缺氧对肺动脉成纤维细胞MMP-2、TIMP-1表达的影响

目的：探讨缺氧对肺动脉成纤维细胞（Fpa）分泌基质金属蛋白酶（MMPs）、金属蛋白酶组织抑制剂（TIMPs）的影响。 方法： 采用酶谱法测定Fpa培养基中MMP-2的酶活性，免疫印迹法检测培养基中MMP-2、TIMP-1 的蛋白水平，免疫组化法测定细胞原位的蛋白表达， RT-PCR法检测mRNA表达量。 结果： 缺氧后Fpa分泌的MMP-2酶活性、细胞内外蛋白表达量、mRNA表达量均下降；而TIMP-1的表达则呈相反变化。 结论： 缺氧可使肺动脉成纤维细胞MMP-2/TIMP-1的表达失衡，可能参与缺氧性肺血管重建。     

机械牵张对兔角膜成纤维细胞细胞外基质基因表达的影响

目的 研究机械牵张与白介素-1β（IL-1β）联合作用对兔角膜成纤维细胞细胞外基质相关基因表达的影响。方法 对原代提取的兔角膜成纤维细胞进行牵张幅度15%、频率0.1 Hz的周期性牵张12、24、36 h,同时给予IL-1β处理,采用实时荧光定量PCR检测细胞基质金属蛋白酶（MMPs）、基质金属蛋白酶的组织抑制剂（TIMP-1）和I型胶原α1（Collagen Iα1）mRNA表达变化水平。结果 IL-1β单独作用可以诱导角膜成纤维细胞MMP-1、MMP-3和MMP-9 mRNA表达;MMP-1和MMP-3 mRNA表达随时间而降低,MMP-9、TIMP-1、Collagen Iα1 mRNA则随时间而增加;IL-1β与机械牵张联合作用使MMP-1、MMP-3、MMP-9 mRNA表达水平上调,TIMP-1和Collagen Iα1 mRNA表达下调,且具有时间依赖。单独机械牵张使Collagen Iα1 mRNA表达下降,IL-1β与机械牵张联合作用使其表达进一步下调,且具有时间依赖性。结论 机械牵张与炎性因子联合作用可加剧角膜组织破坏,促进角膜膨隆的发生发展。     

三氧化二砷对皮肤成纤维细胞、中性粒细胞MMPs活性,TIMP-1及TGF-β1表达的影响

目的：砒石是化腐生肌的常用中药，其主要成分是三氧化二砷（As₂O₃）。本研究通过观察As₂O₃对基质金属蛋白酶（MMPs）活性、基质金属蛋白酶组织抑制因子-1（TIMP-1）及转化生长因子β₁（TGF-β₁）表达影响，探讨化腐中药能否调节胶原代谢，从而治疗慢性皮肤溃疡。方法:明胶酶谱法检测大鼠中性粒细胞（PMNs）来源的MMP-9活性、人成纤维细胞（hFb）分泌的MMP-1、MMP-2的活性，免疫细胞化学法检测hFb TIMP-1、TGF-β₁的表达。结果:As₂O₃浓度在50 mg/L时可以提高大鼠PMNs来源的MMP-9的活性（P<0.01）；在0.8 mg/L可以提高hFb分泌的MMP-1、MMP-2的活性（分别P<0.01）；同时As₂O₃作用于hFb 6 h、12 h、18 h后，TIMP-1、TGF-β₁表达持续降低（P<0.01）。结论:As₂O₃在一定范围内可提高PMNs来源的MMP-9的活性；也可提高hFb分泌的MMP-1、MMP-2的活性，同时抑制hFbTIMP-1、TGF-β₁的表达。提示砷类制剂可通过提高多种MMPs的活性，降低TIMP-1的表达从而发挥化腐作用。     

11-羰基-β-乙酰乳香酸（AKBA）调节基质金属蛋白酶-1,-2,-9的活性(英文)

