下肢慢性静脉溃疡与基质金属蛋白酶相关性的研究进展

下肢慢性静脉溃疡是慢性静脉性疾病的常见和难治并发症,典型表现为下肢慢性溃疡和疼痛.基质金属蛋白酶是一类高度保守的锌依赖的内切蛋白酶,根据底物及结构差异,基质金属蛋白酶分为6类,其中胶原酶、明胶酶、间质溶解素三类在下肢慢性静脉溃疡发生、发展中起重要作用,基质金属蛋白酶1、2、3、8以及基质金属蛋白酶2与基质金属蛋白酶组织抑制因子2比例上调与溃疡愈合延迟有关,基质金属蛋白酶7、10、13和基质金属蛋白酶组织抑制因子1、2上调有利于溃疡愈合,而基质金属蛋白酶9、12在静脉溃疡中的作用尚需进一步研究.结合某些基质金属蛋白酶的功能特征,通过干预其合成及功能,可为下肢慢性静脉溃疡的治疗提供新的靶位.     

基质金属蛋白酶与基质金属蛋白酶抑制剂在光老化中作用的研究进展

紫外线照射会给人体的皮肤造成有害影响,其中,光老化是目前公认的长期暴露于紫外线照射所造成的主要危害之一.紫外线照射导致了基质金属蛋白酶家族的表达增加,其可降解真皮中的胶原及其他细胞外基质蛋白.大量的皮肤正常结缔组织结构的破坏损害了皮肤的功能并引起皮肤的老化.基质金属蛋白酶抑制剂是基质金属蛋白酶家族特异性的内源性抑制剂,并参与调控其在机体组织中的活性变化.二者之间的平衡在皮肤光老化的发生与发展中占有举足轻重的地位.     

Differential expression of matrix metalloproteinases and proteoglycans in Juvenile Hyaline Fibromatosis

Background

Juvenile Hyaline Fibromatosis (JHF) is a rare autosomal recessive disorder, histologically characterized by the production and deposition of an unidentified hyaline material in the skin and other organs. Extracellular matrix molecules are implicated in the development of skin lesion which is debilitating and recurrent and, so far, no treatment is satisfactory.

Objective

To investigate the expression of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and proteoglycans in lesional as compared to site-matched lesion-free skin tissue specimens of a JHF patient, aiming to elucidate the aetiopathological mechanisms involved in the development of JHF skin lesions.

Methods

Gelatinase activity of MMP-2 and MMP-9 was investigated by gelatine zymography. Protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 in skin tissue extracts were measured by ELISA. Gene expression of MMPs, TIMPs and proteoglycans was examined by quantitative RT-PCR.

Results

JHF lesions exhibited significantly higher activity as well as elevated protein and gene expression of MMP-2 and MMP-9, as compared to lesion-free skin tissue specimens. Decorin was downregulated and aggrecan was upregulated in lesional skin, as compared to normal skin.

Conclusion

The results presented in this study indicate that MMPs and proteoglycans may be involved in the pathogenesis of JHF and therefore these molecules may offer alternative targets for pharmacological intervention to achieve more radical and effective treatment.     

Alteration of extracellular matrix modulators after nonablative laser therapy in skin rejuvenation

BACKGROUND: Nonablative laser therapy is widely practised for skin rejuvenation, which stimulates collagen production and dermal matrix remodelling. Matrix remodelling is primarily modulated by a coordinated action of matrix metalloproteinases (MMPs) and their inhibitors, but the effects of nonablative lasers on these matrix modulators are not fully investigated. OBJECTIVES: To evaluate the changes in matrix modulators, such as MMP-1, MMP-2, MMP-3, MMP-9 and MT1-MMP, and their inhibitors (TIMP-1, TIMP-2 and RECK in particular), after nonablative laser treatments of human facial skin. METHODS: Twenty-four adult volunteers received a series of four nonablative laser treatments separated by 3-week intervals on facial skin. Two-millimetre skin punch biopsies were obtained at baseline and 3 weeks after the last treatment. RESULTS: Nonablative laser treatments led to a robust increase in two major dermal matrix components, type I collagen and tropoelastin. Among MMPs tested, levels of MMP-2 mRNA were statistically significantly increased, but the amount of active MMP-2 was rather reduced. More importantly, the expression level of RECK was significantly enhanced by laser treatments. CONCLUSIONS: Clinical outcomes following nonablative laser treatments may result not only from increased biosynthesis but also from decreased degradation, via an induction of RECK expression, of matrix proteins.     

