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1.
Shaking or stirring in a miniaturized device is often applied in the development of protein pharmaceuticals, serving as a test of stability under physical stress conditions also occurring in the real process. The overall purpose of these “stress tests” is to accelerate protein aggregation that could otherwise take place at a much slower rate, thereby enhancing experimental throughput to speed up determination of critical process parameters of stability. Results are useful to determine critical parameters for formulation development. However, shaking differs from stirring in the forces applied on proteins in solution and therefore, there is the possibility that characteristics of protein stability interrogated with each method may not be the same. We have performed a detailed, time-resolved analysis of aggregation of the human growth hormone (hGH), exposed to stirring and shaking in a well-defined and comparable mini-reactor set-up. We show that aggregation of hGH, which in its native form is a monomer, is not the result of protein deamidation, a chemical protein modification that occurs in hGH under the incubation conditions used. We also show that both under stirring and shaking, hGH appears to precipitate into mainly spherical particles in the presence of only small amounts of soluble protein oligomers. The precipitate appears to contain protein that has a substantial amount of hydrophobic surface exposed. An air–liquid interface, constantly renewed by stirring or shaking, was identified to be mainly responsible for aggregation of hGH under each of the stress conditions applied. In hGH, therefore, controlled shaking and stirring lead to identical conclusions about the overall protein stability and the main critical parameter driving the aggregation, thus informing stabilization strategies and formulation development. 相似文献
2.
Cell suspensions of Morinda citrifolia were subjected to turbulent flow conditions in a submerged jet apparatus, to investigate their hydrodynamic shear susceptibility. The suspensions were exposed to repeated, pressure-driven passages through a submerged jet. Two nozzles, of 1 mm and 2 mm diameter, were employed. Average energy dissipation rates were in the range 10(3)-10(5) W/kg and cumulative energy dissipation in the range 10(5)-10(7) J/m3. System response to the imposed conditions was evaluated in terms of suspension viability (determined using a dye exclusion technique) and variations in both chain length distribution and maximum chain length. Viability loss was well-described by a first-order model, and a linear relationship was identified between the specific death rate constant and the average energy dissipation rate. This relationship was consistent with results obtained using the same suspension cultures in a turbulent capillary flow device. Morphological measurements indicated that exposure to the hydrodynamic environment generated in the jet resulted in a significant reduction in both the average and maximum chain lengths, and the reduction in the maximum chain length was identified as an appropriate measure of sustained damage. Analysis of both viability and chain length in terms of cumulative energy dissipated revealed good agreement with results reported by other authors for morphologically different plant cell systems. Copyright 1998 John Wiley & Sons, Inc. 相似文献
3.
Tanaka H 《Biotechnology and bioengineering》2000,67(6):775-790
Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technological problems in the cultivation of the plant cells at high density were investigated. Using "shake" flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen in order to obtain high cell densities in shaken cultures, particle breakdown and damage to the largest cell aggregate group (above 1981 microm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K) for a model solid-liquid system (beta-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in suspension cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 x 10(-3) cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble dispersion). The most suitable bioreactor for culturing plant cells at high density was a jar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium. 相似文献
4.
Proceedings: Characteristics of freeze-dried cells 总被引:4,自引:0,他引:4
Microorganisms have been found to be more sensitive to selective media after freeze-drying. This increased sensitivity can be measured and thus the degree of sublethal injury can be determined as a function of various processing variables. In light of this, the use of selective media for the enrichment and detection of pathogens in freeze-dried products has to be reevaluated; indeed, the literature is now becoming abundant with such evaluations. In addition, the response of freeze-dried microorganisms has been found to be dependent on the medium in which they were grown; the phenomena of “metabolic injury” and “minimal medium recovery” are observed when microorganisms are grown in a complete and minimal medium, respectively. The expression of these two phenomena also can be used to assay for injury.Observations on the effects of freeze-drying on cell viability lead to the conclusion that freeze-drying is a complex stress. Damage to the cellular membrane structure and function, RNA integrity, and, possibly, DNA have been cited. The extrapolation of these macromolecular changes to specific viability responses for the purpose of elucidating the principal site of damage is still difficult. It is our opinion that the pre- and post-freeze-drying conditions to which the microorganisms are exposed can lead to a situation in which a particular macromolecular damage can become dominant over others, depending on the physiology of the cell.This knowledge can not only be applied for the purpose of improving detection of undesirable microbes but also for the preservation of desirable cultures, such as starter cultures in the dairy industry. Finally, the finding that microorganisms leak or release nucleic acids after freeze-drying, as they do after freezing and heating, can be applied to the problem of elimination of undesirable cytoplasmic components of organisms to be used as protein sources (4, 8). 相似文献
5.