目的：降低创面的高MMPs活性是治疗慢性皮肤溃疡的新途径。本研究主要观察11-羰基-β-乙酰乳香酸（AKBA,中药乳香的一种活性成分）对MMP-1、MMP-2和MMP-9活性的调节作用。方法： 人间质胶原酶（MMP-1）或者明胶酶A（MMP-2）被醋酸氨基苯汞（p-aminophenylmercuric acetate, APMA)激活后,与不同浓度的AKBA共同孵育1h,通过底物裂解法观察其活性的改变。MMP-9在中性粒细胞（polymorphonuclear neutrophils,PMNs)中含量丰富,因此以大鼠腹腔PMN作为MMP-9的来源。PMN裂解产物与不同浓度的AKBA共同孵育1 h,通过明胶酶谱法观察其中MMP-9活性的改变。我们建立了3个细胞模型：由TNF-α活化的人皮肤成纤维细胞模型;PMA活化的THP-1细胞模型和成纤维细胞-THP-1共培养细胞模型。AKBA与这3个细胞模型共同孵育24 h后,用ELISA法检测细胞上清中MMP-1、MMP-2和MMP-9的含量,用明胶酶谱法检测细胞上清中 MMP-2、MMP-9的活性。结果： AKBA在0.1-0.8 mmol/L浓度范围内对MMP-1、MMP-2的活性有抑制作用,IC50分别为0.18 mmol/L和0.27 mmol/L;在0.05-0.85mmol/L浓度范围内对MMP-9活性表现出不同程度的抑制作用（P<0.01）,其抑制作用呈浓度依赖性。AKBA促进成纤维细胞分泌MMP-2,但是,对THP-1细胞分泌MMP-9表现出抑制作用。在共培养细胞模型中,AKBA对MMP-1、MMP-2和MMP-9的分泌均表现出抑制作用。结论： AKBA作为乳香的一种活性成分,它对MMPs活性的直接抑制作用和对MMPs分泌的抑制作用可能是中药乳香治疗慢性皮肤溃疡的机制之一。     

N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸对转化生长因子β1介导的心成纤维细胞MMP-1/TIMP-1表达的调节作用

目的:探讨N-乙酰基-丝氨酰-天门冬酰赖氨酰-脯氨酸(AcSDKP)对转化生长因子β_1(TGF-β_1)介导的大鼠心成纤维细胞MMP-1/TIMP-1的调节作用。方法:差速贴壁法分离与获取新生大鼠心成纤维细胞。分别采用免疫细胞化学法和Western印迹法检测心成纤维细胞MMP-1、TIMP-1蛋白表达。结果:TGF-β_1可使心成纤维细胞MMP-1蛋白表达水平下降,而促进TIMP-1蛋白表达,MMP-1/TIMP-1比值下降。AcSDKP可以抑制TGF-β_1对心成纤维细胞MMP-1表达的下调作用,使MMP-1蛋白表达增加,而对TGF-β_1介导的TIMP-1蛋白表达无明显影响,MMP-1/ TIMP-1比值增加。结论:AcSDKP可以通过上调TGF-β_1介导的心成纤维细胞MMP-1蛋白表达并增加MMP-1/ TIMP-1比值,以加速细胞外基质降解,这可能与AcSDKP抗心纤维化的作用有关。     

三氧化二砷对皮肤成纤维细胞、中性粒细胞MMPs活性，TIMP-1及TGF-β1表达的影响

目的:砒石是化腐生肌的常用中药,其主要成分是三氧化二砷(As2O3)。本研究通过观察As2O3对基质金属蛋白酶(MMPs)活性、基质金属蛋白酶组织抑制因子-1(TIMP-1)及转化生长因子β1(TGF-β1)表达影响,探讨化腐中药能否调节胶原代谢,从而治疗慢性皮肤溃疡。方法:明胶酶谱法检测大鼠中性粒细胞(PMNs)来源的MMP-9活性、人成纤维细胞(hFb)分泌的MMP-1、MMP-2的活性,免疫细胞化学法检测hFb TIMP-1、TGF-β1的表达。结果:As2O3浓度在50mg/L时可以提高大鼠PMNs来源的MMP-9的活性(P<0.01);在0.8mg/L可以提高hFb分泌的MMP-1、MMP-2的活性(分别P<0.01);同时As2O3作用于hFb6h、12h、18h后,TIMP-1、TGF-β1表达持续降低(P<0.01)。结论:As2O3在一定范围内可提高PMNs来源的MMP-9的活性;也可提高hFb分泌的MMP-1、MMP-2的活性,同时抑制hFbTIMP-1、TGF-β1的表达。提示砷类制剂可通过提高多种MMPs的活性,降低TIMP-1的表达从而发挥化腐作用。     

