肺癌金属蛋白酶、金属蛋白酶抑制因子表达和细胞凋亡与预后的相关性

目的：研究人肺癌组织中金属蛋白酶，金属蛋白酶抑制因子的表达和细胞调亡与病人预后的关系。方法：应用免疫组化法测MMPs、TIMPs在肺癌标本中的表达、用原位杂交法(ISH)测TIMP1mRNA、用TUNEL法测细胞凋亡。结果：MMP2与MT1-MMP表达十分相近；TIMP1-2在Ⅲ、Ⅳ期肿瘤中强阳性表达明显高于Ⅰ、Ⅱ期肿瘤；NSCLC术后存活4年以下者其MMP1、MT1-MMP、MMP2蛋白强阳性表达率和显著高于4年以上存活者细胞凋亡指数。结论：MMP1-2、TIMP1-2在肺癌组织中的表达强度与肿瘤的分期相关，MMP2与MT1-MMP的表达密切相关且与病人预后有相关性，TIMP3的表达和细胞凋亡与病人预后有相关性。     

子宫腺肌症与肿瘤转移相关基因之间关系的研究

目的：探讨肿瘤转移相关基因在子宫腺肌症发生中的作用。 方法： 采用免疫组织化学方法，对43例子宫腺肌症患者、22例对照组（正常子宫内膜）的nm23-H1、基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）、膜型1-基质金属蛋白酶（MT1-MMP）和基质金属蛋白酶组织抑制因子-1（TIMP-1）的表达进行研究。 结果： 子宫腺肌症中，MMP-2、MMP-9和MT1-MMP的表达水平明显高于对照组（P<0.01），nm23-H1和TIMP-1的表达水平无显著差异（P>0.05）。 结论： MMP-2、MMP-9和MT1-MMP在子宫腺肌症的发病过程中可能起重要的作用。     

胸腺上皮肿瘤组织中基质金属蛋白酶-2活性与Ⅰ型膜型基质金属蛋白酶 mRNA表达的相关性

目的探讨胸腺瘤和胸腺癌中基质金属蛋白酶（MMP）-2、Ⅰ型膜型（MT1）-MMP、金属蛋白酶组织抑制剂（TIMP）-2mRNA的表达和MMP-2蛋白活性的关系。方法分别用real-time逆转录．聚合酶链反应（RT-PCR,Taqman法）、明胶酶谱法和Filmin situ gelatin-Zymography（FIZ）对正常的胸腺组织（2例）、胸腺瘤（12例）和胸腺癌（2例）患者的新鲜肿瘤组织中MMP-2、MT1-MMP、TIMP-2mRNA的表达,pro-MMP-2的活性率及活性蛋白的定位进行测定。结果MMP-2、MT1-MMP及TIMP-2mRNA在Ⅰ期与Ⅱ期和Ⅲ期与Ⅳ期中的表达差异均无统计学意义（P〉0．05）,在Ⅰ～Ⅱ期与Ⅲ～Ⅳ期和胸腺癌3组中差异均有统计学意义（P〈0．01）。在AB、B1型（混合型和淋巴细胞为主型）与B2、B3型（皮质型和多角细胞为主型）以及胸腺癌3组中差异均有统计学意义（P〈0．05）。MMP-2的蛋白活性率（MMP-2／pro—MMP-2＋MMP-2）在Ⅰ～Ⅱ期、Ⅲ～Ⅳ期和胸腺癌各组中差异有统计学意义（P〈0．05）,在AB、B1型与B2、B3型以及胸腺癌各组中的差异均具有统计学意义（P〈0．05）。胸腺瘤各期及各型中MT1-MMP、TIMP-2mRNA与MMP-2蛋白活性表达均呈正相关,且相关程度相似（r=0．7235、r=0．7647、P〈0．005）。MMP-9的蛋白表达在各组间差异均无统计学意义。结论MMP-2、MT1-MMP及TIMP-2的mRNA表达与胸腺瘤临床分期、病理分型相关,MMP-2的活性与MT1-MMP和TIMP-2的表达升高正相关。推测MT1-MMP通过TIMP-2对MMP-2的激活起促进作用。     

