Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.     

Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.     

壳聚糖携载质粒纳米微球体外转染兔关节软骨细胞的实验研究

目的：研究携载质粒的不同分子量的壳聚糖纳米微球的包裹率和保护DNA的能力,镜下观察其大小和形态,观察其对原代兔关节软骨细胞的转染效率。方法：利用酶消化法消化3周龄新西兰大白兔的关节软骨,贴壁培养原代兔关节软骨细胞。购买相对分子量在5K和800K之间的八种壳聚糖,利用表达增强型绿色荧光蛋白的质粒（pEGFP）作报告基因,通过复合凝聚法制备壳聚糖-质粒纳米微球。琼脂糖凝胶电泳、紫外分光光度计分析不同N/P比值对不同分子量壳聚糖和质粒的结合能力及包封率的影响;纳米粒度仪、透射电子显微镜和环境扫描电子显微镜考察纳米微球的粒径分布和形态;荧光显微镜观察壳聚糖纳米微球介导pEGFP在体外培养的兔关节软骨细胞中的表达情况;流失细胞仪计算具体转染效率。结果：①N/P值为4及4以上时,各分子量的壳聚糖可完全包裹质粒成球;N/P值为2时,分子量为5K、50K、85K仅部分包裹质粒,其余可完全包裹;N/P值为1时,各壳聚糖均与质粒部分包裹;N/P值为0.25时,各壳聚糖均与质粒完全分离。②纳米粒度仪分析得出：N/P值为4时,各分子量的壳聚糖纳米微球的平均粒径均在1微米以下,③透射电子显微镜和扫描电子显微镜均可观察到球形或不规则形的大小不同的微球。荧光显微镜可大致观察到绿色荧光蛋白在软骨细胞内表达的表达情况。④流式细胞仪得出具体转染效率,分子量为170K、250K和800K的壳聚糖纳米微球的转染效率均高于5K、50K和85K的壳聚糖纳米微球,其中800K的壳聚糖纳米微球与脂质体相当（差异有统计学意义,P〈0.05）。结论：与脂质体相比,N/P比值为4时,相对分子量为800k的壳聚糖纳米微球可高效转染原代培养的兔软骨细胞,可以作为今后进一步体外、体内实验的首选转染载体。     

壳聚糖携载质粒纳米微球体外转染兔关节软骨细胞的实验研究

目的:研究携载质粒的不同分子量的壳聚糖纳米微球的包裹率和保护DNA的能力,镜下观察其大小和形态,观察其对原代兔关节软骨细胞的转染效率。方法:利用酶消化法消化3周龄新西兰大白兔的关节软骨,贴壁培养原代兔关节软骨细胞。购买相对分子量在5K和800K之间的八种壳聚糖,利用表达增强型绿色荧光蛋白的质粒(pEGFP)作报告基因,通过复合凝聚法制备壳聚糖-质粒纳米微球。琼脂糖凝胶电泳、紫外分光光度计分析不同N/P比值对不同分子量壳聚糖和质粒的结合能力及包封率的影响;纳米粒度仪、透射电子显微镜和环境扫描电子显微镜考察纳米微球的粒径分布和形态;荧光显微镜观察壳聚糖纳米微球介导pEGFP在体外培养的兔关节软骨细胞中的表达情况;流失细胞仪计算具体转染效率。结果:①N/P值为4及4以上时,各分子量的壳聚糖可完全包裹质粒成球;N/P值为2时,分子量为5K、50K、85K仅部分包裹质粒,其余可完全包裹;N/P值为1时,各壳聚糖均与质粒部分包裹;N/P值为0.25时,各壳聚糖均与质粒完全分离。②纳米粒度仪分析得出:N/P值为4时,各分子量的壳聚糖纳米微球的平均粒径均在1微米以下,③透射电子显微镜和扫描电子显微镜均可观察到球形或不规则形的大小不同的微球。荧光显微镜可大致观察到绿色荧光蛋白在软骨细胞内表达的表达情况。④流式细胞仪得出具体转染效率,分子量为170K、250K和800K的壳聚糖纳米微球的转染效率均高于5K、50K和85K的壳聚糖纳米微球,其中800K的壳聚糖纳米微球与脂质体相当(差异有统计学意义,P<0.05)。结论:与脂质体相比,N/P比值为4时,相对分子量为800k的壳聚糖纳米微球可高效转染原代培养的兔软骨细胞,可以作为今后进一步体外、体内实验的首选转染载体。     

