基质金属蛋白酶MMP-2和MMP-9在血液肿瘤发生中的作用

基质金属蛋白酶MMP-2和MMP-9在血管生成、肿瘤生长及转移中起重要作用,主要是通过降解细胞间基质(ECM)和活化生长因子促进癌症进展。本文从分子和细胞水平阐述了MMPs的作用机制、在血液系统肿瘤如急性髓系白血病、急性淋巴系白血病、多发性骨髓瘤和淋巴瘤等中的作用及其表达的最新研究进展。     

基质金属蛋白酶及其抑制剂与恶性肿瘤的侵袭转移

基质金属蛋白酶(matrix metalloproteinases,MMPs)是细胞外基质(extracellular matrix,ECM)降解过程中的重要酶类,基质金属蛋白酶抑制剂(tissue inhibitors of metallopmteinases,TIMPs)是基质金属蛋白酶的天然抑制物,两者平衡关系的破坏与肿瘤的侵袭、转移及预后密切相关,因此,对基质金属蛋白酶及其抑制剂的研究已成为肿瘤研究中的一大热点。     

基质金属蛋白酶在肿瘤侵袭转移作用中的研究

肿瘤细胞的侵袭与转移是肿瘤的恶性生物学行为,其作用机制多样且复杂。基底膜是机体的重要组织屏障,多种基质蛋白酶纷纷参与了肿瘤细胞降解基底膜和细胞外基质的过程,其中以基质金属蛋白酶类的作用最为突出。该文对基质金属蛋白酶类在肿瘤细胞侵袭与转移中的作用机制、相关转录因子以及国内外研发的抑制剂做了综述。     

急性冠状动脉综合征患者血清中基质金属蛋白酶-2的表达

目的探讨急性冠状动脉综合征与血清基质金属蛋白酶-2(MMP-2)的相关性。方法应用酶图(SDS-PAGE en-zymograph)和Western blot方法检测50名急性冠脉综合征患者(27名ST段抬高急性心肌梗塞患者和23名不稳定性心绞痛患者)、20名稳定性心绞痛患者及40名正常对照者的血清MMP-2水平。结果急性心肌梗塞组血清MMP-2水平明显高于不稳定性心绞痛组;急性心肌梗塞组及不稳定性心绞痛组血清MMP-2水平明显高于正常对照组;稳定性心绞痛组与正常对照组差异无显著意义;急性心肌梗塞组及不稳定性心绞痛组血清MMP-2水平高于稳定性心绞痛组。结论急性冠脉综合征患者血清MMP-2水平明显升高,其水平可能与冠状动脉斑块的稳定性相关。     

米非司酮对异位子宫内膜基质金属蛋白酶表达的影响

目的探讨米非司酮对子宫内膜异位症(endometriosis,EMS)患者子宫内膜基质金属蛋白酶(MMP-2,MMP-9mRNA)表达的影响,以探讨米非司酮治疗EMS的可能机制。方法收集子宫内膜异位症患者38例,用宫腔镜分别取患者米非司酮治疗前和治疗后子宫内膜标本38份。采用RT-PCR半定量方法检测内膜标本中MMP-2,MMP-9 mRNA表达率及表达强度。结果所有内膜标本均有MMP-2、MMP-9 mRNA表达,治疗前和治疗后标本中MMP-2、MMP-9mRNA的表达率分别为(97%,68%)和(95%,66%);治疗后子宫内膜中MMP-2、MMP-9mRNA的表达强度低于治疗前(P<0.05)。结论米非司酮可以有效抑制子宫内膜症患者内膜中MMP-2、MMP-9mRNA的表达,降低了内膜的侵袭性,减少了转移到发生,为子宫内膜异位症的治疗提供了新的思路。     

基质金属蛋白酶及其与恶性肿瘤的关系

基质金属蛋白酶(matrixmetal loproteinases,MMPs)是一组结构相近、功能复杂的蛋白水解酶,因其需要Zn2+等金属离子作为辅助因子而得名。MMPs通过参与细胞表面和细胞外基质蛋白的降解或激活过程来调控细胞与细胞之间、细胞与细胞外基质(extracel lular matrix,ECM)之间的相互作用,是正常细胞与肿瘤细胞微环境的重要调节因子。     

VEGF与MMP-9在结肠癌中的表达及临床病理意义

目的探讨VEGF和MMP-9在结肠癌中的表达及临床意义。方法采用免疫组化S-P法检测71例结肠癌组织和18例正常肠粘膜组织中VEGF与MMP-9的表达水平。结果 MMP-9和VEGF在结肠癌组织中的表达明显高于正常肠粘膜(P<0.05),在临床晚期组中表达明显高于早期组(P<0.05),在有淋巴结转移组中表达明显高于无淋巴结转移组(P<0.05)。结论 MMP-9和VEGF在结肠癌组织中的表达和浸润深度、淋巴结转移有关。     

