Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans

Summary This study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and betaglucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and betaglucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.     

Pulsed electromagnetic fields alter phenotypic expression in chondroblasts in tissue culture

We hypothesize that pulsed electromagnetic fields (PEMF) alter phenotypic expression of chondroblasts by promoting the production of alkaline phosphatase (AP) and altering the structure of proteoglycans. Chondroblasts from the hypertrophic zone of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from 16 day chick embryos. Cultures were randomly designated control (C) or experimental (E). E received PEMF for 24 h in a 6 h on, 6 h rest sequence. The controls were in the same incubator shielded by Mu metal. Assays for AP activity were performed and normalized to protein content. Proteoglycan synthesis assay involved labeling with 35S fractionating in a 5% to 20% surcrose gradient determining total protein and chondroitin sulfate content. PEMF showed no change of AP activity on F. A high AP basal activity was found in HC, but was not increased above the control. PEMF increased AP in the SC samples (E/C ratio). The sucrose gradient data showed a shift in peaks for SC only altering the ratio of carbohydrate to protein for the SC. Analysis of carbohydrate and protein indicated that the effect was decreased synthesis or degradation of protein. We conclude that PEMF alters the phenotypic expression of sternal chondroblasts in our in vitro system.     

筋膜皮瓣治疗小腿顽固溃疡9例报告

本文报道应用小腿后侧筋膜皮瓣治疗9例胫前区顽固性溃疡的经验,一期治愈者8例,二次皮瓣转移治愈者1例。随诊6个月至2年,未见溃疡复发。     

组织工程化口腔粘膜的构建技术初步研究

目的　探索组织工程化口腔粘膜的构建方法。方法　用 Dispase中性蛋白酶分离提取未长毛的 SD乳鼠硬腭口腔粘膜细胞 ,用角化细胞培养液对口腔粘膜细胞进行培养 ,以自制藻酸钠薄膜作为支架材料构建组织工程化的口腔粘膜。结果　 Dispase中性蛋白酶可较好地分离提纯口腔粘膜细胞 ,用 Dispase消化酶分离获取上皮后 ,再用胰蛋白酶消化上皮为单细胞的最佳时间为 10 m in,过低的细胞密度不易形成克隆 ,鼠口腔粘膜细胞培养的最适密度为 1.5× 10 5/ cm2 。角化细胞培养液能对鼠口腔粘膜细胞进行培养 ,且没有成纤维细胞污染 ;口腔粘膜细胞在藻酸钠薄膜上生长良好。结论　利用角化细胞培养液作为培养基 ,藻酸钠薄膜作为支架材料可成功构建组织工程化口腔粘膜     

组织工程颅骨缺损修复过程中超微结构观察

目的 用组织工程骨修复SD大鼠颅骨极量骨缺损，并在透射电镜下观察骨修复过程中成骨细胞、血管内皮细胞与陶瓷支架的关系。方法 14只雄性SD大鼠，随机分成实验组和对照组，每组7只。取左侧腔、股骨骨髓，体外诱导培养骨髓基质细胞。实验组将已分化的成骨细胞与含孔磷酸钙陶瓷复合用于修复大鼠自体颅骨极量骨缺损；对照组颅骨缺损处仅置入无细胞的陶瓷材料。分别于术后4、8、12、16、20、24及28周每组各处死1只动物取材，制成脱钙超薄切片，透射电镜下观察成骨细胞、血管内皮细胞与陶瓷残余支架的关系。结果 实验组动物颅骨修复12周前成骨细胞或成骨细胞样细胞毗邻新生小血管、血管内皮细胞存在，16周后成骨细胞转变为骨细胞，并围绕血管被包埋于骨基质及胶原纤维中；陶瓷残余与新生骨间有一条明显的钙化沉积带，大量胶原纤维已伸入到陶瓷支架的纳米级结构中。对照组陶瓷中央部位有大量小血管及血管内皮细胞增生，其周围多为幼稚成纤维细胞，无成骨细胞与血管内皮细胞的依附关系。结论 新型含孔磷酸钙陶瓷作为组织工程细胞支架，其间的纳米级结构有利于骨的再生和血管形成；未见来源于植骨床血管的内皮细胞衍变为成骨细胞。     

Total and free tissue factor pathway inhibitor in pregnancy hypertension.

