纤维取向性对短纤维增强复合材料力学性能的影响

<正> 1.前言短纤维增强复合材料的增强机理经过Cool、Dow、Rosen和Hayashi几人采用单根短纤维模型已弄清。并对多根纤维模型进行了一些研究工作,但对它们的增强机理还难解释清楚。关于长纤维取向对纤维增强复合材料力学性能影响的研究,已有报导。本文通过实验阐明短纤维增强复合材料力学性能与纤维方向之间的关系。采用三种类型的试件:单向、双向和随机方向。从其拉伸实验结果可看出在不同的纤维方向上所得到的杨氏模量和拉伸强度有很大差别。断裂表面用扫描电镜观察,认为影响复合材料断裂的主要因素是纵向纤维的特性。此外,我们基于Cox理论的纤维排列模型证明计算结果与实验结果符合。

Fluid flow induced calcium response in bone cell network

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95-107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18 alpha-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE(2) or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18 alpha-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

分子标记技术在甘兰型杂交油菜育种上的研究与应用

本文应用RAPD分标记技术对我国重要的油料作物“杂油59”杂种种子的Fl代杂种的纯度进了技术鉴定，并完善了这一技术，摸索出这一适合于目前生产应用的实用方法，填补了这一技术在油菜作物应用上的空白。用RFLP技术对我国重要的雄性不育材料“陕2A”细胞质进行了分子水平的鉴定，为证明“陕2A”是一类新型的雄性不育材料提供了重要的实验证据。 对甘蓝型油菜采用DNA快速提取法、酚仿法和CTAB法应用于不同的分子标记分析，实验结果显示： CTAB法适用于样品量大，纯度要术高的RFLP技术，酚仿法适用于引物筛选、DNA模板用量大的PCR反应，而快速提取法特别适合于生产上对种子纯度检测，是生产上推广前景很好的实用技术。 对甘蓝型油菜RAPD技术应用当中PCR体系的建立进行了探讨。实验结果显示：热启动对PCR结果的影响至关重要。而Mg++浓度、dNTP浓度、模板浓度、Tag酶用量对反应结果有不同程度的影响。经过反复实验：当PCR各组分按Mg++，2mM;dNTP，200uM;模板浓度，50ng - lOOng时，PCR的结果最好。PCR反应条件经反复实验后确定为：第一个循环：（热启动）94℃，Imin20sec．OoC 2min循环一次;第二个循环：（解链）94℃ 50sec，（退火）40℃ Imin30sec;（延伸）72℃lmin;循环40次。第三个循环：72℃lOmin，循环1次。反应总体积为20ul时最为适用。 用40个lOmer的RAPD随机引物对“杂油59”的2个亲本“垦C8”和“陕3A” 进行RAPD分析，共出现290条带，分布于3530-220bp之间。引物opA-06、opK-03、opK-13、opj-12出现阳性扩增。经重复实验后确定： opK-03的PCR结果重复性最好，该引物序列为：CCAGCTTAGG。用它对两个亲本进行RAPD分析，PCR结果共出现9条带，其中510bp、260bp为二条特征带。在Fl代中这两条特征带重现性很好。用50个商品用种萌发的F1单株进行验证，检测结果为3个个体没有出现510bp的特征带，4个个体没有出现260bp的特征带，有5个个体出现了其它带，纯度为78%，与生产用种的纯度相符。 通过对“杂优59”不同生育时期及不同取样部位作酯酶同工酶电泳方法与RAPD方法相比较，结果显示：RAPD方法可以弥补同工酶方法的缺限。由于它是基于基因水平的分析技术，可以不受环境条件、发育时期、取材部位等客观条件的限制，并具有取样量小、易操作、费用低、灵敏度高、可以检测出亲缘关系相当近的种闾或种内的材料，具有独到的优点。是值得今后在生产上推广的新技术。 用6个雄性不育材料线粒体的特异探针：ALXR 18（线粒体ATPaseα亚基）;COB 640(脱辅基细胞色素-b);COX -I（细胞色素氧化酶亚基-I）;COX -Ⅱ（细胞色素氧化酶亚基-II;PDC - 12（胡萝卜线粒体随机片断）;C2（玉米线粒随机片断），对“陕2A”，Hybrides Polima，Ogura NSL 94/96, Ogura MLCH036, Ogura NSL, Polima, Fu27,Fu38, Anand等9个材料进行RFLP分析，结果显示：用限制性内切酶EcoR I消化后的DNA与探针COB 640杂交，“陕2A”材料在4.5 kb处缺失，与ALXR 18探针杂交，在4.4 kb、4.2 kb处也明显缺失，证明“陕2A”显然不同与其它不育材料。用ALXR l8为探针，与用内切酶Nc01的酶切片断作Sourthern杂交，在RFLP谱带上6.1 kb、2.4 kb、2.5 kb处明显缺带，进一步为“陕2A”是一种新型的甘蓝型油莱雄性不育系提供了证据。

