逆转录病毒介导的c-met-小干扰RNA抑制肝癌转移的体内研究

我们对c-met与肝癌的临床病理因素的关系进行了分析，明确了c-met与肝癌转移有关联。此前也通过逆转录病毒载体介导，成功建立了表达c-met-siRNA的肝癌细胞株MHCC97-H／c-met-siRNA，并证实逆转录病毒载体能长期、稳定表达针对靶基因c-met的siRNA，因此，本研究拟进一步在体内研究c-met-siRNA对肝癌生长、转移的影响，以期为c-met-siRNA应用于临床防治肝癌转移复发提供实验依据。     

Evaluation of stromal metalloproteinases and vascular endothelial growth factors in a spontaneous metastasis model

This study aims to investigate MMP2 and MT1-MMP protein as well as VEGF-C and VEGF-D mRNA expression in tumor cells and distant organs considered to be targets for metastasis in a tumor spontaneous metastasis model previously described. Cultured tumor cells, able to express pro-MMP2, MMP2, pro-MMP9, and MT1-MMP, develop tumor growth and metastasis, mainly in the liver and spleen, when they are injected in the mammary pad gland of Wistar rats. Immunohistochemical studies of tumor masses showed small groups of tumor cells staining for MT1-MMP but not for MMP2. In the liver, tumor metastatic foci and a stromal positive staining for both MMP2 and MT1-MMP were shown. The spleen and lymph nodes, with only scattered metastatic cells, did not show MMPs immunostaining. Using RT-PCR, a significantly higher VEGF-C and VEGF-D gene expression was shown in the liver of tumor-bearing rats respect to normal rats, whereas spleen and lymph nodes did not show significant differences in mRNA VEGF-C/D levels. Taken together, our results suggest that the stroma microenvironment of target organs for metastasis has the ability to produce MMPs and VEGFs that facilitate the anchorage of tumor cells and promote tumor cell growth and angiogenesis.     

基质金属蛋白酶1与冠状动脉粥样硬化斑块破裂的关系

目的 探讨冠状动脉粥样硬化斑块破裂与基质金属蛋白酶１（ＭＭＰ－１）的关系,以及不稳定斑块中ＭＭＰ－１的来源。方法 收集２０例死于急性心肌梗死、１０例有不稳定心绞痛史,以及１２例有稳定心绞痛史的尸体解剖病例共４２例,从冠状动脉各分支取材,常规病理检查、部分节段行ＭＭＰ－１、平滑肌肌动蛋白、ＣＤ６８、ＣＤ４５ＲＯ和ＣＤ２０洒色。结果 在急性心肌梗死及不稳定心绞痛病例中,均见有斑块破裂伴血栓形成,而在稳     

人大肠癌中基质金属蛋白酶-7 mRNA的表达

目的:探讨基质金属蛋白酶-7(matrix metalloproteinase 7,MMP-7)与大肠癌浸润和转移的关系.方法:用逆转录聚合酶链式反应(RT-PCR)检测大肠癌和正常黏膜、炎性息肉、腺瘤及淋巴结中MMP-7 mRNA的表达.结果:97.4%(37/38)的大肠癌和1例肝转移中MMP-7 mRNA表达阳性,而相应正常黏膜几乎不表达;大肠癌中MMP-7 mRNA的表达水平随Dukes分期的进展而逐渐升高(P＜0.01),A期(0.28±0.08),B期(0.54±0.17),C期(0.55±0.09),D期(0.73±0.06).5例腺瘤MMP-7均为阳性,但表达水平低于癌组织(P＜0.01);3例炎性息肉均未见MMP-7表达.传统组织学检查有癌转移的13个淋巴结MMP-7表达均为阳性;组织学检查未见转移的12个淋巴结MMP-7表达6个阴性、6个阳性,再次行组织学检查于6个MMP-7阳性者中发现3个有微转移.结论:MMP-7的高表达可能在大肠癌的浸润和转移过程中具有重要作用;用RT-PCR方法检测淋巴结中MMP-7mRNA的表达是诊断大肠癌淋巴结微转移的敏感方法.     

Resolution of isoforms of mutalysin II, the metalloproteinase from bushmaster snake venom

Mutalysin II, a zinc endopeptidase possessing direct-acting fibrinolytic activity has been previously purified from bushmaster (Lachesis muta muta) snake venom. We now report a method to isolate two isoforms of natural mutalysin II (mut IIa and mut IIb) using chromatographies on Sephacryl S-200, CM Sepharose CL 6B and Sephadex G-50. The two proteins are monomeric non-glycosylated proteinases with similar molecular masses of 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Both isoforms showed high similarity in all enzymatic properties using fibrinogen, fibrin and dimethylcasein as substrates. Thus, the specific fibrinolytic activity was estimated as 12±1.04 and 11.5±1.02 U/μg for mut IIa and mut IIb, respectively. The antigenic cross-reactivity of both isoforms was examined using rabbit hyperimmune serum or immunoglobulin G anti-mut IIa assays on immunodiffusion microscope slides, indirect enzyme-linked immunoabsorbent assay and western blots. From these experiments it was concluded that the two metalloproteinases mut IIa and mut IIb share identical antigenic structures. Since the stability of mutalysin II is dependent upon the presence of zinc, we examined the EDTA sensitivity of the isoforms of mutalysin II. Thus, the IC₅₀ values (concentration of EDTA to produce 50% inhibition of dimethylcasein hydrolysis) for mut IIa is 180 μM and 165 μM for mut IIb.     

