大鼠肺纤维化模型间质成纤维细胞和肺泡巨噬细胞基质金属蛋白酶2及膜型基质金属蛋白酶mRNA的表达

目的探讨肺间质成纤维细胞和肺泡巨噬细胞基质金属蛋白酶2(MMP-2)和膜型基质金属蛋白酶(MT1-MMP)mRNA在肺纤维化中的表达及意义.方法清洁级SD大鼠50只,随机分为10组,实验组5组气管内注射盐酸博莱霉素A5,对照组5组代以等剂量生理盐水.于注射后1d、3d、7d、14d、28d分离和提取肺间质成纤维细胞及肺泡巨噬细胞,应用逆转录多聚酶链反应(RT-PCR)方法,观察其MMP-2及MT1-MMPmRNA的动态变化.结果(1)博莱霉素作用后1d成纤维细胞MMP-2基因转录就明显增强,达对照组的2.05倍(t=10.667,P＜0.01),且一直维持于高水平,直到用药28d才略下降.而此过程中肺泡巨噬细胞MMP-2mRNA的转录极微弱,仅于实验第1d较对照组增高(t=3.27,P＜0.05).(2)成纤维细胞和肺泡巨噬细胞MT1-MMPmRNA的转录均有增强,前者于实验第14d和第28d时分别为对照组的2.1倍(t=4.823,P＜0.01)和1.8倍(t=4.016,P＜0.01),后者于第28d时为对照组的2.4倍(t=5.851,P＜0.01).结论(1)成纤维细胞不单纯是肺纤维化效应细胞,其通过MMP-2基因转录的增强,参与了肺基膜结构的损伤,参与了肺间质纤维化的启动机制.(2)实验中后期成纤维细胞和肺泡巨噬细胞MT1-MMP表达增强,有助于MMP-2的持续活化,促进肺间质纤维化的进展.     

TIMP-2、MMP-2和MT1-MMP在活化肝星状细胞中的表达及对细胞外基质合成分泌的影响

目的 研究金属蛋白酶组织抑制剂-2(TIMP-2)、基质金属蛋白酶-2(MMP-2)和膜型基质金属蛋白酶-1(MT1-MMP)在活化肝星状细胞(HSCs)中的表达,观察其对细胞外基质(ECM)合成分泌的影响.方法 原代分离培养大鼠HSCs活化后,分别给予40～160 pmol化学合成经修饰抗TIMP-2 siRNA进行干预,检测培养细胞上清液透明质酸(HA)、Ⅲ型前胶原(PCⅢ)和羟脯氨酸(Hyp)的含量,采用荧光实时定量PCR法检测TIMP-2、MMP-2、MT1-MMP、MMP-13、COL Ⅰ和COL Ⅲ mRNA的表达,western印迹检测TIMP-2、MT1-MMP和MMP-13蛋白表达及明胶酶谱法检测MMP-2蛋白表达.结果 应用化学合成经修饰抗TIMP-2 siRNA后,TIMP-2、MMP-2、MT1-MMP、COL Ⅰ和COL Ⅲ的表达明显降低,而MMP-13的表达则明显增加,培养细胞上清液中HA、PCⅢ和Hyp的含量也明显减少.结论 TIMP-2通过MT1-MMP介导MMP-2的活化,抑制TIMP-2的表达,MT1-MMP和MMP-2的表达随之降低,而HSCs合成分泌ECM也相应减少.     

MT1-MMP对人肝癌细胞浸润能力的影响

目的 观察膜型基质金属蛋白酶-1(MT1-MMP)对人肝癌细胞株浸润能力的影响,并初步探讨其作用机制.方法 将稳定表达MT1-MMP基因转染至HepG2细胞中,应用Western印迹法检测MT1-MMP蛋白表达;明胶酶谱分析法检测MMP-2酶原活性;体外侵袭实验检测细胞株浸润能力.结果 重组质粒转染株MT1-MMP蛋白表达水平明显高于空质粒组和未转染组(P＜0.01).明胶酶谱分析实验发现,MT1-MMP组同时检测到72000酶原形式和64000活性形式的MMP-2,另两组只检测到72000酶原形式的MMP-2.体外侵袭实验结果显示,MT1-MMP组细胞穿过基质胶(Matrigel)的细胞数目明显多于其他两组(P＜0.01).结论 MT1-MMP能显著增强肝癌细胞株的浸润能力,其机制主要是通过激活MMP-2-酶原,降解肿瘤周围的基质成分实现的.     