Association of prostaglandin-endoperoxide synthase 2 gene polymorphisms with asthma and atopy in Chinese children

Background: Cyclooxygenase-2 (COX-2) plays essential roles in inflammation. Previous studies have suggested associations between prostaglandin-endoperoxide synthase 2 (PTGS2) polymorphisms and prostaglandins production in asthma. Objective: We have invest

Association of pro staglandin-endoperoxide synthase 2 (PTGS2) polymorphisms and Alzheimer's disease in Chinese

Cyclooxygenase-2 (COX-2, encoded by the gene prostaglandin-endoperoxide synthase 2, PTGS2) is a key enzyme in the conversion of arachidonic acid to prostaglandins. The prostaglandins produced by COX-2 are involved in inflammation and pain response in diff

Consumption of fa cai Nostoc soup: A Potential for BMAA exposure from Nostoc cyanobacteria in China?

Grown in arid regions of western China the cyanobacterium Nostoc flagelliforme - called fa cai in Mandarin and fat choy in Cantonese - is wild-harvested and used to make soup consumed during New Year's celebrations. High prices, up to $125 USD/kg, led to overharvesting in Inner Mongolia, Ningxia, Gansu, Qinghai, and Xinjiang. Degradation of arid ecosystems, desertification, and conflicts between Nostoc harvesters and Mongol herdsman concerned the Chinese environmental authorities, leading to a government ban of Nostoc commerce. This ban stimulated increased marketing of a substitute made from starch. We analysed samples purchased throughout China as well as in Chinese markets in the United States and the United Kingdom. Some were counterfeits consisting of dyed starch noodles. A few samples from California contained Nostoc flagelliforme but were adulterated with starch noodles. Other samples, including those from the United Kingdom, consisted of pure Nostoc flagelliforme. A recent survey of markets in Cheng Du showed no real Nostoc flagelliforme to be marketed. Real and artificial fa cai differ in the presence of beta-N-methylamino-L-alanine (BMAA). Given its status as a high-priced luxury food, the government ban on collection and marketing, and the replacement of real fa cai with starch substitutes consumed only on special occasions, it is anticipated that dietary exposure to BMAA from fa cai will be reduced in the future in China.