The likelihood of microorganisms being transferred to new environments by jet aircraft was investigated. Initial random sampling of the aircraft surface revealed the presence of microorganisms in varying numbers on different aerodynamic surfaces. Bacteria of the genus Bacillus were the most common isolates, comprising approximately one-third of the total organisms found. The most frequently isolated fungi were Cladosporium, Alternaria, Penicillium, and several yeasts. Sampling of surfaces before and immediately after a flight demonstrated that microorganisms were collected during flight in areas protected from the airstream and lost in those areas directly exposed to it. These experiments also showed that the majority of the organisms contaminating the aircraft were acquired from the air at ground level. The placement of microorganisms on the aircraft surface before a flight and determination of their survival after flight indicated that the test organisms were most likely to be transported in the areas protected from the airstream. The organisms showing the best chance of being transferred seem to be the sporeforming bacteria, arthrospore-forming fungi, and some yeasts. All phases of this work showed that microorganisms could be carried by jet aircraft to environments they could not reach by natural means of dispersal. 相似文献
6.
Rao AK Dietrich AK Ziegler YS Nardulli AM 《The Journal of steroid biochemistry and molecular biology》2011,127(3-5):382-389
A number of studies have demonstrated that 17β-estradiol (E(2)) protects the brain from ischemia and yet the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E(2), overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E(2) treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E(2) might increase SOD1 expression in the brain and that this E(2)-mediated increase in SOD1 expression might help to protect the brain from ischemia. We now show that SOD1 is expressed in cortical neurons, that SOD1 expression is increased by exposure of brain slice cultures to E(2), and that the E(2)-mediated increase in SOD1 expression is further augmented by exposure of brain slice cultures to increased superoxide levels or oxygen and glucose deprivation. Importantly, when cortical neurons are exposed to increased superoxide levels and markers of protein and DNA damage, nitrotyrosine and 8-oxoguanine, respectively, are measured, both protein and DNA damage are reduced. In fact, E(2) reduces nitrotyrosine and 8-oxoguanine levels in brain slice cultures regardless of whether they have or have not been exposed to increased superoxide levels. Likewise, when brain slice cultures are treated with E(2) and deprived of oxygen and glucose, 8-oxoguanine levels are reduced. Taken together, these studies provide a critical link between E(2) treatment, SOD1 expression, and neuroprotection and help to define a mechanism through which E(2)-mediated neuroprotection may be conferred. 相似文献
7.
Laminar shear is the primary mechanism of cell damage, limiting flow rate (and hence flux) in crossflow microfiltration of animal cells. Sensitivity to hydrodynamic and interfacial stress is reduced by the addition of 0.1% Pluronic polyol. A critical average wall shear rate of 3000 s(-1) (above which damage occurs) is found for several cell types, including mammalian and insect cells. Hydrodynamic stress also limits the maximum tip speed in a rotary lobe pump to less than 350 cm/s. Turbulent flow in the recirculation loop piping at Reynolds numbers of up to 71,000 does not cause cell damage. Maximum sustainable flux decreases with cell concentration and increases with cell size (in qualitative agreement with the hydrodynamic lift model). A flux of 30 to 75 L/m(2) h (depending on cell size) can be sustained during 20-fold concentration from 2.5 x 10(6) cells/ml, while maintaining high cell viability. 相似文献
8.