非类固醇类抗炎药NS398对肺癌H460细胞RECK及MT1-MMP表达的影响

目的 探讨非类固醇类抗炎药NS398对肺癌H460细胞增殖及其Kazal基序逆向诱导半胱氨酸丰富蛋白(RECK)及膜型基质金属蛋白酶1(MT1-MMP)表达的影响.方法 应用MTT方法检测H460细胞生长的抑制率,用免疫荧光法、Western blot法检测RECK及MT1-MMP蛋白的表达.结果 NS398可抑制H460细胞的增殖,促进RECK蛋白的表达,减少MT1-MMP蛋白的含量,并呈剂量依赖关系.结论 NS398可通过促进肺癌细胞RECK的表达并减少MT1-MMP的含量,进而抑制肺癌H460细胞的生长,这可能是肺癌侵袭转移的一个重要机制.     

基质金属蛋白酶-2,9在白血病中的研究现状

基质金属蛋白酶(MMP)是一类能降解细胞外基质成分的蛋白水解酶,其表达与多种肿瘤的生长、侵袭及转移等密切相关,逐渐成为恶性肿瘤治疗的一个新靶点.近年来MMP-2、MMP-9在白血病方面的研究成为国内外学者研究的热点.本文就MMP-2、MMP-9的结构功能、在白血病细胞侵袭转移中的作用机制、与白血病临床疗效及预后的关系作一综述.     

基质金属蛋白酶2表达与胃癌侵袭及转移的关系

基质金属蛋白酶2（MMP-2）在肿瘤细胞介导的细胞外基质降解中起关键作用。其过度表达与人类多种恶性肿瘤侵袭转移潜能及预后密切相关。MMP-2高度表达的胃癌,其侵袭性和转移性较强,恶性程度较高,患者的预后较差。本文就MMP-2和胃癌侵袭转移的相关性及其作用机制和影响因素作一综述。     

组织蛋白酶B、基质金属蛋白酶-2在非小细胞肺癌中的表达及临床意义

目的探讨组织蛋白酶B(CB)和基质金属蛋白酶(MMP)-2在非小细胞肺癌发生、发展及转移中的作用。方法应用免疫组织化学法检测75例非小细胞肺癌组织及28例良性肺病变中CB和MMP-2的表达情况,并将检测结果与临床病理特征进行综合分析。结果 CB和MMP-2的表达主要定位于细胞质,CB和MMP-2在非小细胞肺癌中表达的阳性率明显高于良性肺病变(P0.05)。非小细胞肺癌中MMP-2蛋白的表达与有无淋巴结转移情况密切相关。肺癌组织中CB、MMP-2的表达水平呈显著正相关。结论 CB和MMP-2在非小细胞肺癌中表达上调,联合检测CB和MMP-2对肿瘤的诊断和预后判断有一定的临床意义。     

基质金属蛋白酶MMP-2与肺癌关系的研究进展

基质金属蛋白酶是一大组金属离子依赖的蛋白酶,它能障解细胞外基质中的各种蛋白成分,在恶性肿瘤的侵袭中具有重要作用,而基质金属蛋白酶的家族中,又以基质金属蛋白酶-2在肿瘤侵袭与转移中的作用最为突出,本文综述了有关基质金属蛋白酶-2与肺癌的相关研究进展．     

MT1-MMP对人肝癌细胞株HepG2生物学行为的影响

目的 观察膜型基质金属蛋白酶-1(MT1-MMP)对人肝癌细胞株HepG2生物学行为的影响.方法 将稳定表达MT1-MMP基因转染至HepG2细胞中,应用RT-PCR、Ⅰ型胶原黏附和Matrigel侵袭小室实验,检测细胞MT1-MMP mRNA水平,体外黏附、侵袭和迁移能力的变化.结果 重组质粒转染株MT1-MMP mRNA表达水平明显高于空质粒组和对照组(P<0.01).MT1-MMP明显提高细胞Ⅰ型胶原黏附、迁移和Matrigel侵袭能力(P<0.01).结论 MT1-MMP过表达可促进HepG2细胞体外侵袭和转移,提示其可作为人肝癌抗侵袭治疗的分子靶点.     

