抑肽酶对新生隐球菌毒力的影响

目的:比较不同浓度抑肽酶对新生隐球菌胞外蛋白水解酶活性及其丝氨酸蛋白酶活力的影响,进一步探讨新生隐球菌分泌的丝氨酸蛋白酶在致病过程中所起的作用和意义。方法:在30℃下,用蛋白琼脂培养基培养并检测菌株产生廓清晕环(CH)大小,用CH值比较胞外蛋白水解酶活性的变化;建立小鼠感染模型,用菌落形成单位(CFU)和阿新蓝染色比较菌株毒力的变化。结果:体外实验中,抑肽酶1.2mU和1.6mU浓度组与对照组的CH值比较,差异有显著性(P<0.01),体内实验中,实验组脑组织匀浆培养产生的CFU多于对照组;实验组脑组织内观察到新生隐球菌。结论:抑肽酶有抑制隐球菌分泌胞外蛋白水解酶和丝氨酸蛋白酶的作用,有望成为潜在的预防和治疗真菌感染的药物。     

真菌病

20061284抑肽酶对新生隐球菌毒力的影响/徐赤宇(二军大附属长征医院皮肤科),温海,王溪涛…∥临床皮肤科杂志.-2006,35(3).-137~139在30℃下,用蛋白琼脂培养基培养并检测菌株产生廓清晕环(CH)大小,用CH值比较胞外蛋白水解酶活性的变化,建立小鼠感染模型,用菌落形成单位(CFU)和阿新蓝染色比较菌株毒力的变化。体外实验中,抑肽酶1·2mU和1·6mU浓度组与对照组的CH值比较,差异有显著性(P<0·01),体内实验中,实验组脑组织匀浆培养产生的CFU多于对照组;实验组脑组织内观察到新生隐球菌。抑肽酶有抑制隐球菌分泌胞外蛋白水解酶和丝氨酸蛋白酶…     

基质金属蛋白酶在银屑病患者皮损中的表达

基质金属蛋白酶(MMP)是一类参与细胞外基质降解的内源性蛋白水解酶,研究表明MMP-1和MMP-3与银屑病有关^[1,2]。为探讨其在银屑病发病机制中的作用,我们采用免疫组化法研究MMP-1、MMP-3在银屑病患者皮损、非皮损和正常人皮肤中的表达。     

地塞米松对瘢痕疙瘩成纤维细胞基质金属蛋白酶1调控的研究

目的：探讨地塞米松对瘢痕疙瘩成纤维细胞基质金属蛋白酶1（又被称为间质胶原酶1）蛋白的分泌合成和mRNA表达的影响。方法：采用酶联免疫吸附实验（ELISA）和Northem杂交技术检测了培养瘢痕疙瘩成纤维细胞基质金属蛋白酶1蛋白的合成分泌和mRNA的表达。结果：培养瘢痕疙瘩成纤维细胞对照组基质金属蛋白酶1蛋白的浓度为（151．02±27．70）pg／mL；加入10^-9M和10^-6M含地塞米松培养基24小时后，基质金属蛋白酶的浓度分别为（121．27±45．57）pg／mL（P〈0．05）和（101．78±35．82）pg／mL（P〈0．01）。加入地塞米松24小时后，培养瘢痕疙瘩成纤维细胞的基质金属蛋白酶1mRNA的表达被显著抑制，经密度扫描定量分析：基质金属蛋白酶1mRNA的表达分别下降了大约36％和53％。结论：地塞米松可抑制培养瘢痕疙瘩成纤维细胞基质金属蛋白酶1蛋白的合成分泌及其mRNA的表达，可能是地塞米松在抗瘢痕疙瘩纤维化作用中，尤其是纤维化形成后，疗效欠佳的原因。     