Effects of rheological properties and mass transfer on plant cell bioreactor performance: production of tropane alkaloids 总被引:1,自引:0,他引:1
Volumetric mass transfer coefficients, K(L)a were measured over an aeration rate range from 0.1 to 1.0 vvm in a 1.2-L draft-tube-type airlift bioreactor for different Datura stramonium cell concentrations and correlated with superficial air velocity and rheological properties of the cell suspension. The measured K(L)a values (17-40 h(-1)) for a cell volume fraction of 0.2 (v/v) were approximately 2 times higher than those for the highest cell concentrations tested (cell volume fraction 0.7-0.8 v/v). Cell suspensions exhibited yield stress and pseudoplastic behavior. This behavior was described by the Casson model. The estimated yield stress values depended upon cell concentration with an exponent of 4.0. An empirical correlation based on the data for plant cell suspensions exhibiting yield stress was developed in order to determine aeration strategy for the plant cell cultivation in draft-tube-type airlift bioreactors: \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm K}_{\rm L} {\rm a} = {\rm A}({\rm U}_{{\rm gr}});{0.3} ({\rm \eta }_{{\rm eff}});{ - 0.4} $$\end{document} Aeration rates above 1.0 vvm caused a significant drop in cell yield and product content. Maximum growth and production were obtained at 0.6 vvm aeration. The cell and product yields obtained at 1.7 vvm were 2.8 times lower than the maximum values (25 g cell DW/L and 73.8 mg tropane alkaloid/L). The effects of the increased aeration rates on cell yield were also evaluated in terms of Reynolds stress. It was found that there was a relation between cell damage and the estimated Reynolds stress. The Reynolds stress estimated for the same aeration rate decreased with increasing cell concentration, suggesting that cells in the cultures at low cell concentrations are subjected to hydrodynamic damage. In the experiments with the cell cultures having a cell concentration of 0.3 (v/v), approximately 70% reduction in cell concentration was observed when the Reynolds stress was increased from 10 to 50 dyn/cm(2). (c) 1993 John Wiley & Sons, Inc. 相似文献
9.
Armeni T Battino M Stronati A Pugnaloni A Tomassini G Rosi G Biagini G Principato G 《Biological chemistry》2001,382(12):1697-1705
Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glutathione peroxidase) were studied. Exposure to H2O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress. 相似文献
10.
F. Garcia-Ochoa E. Gomez A. Alcon V. E. Santos 《Bioprocess and biosystems engineering》2013,36(7):911-925
The specific growth and the xanthan production rates by the bacterium Xanthomonas campestris under different shear levels in shake flasks and in a stirred and sparged tank bioreactor have been studied. The shake flask has been used as a reference for studying the shear effects. An effectiveness factor expressed by the ratio of the observed growth rate and the growth rate without oxygen limitation or cell damage was calculated in both modes of cultures. It was observed that the effectiveness factor was strongly dependent on the operational conditions. A strong oxygen transfer limitation at low stirring rates, indicated by a 54 % decrease in the effectiveness factor was observed. In contrast, at higher stirrer speed, cell damage was caused by hydrodynamic stress in the turbulent bulk of the broth, yielding again a decrease in the effectiveness factor values for stirrer speeds higher than 500 rpm. Cell morphological changes were also observed depending on the agitation conditions, differences in morphology being evident at high shear stress. 相似文献
11.
Minimizing hydrodynamic stress in mammalian cell culture through the lobed Taylor-Couette bioreactor
Robin Sorg Timm Tanzeglock Miroslav Soos Professor Dr. Massimo Morbidelli Arnaud Périlleux Thomas Solacroup Hervé Broly 《Biotechnology journal》2011,6(12):1504-1515
The objective of the present study was to investigate the effect of hydrodynamic stress heterogeneity on metabolism and productivity of an industrial mammalian cell line. For this purpose, a novel Lobed Taylor-Couette (LTC) mixing unit combining a narrow distribution of hydrodynamic stresses and a membrane aeration system to prevent cell damage by bubble bursting was developed. A hydrodynamic analysis of the LTC was developed to reproduce, in a uniform hydrodynamic environment, the same hydrodynamic stress encountered locally by cells in a stirred tank, particularly at the large scale, e.g., close and far from the impeller. The developed LTC was used to simulate the stress values near the impeller of a laboratory stirred tank bioreactor, equal to about 0.4 Pa, which is however below the threshold value leading to cell death. It was found that the cells actively change their metabolism by increasing lactate production and decreasing titer while the consumption of the main nutrients remains substantially unchanged. When considering average stress values ranging from 1 to 10 Pa found by other researchers to cause physiological response of cells to the hydrodynamic stress in heterogeneous stirred vessels, our results are close to the lower boundary of this interval. 相似文献
12.