基质金属蛋白酶-9和组织金属蛋白酶抑制剂-1在食管鳞癌中的表达

目的探讨基质金属蛋白酶-9（MMP-9）和组织金属蛋白酶抑制剂-1（TIMP-1）在食管鳞癌中的表达及其临床意义。方法用免疫组化和Western blot法分别检测41例食管鳞癌患者的癌及相应正常组织中MMP-9和TIMP-1的表达变化。结果食管鳞癌组织中MMP-9阳性表达率与食管癌淋巴结及静脉转移有关；MMP-9的阳性表达率与表达量均显著高于TIMP-1；MMP-9和TIMP-1的表达呈负相关。结论MMP-9与食管鳞癌的侵袭转移有关，其机制可能与食管鳞癌组织中的MMP-9／TIMP-1平衡失调有关；MMP-9与TIMP-1联合检测有助于食管鳞癌生物学行为的判断。     

Chronic hypoxia inhibits MMP-2 activation and cellular invasion in human cardiac myofibroblasts

Cardiac myofibroblasts are pivotal to adaptive remodelling after myocardial infarction (MI). These normally quiescent cells invade and proliferate as a wound healing response, facilitated by activation of matrix metalloproteinases, particularly MMP-2. Following MI these reparative events occur under chronically hypoxic conditions yet the mechanisms by which hypoxia might modulate MMP-2 activation and cardiac myofibroblast invasion have not been investigated. Human cardiac myofibroblasts cultured in collagen-supplemented medium were exposed to normoxia (20% O₂) or hypoxia (1% O₂) for up to 48 h. Secreted levels of total and active MMP-2 were quantified using gelatin zymography, TIMP-2 and membrane-associated MT1-MMP were quantified with ELISA, whole cell MT1-MMP by immunoblotting and immunocytochemistry and MT1-MMP mRNA with real-time RT-PCR. Cellular invasion was assessed in modified Boyden chambers and migration by scratch wound assay.In the human cardiac myofibroblast, MT1-MMP was central to MMP-2 activation and activated MMP-2 necessary for invasion, confirmed by gene silencing. MMP-2 activation was substantially attenuated by hypoxia (P < 0.001), paralleled by inhibition of myofibroblast invasion (P < 0.05). In contrast, migration was independent of either MT1-MMP or MMP-2. Reduced membrane expression of MT1-MMP (P < 0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2. In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI. Regulation of these MMPs remains an attractive target for therapeutic intervention.     

Catechins prevent vascular smooth muscle cell invasion by inhibiting MT1-MMP activity and MMP-2 expression

OBJECTIVE: Regular consumption of green tea is associated with a reduced risk of mortality due to coronary diseases and cancer. The present study examined whether a green tea extract (GTE) inhibits activation of matrix metalloproteinase-2 (MMP-2), a major collagenase involved in vascular remodeling of atherosclerotic plaques, in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The expression of MMP-2 was assessed by Northern and Western blot analyses in human aortic VSMCs. MMP-2 activity was evaluated by zymography, membrane-type1-MMP (MT1-MMP, MMP-14) activity by an enzymatic assay, and cell invasion by a modified Boyden chamber assay. The thrombin-induced activation of secreted MMP-2 was abolished by GTE and the green tea polyphenols (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG). GTE reduced the expression of MMP-2 mRNA and protein. GTE, EGCG and ECG directly inhibited cell-associated MT1-MMP activity, the physiological activator of MMP-2, in a reversible manner. Thrombin-stimulated VSMCs invasion was abolished by EGCG and ECG, and reduced by GTE. CONCLUSIONS: GTE inhibits thrombin-induced VSMCs invasion most likely by preventing MMP-2 expression and its activation by a direct inhibition of MT1-MMP. The ability of green tea to prevent cell invasion and matrix degradation might contribute to its protective effect on atherosclerosis and cancer.     

Induction of membrane-type matrix metalloproteinase-1 stimulates angiogenic activities of bovine aortic endothelial cells

Matrix metalloproteinases (MMPs) have been reported to play critical roles in endothelial cell migration and matrix remodeling during the angiogenic process. Among these MMPs, membrane-type MMP-1 (MT1-MMP) is an important molecule that can trigger the invasion of tumor cells by activating MMP-2 on their plasma membrane. However, the precise involvement of MT1-MMP in the angiogenic process has not been determined. To investigate the roles of the MT1-MMP by the matrix remodeling of endothelial cells, MT1-MMP expression vector was transfected into bovine aortic endothelial cells (BAECs). Increased expression of MT1-MMP in BAECs enhanced the activation of MMP-2, invasion and migration of BAECs. Moreover, the capacity of tube formation was increased in MT1-MMP transfectants. However, cotransfection with antisense MT1-MMP expression vector abolished the effects of MT1-MMP overexpression. These observations indicate that MT1-MMP is involved in the angiogenic process of endothelial cells in vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is down-regulated in estrogen-deficient rat osteoblast in vivo