人细胞外基质金属蛋白酶诱导因子真核表达质粒的构建及表达

目的构建人细胞外基质金属蛋白酶诱导因子(matrix metalloproteinase inducer,EMMPRIN)真核表达质粒,并在COS-7细胞中表达。方法采用巢式PCR法从人结肠癌SW-480细胞中扩增EMMPRIN基因,插入pEGFP-N1质粒,构建重组真核表达质粒pEGFP-N1-EMMPRIN,转染COS-7细胞,48 h后,于倒置荧光显微镜下观察绿色荧光蛋白的表达,RT-PCR法和Western blot法分别检测转染细胞中EMMPRIN基因mRNA的转录和蛋白的表达。结果重组真核表达质粒pEGFP-N1-EMMPRIN经菌落PCR、酶切及测序证实构建正确,测序结果经比对分析,仅发现1个碱基突变,即534位:GCC→GCT,为无义突变;pEGFP-N1-EMMPRIN转染的COS-7细胞能表达绿色荧光蛋白,且能检测到EMMPRIN基因mRNA的转录和蛋白的表达。结论成功构建了人EMMPRIN真核表达质粒,并在COS-7细胞中表达了EMMPRIN,为进一步研究EMMPRIN在恶性肿瘤发生发展中的作用及其基因治疗奠定了基础。     

COX-2和MMP-7在大肠癌组织中的表达及意义

目的研究环氧合酶-2(cyclooxygenase-2;COX-2)蛋白和基质金属蛋白酶-7(matrix metalloproteinase-7;MMP-7)蛋白在人大肠癌组织中的表达及意义。方法应用免疫组化法检测40例大肠癌及其癌旁正常组织中COX-2及MMP-7蛋白表达水平。结果大肠癌组织中COX-2蛋白表达阳性率87.5%(35/40),明显高于癌旁正常组织25%(10/40)(P<0.01);大肠癌组织MMP-7蛋白表达阳性率85%(34/40),明显高于癌旁正常组织35%(14/40)(P<0.01);COX-2阳性表达组中MMP-7阳性率为91.4%(32/35),COX-2阴性表达组中MMP-7阳性率为40%(2/5),二者差异具有统计学意义(P<0.05)。结论COX-2蛋白可能通过促进MMP-7的表达从而共同促进了大肠癌的生长及转移。     

组织蛋白酶B在甲状腺肿瘤组织中的表达及其意义

目的探讨组织蛋白酶B(CathepsinB,CTSB)在甲状腺肿瘤组织中的表达及其与甲状腺肿瘤发生发展的相关性。方法采用荧光原底物法检测甲状腺癌、甲状腺腺瘤及正常甲状腺各30份组织标本中CTSB的活性,采用Western blot法检测各组织中CTSB蛋白的表达。结果甲状腺癌和甲状腺腺瘤组织中CTSB的活性及蛋白表达水平均显著高于正常甲状腺组织(P<0.05),而甲状腺癌组织中二者均显著高于甲状腺腺瘤组织(P<0.05)。结论 CTSB的表达与甲状腺肿瘤的发生发展密切相关。     

Lewis (y) Antigen Overexpression Increases the Expression of MMP-2 and MMP-9 and Invasion of Human Ovarian Cancer Cells

Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable cell line constitutively expressing Lewis (y) antigen (RMG-1-hFUT) by transfecting the cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1, and investigated whether Lewis (y) antigen regulates the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and tissue inhibitors of metalloproteinases (TIMP-1) and TIMP-2. We found that RMG-1-hFUT cells exhibited higher invasive capacities than their control cells. In addition, expression of TIMP-1 and TIMP-2 was down-regulated and expression of MMP-2 and MMP-9 was up-regulated. Anti-Lewis (y) antigen antibody treatment significantly reversed the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9. Taken together, we provide the first evidence that down-regulation of TIMP-1 and TIMP-2 and up-regulation of MMP-2 and MMP-9 represents one of the mechanisms by which Lewis (y) antigen promotes cell invasion.     

Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.     

Association of MMP-2 and MMP-9 Polymorphisms with Diabetes and Pathogenesis of Diabetic Complications

Type 2 diabetes mellitus (T2D) affects millions of people around the world, and its complications have serious health consequences. In addition to external factors, the causes of morbidity and increased risk were also sought in the variability of the human genome. A phenomenon that can answer these questions is the occurrence of single-nucleotide polymorphisms (SNP). They constitute a field for research into genetic determinants responsible for the increase in the risk of the discussed metabolic disease. This article presents the outline of two enzymes: metalloproteinases 2 and 9 (MMP-2, MMP-9), their biological activity and the effect caused by differences in individual alleles in the population, as well as the reports on the importance of these DNA sequence variations in the occurrence of diabetes mellitus type 2 and associated conditions. The results of the conducted research indicate a relationship between two MMP-2 polymorphisms (rs243865, rs243866) and two MMP-9 polymorphisms (rs3918242, rs17576) and the presence of T2D. This could offer a promising possibility to use them as predictive and diagnostic markers. However, due to the low number of reports, more research is needed to clearly confirm the link between these SNPs and diabetes.     