早期妊娠的丢失与MMP-2、MMP-9及TIMP-1、TIMP-2的研究进展

基质金属蛋白酶（matrix metalloproteinases，MMPs）是参与细胞外基质（extracellular matrix，ECM）降解和重塑正常组织的重要酶类，基质金属蛋白酶的组织抑制物（tissue inhibitor of metalloproteinases，TIMPs）是MMPs活性的特异性抑制剂，在正常情况下MMPs和TIMPs维持着一个动态平衡。     

基质金属蛋白酶9在多囊卵巢综合征患者血清中表达增高

目的 近来有研究提示，基质金属蛋白酶（matrix metalloproteinases，MMPs）可能通过调节卵巢内细胞外基质成分的降解和重建，参与多囊卵巢综合征（polycystic ovary syndrome，PCOS）的病理发生。本研究旨在探讨MMP-9在PCOS发病中可能的作用机制。方法 采用病例一对照研究，ELISA法检测PCOS患者和正常对照组血清MMP-9及其抑制因子TIMP-1（tissue inhibitor of metalloproteinase-1，TIMP-1），胰岛素样生长因子I（insulin—like growth factor—I，IGF—I）和胰岛素样生长因子结合蛋白1（insulin—like growth factor binding protein-1，IGFBP-1）的蛋白表达水平。结果 PCOS患者血清中MMP-9浓度和MMP-9／TIMP-1比值明显高于对照组。在PCOS组，血清IGFBP-1水平与MMP-9呈负相关。结论 MMP-9的高表达可能通过调节IGFs的生物利用度参与PCOS的病理发生，其意义和解释需要进一步研究。     

AcSDKP对大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的影响

目的探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)对10%血清和血小板源性生长因子(PDGF)诱导的大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的调节作用。方法明胶酶谱法检测心成纤维细胞MMP-2、MMP-9的活性。Western blot法检测心成纤维细胞MMP-1的表达。结果10%血清和PDGF使心成纤维细胞MMP-2、MMP-9活性增强,也促进MMP-1的表达;AcSDKP能够进一步增加由10%血清和PDGF诱导的心成纤维细胞MMP-2、MMP-9的活性,并促进MMP-1的表达。结论AcSDKP上调了由PDGF介导的心成纤维细胞MMPs活性或表达,这可能与AcSDKP抗心肌纤维化的作用相关。     

高氧、维甲酸对早产大鼠肺成纤维细胞MMP-2及TIMP-2表达的调控

目的:探讨维甲酸（RA）对高氧暴露下早产大鼠肺成纤维细胞（LFs）基质金属蛋白酶-2（MMP-2）及其特异性组织抑制物-2（TIMP-2）表达的影响。方法:建立原代培养的早产大鼠LFs高氧暴露模型,采用半定量RT-PCR方法检测MMP-2和TIMP-2 mRNA表达,明胶酶谱法检测MMP-2酶原和活酶表达, Western blotting检测其磷酸化和总的ERK1/2、JNK1/2、p38和c-Jun表达。结果:（1）与对照组比较,高氧可促进早产大鼠LFs MMP-2 mRNA及其酶原和活酶表达（P<0.05,P<0.01）,同时使其p-ERK1/2、p-JNK1/2、p-p38和p-c-Jun表达水平显著提高（P<0.01,P<0.05）;（2）RA能不同程度下调高氧诱导的早产大鼠LFs MMP-2 mRNA高表达和明显降低其p-JNK1/2、 p-p38和p-c-Jun表达（P<0.01,P<0.05）,但进一步提高p-ERK1/2表达;（3）高氧、RA对TIMP-2 mRNA和总ERK1/2、JNK1/2 、p38及c-Jun表达无明显影响。结论:高氧暴露通过激活MAPKs信号转导通路（主要是JNK和p38）使c-Jun磷酸化水平提高,促进MMP-2表达和激活;RA通过抑制JNK和p38磷酸化,下调MMP-2表达与活化,从而拮抗高氧诱导的肺损伤。     

Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression

Aims:  Keloid is characterized by excessive deposition of collagen, resulting from aberrant extracellular matrix (ECM) production and degradation. The aim was to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in pathological wound healing in keloids.
Methods and results:  Semiquantitative analysis of 60 keloid tissue samples and 25 mature scar tissue samples demonstrated significantly increased expression of MMP-2, TIMP-2 and TIMP-3 in keloids compared with mature scars. Within keloid regions, MMP-2 expression was significantly higher in collagen bundle regions than in non-collagen bundle regions. Double immunofluorescence revealed that keloid fibroblasts between collagen bundles exhibited MMP-2, TIMP-2 and membrane-type 1 MMP (MT1-MMP) co-expression, whereas only MMP-2 expression was evident on the edge of collagen bundles. Western blot analysis and gelatin zymography of 13 keloid-derived fibroblasts (KFbs) and six normal skin dermal-derived fibroblasts (NFbs) demonstrated that unstimulated KFbs exhibited significantly increased MMP-2 activity and expression compared with NFbs under the same conditions.
Conclusions:  These results together indicate that MMP-2 activity can be promoted in keloid fibroblasts between collagen bundles in cooperation with TIMP-2 and MT1-MMP. This could contribute to remodelling of collagen bundle regions and invasion of fibroblasts into peripheral normal regions through promoted degradation of ECM.     

CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

Chronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1β, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1β and TNF-α or down-regulated by IFN-γ. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.     

Platelet-derived growth factor and transforming growth factor-β modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells

Background Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been suggested to be involved in the pathogenesis of asthma. Their expression in airway smooth muscle (ASM) cells could be involved in collagen turnover and migration of these cells and thus may contribute to airway remodelling.
Objective To examine the effect of pro-fibrotic growth factors TGF-β and platelet-derived growth factor (PDGF) on the expression of MMPs/TIMPs in cultured human ASM cells and to examine the role of MMP in the migration of ASM cells.
Methods ASM cells were stimulated with TGF-β and/or PDGF. Expression and activity of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 were evaluated by quantitative RT-PCR, Western blot and zymography. Modified Boyden-chamber migration assay was performed to investigate the effect of secreted MMP-3 and TIMP-1 on ASM-cell migration.
Results PDGF strongly up-regulated the expression of MMP-1 at mRNA and protein levels. PDGF, when combined with TGF-β, caused synergistic up-regulation of MMP-3. TIMP-1 was additively up-regulated by TGF-β and PDGF. These growth factors had no effect on the expression of MMP-2 and TIMP-2. U0126, an extracellular signal-regulated kinase (ERK) pathway inhibitor, inhibited the up-regulation of MMP-1 by PDGF. The synergistic/additive up-regulation of MMP-3 and TIMP-1 was inhibited by U0126 and SB431542, a Smad pathway inhibitor. Supernatant from ASM cells in which MMP-3 production was knocked down by RNA interference showed a decreased migratory effect on ASM cells, whereas supernatant from cells with suppressed TIMP-1 expression resulted in increased migration.
Conclusion Our results suggest that PDGF with/without TGF-β could facilitate migration of ASM cells by modification of MMP–TIMP balance through the ERK pathway.     

Suppressive effect of leflunomide metabolite (A77 1726) on metalloproteinase production in IL-1beta stimulated rheumatoid synovial fibroblasts

Leflunomide, an isoxazol derivative structurally unrelated to other immunomodulatory drugs, has proven to be efficacious in the treatment of rheumatoid arthritis (RA). This study was conducted to elucidate the mechanism by which leflunomide mediated antirheumatic effects. We investigated the effects of A77 1726, leflunomide's active metabolite, on mitogen-activated protein kinase (MAPK) activation in IL-1beta-stimulated rheumatoid synovial fibroblasts. The effects of A77 1726 on the secretion of matrix metalloproteinases (MMPs) from rheumatoid synovial fibroblasts were also examined. A77 1726 partially suppressed IL-1beta-induced ERK1/2 and p38 kinase activation. In contrast, A77 1726 efficiently suppressed IL-1beta-stimulated JNK1/2 kinase activation. Although no suppressive effect was demonstrated on MMP-2, A77 1726 markedly inhibited MMP-1, 3, and 13 secretions from IL-1beta-stimulated rheumatoid synovial fibroblasts. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was constitutively produced from rheumatoid synovial fibroblasts and the suppressive effects of A77 1726 on TIMP-1 production were minimal. Our results suggest that the suppression of the MAPK signalling pathway and MMP synthesis in rheumatoid synovial fibroblasts is a possible mechanism for the inhibitory activity of leflunomide against rheumatoid arthritis.     

