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。     

TLR4介导高迁移率族蛋白致系统性红斑狼疮肾损害的作用研究

目的 探讨高迁移率族蛋白1(HMGB1)致红斑性狼疮肾损害的作用机制与Toll样受体4(Toll-like receptor 4,TLR4)表达的相关性.方法 ELISA检测12例健康对照组、16例系统性红斑狼疮(systemic lupus eqrthematosus,SLE)无肾脏损害和18例狼疮性肾炎(lupus nephritis,LN)患者血清中HMGB1、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶组织抑制剂-2(TIMP-2)的表达情况;流式细胞术检测外周血CD3/TLR4和CD14/TLR4表达情况;分离外周血单个核细胞(PBMC),RT-PCR检测HMGB1 mRNA的表达变化.结果 HMGB1 mRNA相对表达量及血清中HMGB1蛋白在LN组明显高于SLE组和健康对照组;流式细胞术显示CD14+的单核细胞表面HMGB1受体TLR4在LN组表达最高(P＜0.05),且与尿蛋白呈正相关(P＜0.01);LN患者血清中MMP-2和TIMP-2蛋白的浓度明显低于SLE和健康对照组,同时MMP-2/TIMP-2比值下降.HMGB1 mRNA及CD14+/TLR4+与MMP-2/TIMP-2比值均呈显著负相关;LN组患者血清中HMGB1蛋白水平与蛋白尿呈正相关,与MMP-2/TIMP-2比值呈显著负相关.结论 HMGB1是狼疮性肾炎发病中的重要细胞因子;HMGB1可能部分通过TLR4激活PBMC,降低MMP-2/TIMP-2的活性,从而引起蛋白尿.     

PPAR-γ在LDL诱导的大鼠系膜细胞增殖和基质硬化中的作用

目的探讨PPAR-γ在LDL诱导的系膜细胞增殖和基质硬化中的作用。方法以体外培养的大鼠系膜细胞为研究对象,应用不同浓度的LDL刺激系膜细胞。应用MTT法检测细胞增殖;应用RT-PCR的方法检测LDL对MMP-2、TIMP-2、PPAR-γmRNA表达的影响;应用WesternBlot的方法检测LDL对MMP-2、TIMP-2、PPAR-γ蛋白表达的影响。结果以浓度为3.125~100μg/ml的LDL刺激系膜细胞,可促进系膜细胞的增殖,在LDL3.125~50μg/ml的浓度范围内,系膜细胞的增殖和LDL的浓度呈正相关(r=0.865,P<0.05);以浓度为12.5~100μg/ml的LDL刺激系膜细胞,可下调MMP-2/TIMP-2mRNA和蛋白的表达(P<0.01);以浓度为6.25~100μg/ml的LDL刺激系膜细胞,可下调PPAR-γmRNA和蛋白的表达(P<0.01)。结论在LDL诱导下,PPAR-γ的下调促进了系膜细胞的增殖和系膜基质的增生。     

压应力对人软骨终板细胞MMP-1、3、13和TIMP-1表达的影响研究

目的观察压应力对体外培养人软骨终板细胞分泌基质金属蛋白酶-1、3、13(MMP-1、MMP-3、MMP-13)和基质金属蛋白酶组织抑制因子1(TIMP-1)的影响。方法将6个月意外流产胎儿软骨终板,进行软骨终板细胞的分离、培养。将第2代软骨细胞按实验要求种板培养,分别施加周期性压应力(压力大小0.8 MPa,频率为0.1Hz),分别连续加载2 h、6 h和12 h。根据压应力作用时间分为2 h组、6 h组和12 h组,以空白组为对照,测定各组细胞上清液中MMP-1、MMP-3、MMP-13和TIMP-1的表达。结果贴壁后的软骨终板细胞成短梭形、三角形以及多边形,呈典型的"铺路石"状。随着细胞加压时间延长,长梭形细胞数目明显增加,去分化现象明显,老化速度加快,坏死细胞明显增多。施加压应力第3天,6 h组和12 h组MMP-1、MMP-3、MMP-13表达明显增高,TIMP-1明显下降,且加压时间越长,改变越明显。24 h组分别与各组相比较有统计学意义(0.05)。结论长期压应力可使MMP-1、MMP-3、MMP-13表达增高,TIMP-1表达降低,并抑制细胞增殖,同时MMP-1、MMP-3、MMP-13表达水平与压应力作用时间相关。     