妊娠期基质金属蛋白酶的调控及其与胎盘发育的关系

 基质金属蛋白酶（matrix metalloproteinases，MMP） 是一类蛋白水解酶，可以降解多糖以外的细胞外基质（extracellular matrix，ECM）的多种成分，在组织塑型过程中影响着 ECM 的代谢、新生血管的形成等过程。在胎盘发育过程中滋养细胞的入侵包括细胞基质的溶解和重塑，其生物学行为与有着高度侵蚀性的肿瘤细胞相似，又被称为假恶性细胞。MMP 是降解蜕膜基质的重要酶类，可以降解几乎所有基质成分，在胚胎植入、胎盘形成及子宫螺旋动脉重构中发挥重要作用。MMP 在滋养层不同类型细胞中的分布存在特异性，在不同时期胎盘中的表达和活性也有所不同，妊娠期 MMP 的调控直接影响胎盘的发育，对维持正常妊娠至关重要。本文主要就妊娠期 MMP 的调控及其与胎盘发育关系的研究进展做一综述。 1 MMP 分类及特点 MMP 是一族 Zn2+ 依赖性内肽酶，至今已经发现至少26 种^[1]。根据蛋白结构的相似性以及降解底物不同，MMP 通常被分为 5 个亚类：①间质胶原酶（MMP-1、MMP-8和MMP-13），来源于纤维细胞、癌细胞及滋养细胞，降解底物为 Ⅰ~ Ⅲ 型胶原等。②明胶酶（MMP-2、MMP-9），部分来源于滋养细胞、纤维细胞及巨噬细胞等，酶解底物为明胶变性胶原和 Ⅳ 型胶原等。③基质溶解素（MMP-3、MMP-7、MMP-10、MMP-11、MMP-12 和 MMP-26），来源于成纤维细胞和癌细胞等，降解底物为 Ⅲ ~ Ⅴ 型胶原、明胶、蛋白聚糖及糖蛋白等。④膜型金属蛋白酶（MT-MMP），包括 MT1-MMP、MT2-MMP、MT3-MMP、MT4-MMP、MT5-MMP 及 MT6-MMP，存在于肿瘤细胞表面及滋养细胞表面，降解底物为 Ⅳ 型胶原、明胶等。有证据表明，MT1-MMP、MT2-MMP、MT3-MMP 和 MT5-MMP 参与激活 MMP-2^[2]。⑤其他基质金属蛋白酶，包括 MMP-19、MMP-20、MMP-23 等，可以降解抗胰蛋白酶及釉质。     

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。     

MMP-2、MT1-MMP、TIMP-2在胎膜早破中的作用

目的通过检测不同情况产妇胎膜组织中基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)及其激动剂(membrane type 1-matrix metalloprotease MT1-MMP)和其特异性组织抑制剂(tissue inhibitor of matrix metalloproteinases-2,TIMP-2)的表达,以探讨MMP-2在胎膜早破中的作用.方法 RT-PCR和S-P法对20例自发性胎膜早破患者、20例正常分娩产妇的胎膜组织中MMP-2、TIMP-2 mRNA和MT1-MMP蛋白的表达及定位进行检测.结果 (1)MMP-2 mRNA在胎膜早破组表达明显高于正常分娩组,两者比较,差异有显著性(P＜0.05).(2)TIMP-2 mRNA在正常分娩组表达高于胎膜早破组,两者比较差异有显著性(P＜0.05).(3)MT1-MMP蛋白在胎膜早破组表达明显高于正常分娩组,两者比较,差异有显著性(P＜0.05).MT1-MMP在羊膜细胞和绒毛膜的滋养细胞内表达.主要在细胞浆内表达.结论胎膜早破中,MMP-2及其人调节剂的表达失调将可能导致胎膜基质的降解增加并最终导致胎膜的破裂.     