Estrogen receptors in cultured rabbit articular chondrocytes: influence of age

Primary cultures of pubertal and prepubertal rabbit articular cartilage cells were performed. Total homogenates or cell extracts were used to determine the specific binding of 17 beta-estradiol. A comparative study was undertaken with tissue minces homogenized without enzymatic treatment. Scatchard analysis of cell or tissue extracts revealed the presence of a high-affinity receptor with Kd values of 0.55 +/- 0.16 nM and 0.12 +/- 0.03 nM in prepubertal and pubertal rabbit chondrocytes respectively. A significant difference in the affinity of estrogen receptor for its ligand as a function of age was observed. In contrast there was no significant difference in the number of binding sites expressed as fmol per mg DNA between the two age groups. The ligand binding specificity was as expected for an estrogen receptor and the sedimentation coefficient was 3.2 S when analyzed by ultracentrifugation on sucrose density gradient in presence of 0.4 M KCl and 8.1 S in low salt conditions. The binding sites, labeled with [125I]estradiol, were specifically immunoprecipitated by a monoclonal antibody to the estrogen receptor (JS34/32).     

Genotoxic effects of heavy metals in rat hepatocytes

The genotoxic interaction of metals, which are common environmental contaminants, was studied in cultured hepatocytes. Freshly isolated rat hepatocytes were exposed to concentrations of cadmium, copper, silver and lead salts ranging from non-cytotoxic to moderately cytotoxic (as determined by LDH release), and the incorporation of [³H]thymidine into the DNA, as a measure of repair synthesis, was followed. In addition, the uptake of metals by the nuclear fraction was determined using Inductively Coupled Plasma/Mass Spectrometry or atomic absorption spectrophotometry. The evaluation of binding of ¹⁰⁹Cd to the DNA in situ was also attempted. It was observed that after a 20 h exposure period, all the metals investigated were found in the nuclear fraction of hepatocytes, with Ag apparently being accumulated less efficiently. In parallel, Cd (0.18 to 1.8 µM) and Cu (7.9 to 78.5 µM) consistently produced a statistically significant stimulation of [³H]thymidine incorporation into the DNA, in the presence or absence of hydroxyurea while Ag was active only at the highest concentration tested (18.5 µM). In contrast, Pb failed to induce a UDS response at the levels used. Moreover, exposure of hepatocytes to 1.8 µM ¹⁰⁹CdCl₂ for 20 h led to a DNA binding ratio of 0.98 ± 0.23 ng Cd/ µg DNA. The present results support the view that the nucleus may be an important target organelle for metal toxicity.Abbreviations 2-AAF 2-acetylaminofluorene - Cd cadmium - HU hydroxyurea - lCP/MS inductively coupled plasma/mass spectrometry - Hg mercury - Ni nickel - UDS unscheduled DNA synthesis     

