沙棘果汁及叶片中超氧物歧化酶的初步研究

采自辽宁省建平县沙棘果粗提液的材料,采用NBT光化学还原反应及凝胶电泳,研究其SOD活性,同工酶,紫外吸收光谱及其对红松叶和叶绿体类囊体膜冻干粉的有机自由基ESR波峰的抑制作用,证明沙棘果汁内确含有能消除O2-的超氧化物歧化酶(SOD),其研究结果为沙棘作为抗氧化剂、高层次开发利用提供了理论基础。

沙棘果汁及叶片中超氧物歧化酶的初步研究

采自辽宁省建平县沙棘果粗提液的材料 ,采用 NBT光化学还原反应及凝胶电泳 ,研究其 SOD活性 ,同工酶 ,紫外吸收光谱及其对红松叶和叶绿体类囊体膜冻干粉的有机自由基 ESR波峰的抑制作用 ,证明沙棘果汁内确含有能消除 O- 2 的超氧物歧化酶 (SOD) ,其研究结果为沙棘作为抗氧化剂、高层次开发利用提供了理论基础

植物冻害和氧化胁迫——甘蓝叶冻-融循环中超氧的产生

为检验植物冻害的发生和氧化胁迫这一假说 ,在冰冻前把氮蓝四唑 (NBT)真空渗入到甘蓝叶圆片中 .在叶圆片冻 融循环中NBT被还原为甲　 .把其中的单甲　用乙醇提取出来 ,在分光光度计上比色 ,可作为冻 融循环中产生的氧化胁迫的定量指标 .NBT本身作为氧化剂 ,使冻害稍有增加 .作为冰冻保护剂的二甲基亚砜真空渗入叶圆片使其抗冻性显著增加 ,而NBT还原则显著减少 ,表明二甲基亚砜在保护叶组织免受冻害上的作用和它减缓植物组织氧化胁迫的作用有关 .实验结果支持植物冻害的发生和氧化胁迫有关这一假说 .实验还表明还原NBT的还原剂很可能是超氧阴离子自由基 .

Comparative study of paclitaxel physically encapsulated in and chemically conjugated with PEG-PLA

Paclitaxel-loaded poly(ethylene glycol)-b-poly(L-lactide (LA)) (PEG-PLA) micelles were prepared by two methods. One is physical encapsulation of paclitaxel in micelles composed of a PEG-PLA block copolymer and the other is based on a PEG-PLA-paclitaxel conjugate, abbreviated as "conjugate micelles" Their physicochemical characteristics, e.g. critical micelle concentration (CMC), morphology, and micelle size distribution were then evaluated by means of fluorescence spectroscopy, scanning electron microscopy (SEM), and dynamic light scattering (DLS). The results show that the CMC of PEG-PLA-paclitaxel and PEG-PLA are 6.31 x 10(4) and 1.78 x 10(-3) g L-1, respectively. Both micelles assume a spherical shape with comparable diameters and have unimodal size distribution. Moreover, in vitro drug delivery behavior was studied by high performance liquid chromatography (HPLC). The antitumor activity of the paclitaxel-loaded micelles against human liver cancer H7402 cells was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method.

Triblock poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid)/paclitaxel conjugates: Synthesis, micellization, and cytotoxicity

A triblock poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) (PLA-PEG-PLA)/paclitaxel (PTX) conjugate was synthesized by the reaction of carboxyl-terminated copolymer PLA-PEG-PLA with PTX in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. Carboxyl-terminated copolymer PLA-PEG-PLA was prepared by the reaction of the hydroxyl end groups in copolymer PLA-PEG-PLA with succinic anhydride. Its structure was confirmed by NMR and gel permeation chromatography. The PLA-PEG-PLA/PTX conjugates could self-assemble into micelles in aqueous solutions with a low critical micelle concentration. Dynamic light scattering and environmental scanning electron microscopy analyses of the PLA-PEG-PLA/PTX micelles revealed their spherical structure and size of 220 nm. The antitumor activity of the conjugate against woman Hela cancer cells, evaluated by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide method, showed that the conjugates had an antitumor activity similar to that of pure PTX. The obtained PLA-PEG-PLA/PTX conjugates are expected to be used in clinical practice.