新型龙脑靶向非甾体类抗炎药的设计、合成和初步活性评价

非甾体类抗炎药（NSAIDs）是临床较常用的处方药，是高效的止痛、退热和抗炎药。NsADs有广泛的临床适应证，尤其适用于各种急、慢性关节炎，软组织风湿症、运动性损伤、头痛、痛经、拔牙后痛以及癌性疼痛等。因此，NsAIDs一直是世界上处方量最大的药物之一，包括我国在内的各国NSAIDs消耗量都呈明显上升趋势。仅疼痛控制部分，预计2007年就将达到300亿美元［l］。但是，大多数NSAIDs，尤其是我国目前使用的NSAIDs，都有较大的毒性和副作用。最新的分子生物学实验证明，各种NSAIDs起治疗作用的基础是通过抑制环氧合酶（cox），阻断致炎介质前列腺素类化合物的合成。环氧合酶至少包括两种同功酶（可能还有未被发现的新的亚类型），COX-1和COX-2。COX-1主要发挥生理性管家功能；COx-2主要为诱导型，在正常组织内活性极低，当受到某些细胞因子、促有丝分裂物质和内毒素刺激时大量表达，相应引起致炎介质的增加，使炎症加重。这些区别为设计兼具高抗炎活性和低毒性的药物提供了可能。阿司匹林是最早获得应用的NSAIDs，随后又出现一批其它NSADOs，最近美国FDA批准上市的罗非考昔，西乐葆是较好的COX-2选择性抑制剂，但是售价过于昂贵，从费用上考虑较难维持长期服用。至今我国还没有具有自主知识产权的COX-2特异性NSAIDs，服用的NSAIDs中，国外更新淘汰多年的毒副作用较大的药物仍占有较大比重，所以开发具有我们自主知识产权的新型NSAIDs迫在眉睫。鉴于COX-1，COX-2酶晶体结构明确，NSAIDs筛选模型确定，尤其是我国传统的中医药对炎症的独特认识，所以，凭借现代医学和化学知识，结合中国传统的中医药知识开发具有我们自主知识产权的新型NSAIDs是切实可行的。在研究过程中我们发现，在中国传统的中医药体系里，冰片是一种独特的药物，其主要成分为结构明确的龙脑、异龙脑。中医文献对其性质和应用详细的记载表明，其不仅广泛地用于抗风湿，而且其性质符合现代药物概念中的“靶向药物”概念。所以，如果合理设计使龙脑负载有抗炎结构化合物，可能会明显改善原药物的COX-2选择性，从而开发一种或几种COX-2特异性抑制剂。据此，又参考COX-2酶晶体结构，设计合成了其他结构的可能具有抗炎活性的药物分子。小茵香醇是龙脑和异龙脑的同分异构体和结构类似分子，我们选择该分子代替龙脑和异龙脑与布洛芬结合，并进行了活性测试和筛选，以深人了解该类分子。（1）设计并合成了含龙脑、异龙脑和小茵香醇结构的分子。相应结构见Tablel。（2）因为该类分子具有比较高的位阻，所以反应惰性较大，经过实验发现，在丁基锂／四氢吠喃反应体系或二环己基碳二亚胺仁甲氨基毗咙反应体系条件下，反应能够顺利进行。其中，二环己基碳二亚脚二甲氨基毗陡反应体系产率稍高。（3）针对几个主要反应详细探讨了分离纯化条件，为将来的放大实验或规模化制备提供了条件，并对所有产品进行了详细的结构表征。（4）通过“药物对小鼠腹腔巨噬细胞生成COX-1和COX-2的抑制作用”实验，对所获得的化合物进行了初步的活性评价，并得出如下结果：（a）在所设计的分子中，化合物3和化合物8分子表现出一定的COX-2选择性，其IC50COX-1／ IC50COX-2的比值分另为6.663和6.835，稍优于目前使用的第二代NSAIDs。见第七章Tablel，Table 2 and Figurel。（b）在所设计的分子中，化合物13，即布洛芬小菌香醇酷表现出最好的选择性；IC50COX-1／IC50COX-2比值为21.006，普遍超出目前使用的大部分NSAIDs，如果经过进一步的分子设计或修改有可能获得更好的结果。（c）化合物13与化合物9和化合物10相比较，选择性明显更好。即小茵香醇醋比龙脑酷和异龙脑醋表现出更好的靶向性。（d）从得到的结果可以看出，龙脑所负载的分子的选择性普遍比异龙脑要强，对于阿司匹林表现的尤为明显。（e）在中医药的文献和典籍中，关于小茵香醇性质和应用的记载很少，我们的研究结果表明它可能具有潜在的还不为我们所了解的特殊性质，我们的研究也可能会促进对该化合物的研究。（f）以佐剂关节炎模型对乙酰水杨酸龙脑醋进行了活性评价。

