一株放线菌发酵产物抗肿瘤活性的研究

本学位论文主要研究一株放线菌发酵产物的抗肿瘤活性。先对该株放线菌进行活化培养，然后进行大批量发酵，发酵液经过冷冻离心，对离心得到的沉淀和上清液用不同极性的有机溶剂进行萃取，得到六个浸膏样品。对六个样品进行初步抗肿瘤活性检测。 然后对活性浸膏进行分离纯化和活性跟踪。本论文主要进行了如下的工作： 1、对菌种进行活化培养，利用该菌株在280C，200r.min-1条件下进行发酵实验，发酵时间为72h,发酵总量为15L。发酵液经过离心得到上清液和沉淀两部分。 2、分别用石油醚、乙酸乙酯、正丁醇萃取沉淀和上清液，得到编号为1—6的六个浸膏样品，对六个浸膏样品进行初步的细胞毒性和抗HepG2肿瘤活性实验，得出结论为5号样品活性最高。在没有分离纯化的情况下GI50达到0.76µg/mL。 3、对5号样品进行TLC实验，找出能够较好分离5号样品中各组分的溶剂组合，最后得出在氯仿：甲醇=8：1时分离效果较好。然后利用氯仿：甲醇=8：1的溶剂组合作为洗脱剂对5号样品进行过硅胶柱分离纯化并进行活性跟踪分离。 4、对分离纯化后得到的样品进行活性跟踪和结构分析。分离后得到样品A，在其浓度为10µg/ml时，抗肿瘤实验细胞的生长率为73.5%。在浓度为1.0mg/ml时，抗单纯疱疹病毒率（HSVⅡ）为74.5%。结构分析得知其分子式最可能为C41H43N8O4. This dissertation studied about the anti-tumor activity of an actinomycete fermentation product. First, we cultured the actinomycete. Second, we fermented it in large quantities, and then centrifuged the fermentation fluid; the next step is that we extract sedimentation and supernatant in different polar organic solvents, in turn to obtained six samples, which were detected about anti-tumor activity. Last, we purified active sample and tracked activity of it. We carried out the following research work: 1. Activation, culture and screening of the actinomyces was carried on. We used the screening strain to carry on the fermentation when the conditions are 280C，200r.min-1,the fermentation time is 72h. Fermentation fluid volume is 15L.And we obtained sedimentation and supernatant after fermentation fluid was centrifuged. 2. We used Petroleum ether, ethyl acetate, n-butanol separately to extract sedimentation and supernatant, and obtained six samples that were numbered 1-6. From the preliminary cell toxicity and the anti-tumor（HepG2） bioactivity experiment, we found that No.5 sample has the highest activity in the samples; the GI50 was 0.76µg/ml which has not been purified. 3. We Carried on TLC experiment on the No.5 sample, found the solvent composition that can separate each component of the No.5 sample. At last, we found that when the proportions are tri-chloromethane: methyl alcohol = 8:1, the Separation result was the best, and then we used the Solvent composition which proportion are tri-chloromethane: methyl alcohol = 8:1 as eluant to Purify No.5 sample by silica gel column. 4. We tracked the activity of pure sample obtained from Purification and analyzed structure of these substances. We got a compound A after separation, and the cell growth rate was 73.5% when its concentration was 10µg/ml. The anti-virus（HSVⅡ） rate was 74.5% when its concentration was 1.0mg/ml. We analyzed the Structure of A, and informed its molecular formula that was the most likely for C41H43N8O4.