弗林蛋白酶抑制剂对乳腺癌细胞MCF-7迁移的影响

 目的：探讨乳腺癌转移的机制,为深入研究乳腺癌发生、发展机制提供理论基础。方法：不同浓度的弗林蛋白酶(furin)抑制剂处理人乳腺癌细胞MCF-7 48 h。细胞划痕实验（wound healing assay）和细胞趋化实验（Transwell assay）检测MCF-7细胞迁移和侵袭能力。Western blotting 检测细胞迁移相关蛋白膜型基质金属蛋白酶1（MT1-MMP）、血管内皮生长因子（VEGF）-C和VEGF-D水平。酶联免疫吸附法（ELISA）检测细胞培养液中基质金属蛋白酶(MMP)2和9水平。结果：与对照组相比,200 nmol/L的furin抑制剂α1-PDX即对细胞迁移及侵袭起显著抑制作用（均P<0.05）;细胞迁移相关的MT1-MMP、VEGF-C和VEGF-D表达水平均显著降低（P<0.05）;MCF-7细胞上清液中MMP2 和 MMP9的表达均显著降低（P<0.05）。结论：Furin抑制剂通过下调乳腺癌细胞MCF-7的MMPs及VEGFs表达抑制其迁移。     

肺间质纤维化大鼠肺组织基质金属蛋白酶及其组织抑制因子含量变化

观察肺纤维化形成过程中基质金属蛋白酶（Matrix Metallo proteinas简称MMPs)及其组织抑制因子（Tissue inhibitors of Metallo proteinases简称TIMPs)含量的变化,探讨其在肺纤维化发病中的作用。将Wistar大鼠60只,随机均分为对照组及模型组,气管内注入博莱霉素A5 5mg/kg,制备肺间质纤维化动物模型,观察注药后1、3、7、14及28d肺脏病理变化,利用酶谱法及免疫印记法分析肺组织MMP－2、MMP－9、TIMP－1的含量变化。结果显示各模型组pro-MMP-2、MMP－2、TIMP－1蛋白含量均较对照组增加,尤其7、14及28d组MMP－2较前明显增多。而MMP－9变化不很明显。提示在肺纤维化形成过程中,pro-MMP-2、MMP－2及TIMP－1都有所增高,MMP／TIMP比例失衡是最终导致肺间质纤维化形成的重要因素。     

原发性肝细胞癌中膜型金属基质蛋白酶-1的表达及意义

目的 探讨膜型金属基质蛋白酶-1(membrane-type1 matrix metalloproteinase,MT1-MMP/MMP-14)在原发性肝细胞癌(primary hepatocellular carcinoma,HCC)中表达情况及其与肝癌的临床病理特征之间的相关性.方法 对33例HCC的病例资料和5例正常肝脏标本进行免疫组化ImmunoVision法染色.结果 在正常肝组织中,MT1-MMP主要呈现胞质弱表达.HCC中,MT1-MMP在肝癌细胞膜上表达最强.1例肝癌胞核中表达MT1-MMP.胞膜和胞质中MT1-MMP的表达水平与肝癌的临床病理特征无相关性(P＞0.05).结论 HCC肿瘤细胞能够凭借细胞表面MT1-MMP过表达,促进肿瘤浸润和转移,胞核中MT1-MMP过表达与肝组织癌变有关.     