OBJECTIVE: To clarify the role played by tissue factor pathway inhibitor (TFPI) in pregnancy hypertension. METHODS: Using enzyme-linked immunosorbent assays, hemostatic measurements were obtained for women with pre-eclampsia (n=51), nonproteinuric hypertension of pregnancy (n=62), postpartum pre-eclampsia 24 h after childbirth (n=31), and no hypertension (healthy pregnant controls, n=100). RESULTS: There was a significant increase in circulating free TFPI levels in women with pre-eclampsia (9.7+/-6.2 ng/mL) or nonproteinuric hypertension of pregnancy (8.3+/-5.3 ng/mL) compared with healthy controls (5.3+/-2.1 ng/mL). In women with pre-eclampsia the levels remained elevated after placental delivery (10.6+/-4.0 ng/mL). Free protein S levels were significantly higher in women with pre-eclampsia (40.0%+/-10.7%), nonproteinuric hypertension of pregnancy (37.1%+/-12.5%), or postpartum pre-eclampsia (39.3%+/-9.1%) than in healthy pregnant controls (32.2%+/-8.5%). CONCLUSION: Increased levels of the physiologically active free forms of TFPI and free protein S, 2 coagulation inhibitors, may protect women with pregnancy-induced hypertension from the risks of hemostatic activation.     

亚油酸对纤溶酶原激活物抑制剂-1表达的影响及其机制

目的探讨过氧化体增殖物激活型受体α（PPARα）的特异性激活物亚油酸对HepG2细胞1型纤溶酶原激活物抑制剂（PAI-1）mRNA表达及其活性的影响和在该基因转录调控中的作用机制.方法用不同浓度亚油酸为诱导因素刺激HepG2细胞,采用半定量RT-PCR法检测PAI-1mRNA水平,发色底物法检测PAI-1的活性变化.构建四个含PAI-1启动子序列从-804～＋17间不同长度片段驱动的荧光素酶报告基因质粒,体外瞬时转染HepG2细胞,检测荧光素酶的活性.结果与对照组相比,亚油酸组能使HepG2细胞PAI-1mRNA表达及蛋白活性显著升高（P＜0.05,P＜0.01）,且呈一定剂量依赖性;亚油酸诱导可使PAI-1转录活性显著升高（P＜0.01）;与转染质粒PAI-pGL3-A（-804/＋17）相比较,当转染质粒含有PAI-pGL3-B（-636/＋17）、PAI-pGL3-C（-449/＋17）时,荧光素酶活性显著降低（P＜0.01）;共转染PPARα表达质粒（PPARα-pSG5）的细胞在亚油酸诱导下PAI-1转录活性显著升高（P＜0.01）.结论亚油酸可以增加HepG2细胞PAI-1mRNA表达及其蛋白活性,调节PAI-1的基因转录,PPARα参与亚油酸对PAI-1基因的表达调控;在PAI-1启动子-804～-636、-449～-276区域内存在亚油酸作用的调控PAI-1基因表达的序列.     

白细胞介素6、信号传导和转录活化因子3和血管内皮生长因子在人脑胶质瘤中的表达及相关性研究

目的研究人脑不同级别胶质瘤中白细胞介素（IL）-6，信号传导和转录活化因子3（STAT3）和血管内皮生长因子（VEGF）的表达，探讨IL-6、STAT3和VEGF与肿瘤病理级别和侵袭性的关系。方法采用免疫组织化学法，检测70例人脑胶质瘤，10例脑膜瘤和5例正常脑组织中IL-6、STAT3和VEGF的表达。结果胶质瘤中IL-6、STAT3和VEGF的表达水平在高级别组（Ⅲ、Ⅳ级）明显高于低级别组（Ⅰ、Ⅱ级），两组间差异有统计学意义（P〈0．01），STAT3的表达与IL-6和VEGF的表达均呈正相关（P〈0．01）。结论IL-6、STAT3和VEGF的表达与胶质瘤的恶性程度有密切关系；且三者协同在胶质瘤发生、发展过程中起重要作用。三者的相关性证实VEGF基因由STAT3蛋白调节，而STAT3又由IL-6刺激活化。     

