Effects of static and cyclic compressive loading on articular cartilage plugs in vitro

The influence of static and intermittent stress on articular cartilage metabolism was examined in vitro. Full-thickness plugs of cartilage from femoral condyles of normal adult dogs were cultured while static or cyclic stresses were applied for 2 hours. The plugs were then incubated under atmospheric pressure for 2 hours in medium containing radioactive label, to provide measurements of net synthesis of glycosaminoglycan (GAG) or protein. As a control, cartilage from the same knee was cultured in the apparatus at atmospheric pressure. When cartilage plugs were exposed to static stress, or to cyclic stresses at a duty cycle of 60 seconds on/60 seconds off, net GAG synthesis was suppressed to 30—60% of that in controls. In contrast, when a duty cycle of 4 seconds on/11 seconds off was used, GAG synthesis was increased by 34%. The duty cycle which increased GAG synthesis did not affect protein synthesis or tissue contents of DNA, uronic acid, or water. At the cycle which suppressed GAG synthesis, protein synthesis and uronic acid content were decreased, and water content was increased. As judged by uptake of ¹⁴C-aminoisobutyric acid and ¹⁴C-xylose, the above changes in GAG synthesis do not appear to have been due to changes in diffusion of nutrient molecules through the cartilage during loading.     

高糖对大鼠胰星状细胞增殖和细胞外基质合成的影响

目的探讨高糖刺激对大鼠胰星状细胞(pancreatic stellate cell, PSC)增殖和细胞外基质(extracellular matrix, ECM)合成的影响.方法分离培养大鼠PSC,取传第3～5代细胞,分别用正常葡萄糖(5.6 mmol/L葡萄糖)(正常对照)、高葡萄糖(25 mmol/L葡萄糖)(高糖组)和甘露醇(5.6 mmol/L葡萄糖+19.4 mmol/L甘露醇)(等渗对照组)处理5天后,用MTT法、3H-Thymidine掺入和3H-Proline掺入法分别检测细胞增殖、DNA合成和总胶原合成,同时用放免法检测细胞培养上清中Ⅲ型前胶原氨基末端多肽(amino-terminal propeptide of type Ⅲ procollagen, PⅢNP)和透明质酸(hyaluronic acid, HA)含量.结果与等渗对照组比较,高糖组PSC增殖、DNA合成、总胶原合成和细胞上清HA含量均显著增加(P<0.05),但细胞上清PⅢNP含量无显著性差异(P>0.05).结论高糖可刺激PSC增殖与胶原合成,参与PSC活化.体内高糖血症可能是PSC活化的机制之一.     

高糖对大鼠胰星状细胞增殖和细胞外基质合成的影响

目的 探讨高糖刺激对大鼠胰星状细胞(pancreatic stellate cell,PSC)增殖和细胞外基质(extracellular matrix,ECM)合成的影响。方法 分离培养大鼠PSC,取传第3～5代细胞,分别用正常葡萄糖(5.6mmol/L葡萄糖)(正常对照)、高葡萄糖(25mmol/L葡萄糖)(高糖组)和甘露醇(5.6mmol/L葡萄糖+19.4mmol/L甘露醇)(等渗对照组)处理5天后,用MTT法、~3H-Thymidine掺入和~3H-Proline掺入法分别检测细胞增殖、DNA合成和总胶原合成,同时用放免法检测细胞培养上清中Ⅲ型前胶原氨基末端多肽(amino-terminal propeptide of type Ⅲ procollagen,P Ⅲ NP)和透明质酸(hyaluronic acid,HA)含量。结果 与等渗对照组比较,高糖组PSC增殖、DNA合成、总胶原合成和细胞上清HA含量均显著增加(P<0.05),但细胞上清P Ⅲ NP含量无显著性差异(P>0.05)。结论 高糖可刺激PSC增殖与胶原合成,参与PSC活化。体内高糖血症可能是PSC活化的机制之一。     

