基质金属蛋白酶及其抑制剂在器官纤维化中的研究进展

器官纤维化共同的基本特征是ECM过度沉积和组织结构改建 ,而病变组织内ECM降解减少是导致其过度沉积的主要原因之一。正常或异常的ECM代谢基于基质降解限速酶MMPs及其抑制剂TIMPs的平衡或失衡 ,MMPs/TIMPs受多种细胞因子和转录因子的调节 ,对肺、肝、肾等器官纤维化的发生、发展及其临床监测与治疗具有重要意义。     

人组织金属蛋白酶抑制因子-3的分离纯化及其生物学性质

目的:从人胎盘中分离、纯化组织金属蛋白酶抑制因子3(TIMP3),并对其对基质金属蛋白酶1(MMP1)的抑制作用进行评价。方法:利用含4mol/L尿素的TrisHCl缓冲液(pH8.0),从除去多数水溶性蛋白的胎盘匀浆液中提取难溶性蛋白。经CM52阳离子交换树脂和SephacrylS200凝胶过滤两步柱层析纯化后,用SDSPAGE鉴定TIMP3的纯度;用EIA和Westernblot检测TIMP3的含量;用免疫荧光测定法检测TIMP3对MMP1的抑制活性。结果:人胎盘来源的TIMP3由相对分子质量(Mr)为24000的非糖化蛋白和Mr为27000的糖化蛋白组成。非糖基化的TIMP3对MMP1的IC50为1.1×10-10mol/L,糖基化的TIMP3为1.2×10-10mol/L,显著高于重组的TIMP3(IC50为8×10-8)。结论:人胎盘来源的TIMP3由Mr为24000的非糖基化蛋白和Mr为27000的糖基化蛋白两部分组成,二者对MMP1均具有明显的抑制作用。     

心房纤颤模型犬心房组织基质金属蛋白酶9及组织抑制因子1与纤维化的关系***

背景：基质金属蛋白酶及其组织抑制因子在心房组织中的相互作用及动态平衡与心房纤颤的发生及维持密切相关。 目的：构建持续性心房纤颤犬模型,观察其心房肌组织基质金属蛋白酶9及其组织抑制因子1的基因表达与心房纤颤及心肌纤维化的关系。 方法：采用慢性快速心房起搏诱发持续性心房纤颤犬模型,并设置假手术组。通过Masson三色法染色计算胶原容积分数来评估纤维化程度,左心房心肌基质金属蛋白酶9及组织抑制因子1的mRNA水平表达使用反转录聚合酶联反应检测,其蛋白水平表达通过蛋白质印迹法测定。 结果与结论：与假手术组相比,持续性心房纤颤模型组心房肌纤维化程度明显增高,胶原容积分数明显增加(P < 0.01),且基质金属蛋白酶9 mRNA及蛋白表达水平明显增加(P < 0.01),组织抑制因子1的mRNA及蛋白表达水平明显下降        (P < 0.01)。结果证实,心房纤颤心房组织中基质金属蛋白酶9/组织抑制因子1基因表达的调控失衡以及基质金属蛋白酶9活性的增高与组织抑制因子1活性降低可能是影响胶原代谢、促进或抑制心肌纤维化,造成心房纤颤时心房结构重构的分子机制之一。     

芪参二莲汤对肝纤维化大鼠基质金属蛋白酶-1及 抑制因子的影响

目的 研究不同剂量芪参二莲汤对肝纤维化大鼠基质金属蛋白酶-1及抑制因子变化的影响。方法 于2017年6月~8月选择90只SD雄性大鼠作为研究对象,按照体质量随机分为6组,每组15只,其中1组为正常组,其余5组均为肝纤维化模型,将其分为模型组、秋水仙碱组以及芪参二莲汤低剂量组（5 g/kg）、中剂量组（10 g/kg）和高剂量组（15 g/kg）;各给药组分别给予相应的药物灌胃,正常组、模型组则给予生理盐水灌胃;对比各组大鼠MMP-1和TIMP-1水平以及肝纤维化指标水平。结果 正常组与模型组比较,后者的MMP-1、MMP-1/TIMP-1水平明显降低,而TLISA-1水平则明显升高,差异有统计学意义（P＜0.05）;模型组与各给药组比较,各给药组的MMP-1、MMP-1/TIMP-1水平较模型组升高,而TIMP-1水平则较模型组降低,差异有统计学意义（P＜0.05）,且给药组中,以芪参二莲汤高剂量组的变化水平最为显著,差异有统计学意义（P＜0.05）。结论 15 g/kg高剂量的芪参二莲汤用于治疗肝纤维化大鼠,能够有效促进MMP-1表达、减少TIMP-1表达,进而起到逆转肝纤维化的作用。     