C57BL/6小鼠感染新生隐球菌对大脑皮质BDNF和bFGF表达的影响

目的：观察C57BL／6小鼠感染新生隐球菌后对大脑皮质BDNF和bFGF表达的影响，并探讨BDNF和bFGF在新生隐球菌感染过程中的作用及其意义。方法：48只C57BL／6雌性小鼠随机分为对照组、环磷酰胺组和试验组，免疫组化法检测大脑皮质BDNF和bFGF的表达。结果：尾静脉注入新生隐球菌后6h大脑皮质查见新生隐球菌，2h大脑皮质BDNF的表达明显高于对照组和环磷酰胺组（P〈0．01），0.5h大脑皮质bFGF的表达明显高于对照组和环磷酰胺组（P〈0.01）。结论：新生隐球菌感染后大脑皮质BDNF和bFGF表达增高，提示BDNF和bFGF对新生隐球菌性脑膜炎具有修复作用，有望成为潜在的防治新生隐球菌性脑膜炎的药物。     

MMP-2和MMP-9在多发性肌炎/皮肌炎患者单一核细胞中的表达

目的　探讨基质金属蛋白酶MMP-2和MMP-9 与多发性肌炎/皮肌炎（PM/DM）的关系。方法　采用实时荧光定量PCR和Western印迹方法分别检测PM/DM患者外周血单一核细胞（PMBC）中MMP-2和MMP-9的mRNA和蛋白表达水平。结果　MMP-2、MMP-9在PM/DM患者外周血PMBC中的mRNA表达水平明显高于正常人对照组,其中MMP-2为正常人对照组的2.41倍、MMP-9则为1.66倍。Western印迹检测发现MMP-2、MMP-9在PM / DM 患者外周血PMBC中的蛋白表达水平也明显高于正常人对照组,与其mRNA表达结果一致。结论　基质金属蛋白酶MMP-2和MMP-9可能在PM/DM的发生中起作用。     

黑素瘤抑制性活性蛋白及基质金属蛋白酶－9在恶性黑素瘤的表达

目的：探讨黑素瘤抑制性活性蛋白（MIA）及基质金属蛋白酶-9（MMP-9）在恶性黑素瘤中的表达以及作用。方法：应用SP免疫组化技术对38例恶性黑素瘤石蜡标本以及32例色素痣石蜡标本检测MIA及MMP-9表达水平。结果：MIA在所有色素痣中表达阴性,而在原位黑素瘤、侵袭性黑素瘤、有淋巴结转移恶性黑素瘤、无淋巴结转移恶性黑素瘤阳性表达率分别为21.4%,91.6%、94.1%、42.8%,MMP-9在恶性黑素瘤阳性表达率为71.1%,MIA的表达与MMP-9的表达呈正相关。结论：MIA在恶性黑素瘤的发生发展中起着重要作用,这种作用可能是通过上调MMP-9的表达来实现的。MIA有可能成为临床诊断、治疗恶性黑素瘤的有力靶点。     

黑素瘤中VEGF—D，MMP-7和MMP-9的表达

目的：确定黑素瘤组织中血管内皮生长因子D（VEGF—D）、基质金属蛋白酶-7（MMP-7）和MMP-9表达水平及其与肿瘤侵袭和转移的关系。方法：应用免疫组化法检测16例黑素瘤和12例黑素痣组织中VEGF—D,MMP-7和MMP-9蛋白的原位表达,并与临床肿瘤的侵袭和转移进行相关分析。结果：黑素瘤组织中VEGF—D,MMP-7和MMP-9阳性表达率（75％,62．5％和56．3％）明显高于黑素痣组织（25％,16．7％,41．7％）,在有淋巴转移的黑素瘤组织中,VEGF—D,MMP-7和MMP-9的表达（91．7％,66．67％和66．67％）明显高于无淋巴转移的黑素瘤组织（25％,50％和25％）,均P〈0．05。结论：VEGF—D,MMP-7和MMP-9与黑素瘤的侵袭和转移有关,它们的表达水平可作为预测肿瘤转移的指标,可为临床治疗提供靶点。     