MT1-MMP在肿瘤血管新生中的作用

基质金属蛋白酶 (MMPs)是包含至少 2 6个成员的酶家族。它们除降解细胞外基质外 ,还可以作用于其它的蛋白激酶、细胞趋化分子、生长因子、生长因子连接蛋白以及细胞表面分子等。MMPs的活性受组织型金属蛋白酶抑制物 (TIMPs)调控。MMPs和TIMPs在生理状态和癌症进程中的细胞外基质重塑过程中发挥重要作用 ,特别是MMP 2和TIMP 2。它们主要是调节肿瘤进展的早期阶段 ,如肿瘤生成和血管新生。     

Differential expression patterns of MMPs and their role in the invasion of epithelial premalignant tumors and invasive cutaneous squamous cell carcinoma

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumors. MMP-2, MMP-9, TIMP-2, and MT1-MMP are thought to be involved in the process of destruction of basement membranes and stromal invasion by neoplastic epithelial cells. In this study, we investigated the expression and role of MMPs in cutaneous oncogenesis. Tissue microarray consisting of 62 squamous cell carcinomas (SCC), 32 Bowen's disease (BD) samples, 25 normal epidermis samples were obtained for the study. MMP-2,-9, MT1-MMP and TIMP-2 proteins were examined by immunohistochemical staining and mRNA level was detected by quantitative RT-PCR in fresh tissues consisting of 5 cutaneous SCCs and paired normal epidermis samples. Gelatinase activity of MMP-2 and MMP-9 was investigated by gelatin zymography and protein levels of MT1-MMP and TIMP-2 were measured by western blot in 2 human SCC cell lines. The invasive property was evaluated with invasion assays using Transwell filters. SCC exhibited significantly increased MMP-2, MT1-MMP and decreased TIMP-2 mRNA and protein expression compared to that of the normal epithelium. Immunohistochemical staining revealed that MT1-MMP was strongly expressed on the invasive front of SCCs, whereas BD exhibited higher expression around the dyskeratotic cells in the epithelium. In comparison with the expression observed in BD, SCC exhibited significantly increased MMP-2 expression. In addition, high MMP-2 and MT1-MMP expression and low TIMP-2 expression had a significant positive correlation with the invasiveness of SCC cell lines in vitro. Our results revealed significantly increased MT1-MMP and MMP-2 expression and decreased TIMP-2 expression in cutaneous SCC, and the expression correlated with the invasiveness of SCC cell lines. Therefore, the expression of these factors in cutaneous tumors may serve as an indicator of tumor aggressiveness and invasion.     

The metalloproteinase inhibitor TIMP-2 is down-regulated by androgens in LNCaP prostate carcinoma cells

Tissue inhibitors of metalloproteinases (TIMPs) have been shown to perform several biological functions in tumor promotion, principally by their action of inhibiting matrix metalloproteinases (MMPs) at different steps of the metastatic process. In particular, TIMP-2 is involved in a functional complex with the membrane-type 1 (MT1) MMP to convert the secreted MMP-2 progelatinase into the fully active proteolytic enzyme. We used the human, androgen-sensitive prostate carcinoma cell line LNCaP in coculture with the human osteosarcoma cell line OHS to experimentally address the possibility of androgen-dependent regulatory effects on the functional MT1-MMP/TIMP-2/MMP-2 complex upon interaction between prostate carcinoma and osteoblastic cells in metastasis of prostate cancer to bone. In the LNCaP cells a gradual, time-dependent decline in TIMP-2 mRNA expression was observed in the presence of the synthetic androgen analogue R1881 (100 nM), reaching ∼25% of the control level after 48 h of incubation. Consistent with this, the accumulation of secreted TIMP-2 in media from R1881-treated cells was significantly inhibited already after 3 h. Neither MMP-2 gelatinolytic activity nor expression of MT1-MMP was detected in LNCaP cells. In contrast, the OHS cells showed membrane-associated MT1-MMP expression as well as MMP-2 secretion. However, R1881 treatment of the LNCaP/OHS coculture model did not seem to change the overall proteolytic activity of the MT1-MMP/TIMP-2/MMP-2 complex. Hormonal control of TIMP-2 expression in prostate carcinoma cells has not been previously reported, but whether such regulation has any functional role in the development of osteoblastic metastases in prostate cancer is still unclear. This revised version was published online in July 2006 with corrections to the Cover Date.     