基质金属蛋白酶与胎膜早破

胎膜早破（preterm premature of the fetal membranes，PROM）和足月前胎膜早破（P—PROM）是威胁母婴健康的常见并发症。PROM的发生率是10％；P—PROM的发生率是2％-3.5％，30％-40％的早产与此有关，而85％的新生儿发病率和死亡率由早产引起。胎膜破裂后，由于屏障作用消失，将可能引起羊膜腔感染、围产期感染、败血症、新生儿窒息等严重的并发症，如果处理不当可危及母儿安全与健康，因此PROM一直受到产科界的关注。     

基质金属蛋白酶-9及金属蛋白酶组织抑制因子-1在妊娠期高血压疾病患者胎盘组织中的表达

目的通过研究基质金属蛋白酶-9（MMP-9）及金属蛋白酶组织抑制因子-1（TIMP-1）在正常妊娠和妊娠期高血压疾病患者胎盘中的表达,探讨其与妊娠期高血压疾病发病机制的关系。方法收集山西医科大学第一医院产科和山西中医学院中西医结合医院产科住院分娩的产妇胎盘标本共142例,正常妊娠胎盘组织标本40例为对照组,妊娠期高血压疾病患者胎盘组织标本102例为病例组,通过免疫组织化学二步法,对MMP-9和TIMP-1在胎盘组织中的表达进行检测。结果1.MMP-9及TIMP-1在胎盘组织中的表达部位。在正常妊娠胎盘组织的滋养细胞、蜕膜细胞、间质细胞和血管内皮细胞胞浆中均可见MMP-9、TIMP-1的阳性表达,多为中度阳性和强阳性表达,但在妊娠期高血压疾病患者胎盘组织中,MMP-9未见间质细胞和血管内皮细胞有阳性表达,且表达者多为弱阳性。2.MMP-9在妊娠期高血压疾病患者中的阳性表达与正常妊娠组相比,差异有显著性（P〈0.05）,妊娠期高血压、子痫前期轻度、子痫前期重度组间比较差异有显著性（P〈0.05）。即妊娠期高血压疾病患者胎盘中MMP-9的表达显著低于正常妊娠,且随着妊娠期高血压疾病病情加重,MMP-9阳性表达呈明显减少趋势。3.TIMP-1在正常妊娠和妊娠期高血压疾病患者胎盘中的表达相比较,差异无显著性（P〉0.05）。结论MMP-9表达降低、MMP-9与TIMP-1的平衡状态改变可能与妊娠期高血压疾病发病有关,MMP-9及TIMP-1作为一对相互制约的因素在妊娠期高血压疾病发病中可能起重要作用,MMP-9有望成为妊娠期高血压疾病诊断及判断预后的新指标。     

HBV基因组转染对小鼠NIH3T3细胞内基质金属蛋白酶组织抑制因子的转录调控作用

ObjectiveTo understand the mechanism of liver cirrhosis after the infection of hepatitis B virus.MethodsMouse fibroblast NIH3T3 cells were transfected with 3.2 kb HBV DND by exposure of the cells to calcium phosphate.The change of the levels of mRNA for tissue inhibitor of metalloproteinase 1and 2(TIMP1,2) was detected in mouse fibroblast NIH3T3 cells and the cells of transfection with HBV Genome by in situ hybridization.ResultsThe levels of mRNA for TIMP1 and TIMP2 were increased significantly.ConclusionHBV infection can induce the expression of the mRNA for TIMP1 and TIMP2.     