Adiponectin and Leptin Exert Antagonizing Effects on HUVEC Tube Formation and Migration Modulating the Expression of CXCL1, VEGF,MMP-2 and MMP-9

Adiponectin and leptin are two abundant adipokines with different properties but both described such as potent factors regulating angiogenesis. AdipoRon is a small-molecule that, binding to AdipoRs receptors, acts as an adiponectin agonist. Here, we investigated the effects of AdipoRon and leptin on viability, migration and tube formation on a human in vitro model, the human umbilical vein endothelial cells (HUVEC) focusing on the expression of the main endothelial angiogenic factors: hypoxia-inducible factor 1-alpha (HIF-1α), C-X-C motif chemokine ligand 1 (CXCL1), vascular endothelial growth factor A (VEGF-A), matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9). Treatments with VEGF-A were used as positive control. Our data revealed that, at 24 h treatment, proliferation of HUVEC endothelial cells was not influenced by AdipoRon or leptin administration; after 48 h longer exposure time, the viability was negatively influenced by AdipoRon while leptin treatment and the combination of AdipoRon+leptin produced no effects. In addition, AdipoRon induced a significant increase in complete tubular structures together with induction of cell migration while, on the contrary, leptin did not induce tube formation and inhibited cell migration; interestingly, the co-treatment with both AdipoRon and leptin determined a significant decrease of the tubular structures and cell migration indicating that leptin antagonizes AdipoRon effects. Finally, we found that the effects induced by AdipoRon administration are accompanied by an increase in the expression of CXCL1, VEGF-A, MMP-2 and MMP-9. In conclusion, our data sustain the active role of adiponectin and leptin in linking adipose tissue with the vascular endothelium encouraging the further deepening of the role of adipokines in new vessel’s formation, to candidate them as therapeutic targets.     

The Influence of New Silicate Cement Mineral Trioxide Aggregate (MTA Repair HP) on Metalloproteinase MMP-2 and MMP-9 Expression in Cultured THP-1 Macrophages

The aim of the present study was to investigate the new silicate cement mineral trioxide aggregate (MTA Repair HP) with respect to its effect on the inflammation process involving the tooth and periodontal tissues. The composition of MTA Repair HP was supplemented with plasticizer agents which can have a negative effect on the modulation of tooth inflammation. The silicate-based material in question is widely used in regeneration of the pulp-dentin complex, treatment of perforations of various locations in the tooth, as well as in surgical treatment of the complications of periapical tissue. The improved bioceramic restorative cement can affect the expression of metalloproteinases MMP-2 and MMP-9 in monocytes/macrophages involved in modulation of inflammation and regenerative processes of the tooth and periodontal tissues. The novel aspect of the present study lies in the application of the model of THP-1 monocyte/macrophage and applying the biomaterial in direct contact with the cells. Hence, it provides a representation of clinical conditions with respect to regenerative pulp and periodontal treatment with the use of MTA Repair HP. A lack of macrophage activation (as measured with flow cytometry) was found. Moreover, the study identified a lack of expression stimulation of the studied metalloproteinases (with the use of Western blotting and fluorescent microscopy). Similarly, no increase in MMP-2 and MMP-9 concentration was found (measured by ELISA method) in vitro when incubated with MTA Repair HP. Based on the results it can be concluded that new MTA Repair HP does not increase the inflammatory response in monocytes/macrophages associated with the activity of the described enzymes. It can also be speculated that they do not affect the process of dentin regeneration in which MMP-2 and MMP-9 play significant roles.     

Lupinus albus Protein Components Inhibit MMP-2 and MMP-9 Gelatinolytic Activity In Vitro and In Vivo

Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are regarded as important clinical targets due to their nodal-point role in inflammatory and oncological diseases. Here, we aimed at isolating and characterizing am MMP-2 and-9 inhibitor (MMPI) from Lupinus albus and at assessing its efficacy in vitro and in vivo. The protein was isolated using chromatographic and 2-D electrophoretic procedures and sequenced by using MALDI-TOF TOF and MS/MS analysis. In vitro MMP-2 and 9 inhibitions were determined on colon adenocarcinoma (HT29) cells, as well as by measuring the expression levels of genes related to these enzymes. Inhibitory activities were also confirmed in vivo using a model of experimental TNBS-induced colitis in mice, with oral administrations of 15 mg·kg⁻¹. After chromatographic and electrophoretic isolation, the L. albus MMP-9 inhibitor was found to comprise a large fragment from δ-conglutin and, to a lower extent, small fragments of β-conglutin. In vitro studies showed that the MMPI successfully inhibited MMP-9 activity in a dose-dependent manner in colon cancer cells, with an IC50 of 10 µg·mL⁻¹ without impairing gene expression nor cell growth. In vivo studies showed that the MMPI maintained its bioactivities when administered orally and significantly reduced colitis symptoms, along with a very significant inhibition of MMP-2 and -9 activities. Overall, results reveal a novel type of MMPI in lupine that is edible, proteinaceous in nature and soluble in water, and effective in vivo, suggesting a high potential application as a nutraceutical or a functional food in pathologies related to abnormally high MMP-9 activity in the digestive system.     

