甲状旁腺激素调控人成骨样细胞膜型基质金属蛋白酶-1表达

目的 探讨甲状旁腺激素(PTH)对人成骨样MG-63细胞膜型基质金属蛋白酶-1(MT1-MMP)表达的影响及调节骨代谢作用的机制。方法 用PTH(1-34)干预MG-63细胞培养，Northern杂交及Western杂交检测MT1-MMP mRNA与蛋白质表达水平。结果 PTH(1-34)10^-9mmol／L～10^-7mmol／L浓度对MG-63细胞MT1-MMP mRNA及蛋白质表达有抑制作用，并呈剂量依赖性。10^-8mmol／L干预在2～48h内对抑制MT1-MMP mRNA及蛋白质表达抑制有时间依赖性。蛋白激酶A(PKA)抑制剂H-89阻断PTH(1-34)抑制MT1-MMP表达的效应，而蛋白激酶A(PKA)激动剂Forskolin抑制MT1-MMP表达。结论 PTH具有抑制成骨样细胞MT1-MMP表达的作用，其作用途径可能有PKA信号转导途径参与。     

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.     

雌二醇和孕酮对人成骨细胞胰岛素受体底物基因表达的时间与剂量依赖性

目的观察雌二醇和孕酮对人成骨细胞和人成骨肉瘤MG63细胞胰岛素受体底物(IRS)mRNA表达的影响。探讨雌、孕激素参与骨代谢的作用机制。方法用半定量RTPCR检测人成骨细胞和人成骨肉瘤细胞IRS1、IRS2mRNA的表达量。结果雌二醇或孕酮均诱导MG63细胞IRS2mRNA的表达上调,具有剂量依赖性(P<0.05)和时效依赖性(P<0.05)。其中分别以10-8mol/L的雌二醇干预24h和10-8mol/L的孕酮干预12h作用最明显,分别为对照组的(421±68)%和(327±54)%(均P<0.01)。雌二醇或孕酮共同干预进一步促进MG63细胞和人成骨细胞IRS2mRNA表达的上调,分别为对照组的(496±54)%和(452±58)%(均为P<0.01)。雌二醇和孕酮不影响人成骨细胞和MG63细胞IRS1mRNA的表达(P>0.05)。结论雌二醇或孕酮均可使人成骨细胞和MG63细胞的IRS2mRNA表达上调,且存在剂量与时间依赖性;但不影响IRS1mRNA的表达。     

成纤维细胞与肺癌细胞相互作用对膜型基质金属蛋白酶-1及基质金属蛋白酶-2表达的影响

目的 研究胚肺成纤维细胞对肺癌H460细胞膜型基质金属蛋白酶-1(MTl-MMP)、基质金属蛋白酶-2(MMP-2)表达的影响.方法 采用Western blot方法检测各组MT1-MMP的表达.取其上清,采用酶联免疫吸附法检测各组细胞培养液中活性MMP-2的浓度.结果 H460细胞、胚肺成纤维细胞单独培养时均有MT1-MMP表达,但混合培养后MT1-MMP表达增强(P＜0.05).H460细胞、胚肺成纤维细胞单独培养时MMP-2均有分泌,混合培养MMP-2分泌增强(P＜0.05).结论 胚肺成纤维细胞和肺癌H460细胞相互作用能通过上调MT1-MMP、MMP-2的表达从而促进肺癌的侵袭和转移,这可能为肺癌侵袭转移的一个重要机制.     

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.     

雌二醇对人成骨样细胞膜型基质金属蛋白酶1的影响

目的　观察雌二醇 (E2 )对人成骨肉瘤MG 6 3细胞株膜型基质金属蛋白酶 (MT1 MMP)的作用。方法　MG 6 3细胞MT1 MMP蛋白质表达用Western杂交和激光共聚焦显微系统免疫荧光法检测 ,MMP 2活性用明胶酶谱和酶联免疫吸附测定检测。结果　观察到E2 促进MG 6 3细胞MT1 MMP蛋白质表达 ,并呈剂量依赖关系 (P <0 .0 5 ) ,激光共聚焦显微系统免疫荧光法证实E2促进MT1 MMP蛋白质表达增强 ,MT1 MMP蛋白质表达于MG 6 3细胞的细胞膜和细胞质中。E2对MG 6 3细胞MMP 2活性无影响。结论　E2 促进MG 6 3细胞MT1 MMP表达。     

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.     

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.     