急性冠状动脉综合征患者血浆基质金属蛋白酶及其抑制因子的变化

目的：研究急性冠状动脉综合征患者血浆基质金属蛋白酶（MMP-9）及其抑制因子（TIMP-1）的变化。方法：采用夹心酶免疫定量分析技术，测定30例急性冠状动脉综合征患者、29例稳定型心绞痛患者和17例正常对照血浆MMP-9和TIMP-1的变化。结果：3组间年龄、性别、总胆固醇、甘油三脂、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇无显著差异。急性冠脉综合征组高敏C反应蛋白(4.336±1.334) mg/L、MMP-9 (13.145±9.796) μg/L、TIMP-1 (1.363±0.605) μg/L、MMP-9/TIMP-1 (10.013±7.195) 显著高于稳定型心绞痛组[ 分别为(2.205±0.458) mg/L、(2.206±1.996) μg/L、(0.688±0.389) μg/L和(3.249±1.987) ]和正常对照组[ 分别为(1.625±0.434) mg/L、(1.663±1.271) μg/L、(0.583±0.421) μg/L和(5.169±7.416) ]，MMP-9与hs-CRP、TIMP-1、MMP-9/TIMP-1呈正相关。结论：急性冠脉综合征患者存在着由炎症反应导致的以MMP-9升高为主的MMP-9/TIMP-1失衡状态，血浆MMP-9/TIMP-1有可能作为反映急性冠脉综合征患者脆性斑块的指标。     

丁苯酞预处理对脑缺血再灌注损伤大鼠基质金属蛋白酶-9、基质金属蛋白酶组织抑制因子-1和血脑屏障的影响

目的:探讨丁苯酞预处理对脑缺血再灌注损伤大鼠基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶组织抑制因子-1(TIMP-1)和血脑屏障的影响.方法:90只雄性SD大鼠随机分为假手术组,模型组,NBP预处理低剂量组、中剂量组、高剂量组.采用线栓法制作大脑中动脉栓塞(MCAO)模型,缺血2h再灌注24 h后以干湿重法测定脑组织含水量,伊文思蓝(EB)评估血脑屏障的破坏程度,实时PCR检测MMP-9及TIMP-1 mRNA的表达水平.结果:脑缺血再灌注后,脑组织含水量及EB含量明显增加,MMP-9和TIMP-1表达均增强,与假手术组比较差异有统计学意义.丁苯酞预处理各组脑组织含水量及EB含量较模型组明显下降,MMP-9表达显著减少,TIMP-1表达明显增加,丁苯酞预处理中、高剂量组差异无统计学意义.结论:丁苯酞预处理对脑缺血再灌注损伤大鼠可通过调节MMP-9/TIMP-1的表达,降低血脑屏障通透性,减轻脑水肿,发挥预防性保护作用.     

含人基质金属蛋白酶组织抑制因子-1重组腺病毒的构建与体外表达

从人肝癌组织中提取总RNA，RT—PCR合成hTIMP-1的全长cDNA，克隆到腺病毒载体AdEasy系统的穿梭质粒pAdTraek—CMV上，与骨架质粒pAdEasy-1在BJ5183受体菌中进行同源重组，成功构建含hTIMP-1全长eDNA的重组腺病毒载体，经293细胞的包装、扩增，生成含hTIMP-1基因的重组腺病毒AdhTIMP-1并实现体外表达，为进一步研究肝癌浸润和转移机理以及肝癌的基因治疗提供实验基础。     

Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin-induced pulmonary fibrosis