Copper modulation of extracellular matrix synthesis by human articular chondrocytes

OBJECTIVE: The effects of Cu2+ on human articular chondrocytes, arising from both N (normal) and OA (osteoarthritic) cartilage, were investigated "in vitro". METHODS: Chondrocytes, cultured in high density, were incubated with copper chloride (0.01-0.25 microM/mL). Proteoglycan and collagen were assessed by incorporation of [35S]-Sulfate and [3H]-Proline. SDS-PAGE analysis was performed to quantify the ratio of type II to type I collagen. RESULTS: Cu2+ neither increased proteoglycan synthesis by chondrocytes. of origin N or OA, nor influenced their proliferation rate. Collagen synthesis was increased. This effect is time and concentration dependant: in cultures treated for 12 days, collagen synthesis stimulation was +20% and +26% (P < 0.02) in N and OA cultures respectively, the ratio of type II to type I collagen was slightly increased. This effect was more obvious in OA cell lines than in N ones. CONCLUSION: The observations suggest that Cu2+ upregulates collagen anabolism in human articular chondrocytes.     

Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver.

Open fenestrations are a conspicuous feature of sinusoidal endothelial cells and allow free movement of plasma into the space of Disse. In hepatic fibrosis, the number of fenestrations decreases as interstitial collagen increases in the liver, a change that correlates with deposition of extracellular matrix in the space of Disse. In this study, the possibility of a causal relationship between altered fenestral morphology and perisinusoidal matrix has been examined by culturing rat sinusoidal endothelial cells on individual matrix proteins or on a native matrix consisting of human amniotic membrane with interstitial collagen (types I and III) on one side and basement membrane proteins (collagen types IV and V and laminin) on the other. Under culture conditions, individual components of the extracellular matrix failed to maintain fenestrations. A basement-membranelike gel matrix derived from the Engelbreth-Holm-Swarm tumor war similarly ineffective. Fenestral density and porosity (percentage of cell surface occupied by fenestrations) were significantly enhanced, however, when endothelial cells were cultured on the basement-membrane side of human amnion. These data suggest that support of endothelial fenestrations requires a complex matrix. In particular, physiologically derived basement membrane maintains fenestrations, whereas interstitial collagen matrix does not. The loss of fenestrations associated with hepatic fibrosis may be related in part to an accumulation of interstitial collagens in the space of Disse.     

Semicarbazide-sensitive amine oxidase and extracellular matrix deposition by smooth-muscle cells

We have recently reported in vivo disruption of collagen and elastin architecture within blood vessel walls resulting from the selective inhibition of the enzyme semicarbazide-sensitive amine oxidase (SSAO). This study further investigates the effects of SSAO inhibition on extracellular matrix deposition by smooth-muscle cells (SMCs) cultured from neonatal rat hearts. SMCs were characterized, SSAO activity was measured, and soluble and insoluble collagen and elastin in the extracellular matrix (ECM) were quantified. Cultured neonatal rat heart SMC exhibited a monotypic synthetic phenotype that likely represents a myofibroblast. Detectable levels of SSAO activity present throughout 30-d culture peaked at 7–14 d, coinciding with the production of ECM. The addition of enzyme inhibitors and alternate SSAO substrates (benzylamine) produced varied and, in some cases, marked changes in SSAO activity as well as in the composition of mature and soluble matrix components. Similar to our previous in vivo findings, in vitro SSAO inhibition produced aberrations in collagen and elastin deposition by heart SMC. Because changes in SSAO activity are associated with cardiovascular pathologic states, this enzyme may play a protective or modulating role by regulating ECM production during pathologic insult.     