土壤低浓度PAHs胁迫下赤子爱胜蚓差异表达基因初步筛选

为探索土壤低浓度多环芳烃污染的生态毒性及其对土壤生物的致毒机理，本论文初步研究了菲、芘、荧蒽和苯并[a]芘等四种多环芳烃人工土壤污染在0.1mg.kg-1~10.0mg.kg-1浓度水平对赤子爱胜蚓（Eisenia fetida）产卵量、体重变化、排卵激素annetocin基因和翻译控制肿瘤蛋白（translationally controlled tumor protein, TCTP）转录水平的影响，发现在相同的低浓度水平下，只有苯并[a]芘对蚯蚓annetocin前体基因和TCTP基因的表达有显著影响，故其对生物体的生殖风险和致癌风险可能最大。另一方面，低浓度苯并[a]芘对蚯蚓体重和产茧量并无显著影响，这表明基因表达水平作为污染生态监测指标比宏观观测指标更灵敏。 为进一步研究土壤PAHs污染的生态毒性效应，在上述研究基础上，我们采用SSH-PCR的方法构建了人工土壤1.0mg.kg-1苯并[a]芘胁迫下的蚯蚓与对照组蚯蚓之间的消减cDNA文库，随机挑取上调文库301个克隆及下调文库283个克隆进行测序，与NCBI蛋白数据库比对结果表明，其中有391个克隆与已知的75种蛋白质基因显著匹配（期望值< 10-5），其余克隆匹配不显著（期望值> 10-5）或找不到匹配蛋白。显著匹配的基因序列包括：一相解毒酶细胞色素P450，二相解毒酶谷胱甘肽硫转移酶，蛋白质合成所需的核糖体蛋白亚基，参与新合成肽链折叠的热休克蛋白，线粒体基因组编码的呼吸链复合酶体亚基，过氧化物还原蛋白，铁蛋白，钙结合蛋白，半胱氨酸蛋白酶等。表明低浓度苯并[a]芘胁迫引起蚯蚓的生理变化是非常复杂的，涉及污染物降解与解毒、抗氧化保护、能量代谢、蛋白质合成、金属离子调节与蛋白质降解等过程。 Real-time PCR检测验证消减文库中部分差异基因对不同剂量BaP胁迫响应结果表明，各检验基因序列受1.0 mg∙kg-1 BaP 胁迫影响均与消减结果一致，且影响程度均高于0.1 mg∙kg-1浓度水平的BaP；其中，在0.1 mg∙kg-1 BaP胁迫下，过氧化物还原酶PRDX和类似Cyp2R1的P450基因表达未见明显变化。其余的HSP70、HSP90、rpL10、COXⅡ、SCBP、Ferritin等基因在0.1 mg∙kg-1 BaP胁迫组蚯蚓中均有检测到预期表达变化，说明虽然从消减文库中获得的基因在一定的污染物浓度范围内均表现浓度效应，但各个基因对污染物的响应浓度不尽相同。 Real-time PCR检测消减文库中部分差异基因对不同PAHs胁迫响应结果表明，1.0 mg∙kg-1 浓度水平的荧蒽、菲、芘和苯并[a]芘对差异表达基因的影响不尽相同，主要有以下三种情况：(1)广谱响应型：蚯蚓线粒体编码的亚基COXⅡ、可溶性钙结合蛋白、铁蛋白等基因对荧蒽、菲、芘及苯并[a]芘的胁迫均有相似的响应；(2)随芳烃环数而变化型：热休克蛋白HSP70和过氧化物还原酶PRDX表现出响应程度随胁迫多环芳烃的环数增加而提高的现象；(3)仅在苯并[a]芘中有响应型：核糖体蛋白亚基L10和细胞色素P450（类似Cyp2R1）基因，在1.0 mg∙kg-1浓度条件下，它们仅受BaP诱导表达，而芘、菲和荧蒽却没有显示诱导作用。 上述结果表明，在土壤中的低浓度的多环芳烃污染胁迫对蚯蚓的影响是多方面的，这些影响至少涉及能量代谢、污染物降解与解毒、蛋白质合成与修复、信号转导、细胞凋亡、排卵生殖、个体发育等多方面的生理功能。目前蚯蚓基因组还未有完整测序，本文论述的多个差异表达基因是首次在蚯蚓中发现的，这些新发现的基因序列在为低浓度PAHs的生态毒性机理研究提供依据的同时，也为以蚯蚓为模式生物的土壤污染生物监测提供了备选的生物分子标记。另一方面，由于蚯蚓基因组未完整测序，本研究构建的消减文库中仍不少未知功能基因，其功能与调控有待进一步研究。

