猪骨骼肌缺血预适应降低心肌缺血再灌注后基质金属蛋白酶的表达

目的观察猪骨骼肌缺血预适应对心肌缺血再灌注后坏死面积及心肌MMP-2、MMP-9及TIMP-1的影响。方法10只小型猪被随机分为缺血再灌注(I/R)组和远端预适应(RP)组。采用非开胸法建立心脏I/R模型,通过球囊堵塞左股动脉造成骨骼肌短暂缺血。以三硝基四氮唑红确定心肌梗死范围;以免疫组化和RT-PCR法测I/R心肌中MMP-2、MMP-9和TIMP-1表达及骨骼肌RP对此的影响。结果与I/R组相比,骨骼肌RP明显减少心肌梗死范围。在缺血区,RP明显降低MMP-2和MMP-9的表达,增加TIMP-1表达(P<0.05)。且RP明显减弱MMP-2和MMP-9 mRNA的表达,增强TIMP-1的mRNA表达(P<0.05)。结论骨骼肌缺血预适应心肌不仅可减少心肌坏死范围,还可能对心肌间质产生保护作用。     

Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

基质金属蛋白酶3和尿激酶型纤溶酶原激活物受体在2型糖尿病大血管病变中的变化

目的： 探讨基质金属蛋白酶3（MMP-3）和尿激酶型纤溶酶原激活物受体（uPAR）在2型糖尿病大血管病变中的变化。方法: 用ELISA双抗体夹心法测定26例正常对照和39例2型糖尿病患者（其中单纯2型糖尿病15例、合并大血管病变24例）的血浆MMP-3和uPAR水平。结果: 单纯糖尿病组uPAR高于正常对照（P<0.05）而MMP-3无显著差异；合并大血管病变组MMP-3显著高于正常对照和单纯糖尿病组（P<0.01，P<0.01），而uPAR亦高于正常对照及单纯病变组（P<0.01，P<0.05）。2型糖尿病血浆MMP-3水平和uPAR水平呈正相关， LDL-C与uPAR呈正相关且是uPAR主要影响因素。 结论: MMP-3和uPAR与2型糖尿病大血管病变发生密切相关，MMP-3可作为2型糖尿病血管病变的循环标志物。     

琥珀酸美托洛尔HPMC骨架片释放影响因素研究

以羟丙基甲基纤维素（HPMC）为骨架材料，乙基纤维素（EC）为阻滞剂，采用湿颗粒压片法制备琥珀酸美托洛尔亲水凝胶骨架片，考察HPMC用量、HPMC黏度、EC用量、制备方法、压片压力、释放介质及转速对琥珀酸美托洛尔（MS）骨架片体外释药的影响。结果表明，MS骨架片体外释药符合Higuchi方程，药物释放机制是骨架溶蚀和药物扩散的综合效应；HPMC用量与黏度、阻滞剂用量、制备方法、压片压力对释放速率均有显著性影响；释放介质的pH值及转速对释放速率无显著性影响。     

Dithiocarbamates inhibit IL-1-induced cartilage degradation in bovine articular cartilage explants

Interleukin-1-stimulated cartilage degradation in bovine articular cartilage explants is effectively inhibited by several different dithiocarbamates with IC₅₀'s in the micromolar range.accepted by W. B. van den BergSupported by OsteoArthritis Sciences, Inc.     

Preparation of a pure autologous biodegradable fibrin matrix for tissue engineering

Parallel to the growing role of tissue engineering, the need for cell embedding materials, which allow cells to stabilise in a three-dimensional distribution, has increased. Although several substances have been tested, fibrin is thus far the only one that permits the clinical application of cultured tissue. To date, can cause severe immunological side effects. The objective of this study was to explore the practicability of obtaining autologous thrombin from a single patient in an adequate concentration and amount. Fibrinogen was cryoprecipitated from 200 ml of freshly-frozen plasma. Thrombin was isolated from the supernatant through ionexchange chromatography. The thrombin was first bound to Sephadex A-50 and then eluated using 2ml of a salt buffer (2.0M NaCl in 0.015M trisodiumcitrate, pH 7.0). The activity of the thrombin (51 NIH ml⁻¹ to 414 NIH ml⁻¹) reached levels comparable to those in commercially available fibrin glues (4–500 NIH ml⁻¹). The study has shown that it is possible to obtain a sufficient amount of autologous thrombin from a single donor to create a fibrin matrix of high efficiency without the risk of immunological and infectious side effects.     

Matrix metalloproteinases (MMP), EMMPRIN (extracellular matrix metalloproteinase inducer) and mitogen-activated protein kinases (MAPK): Co-expression in metastatic serous ovarian carcinoma