扁平苔藓皮损MMP-2、MMP-9的表达及外周血MMP-9的检测

目的：探讨基质金属蛋白酶（MMP-2、MMP-9）在扁平苔藓皮损及外周血中的表达及意义。方法：应用免疫组化sP法对33例扁平苔藓及20例正常皮肤组织MMP-2、MMP-9的表达进行观察，进一步采用双抗体夹心酶联免疫吸附法检测22例扁平苔藓和16例正常人血清中MMP-9水平。结果：MMP-2在扁平苔藓及正常皮肤组织几乎均无免疫组化着色。MMP-9在扁平苔藓强烈表达于表皮角质形成细胞及真皮淋巴细胞胞浆，其表达显著高于正常皮肤（P〈0．01）。扁平苔藓患者血清MMP-9水平明显高于对照组（P〈0．01）。结论：MMP-9在扁平苔藓基底膜带降解中发挥了作用。     

基质金属蛋白酶与皮肤病相关性

基质金属蛋白酶是降解细胞外基质的一类重要的蛋白水解酶类，基质金属蛋白酶抑制剂为其天然组织抑制物，在多个环节抑制基质金属蛋白酶抑制剂的表达和活性。基质金属蛋白酶抑制剂的表达异常及其与组织金属蛋白酶抑制剂的动态平衡失调可与多种皮肤病发病机制密切相关。基质金属蛋白酶抑制剂及其组织抑制物的深入研究为多种相关皮肤病的发生及发展机制提供了思路，同时也为其临床治疗提供了手段。     

外周血单一核细胞的体外活化对成纤维细胞增殖和基质金属蛋白酶产生的影响

目的 研究不同活化剂对外周血单一核细胞（PBMC）产生基质金属蛋白酶（MMP）的影响以及PBMC活化后培养上清液［称为条件培养基（CM）］对培养的皮肤成纤维细胞增殖活性、产生MMP的影响。方法 抽取、分离健康人PBMC,分为植物血凝素（PHA）组、双抗体组、对照组,分别用活化剂PHA、CD3和CD28双抗体、含10%胎牛血清的RPMI 1640培养基处理细胞,72 h后收集三组CM,并将获得的CM按不同比例稀释后作用于培养的成纤维细胞,以不加CM作为对照组,培养48或24 h。采用噻唑蓝法检测细胞增殖能力,半定量反转录（RT）-PCR法检测细胞MMP-1、MMP-3、MMP-9 mRNA表达水平,酶联免疫吸附实验（ELISA）检测上清液中白介素（IL）-6、MMP-1、MMP-3、MMP-9蛋白含量。采用单因素方差分析、Tukey HSD检验、Games-Howell检验等方法进行统计学分析。 结果 与对照组相比,PHA组PBMC增殖活性及分泌IL-6、MMP-3水平明显增加,差异均有统计学意义（P < 0.05）;3组活化后上清液中均未检测到MMP-1、MMP-9蛋白。对照组、双抗体组、PHA组PBMC中MMP-1 mRNA相对表达水平（以靶基因与β肌动蛋白mRNA之比表示）0.083 ± 0.016、0.188 ± 0.030、0.714 ± 0.104,差异有统计学意义（P < 0.05）,MMP-3、MMP-9 mRNA均无表达。不同稀释度CM刺激成纤维细胞后上清液中可检测到MMP-3蛋白,MMP-3含量以原液CM中最高,1/10 CM刺激时最低;在相同的稀释度刺激时,上清液中MMP-3含量以PHA刺激组最高,对照刺激组最低;不同CM刺激成纤维细胞后上清液中均未检测到MMP-1、MMP-9蛋白。不同处理组成纤维细胞的增殖能力及MMP-1、MMP-3 mRNA表达差异均无统计学意义（P > 0.05）,MMP-9 mRNA无表达。 结论 PBMC活化后可表达MMP-1 mRNA并分泌MMP-3蛋白,PBMC活化后上清液不能刺激成纤维细胞MMP-1、MMP-3、MMP-9 mRNA及蛋白表达,提示炎症细胞可能通过自身产生MMP而发挥作用。     