Suspension cultures of Rubia tinctorum, an anthraquinones (AQs) producer, were grown both in Erlenmeyer flasks at 100 rpm and in a 1.5 L mechanically stirred tank bioreactor operating at 450 rpm. The effect of hydrodynamic stress on cell viability, biomass, and AQs production was evaluated. Cell viability showed a transient decrease in the bioreactor during the first days, returning to the initial values toward the end of the culture time. The biomass obtained in the bioreactor was 29% lower than that attained in the Erlenmeyer flasks. The H2O2 production in the bioreactor (with peaks at 7 and 10 days) was about 15 times higher than that obtained in the flasks. A clear relationship exists between the maximum concentration of H2O2 generated and AQs produced. The AQs content in the bioreactor was 233% higher than that in the Erlenmeyer flasks. The AQs specific productivity in the stirred tank and in the Erlenmeyer flasks was 70.7 and 28.5 micromol/g FW/day, respectively. This production capability was maintained in the regrowth assays. On the other hand, the negative effects of hydrodynamic stress on viability and biomass concentration observed in the bioreactor culture were reverted in the regrowth cultures. It can be concluded that R. tinctorum suspension cultures are able to grow in stirred tanks at 450 rpm responding to the hydrodynamic stress with higher concentrations of AQs, which suggest the possibility of a technological approach taking advantage of this phenomenon. 相似文献
13.
CTC staining and counting of actively respiring bacteria in natural stone using confocal laser scanning microscopy 总被引:1,自引:0,他引:1
A method was established for staining and counting of actively respiring bacteria in natural stone by using the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) in combination with confocal laser scanning microscopy (CLSM). Applying 5 mM CTC for 2 h to pure cultures of representative stone-inhabiting microorganisms showed that chemoorganotrophic bacteria and fungi-in contrast to lithoautotrophic nitrifying bacteria-were able to reduce CTC to CTF, the red fluorescing formazan crystals of CTC. Optimal staining conditions for microorganisms in stone material were found to be 15 mM CTC applied for 24 h. The cells could be visualized on transparent and nontransparent mineral materials by means of CLSM. A semi-automated method was used to count the cells within the pore system of the stone. The percentage of CTC-stained bacteria was dependent on temperature and humidity of the material. At 28 degrees C and high humidity (maximum water holding capacity) in the laboratory, about 58% of the total bacterial microflora was active. On natural stone exposed for 9 years at an urban exposure site in Germany, 52-56% of the bacterial microflora was active at the east, west, and north side of the specimen, while only 18% cells were active at the south side. This is consistent with microclimatic differences on the south side which was more exposed to sunshine thus causing UV and water stress as well as higher temperatures on a microscale level. In combination with CLSM, staining by CTC can be used as a fast method for monitoring the metabolic activity of chemoorganotrophic bacteria in monuments, buildings of historic interest or any art objects of natural stone. Due to the small size of samples required, the damage to these objects and buildings can be minimized. 相似文献
14.
Batch cultures of E. coli TB1/pUC13 were carried out at different oxygen transfer rates (OTR) enhanced by the increase of stirring rate and by the increase of air total pressure of the bioreactor. These two variables showed to have little effect on cell growth but a negative effect on cytochrome b5 (recombinant protein) production. However, this effect was more significant of high stirring rates than for values of pressure up to 0.4?MPa. The effects of stirring and pressure were also investigated for fed-batch mode operation. In this type of cell cultivation high cell densities are reached, thus a high capacity of oxygen supply of the system is required. To compare the two ways of improving OTR, cell behaviour was followed in two bioreactors at different operational conditions giving the same maximum OTR value. The first one operated at a high stirring rate (500?rpm) and at atmospheric pressure (0.1?MPa) and the other one at high air pressure (0.48?MPa) and low stirring rate. The increased pressure seemed to be a better way of ensuring an adequate oxygen supply to a culture of E. coli TB1 cells than an increased stirring rate. For the high pressure experiment a higher cellular density was reached, as well as a higher cyt.b5 expression which led to a 4-fold increase in final productivity. These experiments showed that bioreactor pressurization can be successfully used as a means of enhancing oxygen mass transfer to shear sensitive cell cultures. 相似文献
15.