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸对基质金属蛋白酶-1和基质金属蛋白酶组织抑制因子-1的调节在大鼠矽肺纤维化形成中的作用

目的:探讨N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(AcSDKP)对大鼠肺内基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶组织抑制因子-1(TIMP-1)表达的调节在拮抗矽肺纤维化形成过程中的作用.方法:气管内灌注染尘法制作大鼠矽肺模型,将含有AcSDKP的微量药物释放泵埋入腹腔.实验动物随机分为矽肺模型对照4周组,矽肺模型对照8周组,矽肺模型4周组,矽肺模型8周组,抗纤维化治疗组及预防治疗组.H-E染色和免疫组织化学显色对矽肺纤维化病变和MMP-1和TIMP-1在肺组织内的表达进行形态学观察;免疫印迹法对肺内MMP-1和TIMP-1酶蛋白表达进行检测.结果:与模型对照组比较,矽肺大鼠肺内MMP-1和TIMP-1表达增强.与矽肺模型组比较,AcSDKP能够上调矽肺大鼠肺内MMP-1的表达,下调TIMP-1的表达,使MMP-1/TIMP-1的比值升高.结论:AcSDKP能够促进矽肺大鼠肺内MMP-1的表达,抑制TIMP-1的表达,从而加速了细胞外基质(包括胶原)的降解,这可能与AcSDKP抗矽肺纤维化的作用有关.     

氯喹对体外活化肝星状细胞自噬与胶原代谢的影响

目的:研究不同浓度的自噬抑制剂氯喹(CQ)对活化的大鼠肝星状细胞系HSC-T6中Ⅰ、Ⅲ型胶原表达的影响及可能机制。方法:应用转化生长因子β1(TGF-β1)活化HSC-T6细胞,给予CQ干预24 h。实验分组为:control组、TGF-β1组、TGF-β1+CQ(15μmol/L)组、TGF-β1+CQ(30μmol/L)组和TGF-β1+CQ(60μmol/L)组。采用Western blot技术检测微管相关蛋白轻链3(LC3)比值LC3-Ⅱ/LC3-Ⅰ、自噬靶蛋白P62、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原、Ⅲ型胶原、基质金属蛋白酶13(MMP-13)、金属蛋白酶组织抑制物1(TIMP-1)和TIMP-2的表达情况;免疫细胞化学检测Ⅰ、Ⅲ型胶原的表达;RT-q PCR检测Ⅰ型胶原、Ⅲ型胶原、MMP-13、TIMP-1和TIMP-2mRNA的表达变化。结果:CQ干预后LC3-Ⅱ/LC3-Ⅰ比值明显升高且呈剂量依赖性;P62蛋白表达TGF-β1+CQ组均显著高于TGF-β1组(P0.01)。TGF-β1组的Ⅰ、Ⅲ型胶原表达量较control组显著增加,TGF-β1+CQ组较TGF-β1组也有明显增加。α-SMA的表达在TGF-β1组和TGF-β1+CQ组均显著高于control组(P0.05),而TGF-β1组和TGF-β1+CQ各组之间无显著差异。MMP-13表达在TGF-β1+CQ组较TGF-β1组显著下降(P0.05);TIMP-1和TIMP-2在TGF-β1+CQ组较TGF-β1组显著升高(P0.05),且呈剂量依赖性。结论:自噬抑制剂CQ能显著增加HSC-T6细胞中Ⅰ、Ⅲ型胶原的表达并呈剂量依赖性,这可能与其上调TIMP-1及TIMP-2的表达并抑制MMP-13表达有关。     