Distribution of extractable heavy metals in different soil fractions

Abstract

Due to the difficulties of precisely characterizing environmentally contaminated soil, the effects of heavy metals on plants are studied using uncontaminated soil spiked with known quantities of heavy metals. One problem in using spiked soils is how accurately the distribution of metals mimics stabilized natural soils. We studied the distribution of cadmium, chromium, copper, lead, nickel, and zinc in soil fractions after application in soluble form. The soil samples included a control (an uncontaminated Typic Argiudoll) and two samples spiked with either a moderate or high heavy metal concentration). After application of the salts the soils were subjected to wet/dry cycles over the course of three months. The soils were fractionated using a sequential chemical extraction procedure employing: (1) CaCl₂,(2) NaOH, (3) Na₂EDTA and (4) HNO₃, HCl, and HF. Soil physical separation was carried out by ultrasonic dispersion. The heavy metal levels were determined using ICP-AES. Each heavy metal displayed a unique behavior when added to soil in the form of soluble salts. Cadmium and zinc remained in the soluble fraction, indicating that no equilibrium was attained, while nickel primarily appeared in the insoluble fraction. Chromium, copper and lead were distributed among various soil chemical fractions. The highest levels of all metals appeared in the clay fraction except lead which was mainly present in the silt fraction.     

Removal of heavy metals from industrial wastewater using microbial fuel cell

Removal efficiency of gold from a solution of pure tetrachloroaurate ions was investigated using microbial fuel cell (MFC) technology. The effects of type of catholyte solution and initial gold concentration on the removal efficiency were considered. Due to its presence at high levels in the gold wastewater, the effect of copper ions on the removal efficiency of the gold ions was also studied. The effects of pH and initial biomass concentration on the gold removal efficiency was also determined. The results showed that after 5 h contact time, 95% of gold removal efficiency from a wastewater containing 250 ppm of initial gold ions at ambient temperature using 80 g/L yeast concentration was achieved. After 48 h of the cell''s operation under the same condition, 98.86% of AuCl₄ ^– ions were successfully removed from the solution. At initial gold concentration in the waste solution of 250 ppm, pH 2, and initial yeast concentration of 80 g/L, 100% removal efficiency of the gold was achieved. On the other hand, the most suitable condition for copper removal was found at a pH of 5.2, where 53% removal efficiency from the waste solution was accomplished.     

Accumulative phases for heavy metals in limnic sediments

Data from mechanical concentrates of recent sediments indicate that clay minerals, clay-rich aggregates and heavy minerals are the major carriers of heavy metals in detrital sediment fractions. Hydrous Fe/Mn oxides and carbonates and sulfides, in their specific environments, are the predominant accumulative phases for heavy metals in autochthonous fractions. Sequential chemical extraction techniques permit the estimation of characteristic heavy metal bonding forms: exchangeable metal cations, easily reducible, moderately reducible, organic and residual metal fractions, whereby both diagenetic processes and the potential availability of toxic compounds can be studied. The data from lakes affected by acid precipitation indicate that zinc, cobalt and nickel are mainly released from the easily reducible sediment fractions and cadmium from organic phases. In contrast at pH 4.4, neither lead nor copper seem to be remobilized to any significant extent. Immobilization by carbonate precipitation seems to provide an effective mechanism for the reduction of dissolved inputs 9f metals such as zinc and cadmium in pH-buffered, hard water systems.     

Pesticides and heavy metals in Danish streambed sediment

The role of streambed sediment as a sink for pesticides and heavy metals was investigated in 30 Danish lowland streams. The investigated streams drain catchments varying in hydrology, topography, soil type and land use. The <250 m newly accumulated fraction of the uppermost 1–2 cm layer of streambed sediment was analysed for 19 old and modern pesticides and 9 heavy metals. DDE was present in the sediment of all the streams. Of the herbicides, fungicides and insecticides currently in use, the most frequently detected was diuron (50.0%), fenpropimorph (66.7%) and lambda-cyhalothrin (6.7%), respectively. The pesticides detected in the highest concentration were fenpropimorph (1700 ng g^–1), propiconazole (130 ng g^–1) and isoproturon (110 ng g^–1). The heavy metals are listed in order of increasing median concentration: Cd (0.80 g g^–1), Co (9.1 g g^–1), As (12.0 g g^–1), Ni (19.0 g g^–1), Cr (19.2 g g^–1), Pb (19.7 g g^–1), Cu (20.1 g g^–1), V (28.5 g g^–1), Zn (103 g g^–1). The average number of pesticides detected in the 27 streams draining predominantly agricultural catchments was (3.7±2.0) being higher (p=0.077) than in the three streams draining non-agricultural catchments (1.7±0.6). Pesticides were significantly related to catchment size, soil type and hydrological regime. Several heavy metals (Cr, Cu, Pb, V and Zn) were related to urban activity and soil type.     