CD44v6、MT1-MMP和VEGF在肝细胞肝癌中的表达及意义

采用免疫组化法检测40例肝细胞肝癌(简称肝癌)及10例正常肝组织手术切除标本的血管内皮生长因子(VEGF)、黏附分子(CD44v6)和膜型金属蛋白酶-1(MT1-MMP)的表达情况.结果MT1-MMP、CD44v6和VEGF在肝癌组织中阳性表达明显高于正常肝组织(P均＜0.01);MT1-MMP和VEGF与肿瘤的转移、大小有关,与组织分化程度无关;CD44v6与肿瘤的转移、分化程度有关,与大小肿瘤无关;MT1-MMP与CD44v6、CD44v6与VEGF、MT1-MMP与VEGF在肝癌组织中的表达均呈明显正相关(r=0.506、0.701、0.566,P均＜0.01).提示MT1-MMP、CDd4v6、VEGF联合检测有助于肝癌的生物学行为判断和预后评估.     

内皮素对心脏细胞DNA和蛋白合成的作用

目的观察内皮素-1(ET-1)对心脏心肌细胞和成纤维细胞DNA和蛋白合成作用.方法采用胰酶消化法培养新生Sprague-Dawley(SD)大鼠心脏心肌细胞和成纤维细胞,以3H-胸腺嘧啶核苷(3H-TdR)、3H-亮氨酸(3H-Leu)掺入法测定两种细胞DNA、蛋白合成,Bradford法测两种细胞总蛋白含量,逆转录聚合酶链反应法(RT-PCR)法测心肌细胞心钠素(ANP)mRNA的表达.结果对于心肌细胞,ET-1(10-9～10-7mol/L)显著促进3H-TdR掺入率、较对照组分别增加0.31±0.079(P＜0.01)、1.193±0.267(P＜0.001)、1.336±0.278(P＜0.001),3H-Leu掺入率增加0.223±0.058(P＜0.01)、1.054±0.202(P＜0.001)、1.074±0.204(P＜0.001),呈剂量依赖性,ANPmRNA表达明显增加;对于成纤维细胞,ET-1(10-8 mol/L)促进3H-TdR掺入率,较对照组增加0.248±0.02(P＜0.05)、3H-Leu掺入率较对照组增加0.434±0.041(P＜0.01).结论 ET-1促进心肌细胞肥大和心脏成纤维细胞增殖,且对心肌细胞的作用强于对成纤维细胞的作用.提示ET-1促心肌肥厚主要使促进心肌细胞肥大,对间质纤维化作用较弱.     

Increased extracellular matrix remodeling is associated with tumor progression in human hepatocellular carcinomas.

Matrix metalloproteinase-2 (MMP2) is a key enzyme in the process of extracellular matrix remodeling involved in tumor invasion and metastasis. The activation of MMP2 involves interplay with the membrane type-matrix metalloproteinase-1 (MT1-MMP) and the tissue inhibitor of metalloproteinase-2 (TIMP2). In vitro, activated hepatic stellate cells are a main source of MMP2 and collagen I induces MMP2 activation. The steady-state mRNA levels of MMP2, MT1-MMP, TIMP2, collagen I, collagen IV, and laminin gamma1 were compared with MMP2 activity in 55 hepatocellular carcinomas, 47 matching nontumor biopsies and 19 histologically normal livers. In hepatocellular carcinomas, increased collagen I mRNA levels were strongly associated with those of MMP2 (Spearman R =.74, P <.001), MT1-MMP (R =.65, P <.001) and TIMP2 (R = 0.61, P <.001). MMP2 activity was correlated with the mRNA expression of collagen I (R =.45 P <.01), collagen IV (R =.40, P <.01) and laminin gamma1 (R =.33, P <.05). Unlike collagen IV and laminin gamma1 mRNAs, MMP2, MT1-MMP, TIMP2, collagen I mRNA levels were increased in nonencapsulated compared with encapsulated tumors (P <.05). In addition, MMP2 activity was fourfold higher (P <.01) in tumors arising in cirrhotic livers than in those arising in noncirrhotic livers. Moreover, tumor recurrence was associated with 4.6- and 2.8-fold (P <.05) higher collagen I and MMP2 mRNA levels, respectively, in hepatocellular carcinomas arising in cirrhotic livers. Thus, a high extracellular matrix remodeling favors tumor progression in hepatocellular carcinomas.     

