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.     

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.     

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.     

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)     

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.     

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。     

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。     

Tissue engineering of cartilage with chondrocytes cultured in a chemically-defined, serum-free medium

Cell culture with serum-containing medium has potential problems associated with contamination of infectious agents. This study demonstrates for the first time the feasibility of regenerating cartilage tissues in vivo by implantation of chondrocytes cultured in vitro in a chemically-defined, serum-free medium. Chondrocytes cultured in the serum-free medium grew similarly to those in a serum-containing medium. Implantation of chondrocytes cultured in the serum-free medium and seeded on to polymer scaffolds resulted in the regeneration of cartilage tissues with histological aspects similar to those of cartilage tissues regenerated from chondrocytes cultured in serum-containing medium.     

Standardization of methods of analysis for heavy metals in sediments

In studies of heavy metals in sediments, there is a need for standardization of the procedures for sample collection and preservation, chemical analyses and presentation of results. The method and depth of sampling depend on the aim of the investigation and on local sediment conditions, such as consistency of the sediment, rate of sedimentation, diagnetic processes and bioturbation. Therefore no general recommendations can be given in this respect. During collection and preservation, contamination and loss of constituents ust be avoided. In sediments, the best means for estimating total contents of metals is digestion with HF, in combination with strong acids. Other methods include X-ray fluorescence and neutron activation analyses. The use of HF is considered objectionable by some laboratories. A reasonable alternative is aqua regia. Because variations in granular composition affect metal contents, it is advisable to use the fraction < 63 μm for the analysis. Chemical partition of sediments provides an insight into the source of metallic constituents, and their pathways to deposition areas. A three-step extraction procedure, in the sequence 0.1 M hydroxylamine-HCl, H₂O₂ 30% and HF, is proposed. Finally, attention is paid to the anthropogenic enrichment of metals in sediments. The establishment of base-line levels is discussed.     

环境重金属污染的植物修复及基因工程在其中的应用

随着工业技术的发展,重金属在土壤和水体中的含量越来越高,重金属污染已日益成为威胁人类健康和人类生活质量的严重的社会问题和环境问题。植物修复可部分解决这一问题且正引起人们的普遍关注。但现在发现许多用于修复的超量积累植物生长缓慢、植株矮小、地上部生物量小,成了实际应用中的最大限制。利用基因工程手段改变植物对重金属吸收、转运、积累和忍耐的机制,从而提高植物对重金属的富集能力,将成为今后植物修复领域研究的一个重要方向。     

A depth-dependent model of the pericellular microenvironment of chondrocytes in articular cartilage

Experimental studies suggest that the magnitude of chondrocyte deformation is much smaller than expected based on the material properties of extracellular matrix (ECM) and cells, and that this result could be explained by a structural unit, the chondron, that is thought to protect chondrocytes from large deformations in situ. We extended an existing numerical model of chondrocyte, ECM and pericellular matrix (PCM) to include depth-dependent structural information. Our results suggest that superficial zone chondrocytes, which lack a pericellular capsule (PC), are relatively stiff, and therefore are protected from excessive deformations, whereas middle and deep zone chondrocytes are softer but are protected by the PC that limits cell deformations in these regions. We conclude that cell deformations sensitively depend on the immediate structural environment of the PCM in a depth-dependent manner, and that the functional stiffness of chondrocytes in situ is much larger than experiments on isolated cells would suggest.     

Scandinavian limnochronology of sediments and heavy metals

Sediment and peat accumulation rates as well as heavy metal fluxes are examined in Scandinavian lakes, marine coasts and peatlands. Modern accumulation rates are determined using Pb-210 and other chronological tools. Mean deposition rates in the past 150 ± 20 yrs are 3–95, 40–190 and 1–5 mg·cm^–2·yr^–1 for lakes, marine coasts and peatlands respectively. The Pb-210 fluxes at the investigated sites are quite variable. The lowest values are found for peatlands 0.04–0.20 pCi·cm^–2·yr^–1 while coastal marine environments showed 0.35–0.70 pCi·cm^–2·yr^–1. A wide range of variation of Pb-210 fluxes is obtained for the studied lake sediments 0.01–0.51 pCi·cm^–2·yr^–1.The chronological records of Pb, Zn, Cu, Cd and Hg show that heavy metal fluxes are much higher in coastal marine areas. Inland lakes and ombrotrophic peat-bogs indicate that Pb is introduced exclusively through the atmosphere and decrease exponentially from South to North Scandinavia. However, the other heavy metals are transported to freshwater systems by different hydrologic and atmospheric processes. Zn and Fe exhibit some correlation with lake-drainage area.     