土壤重金属污染的微生物生态效应研究进展

对近年来土壤重金属污染微生物生态效应的研究进展进行了归纳总结,主要从微生物群落特性和微生物生理、生化参数等几个方面进行了阐述。重金属污染土壤后,尤其是高浓度的重金属污染对微生物生物量和群落结构都有破坏作用,但由于微生物群落结构的复杂性和研究方法的片面性,一直是研究的热点和难点,开发更加简便、直接的方法将是对这方面研究的突破。同时,微生物的生理、生化参数是从另一侧面反映重金属污染对微生物的影响,它是对微生物群落特性研究的有利补充,所以不同方法的合理选择和搭配是实验取得预期结果的关键因素之一。     

Inhibition of photosynthesis by heavy metals

Inhibition of photosynthesis by heavy metals is well documented. In this review the results are compared between in vitro experiments on isolated systems (chloroplasts, enzymes ­.), experiments on excised leaves and intact plants and algae in vivo. In vitro experiments suggest potential sites of heavy metal interaction with photosynthesis at several levels of organisation, which are not necessarily confirmed in vivo. Analytical data on subcellular heavy metal level are generally missing to discuss their mechanism of action in the intact organism. In the field factors such as soil characteristics and air pollution have to be taken into account for assessing the mechanism of action of heavy metals on photosynthesis in plants, growing in a polluted erea.paper presented at the FESPP meeting in Strasbourg (1984)     

微藻对污水中重金属的生物吸附(英文)

在我国,水源污染问题异常突出,特别是水体重金属污染情况非常严重,因此,如何有效治理水体重金属污染成为了摆在科技工作者面前十分紧迫的任务。利用微藻生物吸附来治理水体重金属污染是一种经济、简便、有效可行的方法,具有极其广阔的应用前景。论文介绍了我国近年水体污染的状况及水体重金属污染特点;综述了水体重金属污染对水体植物、水体动物以及人类潜在的危害;比较了几种常见治理重金属污染的方法;分析了微藻吸附水体重金属的优点,并阐述了微藻对重金属生物吸附的机理及影响生物吸附过程的外在因素;最后提出了今后的研究发展方向。     

Concentrations of heavy metals in maternal and umbilical cord blood

Concentrations of lead, cadmium, methylmercury and total mercury were measured in maternal and umbilical cord blood using graphite atomic absorption spectrometry. Two essential metals, copper and zinc, were also determined using ion chromatography. Lead, copper and zinc were found to be lower in the cord blood, whereas methylmercury and total mercury were higher in cord blood than in maternal blood. Little differences were noted for cadmium in maternal and cord blood. Significant positive correlations were observed between the concentrations in maternal and cord blood with regard to lead (correlation coefficient, r = 0.44), copper (r = 0.34), zinc (r = 0.29), methylmercury (r = 0.44) and total mercury (r = 0.58). These results suggest that, like essential metals, most heavy metals can move rather freely across the human placenta. The potential health effects of heavy metal transfer from mothers to young infants cannot be discounted.     

土壤重金属生物毒性研究进展

世界范围内土壤重金属污染不断加重,由污染所带来的问题以及如何治理污染已经受到人们越来越多的关注.土壤重金属将对土壤生物产生影响,而土壤生物在重金属的胁迫下也会产生不同的响应.综述了国内外近年来土壤重金属生物毒性的研究进展,介绍了土壤重金属污染对陆地生态系统中植物、动物和微生物生长的影响;土壤重金属生物毒性的影响因素;土壤重金属生物毒性的研究方法;土壤重金属生物毒性的预测模型,最后提出了问题和展望.     

