, Oktay G. Matrix metalloproteinase-2 and -9 activities in human keloids, hypertrophic and atrophic SCarS:a pilot study. Cell Biochem Funct,2009,27(2):81-7.
[78]Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure, function, and biochemistry. Circ Res,2003;92(8): 827-39.
[79]Goede V, Brogelli L, Ziche M, et al. Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1. Int J Cancer,1999,82(5):765-70.
[80]Anders HJ, Sayyed SA, Vielhauer V. Questions about Chemokine and Chemokine Receptor Antagonism in Renal Inflammation, Nephron Exp Nephrol, 2010;114(2):e33-8.
[81]Xia Y, Frangogiannis NG MCP-1/CCL2 as a therapeutic target in myocardial infarction and ischemic cardiomyopathy. Inflamm Allergy Drug Targets,2007;6(2): 101-7.
[82]Tesch G H. MCP-1/CCL2:a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol,2008; 294(4):697-701.
[83]Gharaee KM, Denholm EM, Phan SH. Costimulation of fibroblast collagen and transforming growth factor betal gene expression by monocyte chemoattractant protein-1 via special receptors. J Biol Chem,1996;271(30):17779-84.
[84]Fivenson DP, Faria DT, Nickoloff BJ, et al. Chemokine and inflammatory cytokine changes during chronic wound healing. Wound Repair Regen,1997; 5(4):310-22.
[85]Low QE, Drugea IA, Duffner LA, et al. Wound healing in MIP-lalpha(-/-) and MCP-1 (-/-) mice. Am J Pathol,2001;159(2):457-63.
[86]Zymek P, Winkelmann K, Koerting A, et al Abou-Khamis T, et al. CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res,2005; 96(8):881-9.
[87]Yeh FL, Shen HD, Tai HY. Decreased production of MCP-1 and MMP-2 by keloid-derived fibroblasts. Burns,2009;35(3):348-51.
[88]Lim C P, Phan T T, Lim I J, et al. Cytokine profling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. J Invest Dermatol,2009; 129(4):851-61.
[89]Girardeau S, Mine S, Pageon H, et al. The Caucasian and African skin types differ morphologically and functionally in their dermal component. Exp Dermatol, 2009; 18(8):704-11.
[90]Darbonne WC, Rice GC, Mohler MA. Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin. J Clin Invest,1991;88(4):1362-69.
[91]Mangalmurti NS, Xiong z, HulverM. Loss of red cell chemokine scavenging promotes transfusion-related lung inflammation. Blood,2009;113(5):1158-66.
[92]Vielhauer V, Allam R, Lindenmeyer MT. Efficient renal recruitment of macrophages and T cells in mice lacking the Duffy antigen/receptor for chemokines. Am J Pathol,2009; 175(1):119-31.
[93]Fukuma N, Akimitsu N,Hamamoto H. A role of the Duffy antigen for the maintenance of plasma chemokine concentrations.Biochemical and Biophysical Research Communications,2003;303(1):137-9.
[94]Zarboek A, Sehmolke M, Bockhom SG, el al. The Dufry antigen receptor for chemokines in acute renal failure:A facilitator of renat chemokine presentation. Crit Care Med,2007; 35(9):2156-63.
[1]Marnems AG, Norris JE,Olsen BR, et al. Clinical genetics of familial keloids[J]. Arch Dermatol,2001,137(11):1429-34.
[2]Brown JJ, Ollier W, Thomson W, et al. Bayat A Positive association of HLA-DRB1*15 with Dupuytren's disease in Caucasians[J]. Tissue Antigens, 2008,72(2):166-70.
[3]Butler PD, Longaker MT, Yang GP. Current progress in keloid research and treatment[J]. J Am Coll Surg,2008,206(4):731-41.
[4]Bloom D, et al Heredity of keloids; review of the literature and report of a family with multiple keloids in five generations[J]. N Y State J Med,1956,56(4):511-519.
[5]Omo-Dare P, et al Genetic studies on keloid[J]. J Natl Med Assoc,1975, 67(6):428-32.
