口腔角质形成细胞与成纤维细胞共培养对胶原代谢的影响

目的:了解口腔角质形成细胞与成纤维细胞共同培养对胶原代谢的影响.方法:对口腔粘膜角质形成细胞和成纤维细胞进行了共培养,检测其胶原分泌水平、基质金属蛋白酶的含量及活性、基质金属蛋白酶抑制剂的水平.结果:只有共同培养组才能检测到MMP-9;共同培养组的活化型MMP-2水平与单独培养组相比无显著差异.角质形成细胞与成纤维细胞共同培养组的TIMP-1水平及胶原水平均明显高于单独培养组.结论:口腔角质形成细胞与成纤维细胞共同培养可能主要通过改变基质金属蛋白酶抑制剂的水平而影响胶原代谢.     

运动负荷对骨基质有机成分的影响及其可能机制

骨基质的有机成分主要为骨Ⅰ型胶原基质金属蛋白酶,该酶是细胞外基质降解的重要酶类;基质金属蛋白酶抑制因子则是基质金属蛋白酶活性的抑制剂,它们均为骨代谢过程中的重要标志性物质。本文通过查阅文献资料,对生理、部分病理状态及运动干预条件下,骨基质的Ⅰ型胶原和基质金属蛋白酶及其抑制因子的变化情况进行综述,并对其变化的机制予以阐释。     

基质金属蛋白酶9对糖尿病足溃疡愈合影响的研究进展

在糖尿病足患者溃疡创面分泌物中,基质金属蛋白酶9 (matrix metalloproteinase-9,MMP-9)过高是预测糖尿病足的发生及足溃疡难愈的主要指标,其可能的机制包括:高水平MMP-9降低VEGF的表达、抑制成纤维细胞的生物学行为影响糖尿病足溃疡愈合;失衡的MMP-9/TIMP-1比值影响糖尿病足溃疡愈合。选择性MMP-9抑制剂(包括小分子抑制剂、高级伤口辅料抑制剂、基于干扰基因水平表达的RNA抑制剂)可以作为促进糖尿病足溃疡愈合的手段,但仍需大样本、多中心随机对照试验以及长期随访进一步验证其疗效及安全性。现查阅近年来涉及MMP-9和糖尿病足溃疡相关的文献,综述MMP-9对糖尿病足溃疡愈合的影响及其机制研究进展。     

糜酶转基因小鼠心脏组织中基质金属蛋白酶9的活化

为研究糜酶在心脏中的功能 ,用明胶酶谱法和放免法检测了糜酶转基因小鼠心脏组织中基质金属蛋白酶及糜酶的活力 .糜酶转基因小鼠心脏组织中糜酶样活力较转基因阴性小鼠升高了约80 % ;而其心脏匀浆液凝胶酶谱分析结果显示在 92 k D处明胶酶活力也升高约 30 % ;经 Western印迹鉴定为基质金属蛋白酶 9,而在蛋白水平上与转基因阴性小鼠无显著差异 .结果提示 ,糜酶转基因小鼠心脏组织中糜酶活力的升高可活化基质金属蛋白酶 9,从而影响心脏胶原代谢 .     

阿斯匹林对T H P-1来源巨噬细胞M M P g的表达及其活性的影响

目的：探讨阿司匹林对基质金属蛋白酶9（Matrix Metalloproteinases,MMPs）表达及其活性的影响。方法：应用MTT法观察阿司匹林对THP-1来源巨噬细胞增殖的影响;逆转录聚合酶链反应检测阿司匹林对体外培养的THP-1细胞基质金属蛋白酶9mRNA表达的影响,应用明胶酶谱法观察阿司匹林对体外培养的THP-1来源巨噬细胞基质金属蛋白酶9活性的影响。结果：与对照组相比,不同浓度的阿司匹林（150、3000、600、1200μmol／l）对THP-1采源巨噬细胞增殖无影响;不同浓度的阿司匹林可以抑制PMA诱导的THP-1细胞基质金属蛋白酶9mRNA的水平及其活性水平,且呈浓度依赖性。结论：阿司匹林可以抑制PMA诱导的THP-1巨噬细胞基质金属蛋白酶9的表达,并可能通过这一机制影响动脉粥样硬化斑块的稳定性．     