CD147 siRNA对恶性黑素瘤细胞株A375增殖及CD147表达的影响

目的 研究CD147siRNA转染对恶性黑素瘤细胞株A375中CD147表达及细胞增殖的影响。方法 将前期构建的重组质粒pSUPER/CD147 siRNA稳定转染至恶性黑素瘤细胞株A375中,采用半定量RT-PCR法检测转染细胞中CD147 mRNA的表达。采用MTT法检测转染pSUPER/CD147 siRNA对肿瘤细胞增殖的影响。结果 转染了pSUPER/CD147 siRNA的恶性黑素瘤细胞株A375中CD147 mRNA表达水平显著下调,其在转染后24h、48h和72h的增殖水平均显著下降。结论 siRNA转染能有效抑制恶性黑素瘤细胞株A375中CD147的表达,且能抑制肿瘤细胞的增殖能力。     

SIRT1 modulates expression of matrix metalloproteinases in human dermal fibroblasts

Background SIRT1, an NAD⁺‐dependent histone/protein deacetylase, controls a broad range of cellular functions. Objectives We examined if SIRT1 is involved in the regulation of matrix metalloproteinase (MMP) expression in human dermal fibroblasts. Methods We studied the effect of inhibition of SIRT1 by specific inhibitor and small interfering RNA (siRNA) on MMP‐1 and MMP‐3 expression in human dermal fibroblasts. Results Treatment with a potent and selective inhibitor of SIRT1, EX‐527, increased the basal expression levels of MMP‐1 and MMP‐3 proteins. Knockdown of endogenous SIRT1 by siRNA led to increased expression of MMP‐1 and MMP‐3 at both mRNA and protein levels. SIRT1 knockdown also upregulated MMP protein induction caused by an inflammatory cytokine, interleukin (IL)‐1β. Moreover, treatment with a SIRT1 activator, resveratrol, significantly suppressed IL‐1β‐mediated induction of MMP‐1, which was attenuated by pretreatment with EX‐527. Finally, MMP‐1 promoter activity was increased by EX‐527 in cells treated with or without IL‐1β. Conclusions Our findings suggest that SIRT1 exerts a negative regulatory role in the production of MMP‐1 and MMP‐3 in human dermal fibroblasts.     

地塞米松对瘢痕疙瘩成纤维细胞基质金属蛋白酶1调控的研究

目的：探讨地塞米松对瘢痕疙瘩成纤维细胞基质金属蛋白酶1（又被称为间质胶原酶1）蛋白的分泌合成和mRNA表达的影响。方法：采用酶联免疫吸附实验（ELISA）和Northem杂交技术检测了培养瘢痕疙瘩成纤维细胞基质金属蛋白酶1蛋白的合成分泌和mRNA的表达。结果：培养瘢痕疙瘩成纤维细胞对照组基质金属蛋白酶1蛋白的浓度为（151．02±27．70）pg／mL；加入10^-9M和10^-6M含地塞米松培养基24小时后，基质金属蛋白酶的浓度分别为（121．27±45．57）pg／mL（P〈0．05）和（101．78±35．82）pg／mL（P〈0．01）。加入地塞米松24小时后，培养瘢痕疙瘩成纤维细胞的基质金属蛋白酶1mRNA的表达被显著抑制，经密度扫描定量分析：基质金属蛋白酶1mRNA的表达分别下降了大约36％和53％。结论：地塞米松可抑制培养瘢痕疙瘩成纤维细胞基质金属蛋白酶1蛋白的合成分泌及其mRNA的表达，可能是地塞米松在抗瘢痕疙瘩纤维化作用中，尤其是纤维化形成后，疗效欠佳的原因。     

银屑病患者皮损中CD147、亲环素A和亲环素B的表达

目的 检测CD147、亲环素A和亲环素B在正常人皮肤和银屑病皮损中的表达,探讨它们在银屑病发病机制中的作用。方法 用免疫组化法半定量检测银屑病皮损和正常人皮肤中CD147、亲环素A和亲环素B的表达,用免疫反应强度分布指数(IRIDI)表示。结果 银屑病各组角质形成细胞中CD147和亲环素A的IRIDI均显著高于正常对照组(P值均<0.05)。脓疱性银屑病组角质形成细胞、T细胞和中性粒细胞中CD147和亲环素A的IRIDI均显著高于进展期寻常性银屑病组(P值均<0.05)。寻常性银屑病组角质形成细胞中CD147和亲环素A的IRIDI差异无统计学意义(P值均>0.05),进展期寻常性银屑病组T细胞和中性粒细胞中CD147和亲环素A的IRIDI均显著高于静止期寻常性银屑病组(P值均<0.05)。脓疱性银屑病组T细胞中亲环素B的IRIDI显著高于进展期寻常性银屑病组(P<0.05),进展期寻常性银屑病组T细胞中亲环素B的IRIDI显著高于静止期寻常性银屑病组(P<0.05)。所有组间角质形成细胞及银屑病组间中性粒细胞中亲环素B的IRIDI差异无统计学意义(P值均>0.05)。结论 CD147、亲环素A和亲环素B可能在银屑病的发生发展中起作用。     