Serum Level of MMP-3 and MMP-9 in Patients with Diabetes Mellitus Type 2 Infected with Epstein-Barr Virus

Diabetes mellitus type 2 (DM2) has recently become one of the most important health problems in the world. Patients with DM2 with long-term glycaemia are more likely to become infected than the healthy population. Matrix metalloproteinases (MMPs) play a key role in tissue remodeling during various physiological processes. However, it has been reported that certain MMPs are overexpressed during the development of various human diseases. In this study, we analyzed the levels of MMP-3 and MMP-9 in the serum of DM2 patients with and without Epstein-Barr virus (EBV) infection. The study included 115 patients with DM2 hospitalized in the Internal Ward of the Masovian Specialist Hospital in Radom, Poland, who were divided into two groups: EBV-positive and EBV-negative. The levels of MMP-3 and MMP-9 were tested in the serum of patients using the ELISA method, while the presence of EBV in saliva was tested by polymerase chain reaction (PCR). The presented studies showed a significant difference in the concentration of both MMPs in diabetic patients additionally infected with EBV compared to the group of non-infected individuals. It seems that MMPs may be useful biomarkers in the diagnosis, prognosis, and monitoring of diabetes associated with EBV infection.     

健康牙齿和龋齿成牙本质细胞中MMP-2和MMP-9的活性

目的比较健康牙齿和龋齿成牙本质细胞中基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的活性,以探讨MMP-2和MMP-9在龋病发病机制中的作用。方法培养健康牙齿和龋齿成牙本质细胞,利用酶谱分析细胞培养上清中MMP-2和MMP-9的活性。结果龋齿组中MMP-2和MMP-9的活性显著高于健康组。结论MMP-2和MMP-9在健康牙齿和龋齿成牙本质细胞中的活性不同,提示MMPs可能在龋病的进展过程中发挥作用。     

激活素受体相互作用蛋白2在小鼠Hepa1-6细胞中的表达及调控

目的研究激活素受体相互作用蛋白2(ARIP2)在小鼠肝细胞中的表达及调控。方法采用半定量PCR技术检测ARIP2 mRNA在Hepal-6细胞中转录水平的动力学变化规律及其调控因素。结果Activin A刺激Hepal-6细胞ARIP2 mRNA的转录水平呈时间依赖性升高,刺激早期(4h)无明显变化,12h后显著升高。信号传导激动剂PMA和LPS刺激He- pal-6细胞24h,均可上调ARIP2 mRNA的转录水平,而A23187则抑制其转录。ARIP2过表达明显抑制Hepal-6细胞ActRIIA mRNA的转录水平,对ActRIIB则无影响。结论ARIP2作为激活素信号传导抑制蛋白,其表达受多种因素影响。ARIP2可能通过影响ActRIIA表达,参与激活素作用后期的信号传导负反馈调节过程。     

胰高血糖素基因克隆、表达及鉴定

目的 研制基因工程胰高血糖素,用于治疗因注射胰岛素或口服抗糖尿病药物而引起的严重低血 糖反应。方法 依据人胰高血糖素氨基酸组成序列,采用大肠杆菌出现频率高的氨基酸密码子,体外合成胰高血 糖素基因序列,克隆于ｐＧＥＸ－１Ｎ质粒,转化人ＢＬ２１（ＤＥ３）菌中,对阳性克隆转化质粒进行ＤＮＡ测序。用ＩＰＴＧ诱导 转化的胰高血糖素工程菌,产生谷胱苷肽转移酶的融合蛋白,经谷胱苷肽Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析后,由 Ｆａｃｔｏｒ　Ｘａ酶 进行柱上切割,最后用ＲＰ－ＨＰＬＣ得到纯化产物。产品用质谱法测相对分子质量,并对Ｎ末端１５个氨基酸测序。 结果ＤＮＡ测序显示克隆基因序列正确,产品相对分子质量为３４８６Ｊ末端１５个氨基酸序列与理论值相同,产率为 １．９ｍｇ／Ｌ。结论 获得以可溶性融合蛋白方式表达的胰高血糖素基因工程菌,适于快速生产天然序列重组胰高血 糖素。