[6]宋文刚,宋光江,李桂倍,瘢痕疙瘩四家系遗传学分析[J].中国皮肤性病学 杂志,1992,6(2)：109-10.
[7]Chen Y, Gao JI-I, Liu XJ, et al.Characteristics ofoccurrence for Han Chinese familial keloids[J]. Burns,2006,32(8):1052-9.
[8]Clark JA, Turner ML, Howard L,et a 1.Description of familial keloids in five pedigrees:evidence for autosomal dominant inheritance and phenotypic heterogeneity [J]. BMC Dermatol,2009,9(8):1471-9.
[9]Marneros AG, Norris JE, Watanabe S, et al. Genome scans provide evidence for keloid susceptibility loci on chromosomes 2q23 and 7p11[J]. J Invest Dermatol, 2004,122(5):1126-32.
[10]陈阳,高建华,刘晓军,中国人群瘢痕疙瘩家系与染色体2q23和7p11的连锁分析[J].中国实用美容整形外科杂志,2006,17(3)：221-4.
[11]刘晓军,高建华,宋玫,瘢痕疙瘩候选基因与染色体7p11微卫星扫描及连锁分析研究[J].中国组织工程研究与临床康复,2008,12(28)：5453-6.
[12]刘晓军,高建华,宋玫,瘢痕疙瘩候选基因与染色体2q23微卫星扫描及连锁分析的多家系研究[J].中国组织工程研究与临床康复,2008,12(33)：6483-7.
[13]严欣,高建华,陈阳,一汉族瘢痕疙瘩家系的易感基因定位分析研究[J].中华整形外科杂志,2007,23(1)：32-5.
[14]陈阳,高建华,严欣,中国汉族一瘢痕疙瘩家系易感基因的定位研究[J].中华整形外科杂志,2007,23(2)：137-40.
[15]刘晓军,高建华,中国汉族瘢痕疙瘩家系易感基因位点的定位分析研究[J].中国美容整形外科杂志,2008,19(3)：179-82.
[16]刘晓军,高建华,中国汉族瘢痕疙瘩家系易感基因位点的定位分析[J].中国组织工程研究与临床康复,2008,12(37)：7241-4.
[17]刘晓军,高建华,瘢痕疙瘩家系17号染色体易感基因位点的定位分析术[J].广东医学,2008,29(5)：740-2.
[18]Shih B, Garside E, McGrouther DA, et al. Epub 2009 Dec 11 Molecular dissection of abnormal wound healing processes resulting in keloid disease [J]. Wound Repair Regen,2010,18(2):139-53.
[19]Satish L, Babu M, Tran KT, et al. Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways [J]. Wound Repair Regen,2004,12(2):183-92.
[20]段红杰,高建华,瘢痕疙瘩成纤维细胞p53基因突变检测[J].中国临床康复,2002,6(22)：3358-9.
[21]刘永波,高建华,段红杰,瘢痕疙瘩p53基因突变高发区基因结构的研究[J].中华整形外科杂志,2003,9(4)：258-60.
[22]蒋游晖,刘旺,李友良,瘢痕疙瘩组织p53基因突变的实验研究[J].中国美容医学,2003,11(6)：568-70.
[23]刘晓军,高建华,瘢痕疙瘩家系p53基因高发突变区基因突变检测的实验研究[J].中国美容整形外科杂志,2007,18(2)：108-11.
[24]卓阳,高建华,罗深秋,瘢痕疙瘩发病风险与p53基因第72位密码子多态性的关系[J].中华整形外科杂志,2005,21(3)：201-3.
[25]刘永波,高建华,瘢痕疙瘩临床表型与p53基因72位编码子基因多态性的相关性分析[J].中国修复重建外科杂志,2008,22(12)：1433-6.
[26]严笠,吕晓岩,王春梅,p53基因第72密码子多态性与部分中国人瘢痕疙瘩关系的研究[J].中华整形外科杂志,2007,23(5)：428-30.
[27]鲁峰,高建华,携带Fas基因重组腺病毒治疗瘢痕疙瘩的体外研究[J].中国修复重建外科杂志,2005,19(1)：35-8.