Our previous study showed that estrogen stimulates membrane-type matrix metalloproteinases-1 (MT1-MMP) production in osteoblastic cells culture, but has no effect on MMP-2 and TIMP-2 synthesis. Osteoblast-derived MT1-MMP have been recently implied to play a role in bone metabolism by degrading tumor necrosis factor-alpha (TNF-alpha), resolving extracellular matrix and activating proMMP-2, which requires the process of activation mediated by MT1-MMP/tissue inhibitor of metalloproteinase (TIMP-2) complex on the cell surface. To investigate the mechanism of bone loss following estrogen deficiency, we examined the effects of estrogen on osteoblast synthesis of MT1-MMP, MMP-2 and TIMP-2. In situ hybridization and immunohistochemistry of rat bone samples were used to document the synthesis of MT1-MMP, MMP-2, and TIMP-2 mRNA and protein. Osteoblasts from distal femoral head showed an increase in the pattern of MT1-MMP mRNA and protein production in sham-operated controls and 17beta-estradiol (E2)-treated rats, compared with the ovariectomized group; the synthesis of MMP-2 and TIMP-2 mRNA and protein was unaffected. Our data show a down-regulation of MT1-MMP synthesis by osteoblast in vivo following estrogen withdrawal, and treatment with E2 resulted in induced MT1-MMP expression in vivo. There is evidence suggesting a role for MT1-MMP in the process of bone loss during the pathogenesis of osteoporosis.     

Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase

OBJECTIVE: Heart failure is characterized by an imbalance of matrix synthesis/turnover, finally resulting in fibrosis. Cardiac myocytes and fibroblasts play a pivotal role in the remodeling process. Cardiac remodeling involves the expression of TGF-beta1 and matrix metalloproteinases (MMPs) in cardiac fibroblasts (CFBs). Furin, a subtilisin/kexin-like proprotein convertase (PC), activates TGF-beta1 and membrane-bound MT1-MMP, which facilitates pro-gelatinase A (MMP-2) activation. Even though several reports identified TGF-beta1 as a pro-fibrotic cytokine in the heart, it increases MMP-activity and cell migration/invasion in several cell types. The present study was done to investigate the contribution of TGF-beta1 and furin to CFBs MMP-activity and motility. METHODS AND RESULTS: Stimulation of CFBs from adult Sprague-Dawley rats with TGF-beta1 (20 ng/ml) induced furin, but had no effect on the closely related PC5. Inhibition of furin inhibited angiotensin II-induced TGF-beta1 activation, indicating that TGF-beta1 amplifies its activating convertase in CFBs. Pretreatment of CFBs with TGF-beta1 (20 ng/ml, 24 h) increased their migration by about two-fold (p<0.05), which was accompanied by an enhanced expression and activity of MT1-MMP and MMP-2. Brefeldin A (BFA), a Golgi-disturbing agent, inhibited MT1-MMP activation, indicating that it occurs in the trans-Golgi network (TGN), where furin is concentrated and colocalized with MT1-MMP. Inhibition of furin significantly inhibited TGF-beta1-induced MT1-MMP/MMP-2 activation. Furthermore, inhibition of furin attenuated TGF-beta1-enhanced migration on gelatin-coated membranes (p<0.05). This was comparable to the effects of the MMP-inhibitor GM6001, pointing out that MMPs are major mediators of TGF-beta1-enhanced CFB motility. CONCLUSION: We demonstrate that TGF-beta1 induces MMP-activity in CFBs, thereby facilitating CFBs motility. Furthermore, TGF-beta1 amplifies its activating convertase furin, which is also required for MT1-MMP/MMP-2 activation in CFBs. Thus, furin is central for TGF-beta1 and MT1-MMP activation and might be a novel target in cardiac remodeling.     