红发夫酵母发酵虾青素的研究

红发夫酵母分离于北美西部高山地区和日本一些岛屿上落叶树的渗出液中，因其所产主要色素为在水产养殖、食品和医药工业有广阔应用前景的虾青素而成为研究的热点。本论文对红发夫酵母Phaffia rhodozyma 的生长特性、培养参数与培养基组分对生长和虾青素积累的影响及其优化、虾青素合成的调节控制、虾青素的提取测定及红发夫酵母耐高温菌种的诱变进行了系统的研究。 虾青素是红发夫酵母的胞内色素，要对其进行分析首先要对红发夫酵母进行破壁处理，实验发现二甲亚砜是最有效的破壁溶剂，用氯仿和丙酮可以有效地把类胡萝卜素从二甲亚砜破壁后的红发夫酵母细胞中提取出来。 在固定摇床转速为200 rpm，温度为20 ℃的条件下，当种龄为36 h，以10％的接种量接入装液量为30 mL的250 mL三角瓶，初始pH为5.5时最有利于红发夫酵母的生长及类胡萝卜素的合成。 本实验中红发夫酵母最佳利用碳、氮源分别为蔗糖和蛋白胨，但蛋白胨价格昂贵，不适宜作单一氮源，因此使用硫酸铵和酵母膏作为复合氮源。 本论文采用了BP神经网络结合遗传算法的方法来优化红发夫酵母的发酵培养基，得到红发夫酵母发酵培养基的最佳配比为：蔗糖45.10 g/L、硫酸铵3.00 g/L、硫酸镁0.80 g/L、磷酸二氢钾1.40 g/L、酵母膏3.00 g/L、氯化钙0.50 g/L，使用优化后的培养基发酵类胡萝卜素产量达到8.20 mg/L，干重达到9.47 g/L，类胡萝卜素的产量比起始培养基提高了95.90%，干重提高了89.40%。 从代谢途径出发对红发夫酵母合成虾青素调控调控，选择谷氨酸、乙醇、VB1作为添加剂，通过正交试验设计得出三者添加水平分别为0.2 g/L，0.1% (V/V)，10 mg/L时，类胡萝卜素产量提高了25.73%，达到了10.31mg/L。 通过上述优化培养，本论文中红发夫酵母的虾青素产量从1.33 mg/L提高到9.12 mg/L，产量提高了6.86倍；总类胡萝卜素产量从4.23 mg/L提高到10.31 mg/L，产量提高了2.44倍；细胞干重从5.00 g/L提高到11.35 g/L，提高了2.27倍，总体提高效果显著。 红发夫酵母属于中低温菌，本论文采用紫外复合诱变的方式，通过高温筛选，得到一株能在35 ℃下能生长的突变株，但所产类胡萝卜素中虾青素所占比例很小，可能是诱变改变了红发夫酵母的代谢途径，阻断了虾青素的合成。 Phaffia rhodozyma is a heterobasidiomyceteous yeast that was originally isolated from the slime fluxes of brich tree wounds in mountain regions of northern Japan and southern Alaska. Phaffia rhodozyma produces astaxanthin as its principal carotenoid pigment, which has potential applications in acquaculture, food and pharmaceutical industry. This paper researched ways to break cell, analysis of astaxanthin, characteristics of growth, culture parameters and the effects of components of medium on growth and astaxanthin formation , optimization of culture medium, control of astaxanthin synthesis and mutagenesis of Phaffia rhodozyma. It is necessary to disrupt the yeast cell for extracting astaxanthin considering the yeast accumulating carotenoids in cell. Dimethyisulphoxide was the most effective solvent for breaking the yeast cell; acetone and chloroform were effective to extract carotenoids out of the disrupted cell. The optimum pH for growth and carotenoids synthesis is 5.5, the optimum medium volume is 30 mL (in 250 mL flask), the optimum culture time of inoculum is 36 h, the optimum inoculum concentration is 10%. The research on culture medium showed: sucrose is the best one of 6 carbon sources for growth and astaxanthin synthesis. Peptone is the best nitrogen source for growth and astaxanthin synthesis. Uniform Design was used for trial design of the formula medium components, then back-propagation neural network was established to modeling the relationships between the carotenoid yield and the concentration of medium components. Genetic algorithm (GA) was used for global optimization of the model. The optimum combination of the medium was obtained: sucrose 45.10 g/L, ammonium sulfate 3.00 g/L, magnesium sulfate 0.80 g/L, potassium dihydrogen phosphate 1.40 g/L, yeast extract 3.00 g/L, calcium chloride 0.50 g/L. The yield of carotenoid reached 8.20 mg/L, which was 95.90% higher than that of the original medium. Glu, VB1 and ethanol were selected as fermentation addictives, after Orthogonal Test, the carotenoid contents increased by 25.73% when adding 0.16 g/L Glu, VB1 10 mg/L and ethanol 0.1% (V/V). After the above optimization, the astaxanthin content increased 6.86 folds, which is 9.12 mg/L. The carotenoids content increased 2.44 folds, which is 10.31 mg/L. The biomass increased 2.27 folds, which is 11.35 g/L. Phaffia rhodozyma grows in the mild temperature range of 0 to 27 ℃, in this work, a thermotolerant mutant was selected through UV-irradiation. It can grows at 35 ℃, and showed increased carotenoid content. The optimal growth temperature for this mutant is 30 ℃. But the mutant can only produce carotenoids with little astaxanthin accumulation.