非诺贝特对培养的兔脂肪细胞表达凝血和纤溶因子的影响

目的探讨非诺贝特对培养的兔脂肪细胞表达组织因子(TF)和纤溶酶原激活物抑制剂1(PAI-1)的影响.方法取正常兔脂肪组织分离培养脂肪细胞,以不同浓度(分别为0,1,10和100μmol/L)非诺贝特孵育兔脂肪细胞24 h后收集细胞.RT-PCR测定脂肪细胞TF和PAI-1 mRNA表达.用ELISA方法测定TF和PAI-1浓度.结果不同浓度非诺贝特均可抑制兔脂肪组织细胞TF和PAI-1的表达和蛋白产生,其抑制作用呈浓度依赖性增强.在非诺贝特10μmol/L培养时兔脂肪细胞TF和PAI-1 mRNA分别为(0.504±0.016)和(1.500±0.096),明显低于对照[分别为(0.579±0.018)和(1.607±0.063),均P＜0.01].在非诺贝特100μmol/L培养时兔脂肪细胞TF和PAI-1 mRNA分别为(0.451±0.023)和(1.269±0.084),与对照比显著降低(均P＜0.001).结论非诺贝特能抑制兔脂肪细胞TF和PAI-1 mRNA和蛋白的表达,提示非诺贝特可能具有独立于降脂作用外的抗血栓作用.     

地塞米松对内毒素作用下人脐静脉内皮细胞表达组织因子、凝血酶调节蛋白和蛋白S的影响

目的观察地塞米松(DEX)对内毒素(LPS)作用于人脐静脉内皮细胞(HUVEC)后表达组织因子(TF)、凝血酶调节蛋白(TM)和蛋白S(PS)的影响.方法24孔板培养的第1～5代HUVEC,在含不同浓度LPS和DEX的无血清培养基中培养一定时间后裂解细胞,应用酶联免疫吸附法(ELISA)测定裂解液中的TF、TM和PS含量.结果在LPS刺激下,HUVEC表达TF的量呈剂量依赖性升高,在LPS0.1μg/ml下TF的表达量为对照组的4倍(P＜0.01),同时加入0.5 μg/ml和1.0 μg/ml DEX则可使TF的表达量由128.3±25.7 pg/105细胞分别降至94.9±19.4 pg/105细胞和98.8±7.8 pg/105细胞(P＜0.05);LPS(0.01～10μg/ml)可抑制HUVEC表达TM,在LPS 10 μg/ml下,TM表达量降至对照组的60%(P＜0.05).DEX可拮抗LPS抑制HUVEC表达TM的效应,0.1 μg/ml、0.5 μg/ml和1.0 μg/ml DEX可使LPS(10 μg/ml)作用下TM的表达量由0.282±0.014 ng/105细胞分别增至0.409±0.009、0.462±0.017和0.362±0.019 ng/105细胞(P＜0.05);LPS(0～10 μg/ml)可抑制HUVEC表达PS,在LPS浓度1.0 μg/ml及10 μg/ml时,PS的表达量分别为对照组的43%和38%(P＜0.01).DEX则拮抗LPS抑制HUVEC表达PS的效应,0.5 μg/ml DEX可使LPS刺激下PS的表达量由13.1±4.8%/2×105细胞增至48.5±10.2%/2×105细胞(P＜0.01).结论LPS促进HUVEC表达TF,抑制HUVEC表达TM和PS.DEX能部分拮抗上述作用,提示DEX可能纠正内毒素血症时的高凝状态.