Effects of 17beta-estradiol on the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in human osteoblastic MG-63 cell cultures

Estrogens are important regulators of bone cell function. Osteoblast-derived membrane type 1 matrix metalloproteinses (MT1-MMP) have recently been implied to play an important role in the process of bone resorption by proteolytically activating latent matrix metalloproteinase-2 (proMMP-2) at the cell surface and degrading tumor necrosis factor-alpha (TNF-alpha). In the present study, we observed the effects of 17beta-estradiol (E2) on MT1-MMP production and subsequent activation of latent matrix proMMP-2, and also proMMP-2 secretion in cultures of human osteoblastic MG-63 cells. Western immunoblot analysis showed that treatment with increasing doses of E2 in MG-63 cells caused a dose-dependent increase in expression of MT1-MMP protein. Confocal immunohistochemistry analysis also confirmed that E2 induced MT1-MMP synthesis in MG-63 cells. We found unexpectedly that although MT1-MMP synthesis was up-regulated by E2 in cultures of MG-63 cells, activation of proMMP-2 was unchanged, which can be attributed partly to the undetectable tissue inhibitor of metalloproteinase-2 (TIMP-2) protein in MG-63 cells by Western immunoblotting. ProMMP-2 production was also not influenced by E2. In conclusion, E2 induces MT1-MMP protein expression in MG-63 cells while it is not followed by proMMP-2 activation, E2 may suppress bone resorption by accentuated degradation of TNF-alpha which mediated through increasingly MT1 -MMP production in osteoblastic cells.     

Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation.

Activated hepatic stellate cells (HSCs) are a potential source of gelatinase A, which accumulates in fibrotic livers. Progelatinase A activation requires its binding to a complex of membrane-type matrix metalloproteinase (MT-MMP) and tissue inhibitor of metalloproteinases (TIMP)-2. These studies examine gelatinase A, MT1-MMP, and TIMP-2 synthesis by HSCs during activation in vitro and the potential role of gelatinase A in promoting HSC proliferation. Gelatinase A, MT1-MMP, and TIMP-2 messenger RNA (mRNA) were all upregulated in HSCs activated on plastic over 5 to 14 days. Gelatinase A expression was maximal at 7 days of culture, coinciding with the peak of HSC proliferation and the onset of procollagen I and alpha-smooth muscle actin (alpha-SMA) mRNA expression. Active forms of gelatinase A of 62 kd and 66 kd were secreted by activated HSCs and reached a maximum of 12.1% of total enzyme in 14-day culture supernatants. Treatment of HSCs with concanavalin A (con A) induced activation of MT1-MMP and enhanced secretion of activated gelatinase A, which reached a maximum of 44.4% of the total enzyme secreted into culture supernatants using 30 microgram/mL con A. [(14)C]-gelatin degradation assays confirmed the presence of gelatinolytic activity in activated HSC supernatants, which reached a maximum level at 7 days of culture. Antisense oligonucleotide inhibition of endogenous progelatinase A production, or the MMP inhibitor 1,10-phenanthroline inhibited (3)H-thymidine incorporation into HSC DNA by greater than 50%. We conclude that HSCs produce progelatinase A during activation in vitro and activate this enzyme coincident with MT1-MMP and TIMP-2 synthesis. Gelatinase A activity is required for maximal proliferation of HSCs in vitro suggesting this metalloproteinase is an autocrine proliferation factor for HSCs.     

明胶酶A在急性白血病细胞中的表达及其临床意义

目的　探讨急性白血病 (AL)细胞中明胶酶A(MMP2 )的表达及其临床意义。方法　应用明胶酶谱法检测了 4 6例初治AL患者MMP2的表达 ,同时应用逆转录 PCR(RT PCR)测定了MMP2活化相关基因基质金属蛋白酶抑制剂 2 (TIMP2 )、基质金属蛋白酶 1(MT1 MMP)的表达 ,并在体外进行了白血病细胞株穿膜试验研究。结果　 4 6例AL患者中 2 4例 (5 2 2 % )MMP2表达阳性 ,MMP2阳性组与阴性组发生髓外浸润的比例分别为 5 0 0 %和 18 2 % (P值均 <0 0 5 ) ;外周血幼稚细胞百分率分别为 (77 2 1± 13 9) %和 (6 2 95± 17 2 ) % (P值均 <0 0 1)。同时 4 6例AL患者中TIMP2、MT1 MMP阳性表达分别为 2 7例 (5 8 7% )、33例 (71 7% )。体外穿膜试验证实了MMP2对白血病细胞侵袭能力的影响。结论　活性MMP2可能参与白血病细胞从骨髓释出并浸润组织的过程     