Immunohistochemical expression of matrix metalloproteinase-2 and -9 in melanocytic nevi is altered by ultraviolet B

BACKGROUND: Ultraviolet B (UVB) radiation can cause morphological and biological alterations similar to those of melanoma in situ in irradiated melanocytic nevi. matrix metalloproteinase (MMP-2) and -9 appear to be involved with tumour invasion, the formation of metastases and neoangiogenesis in melanomas. The effects of UVB on the immunohistochemical expression of gelatinases in different cell types in melanocytic nevi have not been completely studied. PURPOSE: Evaluate the effects of UVB on the immunohistochemical expression of MMP-2 and -9 in different cell types in melanocytic nevi. METHODS: Forty-two melanocytic nevi had one half irradiated with 2 MEDs of UVB and were excised 1 week later. Three different observers compared the intensity of the immunohistochemical expression of gelatinases in keratinocytes, melanocytes, endothelial cells and fibroblasts on the irradiated (IS) and non-irradiated sides (NIS). RESULTS: All the cell types showed an increase in the expression of MMP-2 on the IS, especially the epidermal melanocytes. No significant increase in the expression of MMP-9 in keratinocytes was detected on the IS; significant increase was observed in all the remaining cells. CONCLUSIONS: One single irradiation of UVB increases the immunohistochemical expression of gelatinases in almost all evaluated cell lines, with the exception of MMP-9 in keratinocytes.     

VEGF促进人黑素瘤SK-MEL-1细胞侵袭性的自分泌机制初探

目的探讨血管内皮生长因子(VEGF)对人黑素瘤细胞SK-MEL-1侵袭力以及对该细胞中基质金属蛋白酶9(MMP-9)的影响,初步研究VEGF对肿瘤侵袭能力的影响及可能的作用机制。方法使用VEGF体外培养人黑素瘤SK-MEL-1细胞,免疫组化法检测该细胞VEGFR-1水平;通过细胞体外侵袭实验检测细胞侵袭能力的改变,再分别使用含20,40,80μg/LVEGF培养液培养SK-MEL-1细胞,以正常培养SK-MEL-1细胞为空白对照组,使用半定量RT-PCR和Western blot法对4组细胞中MMP-9 mRNA表达和蛋白水平进行分析。结果 VEGFR-1在SKM-EL-1细胞胞核和胞浆中均有表达。外加VEGF培养后,细胞侵袭力明显增强(P<0.01);MMP-9mRNA表达和蛋白水平在外加VEGF组明显高于空白对照组,且高浓度和低浓度组之间差异有统计学意义(P均<0.05)。结论人黑素瘤SK-MEL-1细胞系中存在有自分泌机制,VEGF可通过自分泌机制上调人黑素瘤SK-MEL-1细胞MMP-9mRNA表达及蛋白水平,进而促进肿瘤的侵袭转移。     

Interaction of HSV-1 infected peripheral blood mononuclear cells with cultured dermal microvascular endothelial cells: a potential model for the pathogenesis of HSV-1 induced erythema multiforme