[28]刘永波,高建华,刘晓军,Fas基因和P53基因在瘢痕疙瘩成纤维细胞中的结构异常与其功能的关系[J].中国临床康复,2005,9(18)：106-108.
[29]刘晓军,高建华,瘢痕疙瘩家系Fas基因死亡域突变的实验研究[J].中国修复重建外科杂志,2007,21(7)：698-701.
[30]Laurentaci G. Dioguardi D. HLA antigens in keloids and hypertrophic seals [J], Arch Dermat,1977,113(12):1726-9.
[31]Brown JJ, Ollier WE, Thomson W, Bay at A Positive association of HLA-DRB1*15 with keloid disease in Caucasians[J]. Int J Immunogenet,2008, 35(4-5):303-7.
[32]孙东杰,陈东明,HLA-DR, HLA-DQ分子在瘢痕疙瘩患者外周血单个核细胞中的变化[J].中国美容医学,2008,17(3)：370-3.
[33]Lu WS, Wang JF, Yang S, Association of HLA-DQA1 and DQB1 alleles with keloids in Chinese Hans[J]. J Dermatol Sci,2008,52(2):108-17.
[34]Lu WS, Zuo XB, Wang ZX.Association of HLA haplotype with keloids in Chinese Hans[J]. burns,2011,37(5):794-9.
[35]Van Gersdorff G, Susztak K, Rezvan i F, et al. Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta[J]. J Biol Chem,2000,275(15):11320-6.
[36]Bay at A, Bock O, Mrowietz U, Genetic susceptibility to keloid disease: transforming growth factor beta receptor gene polymorphisms are not associated with keloid disease[J]. Exp Dermatol,2004,13(2):120-4.
[37]Bayat A, McGrouther DA, Ferguson MW, Skin scarring[J]. BMJ,2003, 326(7380):88-92.
[38]Brown JJ, Ollier W, Arscott G, Genetic susceptibility to keloid scarring:SMAD gene SNP frequencies in Afro-Caribbeans[J]. Exp Dermatol,2008,17(7):610-613.
[39]宋玫、高建华,瘢痕疙瘩家系外周血Smad2基因的突变检测[J].中国美容整形外科杂志,2009,20(3)：179-82.
[40]Archer HL, Gupta S, Enoch S, Distinct phenotype associated with a cryptic subtle omeric deletion of 19p13.3-pter[J].Am J Med Genet A,2005,136(1):38-44.
[41]Nakashima M, Chung S, Takahashi A, A genome-wide association study identifies four susceptibility loci for keloid in the Japanese population[J].2010, 42(9):768-71.
[1]Lee JY,Yang CC,Chao SC,Wong TW:histopathological differential diagnosis of keloid and hypertrophic scar. The American journal of dermatopathology 2004,26 (5):379-84.
[2]Ana Cristina Garcia-Ulloa, Oscar Arrieta,Tubal occlusion causing infertility due to an excessive inflammatory response in patients with predisposition for keloid formation.medical hypotheses 2005,65(5):908-14.
[3]Segerer S, Jedlicka J, Wtithrich RP. Atypical chemokine receptors in renal inflammation. Nephron Exp Nephrol 2010; 115(4):89-95
[4]Zlotnik A, Yoshie O, Nomiyama H. The chemokine and chemokine receptor superfamilies and their molecular evolution. Genome Biol,2006,7(12):243.
[5]Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure, function, and biochemistry. Circ Res 2003;92(8):827-39
[6]Goede V, Brogelli L, Ziche M, Augustin HG Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1. Int J Cancer 1999;82(5): 765-70.
[7]Xia Y, Frangogiannis N G. MCP-1/CCL2 as a therapeutic target in myocardial infarction and ischemic cardiomyopathy. Inflamm Allergy Drug Targets 2007:6(2): 101-7.
[8]Tesch G H. MCP-1/CCL2:a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy. Am J Physiol Renal Physiol 2008: 294(4):697-701.
[9]Marra F. Renaming cytokines:MCP-1, major chemokine in pancreatitis. Gut 2005: 54(12):1679-81.