Hideo Tanaka 《Biotechnology and bioengineering》1981,23(6):1203-1218
Using Cudrania tricuspidata cells as model plant cells which have high sensitivity to hydrodynamic stress, technology problems in the cultivation of the plant cells at high density were investigated. Using “shake” flasks on a reciprocal shaker and Erlenmeyer flasks on a rotary shaker and with a high supply of oxygen on order to obtain high cell densities in shaken cultures, particles breakdown and damage to the largest cell aggregate group (above 1981 μm in diameter) occurred and normal cell growth became impeded. The mass-transfer coefficient (K)for a model solid–liquid system (β-naphthol particles and water) in place of a system of plant cells and a liquid medium was proposed as an intensity index of hydrodynamic stress effects on plant cells in subsequent cultures under various conditions in the bioreactor systems. Normal cell growth was obtained under culture conditions for K values less than about 4.4 × 10?3 cm/sec. The characteristics of various bioreactors used until now were investigated by considering the three main technological factors (capacity of oxygen supply, intensity of hydrodynamic stress effects on plant cells, and intensity of culture broth mixing and air-bubble desperation). The most suitable bioreactor for culturing plant cells at high density was ajar fermentor with a modified paddle-type impeller (J-M). The yield of cell mass in the 10-liter J-M (working volume 5 liter) was about 30 g dry weight per liter of medium. 相似文献
16.
F. García Camacho E. Molina Grima A. Snchez Mirn V. Gonzlez Pascual Yusuf Chisti 《Enzyme and microbial technology》2001,29(10):602-610
The harmful effect of direct air sparging on Phaeodactylum tricornutum microalgal cultures was investigated in bubble columns and airlift photobioreactors with various superficial air velocities and two types of spargers which generated different sizes of bubbles. Small bubbles bursting at the surface of the culture were apparently the main cause of cell damage in batch cultures in laboratory-scale bubble columns. Other mechanisms of cell damage also were a contributing factor to the observed cell loss in outdoor pilot-scale bubble columns. Supplementation of the microalgal culture medium with carboxymethyl cellulose at concentrations of 0.02% and greater is shown to protect the algal cells against aeration-induced hydrodynamic stress. 相似文献
17.
Determination of the kinetic parameters of the phenol-degrading thermophile Bacillus themoleovorans sp. A2 总被引:1,自引:0,他引:1
H. Feitkenhauer S. Schnicke R. Müller H. Märkl 《Applied microbiology and biotechnology》2001,57(5-6):744-750
Phenolic compounds are pollutants in many wastewaters, e.g. from crude oil refineries, coal gasification plants or olive oil mills. Phenol removal is a key process for the biodegradation of pollutants at high temperatures because even low concentrations of phenol can inhibit microorganisms severely. Bacillus thermoleovorans sp. A2, a recently isolated thermophilic strain (temperature optimum 65 degrees C), was investigated for its capacity to degrade phenol. The experiments revealed that growth rates were about four times higher than those of mesophilic microorganisms such as Pseudomonas putida. Very high specific growth rates of 2.8 h(-1) were measured at phenol concentrations of 15 mg/l, while at phenol concentrations of 100-500 mg/l growth rates were still in the range of 1 h(-1). The growth kinetics of the thermophilic Bacillus thermoleovorans sp. A2 on phenol as sole carbon and energy source can be described using a three-parameter model developed in enzyme kinetics. The yield coefficient Yx/s of 0.8-1 g cell dry weight/g phenol was considerably higher than cell yields of mesophilic bacteria (Yx/s 0.40-0.52 g cell dry weight/g phenol). The highest growth rate was found at pH 6. Bacillus thermoleovorans sp. A2 was found to be insensitive to hydrodynamic shear stress in stirred bioreactor experiments (despite possible membrane damage caused by phenol) and flourished at an ionic strength of the medium of 0.25(-1) mol/l (equivalent to about 15-60 g NaCl/l). These exceptional properties make Bacillus thermoleovorans sp. A2 an excellent candidate for technical applications. 相似文献
18.
Accumulation of heavy metals in unicellular algae 总被引:2,自引:0,他引:2
19.
Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene 总被引:2,自引:0,他引:2
Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual. All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc. Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene. 相似文献
20.
《Mutation Research Letters》1983,119(3-4):361-369
Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual.All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc.Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene. 相似文献