The effect of herpes virus infection on human dermal microvascular endothelial cells and herpes-virus-1-infected peripheral blood mononuclear cells on human dermal microvascular endothelial cells was studied as a model of herpes-associated erythema multiforme. After infection of human dermal microvascular endothelial cells with native herpes virus and overnight culture, 60%--90% of human dermal microvascular endothelial cells showed cytopathic effects. HLA class I molecules and CD31 (PECAM-1) surface expression in herpes-virus-infected endothelial cells were substantially downregulated, whereas CD54 (ICAM-1) remained unchanged. Cocultivation with herpes-virus-1-infected peripheral blood mononuclear cells left characteristic plaques on the human dermal microvascular endothelial cell monolayer; however, very few human dermal microvascular endothelial cells (1%--3%) were infected. Adhesion molecule expression of human dermal microvascular endothelial cells cocultivated with herpes-virus-infected peripheral blood mononuclear cells demonstrated a 5-fold increase in CD54 expression, a 2-fold increase in HLA class I expression, but no change of CD31 by fluorescence-activated cell sorter analysis. Incubation of human dermal microvascular endothelial cells with ultraviolet-C irradiated herpes-virus-infected peripheral blood mononuclear cells had no effect on morphology or adhesion molecule expression levels. Changes of adhesion molecule expression by direct infection or cocultivation with peripheral blood mononuclear cells (with native and ultraviolet-C inactivated herpes virus infection) were also documented at the mRNA level. Adhesion assays demonstrated an increased binding of herpes-virus-infected peripheral blood mononuclear cells versus noninfected peripheral blood mononuclear cells to noninfected human dermal microvascular endothelial cells. Our results suggest that incubation of herpes-virus-infected peripheral blood mononuclear cells with human dermal microvascular endothelial cells induces significant upregulation of CD54 and major histocompatibility complex class I molecules in the surrounding noninfected human dermal microvascular endothelial cells, which is associated with an increased binding of peripheral blood mononuclear cells. Our in vitro findings may serve as a model for herpes-associated erythema multiforme possibly explaining the dermal inflammatory reaction seen in that condition.     

Expression and modulation of the vitronectin receptor on human dermal microvascular endothelial cells.

Microvascular endothelial cells express a variety of cell-surface integrins in vivo and in vitro with varying affinities for matrix proteins. The vitronectin receptor (VnR), a complex of the alpha v and beta 3 integrin chains, is capable of binding to a variety of matrix proteins that are deposited in injured tissues, including vitronectin, fibrinogen, and thrombin. Staining of frozen sections of human skin with antibodies recognizing the VnR and examination by immunofluorescence microscopy demonstrates staining in a vascular pattern suggesting in vivo expression of the vitronectin receptor on endothelial cells. Examination of pure cultures of human dermal microvascular endothelial cells (HDMEC) by flow-cytometric analysis and enzyme-linked immunosorbent assay confirmed that HDMEC also express cell surface VnR complex in vitro. Stimulation of human dermal microvascular endothelial cells in vitro with agents that stimulate protein kinase C resulted in dose- and time-dependent increases in expression of alpha v and beta 3 integrin chains. Additionally, stimulation with basic fibroblast growth factor induced similar increases, but stimulation with transforming growth factor-beta or interleukin-1 alpha failed to increase VnR expression. Increases in cell-surface VnR expression also correlated with an increased ability of microvascular endothelial cells to bind to vitronectin, but not fibronectin-coated surfaces. Although increases in cell-surface expression of beta 3 paralleled increases in expression of cell-surface alpha v, regulation of mRNA expression was distinct for each chain. These data suggests that microvascular endothelial cells express the VnR complex in vivo, that the cell-surface expression of this integrin on dermal microvascular endothelial cells can be regulated, and that this regulation may be important in cell adherence, cell migration, and wound healing.     

The effect of moesin overexpression on ageing of human dermal microvascular endothelial cells

Abstract:  Senescence of microvascular endothelial cells is known to play an important role in the pathophysiology of vascular diseases related to ageing, but the accurate mechanism or related genes are not known. Moesin, a cytoskeletal protein and the most potent candidate as an ageing-related protein, showed obvious changes in expression when compared before and after ageing. In this study, a lentivirus was used to overexpress moesin in endothelial cells. The expression of cell cycle mediators such as p16, cyclin D1 and cdk4, which can be the markers of ageing, was compared by RNA and was shown to be suppressed in moesin overexpressed endothelial cells. In conclusion, it can be said that the expression of moesin delays senescence of human dermal microvascular endothelial cells and this fundamental discovery can be used as a basis for understanding the mechanism of ageing and age-related diseases.     