原位杂交检测组织金属蛋白酶抑制因子-1mRNA在IgA肾病肾脏中的表达

为探讨组织蛋白酶抑制因子在 Ig A肾病肾小球硬化中的作用 ,本文用原位杂交法研究组织金属蛋白酶抑制因子 -1(TIMP- 1) m RNA在 3种类型 Ig A肾病 (轻度系膜增生型、中度系膜增生型及局灶型 )肾组织中的表达。结果表明 :TIMP-1m RNA原位杂交的阳性信号主要分布于部分肾小管上皮细胞内。在肾小球系膜细胞也可检测到 TIMP- 1m RNA阳性信号 ,但较肾小管弱。中度系膜增生型的 TIMP- 1m RNA阳性信号明显地较其他两型 Ig A肾病为强。以上结果表明 :由肾小管上皮细胞及系膜细胞合成的 TIMP- 1通过抑制基质金属蛋白酶的活性 ,可导致细胞外基质 (ECM)在肾小管间质及肾小球内过度沉积 ,从而对肾小球硬化有促进作用。     

基质金属蛋白酶与心肌重塑

细胞外基质参与和促进了心肌重塑的过程,基质金属蛋白酶是调节细胞外基质重要的酶,基质金属蛋白酶在心肌重塑过程表达变化可分为三个时相,其活性受到信号传导途径、炎症因子和活性氧/活性氮的调节,基质金属蛋白酶可能作为心肌梗塞等疾病治疗的靶标     

葛根素对糖尿病大鼠肾组织MMP-2的表达及活性的影响

为探讨葛根素对糖尿病大鼠肾组织基质金属蛋白酶2(MMP-2)及活性表达的影响,采用单侧肾切除大鼠ip链脲佐菌素诱发糖尿病模型的方法,每日ip葛根素注射液,共16周。采用原位杂交法检测肾小球MMP-2、TIMP-2mRNA表达,流式细胞术和免疫组织化学检测肾皮质MMP-2、TIMP-2及Ⅳ型胶原表达;酶谱分析检测肾皮质MMP-2活性变化。结果发现糖尿病组较对照组肾小球MMP-2mRNA及蛋白表达降低而TIMP-2mRNA及蛋白表达升高,Ⅳ型胶原表达亦增加,MMP-2活性降低,肾功能恶化;葛根素用药组较糖尿病组MMP-2mRNA及蛋白表达升高而TIMP-1、Ⅳ型胶原表达减少,MMP-2活性部分恢复,肾功能改善。表明葛根素可能部分是通过调节肾小球MMP-2蛋白表达及活性的改变从而减轻肾小球细胞外基质沉积,保护糖尿病大鼠的肾功能和形态。     

基质金属蛋白酶

基质金属蛋白酶是一类分解细胞外基质组分的锌蛋白酶⒚它们在有机体生长发育中的细胞外基质逆转与重塑以及疾病中的病理损害起着极为重要的作用⒚基质金属蛋白酶的表达和活性在不同细胞水平受到严密调控,如细胞因子、生长因子以及激素的调节⒚基质金属蛋白酶以酶原形式分泌,随后被其它蛋白酶如胞浆素或非蛋白酶类化学物质如有机汞所激活⒚所有基质金属蛋白酶都受到天然抑制剂 金属蛋白酶组织抑制剂所抑制⒚两者的不平衡导致许多疾病的发生,如肿瘤侵入及转移⒚合成基质金属蛋白酶组织抑制剂所抑制,如 Ｍ ａｒｉｍ ａｓｔａｔ 能控制肿瘤转移的发生及进一步扩散⒚本文将对基质金属蛋白酶的特征、分子区域结构、底物特性、激活机制、调控方式等方面进行最新概述⒚     