Study of the interaction of highly charged ions with SiO2 surface

Highly charged ions (HCIs) AO(q+)/Pbq+ are extracted from ECR source and impacted on solid surface Of SiO2 Sputtering yield as a function of incident angle is measured by multi-channel plate (MCP). The results have been fitted by a new formula. We proposed the cooperation model to explain the formula. The results demonstrate that the potential assisted kinetic sputtering yield increases with the charge state and potential sputtering (PS) could be induced by impact of HCIs. At larger incident angles, the sputtering yield is dominated by elastic collision between HCIs and material atoms. It is found that, smaller the incident angle, larger the contribution from the potential sputtering. (C) 2009 Elsevier B.V. All rights reserved.

Sputtering induced by Arq+ impact on Nb surface

The relative sputtering yield induced by highly charged Arq+ impacting on Nb surface is investigated. The yield increases drastically as the incidence angle increases. A formula Y=A* tan(B) (theta) + C, developed from classical sputtering theory, fits well with the yield. By analysing a series of coefficients A and C extracted by curve fitting, the results demonstrate the presence of a synergy of the linear cascade collision and potential energy deposition.

Half-Lives of Superheavy Nuclei in Z=113 Alpha Decay Chain

The generalized liquid drop model (GLDM), including the proximity effects and centrifugal potential, and the cluster model with Cosh potential are used to study the half-lives of some Z=113 isotopes and their alpha-decay products.The experimental half-lives of (284)113, (283)113, (282)113and their alpha-decay products are well reproduced by the two models when zero angular momenta transfer is assumed. For (278)113 and its alpha-decay products, both the GLDM andthe cluster model could provide satisfactory results if we assume the alpha particle carry five units of angular momenta, which indicates that possible non zero angular momenta transfer and need further experimental measurements with high precision. Finally, we show that half-lives of alpha-decay are quite sensitive to the angular momentum transfers, and a formula could be used to describe the correlation between alpha-decay half-life and angular momentum transfer successfully.

Single particle properties in asymmetric nuclear matter and TBF rearrangement effect

We have developed the formula and the numerical code for calculating the rearrangement contribution to the single particle (s.p.) properties in asymmetric nuclear matter induced by three-body forces within the framework of the Brueckner theory extended to include a microscopic three-body force (TBF). We have investigated systematically the TBF-induced rearrangement effect on the s.p. properties and their isospin-behavior in neutron-rich nuclear medium. It is shown that the TBF induces a repulsive rearrangement contribution to the s.p. potential in nuclear medium. The repulsion of the TBF rearrangement contribution increases rapidly as a function of density and nucleon momentum. It reduces largely the attraction of the BHF s.p. potential and enhances strongly the momentum dependence of the s.p. potential at large densities and high-momenta. The TBF rearrangement effect on symmetry potential is to enhances its repulsion (attraction) on neutrons (protons) in dense asymmetric nuclear matter.

Systematic study on alpha decay half-lives of superheavy nuclei

The a-decay half-lives of a set of superheavy nuclear isotope chain from Z = 105 to 120 have been analyzed systematically within the WKB method, and some nuclear structure features are found. The decay barriers have been determined in the quasi-molecular shape path within the Generalized Liquid Drop Model (GLDM) including the proximity effects between nucleons in a neck and the mass and charge asymmetry. The results are in reasonable agreement with the published experimental data for the alpha decay half-lives of isotopes of charge 112, 114, and 116, of the element 294118 and of some decay products. A comparison of present calculations with the results by the DDM3Y effective interaction and by the Viola-Seaborg Sobiczewski (VSS) formulae is also made. The experimental a decay half lives all stand in between the GLDM calculations and VSS formula results. This demonstrates the possibility of these models to provide reasonable estimates for the half-lives of nuclear decays by a emissions for the domain of SHN. The half-lives of these new nuclei are thus well tested from the reasonable consistence of the macroscopic, the microscopic, the empirical formulae and the experimental data. This also shows that the present data of SHN themselves are consistent. It could suggest that the present experimental claims on the existence of new elements Z = 110 similar to 118 are reliable. It is expected that greater deviations of a few SHN between the data and the model may be eliminated by further improvements on the precision of the measurements.