引经药柴胡对胰腺纤维化大鼠细胞外基质合成调控增效的作用

[目的]观察引经药柴胡对大黄丹参调控胰腺纤维化大鼠细胞外基质(ECM)合成增效的作用。[方法]50只雄性SD大鼠随机分为假手术组、模型组、柴胡组、大黄丹参组和柴胡大黄丹参组,每组10只。假手术组开腹后关腹,其他各组通过结扎胰胆管联合腹腔注射雨蛙素3d建立胰腺纤维化模型;柴胡组、大黄丹参组、柴胡大黄丹参组分别于术前、后3d用对应药物灌胃(剂量分别为1.38g.kg-1.d-1、3.04g.kg-1.d-1、4.11g.kg-1.d-1)。术后第4天取材用苏木精-伊红染色观察胰腺病理学变化,免疫组化检测胰腺α-平滑肌肌动蛋白及Ⅰ型胶原表达,ELISA测定血清纤维连接蛋白(FN)、层结合蛋白(LN)含量。[结果]与模型组相比,柴胡组、大黄丹参组和柴胡大黄丹参组均可改善胰腺病理变化,降低α-平滑肌肌动蛋白、Ⅰ型胶原表达,降低血清LN及FN含量,均P0.01;其中柴胡大黄丹参组FN含量降低更显著。[结论]柴胡、大黄丹参、柴胡大黄丹参均可通过抑制ECM合成来发挥抗胰腺纤维化的作用,其中柴胡大黄丹参在降低血清FN含量方面效果最优,提示柴胡在抑制胰腺ECM合成方面对大黄丹参存在一定的增效作用。     

Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue.     

高糖对人肾小球系膜细胞增殖及细胞外基质的影响

目的 观察不同浓度的葡萄糖对人肾小球系膜细胞增殖的影响及对细胞外基质纤维连接蛋白(Fn),Ⅳ型胶原(ColⅣ)的影响.方法 体外培养人肾小球系膜细胞株,分别加入葡萄糖终浓度为5.5、10、20、30、40 mmol/L进行处理,于不同时间段(12、24、48、72 h)用MTT法检测肾小球系膜细胞的增殖情况,ELISA法检测培养48 h后条件培养基中Fn及ColⅣ的含量.结果 高糖作用12、24、48、72 h均能促进细胞的增殖,且随着葡萄糖浓度升高作用时间的延长促进作用增强.检测细胞上清液的Fn和ColⅣ.与对照组相比,30 mmol/L D-葡萄糖作用48 h后,Fn和ColⅣ表达增高.结论 高浓度葡萄糖对系膜细胞增殖作用具有量效及时效关系,我们选择30 mmol/L浓度的葡萄糖作用48 h为最合适的细胞刺激条件.高浓度葡萄糖可促进系膜细胞细胞外基质的产生,而系膜细胞细胞外基质过度表达,可以直接导致细胞外基质的积聚,从而导致肾小球硬化的发生.     

黄芩甙抗肝星状细胞增殖及胶原合成的作用

目的研究黄芩甙对大鼠肝星状细胞活化、增殖及细胞外基质合成的作用,探讨黄芩甙抗肝纤维化的机制。方法采用胶原酶循环灌流法和密度梯度离心法分离肝星状细胞。不同浓度黄芩甙作用后,通过观察细胞贴壁及细胞形态变化情况来检测细胞活化：采用MTT法检测细胞增殖;免疫细胞化学法检测Ⅰ、Ⅲ型胶原及层粘连蛋白的合成。结果黄芩甙（75～1200μg/mL）可抑制HSC活化,MTT法示75μg／mL、150μg／mL、300μmL、600μg／mL、1200μg／mL黄芩甙作用于HSC的A值分别为（0．060±6．53）×10^-2、（0．052±7．38）×10^9、（0．036±1．26）×10^-3、（0．023±1．72）×10^-3、（0．013±4．01）×10^-3,空白对照组A值为（0．065±1．32）×10^4,F值=1147．611,P〈0．05。黄芩甙可抑制活化的HSC增殖,减少HSCⅠ、Ⅲ型胶原及层粘连蛋白合成。结论黄芩甙抗肝纤维化作用主要机制可能为黄芩甙抑制肝星状细胞活化、增殖,抑制细胞胶原蛋白及糖蛋“等细胞外基质合成.     