Increased expression and cell surface localization of MT1-MMP plays a role in stimulation of MMP-2 activity by leptin in neonatal rat cardiac myofibroblasts

Myocardial matrix remodeling is a well-recognized disease modifier in the pathogenesis of heart failure, although the precise underlying molecular mechanisms remain to be elucidated. Here we investigated the effects of leptin, circulating levels of which are typically increased in obese individuals, on MMP and collagen expression and MMP activity in isolated cardiac myofibroblasts. Neonatal rat myofibroblasts were treated with 6 nM recombinant leptin and the collected supernatant analyzed for MMP-2 activity via gelatin zymography. MMP-2, MT1-MMP and procollagen-I and -III protein expression were determined by western blotting and MMP-2 and MT1-MMP mRNA expression were examined utilizing real-time PCR. Procollagen-I levels were analyzed by confocal microscopy and collagen synthesis was determined through [³H]-proline incorporation. Exposure of myofibroblasts to leptin (24 h) significantly increased MMP-2 activity, while mRNA and protein levels remained unchanged. Leptin also significantly enhanced mRNA and protein expression of MT1-MMP, a known activator of MMP-2. Biotinylation assays indicated increased cell surface expression of MT1-MMP in response to leptin and use of a MT1-MMP inhibitor attenuated the leptin-mediated elevation of MMP-2 activity. Total cellular collagen synthesis was unaffected by leptin treatment, however intracellular procollagen-I protein was significantly increased in treated cells. Furthermore, extracellular soluble procollagen-I was increased, while a decrease in soluble procollagen-III protein was observed in conditioned media. In summary, these findings in isolated cardiac myofibroblasts support the suggestion that leptin may directly influence myocardial matrix metabolism, and this may represent a mechanism contributing to cardiac fibrosis in obese patients with elevated plasma leptin levels.     

MT1-MMP、MMP-2和TIMP-2基因在去卵巢大鼠成骨细胞中的表达

目的　通过观察去卵巢 (OVX)骨质疏松模型大鼠成骨细胞膜型基质金属蛋白酶 1(MT1 MMP)基因的表达 ,探讨绝经后骨质疏松的发病机制。　方法　对OVX大鼠与假手术大鼠进行骨密度和骨组织形态计量学测定。股骨远端行原位杂交检测骨组织MT1 MMP、MMP 2和TIMP 2mRNA表达 ,免疫组化检测骨组织MT1 MMP、MMP 2和TIMP 2蛋白质表达。　结果　OVX大鼠第 3、4腰椎骨密度 (0 14 4± 0 0 11) g cm2 、(0 14 3± 0 0 15 ) g cm2 较对照组 (0 15 8± 0 0 18)g cm2 、(0 16 2± 0 0 17) g cm2 明显减少 ,OVX组骨小梁面积 (9 5 8± 3 39) %、厚度 (40 85± 8 0 4 ) μm和数目 (2 30± 0 4 8)个 mm分别较对照组 (2 0 6 3± 4 8) %、(44 73± 6 8) μm、(4 6± 0 7)个 mm明显下降 ,OVX组骨小梁间隔 (5 85 8± 115 1) μm较对照组 (2 5 4 6± 4 8 0 ) μm明显增宽 ;成骨细胞MT1 MMPmRNA与蛋白质表达下调 ,而MMP 2和TIMP 2之间表达无差异。　结论　雌激素不足可使成骨细胞MT1 MMP基因表达减少 ,可能为绝经后骨质疏松的发病机制之一。     

反义RNA对胃癌细胞MT1-MMP基因表达和侵袭性的抑制作用

目的:膜研究膜型-1基质金属蛋白酶(MT1- MMP)反义RNA对人胃癌细胞BGC823靶基因表达和侵袭特性的影响方法:利用基因重组技术构建人MT1-MMP反义RNA真核表达载体,转染人胃癌细胞BGC823,应用RT-PCR、MTT、明胶酶谱和体外侵袭实验等方法观察人胃癌细胞BGC823转染前后,MT1-MMP mRNA表达水平、细胞生长、明教酶A活性及细胞体外侵袭能力等指标的变化.结果:成功构建了MT1-MMP反义RNA真核表达载体pasMMP14,将其转染胃癌细胞BGC823后,与阴性对照组相比,实验组MT1- MMP mRNA表达水平降低,抑制率为36%.转染48 h,明教酶A的活化受到了明显抑制.转染72 h,细胞增殖明显受抑(t=2.358,P<0.01 vs空白组:t=2.727 P<0.01 vs阴性组).实验组的穿膜细胞数明显低于空白对照组和阴性对照组(t=5.744,P<0.01;t=5.695,P<0.01).结论:反义RNA对人胃癌细胞MT1-MMP基因表达和侵袭能力具有明显的抑制作用,MT1-MMP基因可作为胃癌抗侵袭治疗的分子靶点.