VEGF、MMP-2、MMP-9在卵巢原发性与转移性癌中表达的研究

目的 :探讨VEGF、MMP 2、MMP 9在卵巢原发上皮癌、库肯勃氏瘤中的表达差异及临床病理意义。方法 :采用免疫组化S P染色技术对 83例卵巢癌 (卵巢原发癌 4 5例、库肯勃氏瘤 38例 )进行分析 ,观察VEGF、MMP 2、MMP 9在卵巢癌中的表达。结果 :VEGF在卵巢原发癌和库肯勃氏瘤组织中的表达显著高于正常卵巢组织 (P <0 .0 5 ) ;MMP 2、MMP 9在正常卵巢组织中表达缺如 ;VEGF、MMP 2、MMP 9在卵巢原发癌与库肯勃氏瘤中表达均有显著差异 (P <0 .0 5 ) ;VEGF、MMP 2、MMP 9在库肯勃氏瘤中及卵巢原发癌中 ,任意两指标阳性表达均有正相关性 (P <0 .0 5 )。结论 :VEGF、MMP 2、MMP 9在库肯勃氏瘤的形成机制中起重要作用 ,与肿瘤的浸润转移密切相关 ,并可为鉴别卵巢原发癌与库肯勃氏瘤提供一定的依据     

Role of Matrix Metalloproteinases in Skin Ageing

Matrix metalloproteinases are a family of ubiquitous endopeptidases playing a role in many different physiological and pathological processes in the skin. They are also involved in cutaneous ageing. This review summarizes the features and regulation of these enzymes and presents an overview of the molecular mechanisms of both intrinsic and extrinsic skin ageing presents. The role of matrix metalloproteinases in skin ageing is discussed in detail.     

Temperospatial Expression of Matrix Metalloproteinases 1, 2, 3, and 9 During Early Tooth Development

Odontogenesis involves a complex series of processes including epithelial-mesenchymal interactions, morphogenesis, differentiation, fibrillogenesis, and mineralization. Extracellular (ECM) remodeling plays a critical role in the rapid morphological changes that accompany these events. It is proposed that matrix metalloproteinases (MMPs) participate in the remodeling of tooth-specific matrices that accompanies the developmental events. MMPs are zinc-requiring endopeptidases that are centrally involved in the controlled turnover of ECM components and are key to a varied range of developmental processes. Thus, the aim of this study was to examine the expression of MMPs 1, 2, 3, and 9 within the developing tooth germ of Wistar rats, using immunohistochemical localisation. During the bud stage, MMPs 1, 2, 3, and 9 were expressed within both epithelial and mesenchymal cells. Later on, during the cap stage, differential expression was observed; of note was the expression of MMP 3 within the enamel knot. This study reports the temperospatial expression of MMPs 1, 2, 3, and 9 during early tooth development, and points to them having a key role during this important developmental period.     

Changes in the connective tissue sheath of Wistar rat nerve with aging

The alterations due to aging in the peripheral nerves can affect the physiology of these structures. Thus, the purpose of the present study was to describe the activity of the MMP-2 and MMP-9, as well as the structure and composition of the extracellular matrix of the rat sciatic nerve during maturation and aging. Our results have shown that the extracellular matrix of the sciatic nerve of 30-, 180- and 730-day-old Wistar rats present ultrastructural, morphometrical and biochemical changes during aging. The perineurium was the structure most affected by age, as evidenced by a decrease in thickness and in collagen fibril content. Cytochemical analysis detected proteoglycans in the basal membrane of Schwann cells and around perineural cells, as well as on the collagen fibrils of the perineurium and endoneurium at all ages. Biochemical analyses showed that the quantity of non-collagenous proteins was higher in 730-day-old animals compared to other ages, while the uronic acid content was higher in 30-day-old animals. Morphometrical analysis detected greater numbers of myelinated fibers and increased myelin thickness in 180-day-old animals. Zymography analysis detected greater amounts and activity of MMP-2 and MMP-9 in 180- and 730-day-old animals compared to younger rats. In conclusion, our results showed changes in the structural organization and composition of extracellular matrix of the sciatic nerve during aging, such as increase in the non-collagenous protein content and higher MMP-2 and MMP-9 activity, decrease in uronic acid concentration and in collagen fibril content in the perineurium, as well as degeneration of nerve fibers.     

Elucidating the Function of Non catalytic Domains of Collagenases and Aggrecanases

Metalloproteinases that degrade extracellular matrix molecules play important roles in development and progression of various diseases. Among them, collagenases are unique as they have an ability to degrade triple helical interstitial collagens into 3/4 and 1/4 fragments, a crucial step for collagenolysis in the tissue. Collagenases, consisting of a catalytic domain and a hemopexin domain, requires both domains for collagenolysis. The enzymes unwind triple helical collagen before they hydrolyze the peptide bonds. Aggrecanases are also multidomain metalloproteinases belonging to the ADAMTS family, and the noncatalytic ancillary domains also play an important role in recognition of aggrecan and their activities. Attenuation of collagenase and aggrecanase activities will be achieved by inhibitors or antibodies that interact directly with those noncatalytic ancillary domains (exosite inhibitors). Such molecules will be attractive for therapy as they will be highly selective because they are based on the unique mechanism of each proteinase.