Study on the localization of the electric dipole sources

The research of dipole source localization has great significance in both clinical research and applications. For example, the EEG recording from the scalp is widely used for the localization of sources of electrical activity in the brain. This paper presents a closed formula that describes the electric field of dipoles at arbitrary position, which is a linear transformer called the transfer matrix. The expression of transfer matrix and its many useful characteristics are given, which can be used for the analysis of the electrical fields of dipoles. This paper also presents the closed formula for determining the location and magnitude of single dipole or multi-dipoles according to its electrical field distribution. A calculation result for a single dipole shows that the dipole will be located at the midpoint of a line segment if there are equivalent fields at its two ends.

Study of mass distributions of quasifission products based on dinuclear system

In the concept of dinuclear system, the quasifission rate from Kramers formula has been incorporated in the master equation in order to study the competition between fusion and qusifission. Mass yields of quasifission products of the three reactions Ca-48 + Pu-244, Ca-48 + U-238 and Fe-58 + Th-232 have been calculated, and the experimental data are reproduced very well, which is a critical test for the existing fusion model. Also we have shown the time evolution of the mass distributions of quasifission products, which provides valuable information of the process of competition between fusion and quasifission.

Properties of alpha-decay to ground and excited states of heavy nuclei

Branching ratios and half-lives of alpha-decay to the ground-state rotational bands as well as the high-lying excited states of even-even nuclei have been calculated in the framework of the generalized liquid drop model (GLDM) and Royer's formula that we improved very recently. The calculation covers the isotopic chains from Ra to No in the mass regions 222 <= A <= 252 and 88 <= Z <= 102. The agreement between the calculated results and the experimental data indicates the reliability of investigating the properties of the unfavored alpha-decay with our method, especially the improved Royer's formula, which is very valuable for the analysis of experimental data. In addition, the dependence of half-lives on excitation energies of daughter nuclei has been investigated. It is shown that the influence on half-lives becomes stronger and stronger with the increase of the excitation energies.

Competing fusion and quasifission reaction mechanisms in the production of superheavy nuclei

Within the framework of a dinuclear system model, a new master equation is constructed and solved, which includes the relative distance of nuclei as a new dynamical variable in addition to the mass asymmetry variable so that the nucleon transfer, which leads to fusion and the evolution of the relative distance, which leads to quasifission (QF) are treated simultaneously in a consistent way. The QF mass yields and evaporation residual cross sections to produce superheavy nuclei are systematically investigated under this framework. The results fit the experimental data well. It is shown that the Kramers formula gives results of QF, which agree with those by our diffusion treatment, only if the QF barrier is high enough. Otherwise some large discrepancies occur.

Fine structure of alpha decay to rotational states of heavy nuclei

To gain a better insight into alpha-decay fine structure, we calculate the relative intensities of alpha decay to 2(+) and 4(+) rotational states in the framework of the generalized liquid drop model (GLDM) and improved Royer's formula. The calculated relative intensities of a decay to 2(+) states are in good agreement with the experimental data. For the relative intensities of alpha decay to 4(+) states, a good agreement with experimental data is achieved for Th and U isotopes. The formula we obtain is useful for the analysis of experimental data of alpha-decay fine structure. In addition, some predicted relative intensities which are still not measured are provided for future experiments.

alpha-decay half-lives and Q(alpha) values of superheavy nuclei

The alpha-decay half-lives of recently synthesized superheavy nuclei (SHN) are investigated by employing a unified fission model (UFM) where a new method to calculate the assault frequency of alpha emission is used. The excellent agreement with the experimental data indicates the UFM is a useful tool to investigate these alpha decays. It is found that the alpha-decay half-lives become more and more insensitive to the Q(alpha) values as the atomic number increases on the whole, which is favorable for us to predict the half-lives of SHN. In addition, a formula is proposed to compute the Q(alpha) values for the nuclei with Z >= 92 and N >= 140 with a good accuracy, according to which the long-lived SHN should be neutron rich. Several weeks ago, two isotopes of a new element with atomic number Z = 117 were synthesized and their alpha-decay chains have been observed. The Q(alpha) formula is found to work well for these nuclei, confirming its predictive power. The experimental half-lives are well reproduced by employing the UFM with the experimental Q(alpha) values. This fact that the experimental half-lives are compatible with experimental Q(alpha) values supports the synthesis of a new element 117 and the experimental measurements to a certain extent.