The role of various matrix metalloproteinases (MMP) and tissue Inhibitor of metalloprotelnases-2 (TIMP-2), and the gelatholytic activities of MMP involved in the process of bleomycin-induced pulmonary fibrosis in rabbits were Investigated. Male Japanese white rabbits were intubated with tracheal tubes under anesthesia, and bleomycin hydrochloride in sterile saline or only sterile saline was administered through the tracheal tubes. The animals were killed 1, 3, 7, 14 and 28 days after the administration of bleomycln ( n = 3) or saline ( n = 2). Light microscopic lmmunohistochemlstry for MMP-1 (interstitial collagenase), MMPP (gelatinase A), MMP-9 (gelatinase B) and TIMP-2 was performed. The gelatinolytic activities of lung tissue homogenates were studied by gelatin zymography. In the early stages, the gelatholytic activity of MMP-9 was predominant. MYP-9 localized in the infiltrating neutrophils, macrophages, bronchial and bronchiolar epithelial cells. The alveolar epithelial basement membrane was frequently disrupted in the early stages, where MMP-9 possibly contributed to the disruption. In the late stages, the gelatinolytic activities of the latent and active forms of MMP-2 were predominant, and MMPP localized in the regenerated alveolar epithelial cells in addition to the bronchial epithelial cells. MMP-2, especially its active form, possibly plays a role in alveolar epithelial cell regeneration. The localization of MMP-1 was similar to that of MMP-9. TIMP-2 localized in the epithelial cells and in some fibroblasts in fibro tic lesions. TIMP-2 possibly plays a role in extracellular matrix deposition in balance with MMP.     

基质金属蛋白酶1、3,基质金属蛋白酶组织抑制剂1和miR-29在大鼠肺纤维化中及活性维生素D3处理后的表达与作用

目的:探讨大鼠肺纤维化发生发展中基质金属蛋白酶1(MMP1)、MMP3、基质金属蛋白酶组织抑制剂1(TIMP1)及miR-29的表达和作用以及活性维生素D3[1,25(OH)_2D_3]处理对这些基因表达的影响。方法:雄性SD大鼠随机分为预防组和治疗组。预防组分为对照组Ⅰ、模型组Ⅰ和给药组Ⅰ,治疗组分为对照组Ⅱ、模型组Ⅱ和给药组Ⅱ。模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ经气管注入博莱霉素,对照组Ⅰ/Ⅱ经气管注入生理盐水。预防组和治疗组分别于术后第2和14天腹腔注射给药,给药组给予活性维生素D3,模型组给予活性维生素D3溶剂,对照组给予生理盐水。预防组的各组分别于术后第14、21、28天处死大鼠取材,治疗组的各组分别于术后第21和28天处死大鼠取材。实时定量PCR检测大鼠肺组织中miR-29a及TIMP1 mRNA表达水平,免疫组织化学检测组织中MMP1、MMP3和TIMP1的表达水平。结果:模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ中的MMP1、MMP3和TIMP1的表达均明显高于相同时间点对照组Ⅰ/Ⅱ中的表达,而miR-29a的表达明显低于相应的对照组Ⅰ/Ⅱ;给药组Ⅰ/Ⅱ中MMP1、MMP3和TIMP1的表达均低于相应时间点的模型组Ⅰ/Ⅱ的表达,而给药组Ⅰ/Ⅱ中miR-29a的表达则高于模型组Ⅰ/Ⅱ中的表达。结论:MMP1、MMP3、TIMP1和miR-29在大鼠肺纤维化发生发展中具有重要作用,活性维生素D3可以促进miR-29表达,抑制MMP1、MMP3、TIMP1的表达;可能是通过miR-29调节包括MMP1、MMP3、TIMP1在内的多种靶基因来发挥抑制纤维化的作用。     

Pathogenesis of idiopathic pulmonary fibrosis and its clinical implications

Idiopathic pulmonary fibrosis (IPF) is the most common and lethal form of idiopathic interstitial pneumonia. The disease is thought to arise following an aberrant reparative response to recurrent alveolar epithelial cell injury leading to progressive loss of function. The median survival time is 3–5 years from diagnosis. Cigarette smoking, exposure to organic and inorganic dust and genetic factors have been shown to increase the risk of disease, although the cause of IPF remains elusive and its pathogenesis incompletely understood. In the last decade, several clinical trials evaluating novel therapies for IPF have been conducted but the results have been mostly disappointing. Conversely, compounds that target anti-fibrotic and growth factor pathways have been proven effective in slowing functional decline and disease progression. These promising results notwithstanding, truly effective therapeutic strategies will likely require combinations of drugs in order to target the multitude of pathways involved in disease pathogenesis.     