抗纤复方I号对乙醛刺激的肝星状细胞的细胞外基质及细胞因子分泌的干预作用

目的:研究乙醛对肝星状细胞(HSC) 细胞外基质和细胞因子分泌的影响及中药抗纤复方I号(KXI) 的干预作用.方法:采用大鼠肝脏原位灌流消化法获得并原代及传代培养HSC,大鼠灌以KXI 制备药物血清,以乙醛和药物血清作用于HSC,通过RTPCR 测定细胞内α1(I) 型胶原mRNA 的表达,以放射免疫法和ELISA 法...     

l-Carnitine enhances extracellular matrix synthesis in human primary chondrocytes

Osteoarthritis (OA) is one of the most common degenerative joint disease for which there is no cure. It is treated mainly with non-steroidal anti-inflammatory drugs to control the symptoms and some supplements, such as glucosamine and chondroitin sulphate in order to obtain structure-modifying effects. Aim of this study is to investigate the effects of l-carnitine, a molecule with a role in cellular energy metabolism, on extracellular matrix synthesis in human primary chondrocytes (HPCs). Dose-dependent effect of l-carnitine on cartilage matrix production, cell proliferation and ATP synthesis was examined by incubating HPCs with various amounts of molecule in monolayer (2D) and in hydromatrix scaffold (3D). l-Carnitine affected extracellular matrix synthesis in 3D in a dose-dependent manner; moreover, l-carnitine was very effective to stimulate cell proliferation and to induce ATP synthesis, mainly in 3D culture condition. In conclusion, l-carnitine enhances cartilage matrix glycosaminoglycan component production and cell proliferation, suggesting that this molecule could be useful in the treatment of pathologies where extracellular matrix is degraded, such as OA. To our knowledge, this is the first study where the effects of l-carnitine are evaluated in HPCs.     

肝素对大鼠肝星状细胞生长、细胞外基质和基质金属蛋白酶基因表达的影响

目的 探讨肝素对ＨＳＣ生长、细胞外基质和基质金属蛋白酶基因表达的影响。方法 用肝素和不用肝素对活化的ＨＳＣ进行处理,以直接细胞计数和ＢｒｄＵ标记免疫细胞化学染色检测细胞的生长情况。用免疫细胞化学染色和细胞闰核酸分子杂交分别检测Ｉ、Ⅳ型前胶原蛋白、纤连蛋白和Ⅰ、Ⅳ型胶原、纤连蛋白、基质金属蛋白酶－２及膜型基质金属蛋白酶基因的表达,并用酶图法检测基质金属蛋白酶－２的活性。结果 肝素使血清所致的ＨＳＣ生     

白藜芦醇通过调节SIRT1活性促进退变椎间盘终板软骨细胞合成细胞外基质

目的探讨白藜芦醇(Res)干预对退变椎间盘终板软骨细胞(DEC)合成细胞外基质的影响。方法老年腰椎间盘突出症患者的终板软骨细胞培养、传代,P2代细胞随机分为5组,即Res组、二甲基亚砜对照组、Res+尼克酰胺(Nam)组、Res+siRNA转染组及空白对照组。各组进行对应处理后检测沉默信息调节因子2相关酶1(SIRT1)、Ⅱ型胶原(COLA2)、蛋白聚糖聚合物(aggrecan)及SIRT1 mRNA表达水平。结果 Res干预上调DEC SIRT1 mRNA及蛋白的表达,促进细胞外基质(COLA2、aggrecan)的合成;Nam及siRNA干预分别有效抑制了SIRT1酶活性及SIRT1 mR-NA的表达,并抑制了细胞外基质的合成。结论 Res可促进DEC合成细胞外基质,抑制椎间盘软骨终板退变,其机制与调节SIRT1活性有关。     