皱纹盘鲍血细胞内活性氧抗病机制及几种环境污染物及其影响的研究

本研究首次揭示了皱纹盘鲍(Haliotis discus hannai) 血细胞内存在着利用活性氧（Reactive oxygen species, ROS）的抗病机制。为今后我国研究贝类的活性氧抗病机制和筛选免疫药物提供了理论基础。本研究主要结果如下：1. 利用鲁米诺（Luminol, 3-氨基邻苯二甲酰肼）为依赖的化学发光法在体外条件下用不同刺激物（酵母细胞和酵母聚糖）对皱纹盘鲍血细胞进行刺激，测定血细胞吞噬活动中历经呼吸爆发产生活性氧的化学发光反应。结果表明皱纹盘鲍血细胞在体外条件下，经刺激物诱导吞噬活动中有明显的呼吸爆现象和很强的活性氧产生。不同有刺激物诱导血细胞产生的化学发光强度不同；同一刺激物的不同处理和不同浓度对血细胞产生活性氧的化学发光强度的影响不同。刺激物经皱纹盘鲍自体血清调理和未经调理对血细胞刺激所产生的化学发光强度不同。SOD和NaN_3对皱纹盘鲍血细胞吞噬过程中活性氧产生的化学发光有抑制作用。上述结果证明了皱纹盘鲍血细胞内存在有象高等动物血细胞内一样的MPO-H_2O-卤素系统的氧化性抗病机制，即在血细胞吞噬异物过程中能够释放活性氧（ROS）对异物进行杀灭的功能。2. 利用活性氧清除剂（过氧化氢酶、SOD、苯甲酸钠、2，5-二甲基呋喃、NBT和EDTA等）对皱纹盘鲍血细胞释放的活性氧进行分类测试，结果表明活性氧清除剂对皱纹盘鲍血细胞吞噬的化学发光都有明显的抑制作用，从而证明皱纹盘鲍血细胞能够释放的活性氧的种类有：超氧阴离子（O_2~-），过氧化氢（H_2O_2），羟自由基（OH~·）和单线态氧（~1O_2）。3. 在体外条件下利用化学发光法定量地研究了不同种类和不同浓度的农药：对硫磷（Parathion）、敌敌畏（Dichlorovos)、乐果（Dimethoate)、2，4-D 丁酯(2,4-D butylester)和甲胺磷（Methamidophos）对皱纹盘鲍血细胞氧化性抗病机制的影响。结果显示不同农药对皱纹鲍血细胞产生ROS的影响程度不同，及同一种农药的不同浓度的浸泡1h或浸泡12h处理皱纹盘鲍血细胞都能够抑制血细胞吞噬时的化学发光，表明农药能够抑制皱纹敌国鲍血细胞吞噬活动中的活性氧（ROS）的产生，而且这种抑制作用随着农药浓度的增加而加强。几种农药抑制皱纹盘鲍血细胞产生活性氧（ROS）的强度不同：2，4D-丁酯，敌敌畏和乐果的抑制作用强于对硫磷和甲胺磷。同时测定了不同浓度的农药浸泡12h后的皱纹盘鲍因细胞吞噬酵母细胞的吞噬百分率和吞噬指数，结果显示多数农药在低浓度时能够提高皱纹盘鲍血细胞的吞噬百分率和吞噬指数，而在高浓度时则能抑制血细胞吞噬酵母细胞的活力而降低血细胞的吞噬百分率的吞噬指数。4. 在体外条件下利用化学发光法定量地研究了不同种类和不同浓度的重金属：铭（Cr）、镉（Cd）、汞（Hg）、铅（Pb）、铜（Cu）和锌（Zn）对皱纹盘鲍血细胞氧化性抗病机制的影响。结果显示不同种类和不同浓度的重金属浸泡1hr.处理皱纹盘鲍血细胞都不同程度地抑制了血细胞吞噬时的化学发光，表明重金属能够抑制皱纹盘鲍血细胞吞噬活动中的活性氧（ROS）的产生，而且这种抑制作用随着重金属浓度的增加而加强。不同的重金属抑制强度不同，从强到弱依次为Hg > Cd > Cu > Pb > Cr > Zn. 研究表明六种重金属中，Hg对皱纹盘鲍血细胞的免疫毒性最大。

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

中国对虾血细胞体外吞噬活动中活性氧产生的研究

该文首先对中国对虾血细胞体外短时培养条件进行了摸索,以期为后来在体外研究中国对虾血细胞吞噬活动中ROS的产生建立基础.在上在实验的基础上,该文利用化学发光法对中国对虾血细胞体外吞噬过程中ROS的产生进行了研究,试图了解血细胞吞噬活动中的化学发光现象.最后利用NBT还原法研究了中国对虾血细胞体外吞噬过程中O<'-><,2>的产生化及一些环境污染物如重金属离子和农药对其吞噬活动中O<'-><,2>的产生影响.

Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection

The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was Studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis Of L-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F chinensis increased slightly from 0 to 12 h postchallenge, indicated by the variations Of L-Citrulline (from 11.15 +/- 0.10 to 12.08 +/- 0.64 mu M) and total nitrite/nitrate concentrations (from 10.45 +/- 0.65 to 12.67 +/- 0.52 mu M). Then it decreased sharply till the end of the experiment (84 h postchallenge), the concentrations Of L-Citrulline and total nitrite/nitrate at 84 It were 1.58 +/- 0.24 and 2.69 +/- 0.70 mu M, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72 It postchallenge, the concentrations Of L-Citrulline and total nitrite/nitrate increased from 7.82 +/- 0.77 at 0 h to 10.79 +/- 0.50 mu M at 72 h, and from 8.98 +/- 0.43 at 0 h to 11.20 +/- 0.37 mu M at 72 h, respectively. Then it decreased till the end of the experiment (216 h postchallenge), and the concentrations of L-Citrulline and total nitrite/nitrate at 216 h were 5.66 +/- 0.27 and 4.68 +/- 0.16 mu M, respectively. More importantly, an apparent increase of I-PS-stimulated NOS activity was observed in M japonicus at 48 h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the Cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, Whose accumulative mortality reached 100% after 240 h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F chinensis and M. japonicus to WSSV infection. (c) 2005 Elsevier Inc. All rights reserved.

Bis(2,3,5-triphenyltetrazolium) tetrachloridocadmate(II)

The title compound, ( C19H15N4)(2)[ CdCl4], a salt comprising two 2,3,5-triphenyl-substituted tetrazolium cations and a tetrachloridocadmate(II) anion, was synthesized by hydrothermal methods. In the anion, the Cd-II ion is tetrahedrally coordinated by four chloride anions. In the crystal structure, four cations and two anions pack into inversion-related subunits linked by C-H center dot center dot center dot Cl and offset pi-stacking interactions.Each of these subunits is surrounded by six others. Intermolecular pi-pi stacking interactions between phenyl rings are observed along the a axis, with perpendicular distances between the ring planes of 3.6015 and 3.6934 angstrom.