Fibrin and collagen differentially regulate human dermal microvascular endothelial cell integrins: stabilization of alphav/beta3 mRNA by fibrin1

Integrin alphavbeta3 is specifically but transiently expressed on the tips of capillary sprouts as they invade the fibrin clot during angiogenesis of cutaneous wound repair. Specific blocking of alphavbeta3 function inhibits granulation tissue formation in cutaneous wounds. The mechanisms of regulation of alphavbeta3 expression on human dermal microvascular endothelial cells, however, have not been fully delineated. As alphavbeta3 was highly expressed on capillary sprouts in 5 d wounds rich in fibrin, but was almost undetectable on blood vessels in 7 d wounds rich in collagen, we hypothesized that the extracellular matrix environment could regulate human dermal micro- vascular endothelial cell alphavbeta3 expression. To address this, human dermal microvascular endothelial cells were cultured on surfaces coated with collagen, fibronectin, and gelatin, and mRNA levels of integrin alphav/beta3 were determined. Compared with human dermal microvascular endothelial cells on collagen, mRNA levels of alphav/beta3 were higher in human dermal microvascular endothelial cells on fibronectin and on gelatin. To simulate the in vivo environment better, human dermal microvascular endothelial cells cultured on collagen were overlaid by fibrin or collagen gels prior to assessment of alphav/beta3 mRNA levels. alphav/beta3 mRNA levels were higher in human dermal microvascular endothelial cells surrounded by a three-dimensional fibrin gel compared with a collagen gel, whether angiogenic factors were present or absent. As modulation of mRNA stability is a potential regulatory mechanism for integrin expression, integrin subunit mRNA stability was assessed. beta3 mRNA decayed much faster than alphav, alpha2, and beta1 mRNA. Three-dimensional fibrin gels enhanced alphav/beta3 mRNA stability compared with collagen gels. We propose that the provisional matrix molecules in the wound clot regulate angiogenesis associated with cutaneous wound repair through their modulation of integrin receptor expression.     

Matrix metalloproteinases in the progression and regression of Kaposi's sarcoma

BACKGROUND: Matrix metalloproteinases (MMPs) are associated with Kaposi's sarcoma (KS) tumorigenesis. To date, only a few MMPs have been studied in KS lesions. Their role in KS regression has not been investigated. The aim of this study was to evaluate the expression of multiple MMPs in developing and pharmacologically regressed KS lesions. METHODS: Nine samples of acquired immune deficiency syndrome (AIDS)-related and classic cutaneous KS lesions at various histological stages were studied. Regressing KS lesions from three patients treated with systemic therapy were procured after one and two cycles of chemotherapy. Tissue sections from all specimens were immunostained using monoclonal antibodies to MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, and MMP-14. RESULTS: KS lesional cells were immunoreactive for all MMPs, except MMP-14. Admixed inflammatory cells were immunoreactive for MMP-1, MMP-2, MMP-7, MMP-9, and MMP-13. The MMP immunoprofile in residual KS lesional cells was unaltered in regressed lesions. Increased extracellular matrix (ECM) and macrophage immunoreactivity for MMPs was identified in regressed specimens. CONCLUSIONS: These data show that developing KS lesional cells express collagenases (MMP-1, MMP-13), gelatinases (MMP-2, MMP-9), stromelysin-1 (MMP-3), and matrilysin (MMP-7) but not the membrane-type MMP-14. This MMP expression profile is retained by residual KS cells and also expressed by infiltrating macrophages in regressed KS lesions. Pantanowitz L, Dezube BJ, Hernandez-Barrantes S, Tahan SR, Dabbous MK. Matrix metalloproteinases in the progression and regression of Kaposi's sarcoma.