银屑病患者CD147、亲环素A和亲环素B表达

目的 探讨CD147、亲环素A和亲环素B存银屑病发病机制中的作用.方法 用RT-PCR半定量检测40例寻常性银屑病、15例脓疱性银屑病患者和20例正常人外周血T细胞和中性粒细胞中CD147、亲环素A和亲环素B mRNA的表达水平.结果 在T细胞中,寻常性银屑病组CD147、亲环素A和亲环素B mRNA表达水平分别为1.231±0.128、1.254±0.096和1.667±0.166,显著高于正常人对照组(分别为0.983±0.105、1.084±0.070和1.386±0.152,P值均<0.01),且患者组三者表达水平均与其相应的PASI积分呈正相关(r值分别为0.544、0.643和0.668,P值均<0.01);脓疱性银屑病组三者表达水平分别为1.418±0.117、1.760±0.160和1.959±0.156,显著高于寻常性组(P值均<0.01).寻常性银屑病组中性粒细胞CD147 mRNA表达水平(2.118±0.278)显著高于正常人对照组(1.393±0.144,P<0.01),且与其相应的PASI积分呈正相关(r值为0.618,P<0.01);脓疱性银屑病组中性粒细胞CD147的表达水平(3.072±0.371)显著高于寻常性银屑病组(P<0.01).寻常性银屑病组亲环素A和亲环素B mRNA表达水平分别为1.127±0.086和1.081±0.124,与正常人对照组比较差异无统计学意义(P值均>0.05);并与其相应的PASI积分无线性相关关系(r值分别为0.290和0.144,P值均>0.05).脓疱性银屑病组二者的表达水平(分别为1.116±0.075和1.096±0.133)与寻常性银倩病组比较差异无统计学意义(P值均>0.05).结论 CD147、亲环素A和亲环素B可能在银屑病疾病活动和发展过程中起作用.     

Interleukin-6 (IL-6) modulates migration and matrix metalloproteinase function in dermal fibroblasts from IL-6KO mice

BACKGROUND: Interleukin-6-deficient (IL-6KO) mice display significantly delayed cutaneous wound healing characterized by decreased re-epithelialization, granulation tissue and wound closure. Dermal fibroblasts are one of the principal cell types found in granulation tissue and mediate numerous processes during healing. OBJECTIVES: To investigate the effects that IL-6 might have on granulation tissue formation and fibroblast motility. As fibroblast motility is associated with matrix metalloproteinase (MMP) activity, the expression of MMP-2 and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 were assessed. METHODS: Punch biopsies (4 mm) were performed in the skin of IL-6KO and C57BL/6 mice. The expression of MMP-2, TIMP-1 and -2 in wound tissue was monitored over time. Cellular infiltration and granulation tissue formation was monitored by subcutaneous implantation of polyvinyl alcohol (PVA) sponges. A free-floating collagen lattice model was also used to investigate the direct effects of IL-6 treatment on isolated IL-6KO fibroblasts. The expression of MMP-2, and the inhibitors TIMP-1 and -2, were assessed via real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: IL-6KO wounds showed impaired granulation tissue formation 5 days postwounding and fewer fibroblasts had populated the PVA matrices 7 days after implantation in IL-6KO mice compared with wild-type C57BL/6. The mRNA and protein expression of MMP-2 and TIMP-2 mRNA was increased in IL-6KO mice compared with wild-type mice beyond 1 day postwounding, while the expression of TIMP-1 mRNA was transiently higher in IL-6KO only 3 days postwounding. Treatment of collagen lattices with various concentrations of rmIL-6 again showed a dose-response decrease in mRNA and protein expression of MMP-2 and TIMP-2 protein expression, compared with saline control, while TIMP-1 did not appear to be significantly modulated. CONCLUSIONS: These results indicate that IL-6 influences the function of fibroblasts in wounds, and one mechanism of this regulation may be through the modulation of MMP-2 and TIMP proteins.     