Inhibition of bleomycin-induced pulmonary fibrosis in mice by the matrix metalloproteinase inhibitor batimastat

Bleomycin-induced pulmonary fibrosis is known to be associated with the increased activity of two gelatinases, matrix metalloproteinase (MMP)-2 and MMP-9, in bronchoalveolar lavage (BAL). This study has investigated the effect of a synthetic inhibitor of MMP, batimastat, on the development of pulmonary fibrosis induced by bleomycin administration in mice. Animals were intranasally instilled with saline or bleomycin (0.5 mg in 100 microl per mouse). Batimastat (30 mg/kg) or vehicle alone was administered by intraperitoneal injection 24 h and 1 h before saline or bleomycin instillation, and then daily at the same dosage until the end of the study. Fifteen days after bleomycin administration, BAL was performed and the lung was removed. Treatment of mice with batimastat significantly reduced bleomycin-induced lung fibrosis, as shown in the lung by histopathological examination and by a decrease in hydroxyproline levels. Batimastat also prevented the increase in BAL macrophage and lymphocyte numbers, whereas it did not show any effect on the increased expression of active transforming growth factor-beta (TGF-beta) in BAL. Batimastat treatment was effective in reducing MMP-2 and MMP-9 activity as well as the tissue inhibitor of metalloproteinase-1 (TIMP-1) level in BAL. These results suggest that administration of the MMP inhibitor batimastat is useful in preventing experimental pulmonary fibrosis induced by bleomycin and raises the possibility of a therapeutic approach to human pulmonary fibrotic disease.     

小鼠肺纤维化模型中基质金属蛋白酶-9及其抑制剂表达水平的变化

目的 :研究小鼠博来霉素在肺纤维化模型中肺泡巨噬细胞(AM)分泌的基质金属蛋白酶 9(MMP 9)和基质金属蛋白酶抑制剂 1(TIMP 1)的表达随着时间的变化 ,观察肺纤维化中MMP 9和TIMP 1的表达是否存在失衡。方法 :以博来霉素诱导建立小鼠肺纤维化模型 ,采用HE染色观察肺部胶原沉积状况。选用抗CD6 8mAb ,采用微小免疫磁珠法分离纯化肺泡灌洗液中的AM ,用Hoechst 332 5 8染色AM ,在下光镜高倍视野计数法评估AM的纯度和活性 ,用EIA法检测AM上清液中MMP 9和TIMP 1的表达。结果 :肺组织切片HE染色显示小鼠肺部胶原沉积在 1、3、7、14、2 8d呈逐渐加重趋势。粗分离的AM纯度为 (82 .5± 2 .5 ) %。采用微小免疫磁珠法分离肺泡灌洗液中的AM纯度可达到 (99.3± 0 .7) % (P <0 .0 5 )。纯化后的细胞存活率为 (92 .5± 1.8) % ,与纯化前的 (92 .7± 2 .0 ) % ,相差不大 (P >0 .0 5 )。随着小鼠肺间质纤维化的进展 ,MMP 9的分泌量逐渐减少 ,而TIMP 1的表达量逐渐增加。结论 :在小鼠肺纤维化中AM分泌的MMP 9和TIMP 1之间存在失衡 ,表明基质降解系统受到抑制     

Elevated matrilysin levels in bronchoalveolar lavage fluid do not distinguish idiopathic pulmonary fibrosis from other interstitial lung diseases