辽北农田土壤农药残留特征及其对农产品安全影响

农药对产地环境，特别是对土壤的广泛污染严重威胁农产品安全和人类健康。因此，本文采用建立的除草剂和有机氯农药（OCPs）残留分析方法，开展了辽北地区土壤农药残留特征、阿特拉津和乙草胺田间消解动力学、土壤农药残留对农产品安全影响等方面研究。主要研究结果如下： 1. 分别建立了土壤、大米、蔬菜、玉米中3种除草剂和8种OCPs多残留分析方法。方法检出限介于0.04~1.30 ng•g-1之间；11种农药在0.01 (0.02)~1.0 (2.0) mg•L-1范围内线性良好，相关系数介于0.9963-0.9998之间；平均回收率介于71%-117%之间、相对标准偏差小于14.4%。 2. 阿特拉津和乙草胺在辽北农田土壤普遍残留；丁草胺、六氯苯、狄氏剂和艾氏剂在部分土壤有残留；乙草胺和丁草胺相对其它农药残留较高；阿特拉津、六氯苯、狄氏剂和艾氏剂残留量与相关报道和标准相比较低。除艾氏剂外，检出农药残留量经Box-Cox变换后，均服从正态分布。阿特拉津、乙草胺、丁草胺、六氯苯在不同土壤利用类型之间存在显著差异（P<0.05）。 3. 玉米地土壤中阿特拉津和乙草胺消解动态符合一级反应动力学模式，阿特拉津消解半衰期在12.2~59.8d之间，乙草胺在18.5~54.6d之间。喷施地阿特拉津和乙草胺消解速率约为对照地的2~5倍，且喷施量越大，消解越快。 4. 11种农药在辽北蔬菜、大米、玉米中残留较低，仅阿特拉津、六氯苯、乙草胺和丁草胺在部分农产品中有残留，其在土壤中残留通过蔬菜、大米和玉米给消费者带来的总膳食风险较低。大田试验进一步说明在试验区域喷施4倍最大推荐剂量阿特拉津或乙草胺也不会对玉米安全产生影响。