Triggerlike stimulation of cholesterol accumulation and DNA and extracellular matrix synthesis induced by atherogenic serum or low density lipoprotein in cultured cells

A 72-hour incubation of cultured cells with blood sera or plasma of patients suffering from coronary heart disease (CHD) with angiographically assessed coronary atherosclerosis caused a threefold to fourfold elevation of intracellular cholesterol. An elevated cholesterol level in the cells precultured with patients' sera was retained several days after the removal of the examined serum from culture. The accumulation of intracellular cholesterol was accompanied by enhanced synthesis of DNA, total protein, collagen, sulfated glycosaminoglycans, and hyaluronic acid. Enhanced DNA and total protein synthesis was retained for at least 9 days after the serum had been removed from culture. The obtained results suggest that the sera of CHD patients possess an atherogenic potential that manifests itself at the arterial cell level in the stable stimulation of atherosclerotic cellular processes: proliferation, lipidosis, and fibrosis. The examined sera of healthy donors were devoid of such an atherogenic potential. The low density lipoprotein (LDL) fraction (density, 1.030-1.050 g/cm3) obtained from an atherogenic serum had the same atherogenic potential as a whole serum. Atherosclerotic alterations in cultured intimal cells caused by atherogenic LDL were retained for at least 3 days after the removal of the lipoprotein from culture. Preincubation of intimal cells with LDL obtained from healthy donors had no effect on the intracellular cholesterol level or the synthesis of DNA and extracellular matrix. One may assume that the atherogenic potential of CHD patients' sera is related to the presence of LDLs that are qualitatively different from the LDL of healthy subjects.     

Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions

Objectives. The purpose of this study was to examine the proliferative capacity and extracellular matrix synthesis of human coronary plaque cells in vitro.Background. Common to both primary atherosclerosis and restenosis are vascular smooth muscle cell proliferation and production of extracellular matrix proteins. The applicability to humans of experimental animal models of these processes has been questioned.Methods. Primary atherosclerotic and restenotic lesions were excised by percutaneous directional coronary atherectomy in 93 patients. Smooth muscle cells were cultivated by an explant technique and identified by their morphology in culture, ultrastructural features under electron microscopy and immunostaining using monoclonal antibodies to smooth muscle cell alpha-actin. Proliferation in secondary culture was assessed with growth curves and the synthesis of collagen and sulfated glycosaminoglycans by the incorporation of ³H-proline and ³⁵S-sulfate, respectively. These studies were also performed in cells derived from human umbilical artery media.Results. Success rates for primary (45%) and secondary (12%) culture of coronary cells were not influenced by clinical variables or lesion category. Primary culture success was improved by the presence of organized thrombus in the plaque and in relation to increased maximal cell density of the atherectomy specimen. Restenotic cells displayed more rapid growth than did cells of primary atherosclerotic origin, which grew in a manner similar to that of umbilical artery cells. Mean calculated population-doubling for the three cell groups were 52 h (95% confidence interval [CI] 48 to 58 h), 71 h (95% CI 62 to 83 h) and 74 h (95% CI 65 to 84 h), respectively. Restenotic and primary atherosclerotic cells did not differ in the synthesis of collagen ([mean ± SEM] 0.034 ± 0.004 vs. 0.033 ± 0.004 nmol isotope· μg protein⁻¹, p = NS) or sulfated glycosaminoglycans (11.47 ± 1.07 vs. 15.37 ± 3.10 nmol isotope· μg protein⁻¹, p = NS), but the coronary cells synthesized significantly more collagen and sulfated glycosaminoglycans than did umbilical artery cells (0.019 ± 0.004 and 5.43 ± 1.00 nmol isotope· μg protein⁻¹, respectively, both p < 0.05).Conclusions. These data indicate that increased smooth muscle cell proliferation contributes to coronary restenosis in humans and support the concept that the extracellular matrix synthesis of adult smooth muscle cells is important to lesion formation.