Activation or suppression of intracellular signaling via the mitogen-activated protein kinase (MAPK) family has been linked to expression of matrix metalloproteinases (MMP) in experimental models, but this association has not been demonstrated in clinical material. The objective of this study was to investigate the possible association between expression and activity of MMP, expression of the MMP inducer EMMPRIN, and the expression (level) and phosphorylation status (activity) of the extracellular-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) and high osmolarity glycerol response kinase (p38) in effusions from patients diagnosed with serous ovarian carcinoma. MAPK level and activity were studied in 55 effusions using immunoblotting. MMP-1, MMP-2, MMP-9 and EMMPRIN expression was studied using immunocytochemistry (ICC) and mRNA in situ hybridization (ISH). The gelatinolytic activity of MMP-2 and MMP-9 was measured by zymography. ERK and phospho-ERK (p-ERK) were detected in 54/55 (98%) and 50/55 (91%) specimens, respectively. JNK and p-JNK were detected in 53/55 (96%) and 38/55 (69%) specimens, respectively. p38 was expressed in 54/55 (98%) specimens, and its phosphorylated form was found in 51/55 (92%). MMP-2 mRNA expression (P=0.048), protein expression (P=0.046) and gelatinolytic activity (P=0.039) correlated with ERK phosphorylative activity. MMP-2 activity also correlated with p38 activity (P=0.017). MMP-9 protein expression correlated with phosphorylation of p38 (P=0.046), but enzyme activity showed inverse relationship with both p-ERK (P=0.05) and p-p38 (P=0.033) expression. EMMPRIN expression correlated with MMP-1 (P<0.001), MMP-2 (P=0.042) and MMP-9 (P=0.029) expression, as well as with ERK activity (P=0.001). Our results present the first evidence of a possible link between MAPK signaling and MMP expression and activity in vivo. These data may expand our understanding regarding the mechanisms by which MMP synthesis is regulated in effusions and possibly affect treatment strategies for this form of malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

含人基质金属蛋白酶组织抑制因子-1重组腺病毒的构建与体外表达

从人肝癌组织中提取总RNA，RT—PCR合成hTIMP-1的全长cDNA，克隆到腺病毒载体AdEasy系统的穿梭质粒pAdTraek—CMV上，与骨架质粒pAdEasy-1在BJ5183受体菌中进行同源重组，成功构建含hTIMP-1全长eDNA的重组腺病毒载体，经293细胞的包装、扩增，生成含hTIMP-1基因的重组腺病毒AdhTIMP-1并实现体外表达，为进一步研究肝癌浸润和转移机理以及肝癌的基因治疗提供实验基础。     

Matrix Metalloproteinase and Cytokine Profiles in Monocytes over the Course of Stroke

Stroke is a common cause of death and disability in our society. Stroke is associated with changes in immune responses within the central nervous system as well as systemically. The cells contributing to such changes as well as the factors contributing to formation of the inflammatory infiltrate observed in stroke remain to be clarified. In this study, blood monocytes and corresponding mononuclear cells (MNC) were separated and examined in parallel within 4 days and 1–3 months after onset of ischemic stroke. Numbers of TNF--, IL-12-, IL-6-, and IL-10-secreting cells and of cells expressing mRNA for matrix metalloproteinase (MMP)-1, -2, -7, -9 and tissue inhibitor of MMP (TIMP)-1 were studied. The TNF--, IL-12-, and IL-6-secreting monocytes and MNC were elevated during the acute phase compared to healthy controls. Such differences were not observed when stroke patients were examined during convalescence. The IL-10-secreting monocytes did not change over the course of stroke. Levels of monocytes expressing MMP-1, MMP-7 and TIMP-1 mRNA were elevated in the acute phase of stroke patients compared to convalescence and healthy controls, as were levels of MMP-1, -2, -7, -9 and TIMP-1 mRNA expressing blood MNC. The MMP-2 and -9 activity as measured by zymography also was higher in MNC supernatants in the acute phase of stroke compared to convalescence. The high levels of proinflammatory cytokines and MMPs in blood monocytes and MNC further demonstrate the presence of systemic aberrations in the acute phase of stroke. Such changes may contribute to the influx of blood-borne cells into the ischemic lesions during the acute phase of stroke.     

Evaluation of EDTA and fish skin extract in primary culture of fish liver cells

The isolation, primary culture and attachment of liver cells to the substratum from a tropical catfish (Heteropneustes fossilis) using ethylenediaminetetraacetic acid (EDTA) as isolating agent of liver cells and skin extract (SE) from fish as attachment substrate for the primary culture of liver cells has been standardised. A suitable temperature for such cultures has also been determined. Attachment efficiency of the liver cells in culture and their intracellular lactate dehydrogenase (LDH) activity have been taken as parameters for characterization of the primary culture. Disaggregation of liver cells with EDTA is very potent to isolate substantial rumber of cells from the liver of H. fossilis. An ideal concentration of EDTA for liver cell isolation has been standardized. Matrix prepared from carp and catfish skin at different pH (2.0, 4.0, 6.0 and 8.0) were also evaluated for liver cell culture by considering the attachment efficiency of the cells over the substratum as well as retention of intracellular LDH enzyme after 48 hours of seeding. Matrix of carp skin was compared with that of catfish as suitable substrate for primary culture of fish liver cells. It has been found that the SE prepared at pH 4.0, both from carp and catfish skin, performed better than those at other pHs. At the same time, the matrix of carp skin was found to be better than that of catfish skin. Cultures were incubated separately at 17 and 23 °C in air atmosphere. Incubation temperature at 23 °C was found to be more suitable than that at 17 °C. The percent of detached/unattached cells showed only marginal variation between two temperatures but LDH-activity recorded drastic reduction (between 50 to 75%) depending upon the pH of the matrix during preparation. Our finding establishes despensibility of enzyme (collagenase/trypsin) for cell isolation in catfish. Our studies also exhibit that carp skin extract performs better than catfish skin extract in terms of attachment efficiency as well as intracellular LDH activity. This study indicates that no species/generic barrier exists in matrix between catfish and carp.