Gelatinase expression and activity in the synovium and skin of patients with erosive psoriatic arthritis

OBJECTIVE: Matrix metalloproteinases (MMP), especially the gelatinases (MMP2, MMP9), have been implicated in several features of inflammatory arthritis including angiogenesis and bone erosions. We examined the expression and activity of the gelatinases and their regulators in psoriatic skin and synovium using tissue immunohistochemistry and a sensitive tissue based zymographic technique. METHODS: Lesional and perilesional skin biopsies and synovial samples obtained by closed needle biopsy from 15 patients with erosive psoriatic arthritis (PsA) were analyzed by immunohistochemistry for the expression of the gelatinases and their regulators, tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1, TIMP-2) and membrane type metalloproteinases (MTI-MMP. MT2-MMP), using immunohistochemistry. Synovial tissue from 8 patients with erosive rheumatoid arthritis (RA) was used as a comparison. MMP tissue expression was quantified by 2 blinded independent observers. Immunohistochemical data are reported as the mean percentage positive cells per total nucleated cells in 10 high power fields +/- SD. Functional activity of the gelatinases was measured using a sensitive tissue based zymography technique and corrected for protein content. Zymography data are presented as ng/mg +/- SE. RESULTS: MMP expression was greater in the synovial lining layer (LL) than in the synovial sublining layer (SL) in both PsA and RA tissue for most MMP except collagenase I (MMPI), which was equally distributed between the LL and SL. Expression of MMP or their regulators did not differ between PsA synovial membrane (SM) and RA SM in LL. Moreover, although latent gelatinase A (MMP2) staining in PsA SM was equivalent to RA SM, increased gelatinase A activity was found in PsA SM over RA SM using zymography L82.4 (SD 62.8) vs 10.1 (SD 1.7); p = 0.02]. Compared to PsA SM, lesional skin had lower levels of MT1-MMP (MMP14) (1.4 +/- 1.7 vs 15.7 +/- 8.4; p = 0.009) and MT2-MMP (MMP15) (12.1 +/- 8.7 vs 21.6 +/- 9.9; p = 0.001). CONCLUSION: We characterized the expression and activity of gelatinases in PsA and demonstrate that gelatinase activity in SM of PsA patients with erosive disease is comparable to if not greater than that in RA synovium.     

Expression of the melatonin receptor (MT) 1 in benign and malignant human bone tumors

The beneficial effects of melatonin on bone homeostasis have been shown in various diseases. As this indoleamine causes dose-dependent modulation of bone-forming osteoblast and bone-resorbing osteoclast activities by receptor-independent and -dependent pathways, we investigated the expression of G-protein-coupled melatonin receptors (MTs) in malignant and non-malignant human bone lesions. By TaqMan polymerase chain reaction (PCR), we analyzed 30 specimens from osteosarcoma and 11 from benign bone tumors for MT1-mRNA expression. Furthermore, we determined mRNA expression levels of the osteoclast activity-stimulating receptor activator of nuclear factor-kappa B ligand (RANKL) and its counterpart osteoprotegerin (OPG). Although mean MT1-mRNA levels were similar (P = 0.596) in malignant (4.39 +/- 4.98-fold) and benign samples (4.64 +/- 6.81-fold), the highest MT1-mRNA levels (up to 27-fold) were observed in individual osteosarcomas, particularly, in two specimens of patients with local recurrence of the tumor. Moreover, mean RANKL- and OPG-mRNA levels were similar in malignant and benign specimens (RANKL: 7.38 +/- 9.61-fold versus 3.57 +/- 3.11-fold, P = 0.207; OPG: 23.45 +/- 32.76 versus 8.07 +/- 7.23-fold, P = 0.133). Again, highest RANKL- and OPG-mRNA levels (up to 41- and 160-fold, respectively) were observed in individual osteosarcomas. Expression of MT1-mRNA was confirmed in two human osteosarcoma cell lines (HOS, MG63). High expression levels of MT1-mRNA together with low OPG-mRNA were found in both osteosarcoma cell lines, while in normal human osteoblasts and bone marrow stromal cells, high OPG-mRNA levels were associated with low MT1-mRNA levels. These data on the abundant expression of MT1-mRNA in human bone tumors and osteosarcoma cells lines suggest an important role for MT1 in bone pathology.     

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.