具有环氧化酶-2选择性抑制活性的多取代蒽醌的合成

1-甲基-2-甲氧羰基-3, 6, 8-三羟基-7-甲氧基蒽醌是从唐菖蒲干球茎中分离到的具有环氧化酶-2选择性抑制活性的多取代蒽醌类化合物。本文试图合成该化合物，实现了其类似物的合成，同时发现了几个未见报道的反应。 1．通过Diels-Alder 反应合成了关键中间体——3-甲基-5-羟基-1, 2, 4-苯三甲酸三甲酯，1-COOMe选择性水解产物与1, 2, 3-三甲氧基苯进行分子间Friedel-Crafts反应的产物再进行分子内Friedel-Crafts反应得到了目标产物的类似物1-甲基-2-甲氧羰基-3-羟基-6，7，8-三甲氧基蒽醌（路线1）。目标产物及其它类似物的合成正在进行中。 2．以乙酰乙酸甲酯和巴豆醛为原料，经过Michael加成、分子内的Aldol反应、芳香化、选择性甲酰化和还原反应，得到关键中间体2-甲基-3-羟甲基-6-甲氧基苯甲酸甲酯及其衍生物。通过该化合物与3，4，5-三甲氧基苯甲酸甲酯进行Friedel-Crafts烷基化反应得到了多取代的二苯基甲烷衍生物，拟进一步关环合成目标化合物（路线2）。 3．发现邻甲氧基苯甲酸甲酯中酯甲基可以被正丁基锂和仲丁基锂中烷基交换生成相应的酯，反应的机理不明确。当使用叔丁基锂时，得到的是邻甲氧基苯基叔丁酮，这个方法可以用来合成芳基叔丁酮类化合物。 4．以2-苄氧基-6-甲基苯甲酸甲酯为原料进行氯甲基化反应时，以苯和二氯乙烷作溶剂，发生了苄基的迁移和芳环的偶联，分别得到2，2'-二甲基-3，3'-二甲氧羰基-4，4'-二羟基联苯和2，2'-二甲基-3，3'-二甲氧羰基-4，4'-二羟基-5，5'-二苄基联苯。这是对称联苯合成的新方法。 5．水杨酸羟基邻对位的选择性甲酰化可以分别通过水杨酸和水杨酸甲酯用HMTA/CF3COOH来实现。 6．Lewis酸催化3，4，5-三甲氧基苄醇环化成1, 2, 3, 6, 7, 8, 11, 12, 13-nonamethoxyl-10,15-dihydro-5H-trbibenzo [a, d, g] cyclononene (NDTC)，产率(54%)高于已有方法（12%）。 Methyl 3,6,8-trihydroxy-7-methoxy-1-methylanthraquinone-2-carboxylate is a new COX-2 selective inhibitor isolated from Gladiolus gandavensis. Two strategies were investigated to synthesis this compound, in which some important reactions were discovered. 1. The key intermediate 5-hydroxy-3-methylbenzene-1,2,4-tricarboxylic acid 2,4-dimethyl ester was prepared via Diels-Alder reaction followed by selective hydrolysis of 1-COOMe. This compound was coupled with 1,2,3-trimethoxybenzene and the product undergo intramolecular Friedel-Crafts reaction to give methyl 3-hydroxy-5,6,7-trimethoxy-1-methylanthraquinone-2-carboxylate (1st route). The target compound and other analogues are being prepared with the same procedure. 2. The key intermediates methyl 3-hydroxymethyl-6-methoxy-2-methylbenzoate and its derivatives were prepared starting from crotonaldehyde and methyl acetoacetate via Michael addition, intramolecular aldol reaction, aromatization, formylation and reduction. The intermediates were coupled respectively with derivatives of gallic acid to give polysubstituted diphenylmethane. However, attempts to cyclize these compounds to the target compounds and analogues were not successful (2nd route). 3. In the process for ortho-lithiation of methyl 2-methoxybenzoate, the substrate converted respectively to n-butyl 2-methoxybenzoate and sec-butyl 2-methoxybenzoate when n-BuLi and sec-BuLi were used. However, tert-BuLi reacted with methyl 2-methoxybenzoate afford 2-methoxyphenyl tert-butyl ketone, which could be used to synthesize aryl tert-butyl ketones. 4. The transformtion of methyl 2-benzoxy-6-methylbenzoate to dimethyl 4,4'-dihydroxy-2,2'-dimethylbiphenyl-3,3'-dicarboxylate in benzene, and dimethyl 5,5'-dibenzyl-4,4'-dihydroxy-2,2'-dimethylbiphenyl-3,3'-dicarboxylate in 1,2-dichloroethane in the presence of ZnCl2 provides a new method for the synthesis of symmetric biphenyl. 5. The formylation of salicylic acid at C-5 and methyl 2-hydroxybenzoate at C-3 could be regioselectively realized by using HMTA/CF3COOH. 6. Racemic 1, 2, 3, 6, 7, 8, 11, 12, 13-nonamethoxyl-10, 15-dihydro-5H-trbibenzo [a, d, g] cyclononene was prepared via Lewis acids catalyzed trimerization of 3, 4, 5-trimethoxylbenzyl alcohol with yield (54%) higher than the reported procesure (12%).