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。     

不同加载时间流体剪切力对成骨细胞中基质金属蛋白酶-1和基质金属蛋白酶抑制剂-1表达的影响

目的:研究加载不同时间流体剪切力对成骨细胞基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶抑制因子(TIMP-1)表达的影响。方法:利用自行设计的平行平板流体剪切力加载装置,对MC3T3-E1成骨细胞施加12 dyn/cm2流体剪切力0、15、30、45、60 min,采用蛋白免疫印记实验检测MMP-1和其抑制剂TIMP-1的表达水平。结果:对体外培养的MC3T3-E1细胞加载12 dyn/cm2流体剪切力,随着加载时间的延长,MMP-1表达上调,TIMP-1表达水平下调,在45 min左右达到高峰。结论:加载不同时间的12 dyn/cm2流体剪切力能够上调MC3T3-E1成骨细胞MMP-I的表达,下调TIMP-1的表达,加载45 min最合适。     

基质金属蛋白酶-2、3及其组织抑制剂TIMP-1在子宫内膜异位症中的表达

目的探讨基质金属蛋白酶(MMP-2、MMP-3)及其抑制剂(TIMP-1)在子宫内膜异位症发生及发展中的作用.方法采用免疫组化SP法分别测定MMP-2、MMP-3 、TIMP-1在卵巢子宫内膜异位症异位内膜60例(A组),子宫腺肌症异位内膜40例(B组),子宫肌瘤子宫内膜30例(对照组C)的表达强度.结果 A、B组中MMP-2、MMP-3的表达强度明显高于对照组(P<0.05)而TIMP-1的表达明显低于对照组(P<0.05);A、B组间MMP-2、MMP-3 、TIMP-1 的表达无明显差异(P>0.05).结论在子宫内膜异位症中MMP-2、MMP-3的过度表达及TIMP-1的低表达可能与内异症的发生与发展有关.     

丹参单体IH764-3促进H2O2刺激的鼠肝星状细胞胶原降解

目的观察丹参单体IH764-3对鼠肝星状细胞株(HSCs)基质金属蛋白酶-13(MMP-13)及其组织抑制因子(TIMP-1)表达的影响。方法应用体外细胞培养技术,用RT-PCR检测HSCsMMP-13mRNA水平;原位杂交和Westernblotting技术分别检测上述细胞TIMP-1mRNA和蛋白水平。结果IH764-3干预2h组MMP-13mRNA的表达强度明显上调,同时TIMP-1mRNA表达受抑制;IH764-3干预24h组TIMP-1蛋白表达受抑制。结论丹参单体IH764-3诱导HSCsMMP-13表达,抑制其TIMP-1表达是其抗肝纤维化的作用机制之一。     

MMP-3和TIMP-3在卵巢癌中的表达及其意义

目的探讨MMP-3和TIMP-3在卵巢癌的表达及其意义。方法本文采用光镜、透射电镜和免疫组化方法对27例卵巢癌组织中的MMP-3及TIMP-3表达进行检测。结果结果表明,临床分期为晚期的卵巢癌组织中MMP-3阳性细胞的数密度和面密度明显高于早期,而TIMP-3则低于早期。电镜下,早期淋巴细胞和树突状细胞浸润较多,癌细胞没有穿过基底膜;晚期淋巴细胞和树突状细胞浸润较少,可见癌细胞穿基底膜。结论MMP-3和TIMP-3的表达程度及MMP-3/TIMP-3的比值,可作为判断卵巢癌病程的指标。     

Sputum matrix metalloproteinase-9: tissue inhibitor of metalloproteinase-1 ratio in acute asthma