A study of matrix metalloproteinase expression and activity in atopic dermatitis using a novel skin wash sampling assay for functional biomarker analysis

Background Atopic dermatitis (AD) is a chronic inflammatory skin condition that is characterized by a defective skin barrier. Despite the well‐recognized role of proteases in skin barrier maintenance, relatively little is known of the contribution made by matrix metalloproteinases (MMPs) to the inflammatory process in AD. Objectives To test a simple, novel ex vivo bioassay technique in an analysis of the MMPs present in wash samples taken from the skin surface of patients with AD. Methods Saline wash samples were collected from eczematous and unaffected areas of the skin of patients with AD and from the skin of normal controls. Wash samples were analysed for their MMP content using a functional peptide cleavage assay, gelatin zymography and an antibody array. Results Using a functional substrate cleavage assay, skin wash samples from AD lesions were shown to contain 10‐ to 24‐fold more MMP activity than those from normal control skin (P < 0·02) and fivefold more than those from unaffected AD skin (P < 0·05); this activity was inhibited by a broad‐spectrum MMP inhibitor Ro 31‐9790. Gelatin zymography and antibody array analysis revealed substantial levels of MMP‐8 (neutrophil collagenase) and MMP‐9 (92‐kDa gelatinase) in AD skin wash samples as well as lower levels of MMP‐10 (stromelysin 2) and tissue inhibitor of metalloproteinases (TIMP)‐1 and TIMP‐2; low levels of MMP‐1 (fibroblast collagenase), MMP‐3 (stromelysin 1) and TIMP‐4 were also detected. Conclusions A simple skin wash technique suitable for the quantitative and functional analysis of biomolecules in AD is described. Using this method we show that MMPs, and in particular MMP‐8 and MMP‐9, represent an important potential component of the pathology of AD. The method is expected to prove useful in advancing our understanding of AD and in identifying biomarkers for the evaluation of new therapies.     

Immunohistochemical expression of matrix metalloproteinases in the granulomatous rosacea compared with the non‐granulomatous rosacea

Background There is a granulomatous variant which is recognized in the rosacea spectrum. However, the pathogenesis of granuloma formation in rosacea has not been clearly demonstrated. Matrix metalloproteinases (MMPs) are required for recruitment of inflammatory cells and for tissue remodelling, making way for the development of well‐organized granuloma. Objective The aim of this study was to investigate the expression of transforming growth factor (TGF)‐β, TGF‐β type II receptor (TβRII), Tumour necrosis factor (TNF)‐α, MMP‐1, 2 and 9 in the granulomatous rosacea (GR) compared with the non‐granulomatous rosacea (NGR) and test the hypothesis that the changes of these profiles in GR would be related with chronic ultraviolet radiation (UVR)‐exposure. Methods Facial skin samples were obtained from 20 patients with GR and NGR (control group). The sections were stained using haematoxylin and eosin, Verhoeff’s elastic stain, and antibodies to TGF‐β, TβRII, TNF‐α, MMP‐1, ‐2 and ‐9. Results The amount of elastotic material was significantly increased in the dermis of GR lesions. Expression of TGF‐β was significantly decreased in the epidermis of GR lesions compared with NGR lesions. In addition, the expression of MMP‐9 was significantly increased in the dermis of GR lesions compared with NGR lesions, especially at the centre of the granuloma on a semi‐quantitative analysis. MMP‐2 expression was also increased in GR lesions, although the difference between the two groups was not statistically significant. Conclusions The results of this study suggest that the increased expression of MMPs in the dermis may participate in granuloma formation of GR in association with UVR.     

基质金属蛋白酶-8在寻常型银屑病患者血清及皮损中的表达研究

 目的:探究基质金属蛋白酶-8（MMP-8）在寻常型银屑病（PV）炎症反应中的作用。方法:将20例PV患者根据银屑病面积和严重程度指数（PASI）分为轻度组6例、中度组6例及重度组8例。用ELISA法检测并比较PV患者组及正常对照组(12例)血清中MMP-8浓度;用荧光定量PCR扩增并比较不同病情严重程度组间皮损处以及外观正常的周边皮肤MMP-8相对表达量;用免疫组化法观察MMP-8在皮损部位的分布状况。结果:血清MMP-8浓度比较：轻度组明显低于对照组(t=2.54,P=0.022）、重度组（t=2.69,P=0.020）。PV患者皮损部位MMP-8表达明显高于皮损相邻部位外观正常的皮肤（t=2.91,P=0.009）;重度组PV皮损部位MMP-8表达明显高于轻度组（t=3.45,P=0.005）及中度组（t=2.74,P=0.018）。在PV皮损部位,MMP-8主要表达于表皮基底细胞及棘细胞,而在皮损周边部位,MMP-8在基底细胞中表达较明显。真皮中MMP-8主要表达于小血管处。结论:MMP-8在炎症反应较强的皮损中表达上升,推测其通过调控炎症反应及促进血管新生等参与PV的免疫病理进程。     