窖蛋白-1、基质金属蛋白酶-2与乳腺肿瘤的侵袭和转移

窖蛋白(caveolin)是分子量为21～24 kD的整合膜蛋白,是胞膜窖(caveolae)的标志性结构分子,其家族成员窖蛋白-1(caveolin-1,Cav-1)参与细胞内许多重要的生命活动.近来研究发现,窖蛋白-1与乳腺上皮细胞转化及乳腺癌的发生密切相关.基质金属蛋白酶(matrix metalloproteinases,MMPs)是基质降解代谢的主要酶类,几乎能降解细胞外基质和基底膜的所有成分,其家族成员明胶酶A(MMP-2)在乳腺癌的浸润和转移过程中起重要作用.新近发现,窖蛋白-1与基质金属蛋白酶-2在胞膜窖中共定位,窖蛋白-1通过抑制基质金属蛋白酶-2的激活来抑制乳腺癌的侵袭和转移,起到肿瘤抑制因子的作用.本文对窖蛋白-1与基质金属蛋白酶-2各自在乳腺肿瘤侵袭转移中的作用及两者关系的研究进行综述.     

Characterization of a lympho-inhibitory peptide produced by Mycoplasma bovis

Mycoplasma bovis is able to inhibit the mitogen-induced proliferation of bovine lymphocytes. Herein is described the isolation of an immuno-inhibitory peptide from M. bovis. Using size exclusion chromatography, three lympho-suppressive fractions were isolated from M. bovis free supernatant. MALDI-TOF analysis revealed a common peak throughout the suppressive fractions. The purest of these fractions was subjected to N-terminal sequencing, revealing an 84% homologous match with the C-terminus of the M. bovis surface protein VspL (variable surface protein-L). A recombinant of the 26 amino acid peptide was also able to suppress Concanavalin A (ConA)-induced proliferation of bovine lymphocytes. This describes a unique immunosuppressive peptide produced by the bovine respiratory pathogen, M. bovis.     

Proliferation and collagen production of human patellar tendon fibroblasts in response to cyclic uniaxial stretching in serum-free conditions

We studied the effect of cyclic mechanical stretching on the proliferation and collagen mRNA expression and protein production of human patellar tendon fibroblasts under serum-free conditions. The role of transforming growth factor-beta1 (TGF-beta1) in collagen production by cyclically stretched tendon fibroblasts was also investigated. The tendon fibroblasts were grown in microgrooved silicone dishes, where the cells were highly elongated and aligned with the microgrooves. Cyclic uniaxial stretching with constant frequency and duration (0.5 Hz, 4 h) but varying magnitude of stretch (no stretch, 4%, and 8%) was applied to the silicone dishes. Following the period of stretching, the cells were rested for 20 h in stretching-conditioned medium to allow for cell proliferation. In separate experiments, the cells were stretched for 4h and then rested for another 4 h. Samples of the medium, total cellular RNA and protein were used for analysis of collagen and TGF-beta1 gene expression and production. It was found that there was a slight increase in fibroblast proliferation at 4% and 8% stretch, compared to that of non-stretched fibroblasts, where at 8% stretch the increase was significant. It was also found that the gene expression and protein production of collagen type I and TGF-beta1 increased in a stretching-magnitude-dependent manner. And, levels of collagen type III were not changed, despite gene expression levels of the protein being slightly increased. Furthermore, the exogenous addition of anti-TGF-beta1 antibody eliminated the increase in collagen type I production under cyclic uniaxial stretching conditions. The results suggest that mechanical stretching can modulate proliferation of human tendon fibroblasts in the absence of serum and increase the cellular production of collagen type I, which is at least in part mediated by TGF-beta1.     