BACKGROUND: The ratio of matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) may be a marker of the balance between airway tissue destruction and repair. TIMP-1 may potentially contribute to the pathogenesis of increased submucosal extracellular matrix deposition in asthma. OBJECTIVE: Our purpose was to assess the variation in sputum MMP-9 and TIMP-1 during acute asthma. METHODS: We evaluated the MMP-9 and TIMP-1 balance in sputa of 16 asthmatic patients admitted with spontaneous exacerbation, conducting measurement before (day 1) and after methylprednisolone infusion therapy (days 2, 3, 5, and 7), and on remission days. RESULTS: Peak expiratory flow and eosinophilic cationic protein levels were significantly (P <.05) improved within 7 days in all patients. Sputum MMP-9 levels on day 2 tended to be lower than on day 1, but not significantly. Zymography revealed that the main enzyme was identified immunologically as MMP-9, and gelatinase activity on day 1 had a tendency to decrease for the following 7 days. The TIMP-1 levels gradually increased until day 5, were significantly (P <.05) high on day 5, and decreased on day 7. The MMP-9/TIMP-1 molar ratios were significantly (P <.05) decreased on days 2, 3, 5, and 7 compared with day 1. Sputum levels of MMP-9 and TIMP-1 and the MMP-9/TIMP-1 molar ratios on day 1 were significantly higher (P <.02) than those on remission days. CONCLUSIONS: An imbalance between MMP-9 and TIMP-1 was present in acute asthma, with an excess of MMP-9 resulting in a high ratio of MMP-9/TIMP-1 before treatment, and over time with glucocorticosteroid the TIMP-1 levels rose, dropping the ratio of MMP-9/TIMP-1. It was suggested that overproduction of MMP-9 and TIMP-1 after asthma exacerbation might contribute significantly to airway tissue remodeling and that TIMP-1 production in acute asthma might not be suppressed by glucocorticosteroid.     

蜕膜组织MMP-9/TIMP-3水平与自然流产关系

目的研究蜕膜组织中MMP-9／TIMP-3之间的平衡与自然流产发生的关系。方法用免疫组化S-P法测定30例自然流产患者与20例正常妊娠者蜕膜组织中MMP-9／TIMP-3的表达。结果研究组蜕膜细胞MMP-9表达阳性率为76．7％，高于对照组(55．0％，P-0．02)，两组蜕膜细胞TIMP-3的表达差异无显著性。结论自然流产患者蜕膜组织中MMP-9的表达增高，TIMP-3表达正常所导致的MMP-9／TIMP-3比例升高，在自然流产的发生中起重要作用。     

基质金属蛋白酶-9及其组织抑制物-1与颈动脉粥样斑块稳定性相关研究

目的: 探讨基质金属蛋白酶-9（MMP-9）、基质金属蛋白酶抑制剂-1（TIMP-1）、MMP-9/TIMP-1与脑梗死患者颈动脉粥样斑块稳定性的相关性。方法: 80例动脉粥样硬化性脑梗死患者按TCD微栓子检测结果分为微栓子阴性组70例和微栓子阳性组10例，20例正常人作为对照组，分别测定血浆MMP-9、TIMP-1水平。结果: 脑梗死患者血浆MMP-9、TIMP-1水平明显高于正常对照组(P<0.01)，相关性分析发现MMP-9水平与TIMP-1水平呈正相关（r=0.76，P<0.01）。MMP-9/TIMP-1比值仅在微栓子阳性组明显增高。结论: 血浆MMP-9参与了脑梗死的病理生理过程，血浆MMP-9和MMP-9/TIMP-1与颈动脉粥样斑块的不稳定性呈正相关。     

Relationship between matrix metalloproteinases MMP-2, MMP-9, tissue inhibitor of matrix metalloproteinases-1 and IL-5, IL-8 in nasal polyps