人载脂蛋白APOA5的克隆表达纯化及相互作用蛋白研究

人类的载脂蛋白A5（apolipoprotein A5，APOA5）是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级，但能显著影响血浆三酰甘油水平，对血脂代谢具有重要意义，可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低，直接从血浆中分离纯化很困难，国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性，探讨其与TG代谢中的其它关键成分之间的相互关系，揭示其在脂类代谢相关疾病中的重要地位，必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术，诱导表达纯化APOA5蛋白，免疫动物制备多克隆抗体，为进一步研究人肝脏细胞中APOA5的相互作用蛋白，研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能，我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术，免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系，为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术，从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD，构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21（DE3），成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列，用镍离子亲和柱进行纯化和复性后，获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔，获得了高效价的兔抗人APOA5多克隆抗体，Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒，经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功，并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白，与预测的分子量APOA5（41 kD）/lamda cI （27 kD）一致。自激活实验证明诱饵蛋白不能单独激活报告基因，可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选，分离出10个阳性克隆。对结果进行生物信息学分析，得到7个与APOA5相互作用的蛋白，其中BI1为细胞凋亡调节因子；ATP6、CYTB、ND2、COX-1为线粒体表达蛋白； ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1，利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5，并验证其表达。通过免疫荧光细胞内共定位研究发现，靶蛋白APOA5主要分布于胞浆，与BI1在HEK293细胞有共定位，即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段，在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果，为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

Light alkenes preparation by the gas phase oxidative cracking or catalytic oxidative cracking of high hydrocarbons

The preparation of light alkenes by the gas phase oxidative cracking (GOC) or catalytic oxidative cracking (COC) of model high hydrocarbons ( hexane, cyclohexane, isooctane and decane in the GOC process and hexane in the COC process) was investigated in this paper. The selection for the feed in the GOC process was flexible. Excellent conversion of hydrocarbons ( over 85%) and high yield of light alkenes ( about 50%) were obtained in the GOC of various hydrocarbons including cyclohexane at 750 degreesC. In the GOC process, the utilization ratio of the carbon resources was high; CO dominated the produced COX (the selectivity to CO2 was always below 1%); and the total selectivity to light alkenes and CO was near or over 70%. In the COC of hexane over three typical catalysts (HZSM-5, 10% La2O3/HZSM-5 and 0.25% Li/MgO), the selectivity to COX was hard to decrease and the conversion of hexane and yield of light alkenes could not compete with those in the GOC process. With the addition of H-2 in the feed, the selectivity to COX was reduced below 5% over 0.1% Pt/HZSM-5 or 0.1% Pt/MgAl2O4 catalyst. The latter catalyst was superior to the former catalyst due to its perfect performance at high temperature, and with the latter, excellent selectivity to light alkenes ( 70%) and the conversion of hexane (92%) were achieved at 850 degreesC ( a yield of light alkenes of 64%, correspondingly).

《保护生态学:生物圈和生物存活》简介

<正> 保护生态学从1969年沿用开始,一直作为一个概念和术语,没有发展成为一门有明确研究对象、范畴和方法论的独立学科。直到1993年由George W.Cox编辑,由Wm.C.Brown Publishers出版的《保护生态学:生物圈和生物存活》(Conservation Ecology:Biosphere and Biosurvival)才奠定了保护生态学理论和实践的基础。该书中G.W.Cox将传统生态学与其它相关学科的相关知识结合起来,运用了保护生物学的理论和方法。全书短小精悍,结构明快,共352页分为三大部分。第一部分回顾了生态学的概念,介绍了生态学的发展历史。接着介绍了"绝灭生态学"(The ecology of extinction),作者回顾了过去及近代生物的绝灭率,绝灭物种的生活史足迹等,认为:近代物种绝灭率由于人