Inhibition of histone deacetylases antagonized FGF2 and IL-1β effects on MMP expression in human articular chondrocytes

Fibroblast growth factor-2 (FGF2) and interleukin-1β (IL-1β) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1β, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1β on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1β in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1β both increased MMP-1 and -13 expression, while IL-1β also increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS-5, which was not affected by HDAC inhibition. FGF2, IL-1β, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1β induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity.     

Kinetics of matrix metalloproteinases and their regulatory factorsin mercuric chloride-induced tubulointerstitial fibrosisin Brown Norway rats

We investigated the kinetics of matrix metalloproteinases (MMPs) and their regulatory factors mRNAs in the kidneys of mercuric chloride-treated Brown Norway rats. The expression of MMP-1 mRNA remained at lower levels than control, while other MMPs mRNAs were upregulated. The expression of tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA showed significant upregulation. On the other hand, the expressions of TIMP-2 and TIMP-3 mRNAs were not significantly changed. In the plasmin-dependent pathway, the expression of plasminogen activator inhibitor (PAI-1) mRNA was continuously increased, while the expression of urokinase-type plasminogen activator (uPA) mRNA was not increased. The signals of TIMP-1 and PAI-1 mRNAs examined by in situ hybridization, were localized in the regenerative epithelial cells of the proximal tubules. In conclusion, these findings suggest that the activity of MMPs may bealtered by MMP-1 downregulation and inhibition of MMP activity by PAl-1 and TIMP-1 generated from tubular epithelial cells.     

PRL-2基因转染对肝细胞基质金属蛋白酶及其抑制剂表达的影响

目的:研究PRL-2基因增强肿瘤细胞侵袭及转移能力的机制。 方法:采用脂质体转染的方法将PRL-2基因表达质粒转染至正常永生化肝细胞系CL1中，G418筛选阳性克隆。应用明胶酶谱法检测转染肝细胞分泌MMPs 酶谱变化，Western blotting 及RT-PCR检测转染肝细胞MMP-2、MMP-9、TIMP-1和TIMP-2的变化。以PRL-2磷酸酯酶特异性抑制剂处理转染细胞，观察抑制PRL-2活性对上述指标的影响。 结果:经过 8周G418筛选及RT-PCR和Western blotting鉴定,获得稳定表达PRL-2的细胞亚系PRL-2-CL1。转染后的CL1细胞分泌MMP－9 、活性型MMP－9 和MMP－2 ,均显著高于转染前CL1细胞的MMPs 分泌(P<0.01);使用特异性抑制剂后， MMP－9 、活性型MMP－9 和MMP－2活性显著降低(P<0.01)。Western blotting及RT-PCR检测显示PRL-2-CL1细胞MMP2、MMP9蛋白及mRNA含量均较转染前显著升高(P<0.05)，TIMP-2则显著降低(P<0.05)；使用抑制剂后，可以逆转上述变化。 结论:PRL-2在永生化肝细胞中获得稳定、高效表达，PRL-2基因增强肝细胞侵袭及转移能力与其提高细胞MMP2、MMP9表达，降低TIMP-2有关。     

基质金属蛋白酶及其组织抑制因子在人前列腺组织及各种类型前列腺细胞中的表达

目的：研究基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）在人前列腺组织及各种类型细胞中的表达。方法： 用半定量RT－PCR的方法，对癌变和非癌变部分的前列腺组织、原代培养的平滑肌细胞、成纤维细胞、上皮细胞以及4种前列腺上皮细胞系（BPH－1、LNCaP、DU－145和PC－3）中MMP2、MMP7和MMP9、膜型基质金属蛋白酶1和3（MT1－MMP和MT3－MMP）及其组织抑制因子1和2（TIMP－1和TIMP－2）的mRNA 水平进行了测定。结果：MMP-2主要在前列腺基质细胞中表达；MMP-7和MMP-9则在前列腺上皮细胞中有较高的表达；MT1-MMP、MT3-MMP、TIMP-1和TIMP-2在前列腺基质细胞和上皮细胞中均有表达，但MT1-MMP和MT3-MMP在成纤维细胞中的表达量较高；另外，各种基质金属蛋白酶及其组织抑制因子在各种前列腺细胞系中也存在差异表达。结论： MMPs和TIMPs在前列腺组织及其各种类型细胞中的差异表达提示：它们可能在前列腺癌的转移中起着不同的作用。