Microarray studies have shown that matrilysin or matrix metalloproteinase (MMP)-7 is highly upregulated in the lungs of patients with idiopathic pulmonary fibrosis (IPF), but MMP-7 protein expression has not been systematically compared between IPF and other interstitial lung diseases. MMP-7 levels in bronchoalveolar lavage fluid (BALF) were compared to corresponding samples from nonspecific interstitial pneumonia (NSIP), sarcoidosis, and healthy controls. MMP-7 levels in the BALF were determined by ELISA and localization of MMP-7 in the lung tissue by immunohistochemistry. MMP-7 was similarly elevated in the BALF of all these disorders compared to healthy controls (p=0.007). Even control subjects with prolonged cough displayed a tendency towards elevated MMP-7 expression. There was a negative correlation between BALF MMP-7 levels and forced expiratory vital capacity (r=-0.348, p=0.02, n=42). In IPF lung, MMP-7 immunoreactivity appeared predominantly in the fibrotic parenchyma and arterial wall. In sarcoidosis and NSIP, prominent MMP-7 immunoreactivity was found in areas of inflammation. These results demonstrate that elevated BALF MMP-7 is not restricted to IPF alone but is also observed in other interstitial lung diseases and cannot be used as a differential diagnostic marker for IPF.     

Evolution of three patterns of intra-alveolar fibrosis produced by bleomycin in rats

In pulmonary fibrosis, it is known that fibrotic changes develop in the intra-alveolar spaces and that intra-alveolar fibrosis can be classified into three patterns, namely intraalveolar buds, mural incorporation and obliterative changes. In order to clarify the evolution of intra-alveolar fibrosis, immunohistochemical studies of extracellular matrix proteins and electron microscopic observations were made of the lungs of rats given a single intretracheal instillation of bleomycin. All three patterns of fibrosis developed in this model. Intra-alveolar buds changed into globular lesions with dense collagen deposition, the surface of which was covered by alveolar epithelium. Electron microscopy revealed that the buds often contained spiraling collagen fibrils and numerous microfibrils, but not mature elastic fibres, beneath the regenerating epithelial lining cells; the epithelial basement membranes were discontinuous. In contrast, mural incorporation and obliterative changes ware associated with alveolar structural remodeling. Electron microscopically, these lesions had bundles of normal collagen fibrils, small elastic fibers, and continuous epithelial basement membranes. These results indicate that: (i) intra-alveolar buds, that become intra-alveolar collagen globules, with an unusual extracellular matrix, do not contribute to alveolar structural remodelling; and (ii) areas of mural incorporation and obliterative changes have the usual type of extracellular matrix and are essential for alveolar structural remodeling.     

The calpain inhibitor calpeptin prevents bleomycin-induced pulmonary fibrosis in mice

Pulmonary fibrosis is characterized by progressive worsening of pulmonary function leading to a high incidence of death. Currently, however, there has been little progress in therapeutic strategies for pulmonary fibrosis. There have been several reports on cytokines being associated with lung fibrosis, including interleukin (IL)‐6 and transforming growth factor (TGF)‐β1. We reported recently that two substances (ATRA and thalidomide) have preventive effects on pulmonary fibrosis by inhibiting IL‐6‐dependent proliferation and TGF‐β1‐dependent transdifferentiation of lung fibroblasts. Rheumatoid arthritis is a chronic autoimmune disorder, and its pathogenesis is also characterized by an association with several cytokines. It has been reported that calpain, a calcium‐dependent intracellular cysteine protease, plays an important role in the progression of rheumatoid arthritis. In this study, we examined the preventive effect of Calpeptin, a calpain inhibitor, on bleomycin‐induced pulmonary fibrosis. We performed histological examinations and quantitative measurements of IL‐6, TGF‐β1, collagen type Iα1 and angiopoietin‐1 in bleomycin‐treated mouse lung tissues with or without the administration of Calpeptin. Calpeptin histologically ameliorated bleomycin‐induced pulmonary fibrosis in mice. Calpeptin decreased the expression of IL‐6, TGF‐β1, angiopoietin‐1 and collagen type Iα1 mRNA in mouse lung tissues. In vitro studies disclosed that Calpeptin reduced (i) production of IL‐6, TGF‐β1, angiopoietin‐1 and collagen synthesis from lung fibroblasts; and (ii) both IL‐6‐dependent proliferation and angiopoietin‐1‐dependent migration of the cells, which could be the mechanism underlying the preventive effect of Calpeptin on pulmonary fibrosis. These data suggest the clinical use of Calpeptin for the prevention of pulmonary fibrosis.