Impact of bioavailable heavy metals on bacterial activities in coastal marine sediments

Total heavy metal concentrations in marine sediments are not sufficient to reliably predict detrimental biological effects. Here we provide evidence that only bioavailable heavy metals have a significant impact on benthic microbial loop functioning. Sediment samples collected along 250 km of the Apulian coast (Mediterranean Sea) were analysed for total and bioavailable heavy metals (Cr, Cd, Pb and Cu), organic matter content, bacterial abundance, biomass and carbon production and -glucosidase activity. Sampling strategy was specifically designed to cover a wide range of environmental conditions and types of anthropogenic influences. Total heavy metal concentrations in the sediments were tightly coupled with organic matter content, whereas bioavailable heavy metal concentrations displayed an opposite pattern. Bioavailable Cr concentrations were up to 10-fold higher than values observed for the other bioavailable metals and significantly inhibited benthic bacterial metabolism and turnover. Results from this study suggest that functional microbial variables are highly sensitive to heavy metal contamination and could be used as bioindicators of stress conditions in coastal sediments.     

The determination of heavy metals in water,suspended materials and sediments from Langat River,Malaysia

The distributions of heavy metals in the Langat River were studied for a period of six months between September 1984 and February 1985. Heavy metals such as arsenic, cadmium, cerium, cobalt, chromium, caesium, lanthanum, rubidium, antimony, scandium, thorium and zinc were determined in water, suspended materials and sediment samples from the Langat River by neutron activation and atomic absorption spectrometry. The concentrations of arsenic, cadmium, cerium, cobalt, scandium, antimony, and zinc were generally highest in the suspended materials, whereas the concentrations of chromium, rubidium and thorium were always highest in the sediments: Arsenic concentrations in the river were slightly higher than the natural concentration, while other elements were generally at their natural concentration levels. The use of arsenical herbicides in plantations along the river could be a source of arsenic pollution.     

Removal of heavy metals from water effluents using supermacroporous metal chelating cryogels

Applications of IDA in, for example, immobilized metal ion affinity chromatography for purification of His-tagged proteins are well recognized. The use of IDA as an efficient chelating adsorbent for environmental separations, that is, for the capture of heavy metals, is not studied. Adsorbents based on supermacroporous gels (cryogels) bearing metal chelating functionalities (IDA residues and ligand derived from derivatization of epoxy-cryogel with tris(2-aminoethyl)amine followed by the treatment with bromoacetic acid (defined as TBA ligand)) have been prepared and evaluated on capture of heavy metal ions. The cryogels were prepared in plastic carriers, resulting in desired mechanical stability and named as macroporous gel particles (MGPs). Sorption and desorption experiments for different metals (Cu2+, Zn2+, Cd2+, and Ni2+ with IDA adsorbent and Cu2+ and Zn2+ with TBA adsorbent) were carried out in batch and monolithic modes, respectively. Obtained capacities with Cu2+ were 74 μmol/mL (TBA) and 19 μmol/mL gel (IDA). The metal removal was higher for pH values between pH 3 and 5. Both adsorbents showed improved sorption at lower temperatures (10°C) than at higher (40°C) and the adsorption significantly dropped for the TBA adsorbent and Zn2+ at 40°C. Desorption of Cu2+ by using 1 M HCl and 0.1 M EDTA was successful for the IDA adsorbent whereas the desorption with the TBA adsorbent needs further attention. The result of this work has demonstrated that MGPs are potential treatment alternatives within the field of environmental separations and the removal of heavy metals from water effluents.     

牡蛎对重金属生物富集动力学特性研究

应用半静态双箱模型室内模拟了牡蛎对四种重金属 (As、Hg、Cd、Pb)的生物富集实验 ,通过非线性拟合得到了牡蛎富集重金属的吸收速率常数 k1 、排出速率常数 k2 、生物富集因子 BCF、生物学半衰期 B1 /2 等动力学参数。结果表明 :吸收速率常数 k1及生物富集因子 BCF均随着外部水体浓度的增大而减小而平衡状态下生物体内金属含量 CAmax随着外部水体浓度的增大而增大 ,且成显著正相关 ,这说明牡蛎比较理想的重金属 Hg、Cd、Pb污染的指示生物。