BACKGROUND: Nasal polyps (NP), a subgroup of chronic rhinosinusitis, are characterized by interleukin 5 (IL-5) mediated infiltration of eosinophils in sinus mucosa, leading to pseudostratified ciliated columnar epithelium, thickening of the epithelial basement membrane and tissue edema. Matrix metalloproteinases (MMP) constitute a large group of Zn2+ dependent endopeptidases with the ability to degrade extracellular matrix and are possibly responsible for the development of tissue edema in chronic sinusitis. OBJECTIVE: The aim of this study was to determine the expression of MMP-2, MMP-9 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) mRNA and to locate the distribution of MMP-2, MMP-9 and TIMP-1 by immunohistochemistry in ethmoid sinus mucosa in NP. Furthermore the correlation between IL-5 or IL-8 and MMP-2, MMP-9 or TIMP-1 is examined. METHODS: Nasal polyps of 33 patients and 18 specimens of inferior turbinate mucosa were examined by real time RT-PCR for MMP-2, MMP-9, TIMP-1, IL-5 and IL-8 mRNA expression. Immunohistochemical labeling for MMP-2, MMP-9 and TIMP-1 was performed. RESULTS: Differences between both locations were detectable for MMP-9 (P < 0.001) and IL-5 (P=0.003) but not for MMP-2 (P=0.278), TIMP-1 (P=0.515) and IL-8 (P=0.386). Correlation was detected only between TIMP-1 and IL-5 (r=0.422, P =0.014). Cytoplasmic staining of MMP-2 was present in the apical part of the ciliated cells, submucosal glands and in smooth muscle cells. Matrix metalloproteinase-9 was expressed in surface epithelium, in seromucous glands and in polymorphonuclear cells. CONCLUSIONS: Expression of MMP-9 and IL-5 mRNA are associated with NP. The correlation between IL-5 and TIMP-1 indicates the role of TIMP-1 in maintaining the homeostasis in NP.     

Imbalance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in toluene diisocyanate-induced asthma

BACKGROUND: Toluene diisocyanate (TDI)-induced asthma is an inflammatory disease of the airways characterized by airway remodelling due, at least in part, to an excess of extracellular matrix deposition in the airway wall. The ratio of matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) may be a marker of the balance between airway tissue destruction and repair. OBJECTIVE: We determined whether an imbalance of the MMP-9 : TIMP-1 molar ratio is present before and/or after challenge with TDI. METHODS: We used a murine model of TDI-induced asthma to evaluate the MMP-9 and TIMP-1 balance in the lung. RESULTS : The expression of MMP-9 and TIMP-1 mRNAs and proteins in the lungs increased at 7 h after TDI inhalation and continued for up to 72 h. Immunohistochemical and immunocytological analyses in the lungs of TDI-exposed mice revealed increases of immunoreactive MMP-9 and TIMP-1. There were significant correlations between the levels of MMP-9 or TIMP-1 and the number of neutrophils, lymphocytes, or eosinophils. The molar ratio of MMP-9/TIMP-1 significantly decreased at 7 h after TDI inhalation and continued up to 72 h. CONCLUSION: These data suggest that TDI-induced asthma may be associated with an imbalance between MMP-9 and TIMP-1, which could be useful as a marker of airway inflammation and airway remodelling in this disease.     

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。     

Investigation of Matrix Metalloproteinase -1, 2 and tissue inhibitor of matrix metalloproteinases-1 in Endometriosis

Objective: To explore the role of matrix metalloproteinase-1,2 (MMP-1, MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in endometriosis. Methods: The eutopic and ectopic endometria from 40 subjects suffering from endometriosis and regular.endometria from 40 subjects (excluding endometriosis) were collected and examined by in situ hybridization technology and western blot assay. Results: Both expressions of MMP-1 and -2 were stronger in ectopic endometrium and eutopic endometrium than in normal endometrium. On the contrary, the expression of TIMP-1 in ectopic endometrium and eutopic endometrium was lower. The differences were significant (P ＜ 0.01). Moreover, there was no relationship among the expressions of MMP-1, 2 and TIMP-1 in ectopic endometrium. Conclusion: The expressions of MMP-1, 2 and TIMP-1 lose balance and lack of periodic changes in ectopic endometrium , which explains the biological invasive behavior of endometriosis. It was suggested that regulating the balance between the MMPs and TIMP-1 should be an ideal therapeutic target to endometriosis.