Transformation-specific matrix metalloproteinases (MMP)-7 and MMP-13 are expressed by tumour cells in epidermolysis bullosa-associated squamous cell carcinomas

BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) have an increased risk of developing rapidly progressive and metastatic cutaneous squamous cell carcinomas (SCC). It is unclear why these SCC behave more aggressively than sporadic SCC. Matrix metalloproteinases (MMP) are a family of endopeptidases that contribute to growth, invasion and metastasis of SCC. The role of MMP in RDEB-associated SCC is not known. OBJECTIVES: To investigate the expression of MMP-7, MMP-13 and MMP-9 in RDEB-associated SCC in comparison with sporadic SCC and Bowen's disease. METHODS: Immunohistochemical analysis of 25 RDEB-associated SCC, 61 sporadic SCC and 28 sporadic lesions of Bowen's disease was carried out using monoclonal antibodies for MMP-7, MMP-9, MMP-13 and E-cadherin and syndecan-1. RESULTS: MMP-7 was detected in all RDEB-associated SCC, in tumour cells within the invasive edge, where E-cadherin and syndecan-1 were markedly diminished or absent. MMP-7 expression was also observed in 98% of sporadic SCC and in 68% of Bowen's diseases. MMP-7 staining was significantly stronger in RDEB-associated SCC than in sporadic SCC, and was most abundant in poorly differentiated tumours. MMP-13 was detected in tumour cells in 96% of RDEB-associated SCC and in all sporadic cutaneous SCC. MMP-9 was detected in the inflammatory cells in all SCC examined. CONCLUSIONS: These results identify MMP-7 and MMP-13 as tumour cell-specific markers for SCC progression and as potential therapeutic targets in RDEB-associated SCC. The pattern of immunolabelling suggests that MMP-7 may shed E-cadherin and syndecan-1 from the SCC cell surface.     

Gene expression of types I and III collagen, decorin, matrix metalloproteinases and tissue inhibitors of metalloproteinases in skin fibroblasts from patients with systemic sclerosis

Cultured fibroblasts from patients with systemic sclerosis (SSc) and normal individuals were examined for gene expression of types I and III collagen, decorin, matrix metalloproteinases (MMP) MMP-1, MMP-2, and MMP-3, tissue inhibitors of metalloproteinases (TIMP) TIMP-1 and TIMP-2, urokinase- and tissue-type plasminogen activators (u-PA and t-PA). Fibroblasts from patients with early stage SSC (less than 1 year duration of disease) exhibited higher levels of types I and III procollagen, decorin, MMP-1, MMP-3, TIMP-1, and PAs than those from normal individuals. The gene expression of procollagen α1(I) and TIMP-1 mRNAs were increased, but those of decorin, MMP-1, MMP-2, and MMP-3 were decreased, in fibroblasts from SSc patients with mid-stage SSc (2 to 4 years duration) as compared with those from normal individuals. In contrast, no significant difference in gene expression was found between fibroblasts from normal individuals and from patients with late-stage SSc (more than 6 years duration). These results suggest that gene expression of collagen, decorin, and degrading factors is dynamically modulated during fibrillogenesis. The responses of procollagen α1(I) mRNA to IL-1 and TGF-β were lower in fibroblasts from SSc patients with early and mid-stage disease, but not in those from patients with-late stage disease, than in control fibroblasts, which indicates that these cytokines may be involved in the earlier phases of fibrosis in SSc. Received: 30 August 1996     

Squamous cell carcinoma complicating chronic venous leg ulceration: a study of the histopathology, course and survival in 25 patients

We have studied 25 cases of squamous cell carcinoma in chronic venous leg ulcers. Twenty-three of the patients were dead and two were alive. The mean age at cancer diagnosis was 78.5 years. The median survival was 1 year. Eleven tumours were well-differentiated, 10 moderately and four poorly. All patients with a poorly differentiated tumour died within a year. Metastases were certain in eight cases. The disease was lethal in 10 cases which included all poorly differentiated tumours. The survival of the study group was significantly shortened compared with a control group of patients with lower limb non-melanoma skin cancer (n = 433) from the Swedish Cancer Registry (P = 0.0084). When diagnosed, squamous cell carcinoma in chronic leg ulcers merits a thorough investigation of the degree of differentiation and spread. Assertive treatment is indicated as poorly differentiated tumours and some moderately differentiated tumours may be fatal.