Review: Nutritional ecology of heavy metals

The aim of this review is to focus the attention on the nutrition ecology of the heavy metals and on the major criticisms related to the heavy metals content in animal feeds, manure, soil and animal-origin products. Heavy metals are metallic elements that have a high density that have progressively accumulated in the food chain with negative effects for human health. Some metals are essential (Fe, I, Co, Zn, Cu, Mn, Mo, Se) to maintain various physiological functions and are usually added as nutritional additives in animal feed. Other metals (As, Cd, F, Pb, Hg) have no established biological functions and are considered as contaminants/undesirable substances. The European Union adopted several measures in order to control their presence in the environment, as a result of human activities such as: farming, industry or food processing and storage contamination. The control of the animal input could be an effective strategy to reduce human health risks related to the consumption of animal-origin products and the environmental pollution by manure. Different management of raw materials and feed, animal species as well as different legal limits can influence the spread of heavy metals. To set up effective strategies against heavy metals the complex interrelationships in rural processes, the widely variability of farming practices, the soil and climatic conditions must be considered. Innovative and sustainable approaches have discussed for the heavy metal nutrition ecology to control the environmental pollution from livestock-related activities.     

淮河安徽段水及沉积物中重金属的研究

对采集淮河安徽段水及沉积物样品,利用ICP-AES进行重金属分析,结果表明砷、铅、铬超标,在沉积物中的含量最多。研究区域的重金属开始富集于生物体内,已对水生态系统及水产品安全产生影响,应进一步加强检测工作,以便及时采取相应防治对策。     

施污土壤重金属有效态分布及生物有效性

以城市污泥为研究对象,将城市污泥与土壤按照一定的质量比配成污泥混合土壤.采用6种不同性质提取剂(0.05mol/L EDTA、0.1 mol/L CH3 COOH、0.01 mol/L CaC12、1 mol/L CH3COONH4,0.05 mol/L NaHCO3和0.05 mol/L Tris-HCl)分别对污泥混合土壤中重金属(Cd、Pb、Cu和Zn)的螯合态、酸溶态、中性交换态、中性结合态、碱性交换态和蛋白质结合态进行提取,考察污泥的添加对土壤中不同形态重金属的消长规律.通过黑麦草盆栽试验,探究污泥混合土壤中不同形态重金属的植物可利用性.结果表明:污泥混合土壤中重金属螯合态比例较大,占总量的20.3％-40.0％;其次为酸溶态和中性结合态,而中性交换态、碱性交换态和蛋白质结合态的含量较低.污泥的添加促进了黑麦草对Cd、Cu和Zn的吸收,在污泥添加率为44.4％时根部对其吸收量达最大,分别较CK处理增加了0.3、2.3和6.5倍.抑制了对Pb的吸收,在污泥添加率为37.5％时,根部对Pb的吸收较CK处理下降0.4倍.Pearson相关系数分析结果表明:污泥混合土壤中以螯合态、酸溶态和中性结合态存在重金属可被黑麦草吸收利用.     

外生菌根与植物抗重金属胁迫机理

外生菌根是外生菌根真菌和植物营养根形成的共生体,能够增加植物对污染胁迫的抵抗能力。本文综述了20多年来国内外研究外生菌根增加植物抗重金属毒害的成果,指出了外生菌根在植物抗重金属毒害中的积极作用,并概括其抗性的主要机理为：外延菌丝的吸收作用;菌根分泌物的调节与螯合作用;菌根菌套或哈蒂氏网吸收过滤有毒金属;菌根菌套的疏水性作用。在研究外生菌根抗重金属毒害机理的基础上,提出了该领域今后的研究前景。