Interleukin-2 inhibits 3T3 fibroblast proliferation

In order to clarify the role played by interleukin-2 (IL-2) in the regulation of fibroblast function, we investigated the effect of rat IL-2 and human recombinant IL-2 on 3T3 fibroblast proliferation and collagen synthesis. Fibroblasts were incubated with various concentrations of IL-2 for different periods of time. IL-2 was found to decrease in time- and dose-dependent manner the proliferation of 3T3 fibroblasts. This effect correlated with ability of IL-2 to enhance PGE₂ production by 3T3 fibroblasts. When 3T3 fibroblasts were cocultured with rat peritoneal mast cells (MC), the growth-inhibiting effect of IL-2 was significantly less pronounced. Treatment of the cultures with IL-2 had no effect on collagen production by both 3T3 fibroblasts and fibroblasts cocultured with MC. In conclusion, in this study we provide evidence that IL-2, the key cytokine in T-cell growth and differentiation, can affect fibroblast functions.     

Thrombin stimulation of matrix fibronectin

Trypsin, thrombin, and peptide analogues of the new amino terminus of the proteolyzed thrombin receptor, SFLLRN and SFLLRNPNDKYEPF, stimulated embryonic fibroblasts cultured as 3-dimensional tissue-like aggregates to elaborate a fibronectin-rich extracellular matrix. Enzymatically inactive thrombin and the control peptide FLLRN failed to stimulate matrix production. The induction of cell proliferation correlated with production of the fibronectin matrix. The regions of active cell proliferation in the fibroblast aggregates co-localized with the matrix and peptide analogues of the RGD cell-adhesion site of fibronectin reversibly inhibited the accumulation of the fibronectin matrix and the stimulation of cell proliferation by SFLLRN. Two different preparations of the fibronectin matrix stimulated cell proliferation in aggregates cultured in growth factor-free medium. We suggest that the stimulation of matrix production is a necessary event for mitogenic signaling in mesenchymal tissue. The tight coupling between the matrigenic and mitogenic activities of growth factors was absent in monolayer cultures of chick embryonic fibroblasts since thrombin and trypsin induced proliferation of monolayer-cultured cells without inducing the production of a fibronectin matrix. © 1996 Wiley-Liss, Inc.     

Isolation of a 19-kDa mycobacterium,bovis-specific antigen,different from MPB70/80, by chromatofocusing

Two antigens, 19-kDa each, were purified from Mycobacterium bovis culture filtrate protein extract by chromatofocusing. Antigen I had a 4.5 pI, and its amino terminal (DPVDAVINTTCNYGQVVAALNATDP) showed a 100% homology with the hypothetical protein Rv1174c. Antigen II had a pI of 6.0 pI and its amino terminal (GDLVGPGCAEYAAANPTGPASVQGM) showed a 100% homology with M. bovis MPB70/80. Antigen I is a hetero-dimer formed by a glycosylated, 10.5-kDa, monomer and a non-glycosylated 8-kDa monomer with identical amino terminal sequences. Both antigens were recognized by the sera of PPD+ animals, but antigen I did not crossreact with sera of human PPD+ individuals. Antigen I was a weak inducer of lymphocyte proliferation and IFN-gamma production. Our results show that M. bovis expresses a 19 kDa glycoprotein, homologue to the product of M. tuberculosis gen Rv1174c, which may prove useful for bovine TB diagnostic assays.     

Effect of PGE2 and indomethacin on human fibroblast collagen production

Fibroblasts can synthetize prostaglandins (particularly PGE2) "in vitro" but it still remains unclear what role they play in the regulation of fibroblast proliferation and collagen production. We report here the effect of PGE2 and indomethacin on collagen synthesis by cultured human dermal fibroblasts. PGE2 (range: 1-300 pmoles/ml) and indomethacin (range: 0.0025-1.0 micrograms/ml) did not significantly affect fibroblast collagen production, when added for 24 hours at 37 degrees C to the cultures, in comparison to controls (fibroblasts incubated for 24 hours at 37 degrees C in medium only). Prostaglandins probably modulate collagen synthesis, as described in a previous report, by means their effect on cell proliferation. It appears they do not affect the intracellular mechanism of collagen production.     

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR1). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR1-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR1-specific agonists and inhibitors were used to demonstrate that PAR1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR1 and not PAR2. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.     

Modulation of fibroblast functions by interleukin 1: increased steady- state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.