EMMPRIN-mediated MMP regulation in tumor and endothelial cells

Tumor invasion and metastasis are multistep processes which require extracellular matrix remodeling by proteolytic enzymes such as matrix metalloproteinases (MMPs). The production of these enzymes is stimulated by many soluble or cell-bound factors. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) is known to increase in vitro stromal cell production of MMP-1, MMP-2 and MMP-3. In this study, we demonstrated that EMMPRIN-transfected MDA-MB-436 tumor cells displayed a more invasive capacity than vector-transfected cells in a modified Boyden chamber invasion assay. Using gelatin zymography and protein analyses, we showed that EMMPRIN-transfected cancer cells produced significantly more latent and active MMP-2 and MMP-3 than vector-transfected cancer cells. We found that EMMPRIN did not regulate MMP-1, MMP-9, membrane type-1 MMP (MT1-MMP) expression and had also no effect on the production of the specific tissue inhibitors of MMPs (TIMPs), TIMP-1 and TIMP-2. We also demonstrated that tumor-derived EMMPRIN stimulated MMP-1, -2, and -3 without modification of MMP-9, MT1-MMP, TIMP-1 and TIMP-2 production in human umbilical vein endothelial cells (HUVEC). These data provide support for the role of EMMPRIN in tumor invasion, metastasis, and neoangiogenesis by stimulating extracellular matrix remodeling around tumor cell clusters, stroma, and blood vessels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gelatin zymography and substrate cleavage assays of matrix metalloproteinase-2 in breast carcinoma cells overexpressing membrane type-1 matrix metalloproteinase

Gelatin zymography is the common method for examining matrix metalloproteinase-2 (MMP-2) in cells and media samples. Activation of the latent MMP-2 zymogen involves its binding to the cell surface MT1-MMP*TIMP-2 (membrane type-1 matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase-2) complex with subsequent cleavage of proMMP-2 by TIMP-2-free adjacent MT1-MMP. This is followed by autolytic maturation of the activation intermediate and the release of the mature MMP-2 species from cell surfaces into the extracellular milieu. To observe the MMP-2 activation pathway in more detail, proMMP-2-deficient MCF7 breast carcinoma cells expressing MT1-MMP were incubated with excess proMMP-2 to saturate the available MT1-MMP*TIMP-2 surface receptors. After removal of the unbound material, the kinetics of proMMP-2 activation and MMP-2 release from cells into media was monitored by gelatin zymography and substrate cleavage. Our observations demonstrate that gelatin zymography is insufficient for providing meaningful information about the status of MMP-2. The proteolytically competent mature MMP-2 moiety alone, but not in its complex with TIMP-2, was released from the cells. In tissue culture conditions, the enzyme's proteolytic activity was suppressed in the next 30 to 60 minutes by tissue inhibitors of MMPs, especially by TIMP-1. The picture emerges that there is a likely temporal regulation of MMP-2 activity by TIMPs in tumor cells. These relatively rapid changes of the MMP-2 status cannot be detected by gelatin zymography. Additional studies are needed to examine the significance of this phenomenon in vivo.     

Protein kinase C activation by phorbol ester increases in vitro invasion through regulation of matrix metalloproteinases/tissue inhibitors of metalloproteinases system in D54 human glioblastoma cells

To elucidate possible mechanisms of phorbol 12-myristate 13-acetate (PMA) induced in vitro invasiveness of glioblastoma cells, we examined expression levels of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 using Western blotting and gelatin zymography assay, and found that PMA induced the secretion of MMP-9, activated MMP-2 proenzyme to fully active form of 59 kDa, down-regulated the TIMP-1 and TIMP-2 secretion, and increased MT1-MMP on the cell surface. However, PKC inhibitor Go 6983 reversed all of these effects brought about by PMA. We, therefore, conclude the activation of PKC by PMA in these cells plays a critical role in the regulation of MMPs/TIMPs system, which has a major role in tumor invasion and metastasis.