[10]Gharaee-Kermani M, Denholm E M, Phan S H. Costimulation of fibroblast collagen and transforming growth factor betal gene expression by monocyte chemoattractant protein-1 via special receptors. J Biol Chem 1996:271(30): 17779-84.
[11]Fivenson DP, Faria DT, Nickoloff BJ, Poverini PJ, Kunkel S, Burdick M, et al. Chemokine and inflammatory cytokine changes during chronic wound healing. Wound Repair Regen 1997;5(4):310-22.
[12]Low QE, Drugea IA, Duffner LA, Quinn DG, Cook DN, Rollins BJ, et al. Wound healing in MIP-lalpha(-/-) and MCP-1 (-/-) mice. Am J Pathol 2001;159(2):457-63.
[13]Zymek P, Winkelmann K, Koerting A, Ren G, Abou-Khamis T, et al. CCL2/monocyte chemoattractant protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res 2005;96(8):881-9.
[14]Yeh FL, Shen HD, Tai HY. Decreased production of MCP-1 and MMP-2 by keloid-derived fibroblasts. burns 2009;35(3):348-351.
[15]Lim C P, Phan T T, Lim I J, Cao X. Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. J Invest Dermatol 2009:129(4):851-61.
[16]Girardeau S, Mine S, Pageon H, Asselineau D. The Caucasian and African skin types differ morphologically and functionally in their dermal component. Exp Dermatol 2009:18(8):704-11.
[17]Liao WT, Yu HS, Arbiser JL, Enhanced MCP-1 release by keloid CD 14+ cells augments fibroblast proliferation:role of MCP-1 and Akt pathway in keloids. Experimental Dermatology 2010; 19(8):142-50.
[18]Shaftel SS, Carlson TJ, Olschowka JA, Kyrkanides S, Matousek SB, O'Banion MK. Chronic interleukin-lbeta expression in mouse brain leads to leukocyte infiltration and neutrophil-independent blood brain barrier permeability without overt neurodegeneration. J Neurosci.2007;27(35):9301-9.
[19]Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, Moldawer LL, Nathan CF, Lowry SF, Cerami A. Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. J Exp Med. 1988;167(2):570-81.
[20]Nirodi CS, Devalaraja R, Nanney LB, Arrindell S, Russell S, Trupin J, Richmond A:Chemokine and chemokine receptor expression in keloid and normal fibroblasts. Wound Repair Regen 2000,8(5):371-82.
[21]Koch AE, Polverini PJ, Kunkel SL, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science,1992,258(5089):1798-801.
[22]Kelly M, Kolb M, Bonniaud P, et al. Re-evaluation of fibrogenic cytokines in lung fibrosis. Curr Pharm Des,2003,9(1):39-49.
[23]Gira AK, Brown LF, Washington CV, Cohen C, Arbiser JL. Keloids demonstrate high-level epidermal expression of vascular endothelial growth factor. Am Acad Dermatol 2004;50(6):850-3.
[24]Lim CP, Phan TT, Lim IJ, Cytokine profiling and Stat3 phosphorylation in epithelial-mesenchymal interactions between keloid keratinocytes and fibroblasts. Epub 2009; 129(4):851-61.
[25]Weber KS, Nelson PJ, Grone HJ, expression of CCR2 by endothelial cells: implications for MCP-1 mediated wound injury reparir and in vivo inflammatory activation of endothelium. Atterioscler thromb vasc boil 1999;19(9):2085-93.
[26]Liu L, Callahan MKJ Huang DJ et al. Chemokine receptor CXCR3:an unexpected enigma. Curr Top Dev BiolJ 2005,68:49-181.
[27]Cecelia C. Yates, Diana Whaley, Priya Kulasekeran*,Delayed and Deficient Dermal Maturation in Mice Lacking the CXCR3 ELR-Negative CXC Chemokine Receptor.The American Journal of Pathology 2007,171(2):484-95.
[28]Cecelia C. Yates,Priya Krishna,Diana Whaley, Lack of CXC Chemokine Receptor 3 Signaling Leads to Hypertrophic and Hypercellular Scarring.The American Journal of Pathology 2010;176(4):1588-91.