Cancer classification based on direction-basis-function neural networks

Automatic molecular classification of cancer based on DNA microarray has many advantages over conventional classification based on morphological appearance of the tumor. Using artificial neural networks is a general approach for automatic classification. In this paper, Direction-Basis-Function neuron and Priority-Ordered algorithm are applied to neural networks. And the leukemia gene expression dataset is used as an example to testify the classifier. The result of our method is compared to that of SVM. It shows that our method makes a better performance than SVM.

MAPK在天花粉蛋白抗HIV-1中的作用以及syncytin基因在白血病细胞中的表达

本学位论文主要包括两部分的内容： 一是关于MAPK信号转导在天花粉蛋白（trichosanthin, TCS）抗人类免疫缺陷病毒-1（HIV-1）中的作用的研究。TCS是I型核糖体失活蛋白（RIP），分子量27Kd，可从传统的中期流产和抗绒癌中药栝楼根块茎（天花粉）中提纯获得。该蛋白具有抗HIV-1活性，但其机制尚不清楚。本文用JNK抑制剂CEP-11004，预处理宿主细胞，检测其对TCS抗HIV-1的影响。用以下两种方法检测病毒的复制：一是用ELISA方法检测细胞培养上清中p24抗原的水平，二是检测上清中病毒粒子的逆转录酶（RT）活性。结果显示，TCS剂量依赖性地抑制HIV-1在C8166细胞中的复制。在TCS实验浓度下，HIV-1的复制水平平均为68 ± 4％（p24抗原检测）和52 ± 4％（RT活性检测）。如果用0.4μM CEP-11004对C8166细胞预处理2小时，TCS似乎失去了抗HIV-1活性，HIV-1的复制水平分别恢复为101 ± 4％和101 ± 7％。但无论是p24抗原检测还是RT活性检测，当不含TCS时，CEP-11004预处理病毒宿主细胞，本身并不影响病毒粒子的复制。这说明CEP-11004能够拮抗TCS的抗病毒活性，或者说CEP-11004抑制的信号转导途径的某些信号分子，与TCS的抗HIV-1活性相关。Western Blot方法检测的结果也证明，TCS能够以时间依赖和剂量依赖的方式激活JNK激酶，0.4μM的CEP-11004能有效抑制JNK的磷酸化。因此，TCS与它激活MAPK信号转导途径有关。 二是关于人类内源性病毒HERV-W家族囊膜蛋白基因syncytin在白血病细胞中的表达的研究。该基因在人的胎盘组织中特异性表达，可能与合胞滋养层的形成有关。另外也少量表达于睾丸组织。本论文采用实时定量RT-PCR的方法证明，syncytin能够在白血病细胞系中表达。进一步的检测还表明，syncytin的mRNA也表达于白血病/淋巴瘤患者的外周血细胞，而不表达于作为对照的10名健康志愿者的血细胞。在15名不同类型的白血病/淋巴瘤患者中，有11名有syncytin的表达。细胞系的表达相对稳定，与C8166细胞系的表达量相比较，介于0.5-2.0倍之间；而在白血病患者外周血细胞中的表达则介于0.8-21.7倍不等。上述结果提示，syncytin可能与白血病的形成有关。

合胞素在白血病和淋巴瘤细胞中的表达及意义研究

人类内源性病毒(HERVs)是远古具有感染能力的逆转录病毒整合于人类基因组的遗迹, 大约占人类基因组的3%~8%，至少包括31个家族。它们通过不同的途径参与人类各种生理和病理活动的调节过程。大多数人类内源性病毒的基因, 由于基因的突变或部分缺失而失去了转录的能力。但有些基因依然保持完整的开放读码框, 能翻译成有功能的蛋白, 如HERV-W家族的囊膜蛋白合胞素基因, 可以被转录并翻译成有功能的蛋白质。目前合胞素的生理功能以及在各种病理过程中的作用机制仍然不清楚。国内对于合胞素的功能研究处于起步阶段，尚无商品化试剂，而且也没有合胞素抗体制备的报道。我们通过PCR扩增人合胞素基因编码区的DNA片段，将其克隆入原核表达质粒pET30a (+)，转化大肠杆菌 BL21，诱导产生了合胞素-His融合蛋白。采用割胶回收的方法纯化目的蛋白, 免疫新西兰白兔, 制备了多克隆抗体。最后通过ELISA、Western-Blot和免疫组织化学等方法检测抗体的效价和特异性。我们成功表达并纯化了合胞素-His融合蛋白, SDS-PAGE分析表明融合蛋白主要以包涵体形式存在；ELISA法测定抗体效价为 1:10 000；Western-Blot和免疫组织化学结果显示所制备的抗体能特异性识别合胞素蛋白，为下一步研究合胞素的生物学功能奠定了基础。我们首次发现，合胞素能够在白血病和淋巴瘤细胞系中表达。利用我们制备的抗合胞素多克隆抗体，我们进一步检测该基因是否在白血病和淋巴瘤患者的外周血中表达，所有患者的外周血标本，均来自云南省有关医院。作为对照，我们还检测了20 名健康志愿者的外周血细胞。实验证明，合胞素基因(包括 mRNA 和蛋白)也在白血病患者的外周血细胞表达，而不表达于健康志愿者的血细胞。在30 名不同的白血病和淋巴瘤患者中，有22 名有合胞素的表达。荧光实时定量RT-PCR 的方法比较了合胞素在细胞系和白血病患者外周血中表达的相对定量，发现合胞素基因在所检测的5 种淋巴细胞系、3 种粒细胞系和1 种淋巴瘤细胞系中都有相对稳定的表达，表达水平与C8166 细胞系相比，介于0.5-2 倍之间。而在白血病患者中的表达则介于1.8-33.4 倍不等。我们的结果提示，合胞素可能与白血病的形成有关，因为该基因表达的囊膜蛋白具有很强的促细胞融合活性，含有具有免疫抑制活性的肽段，而且已发现某些与其类似的蛋白有致瘤能力。

中药蜘蛛香的化学成分研究及以羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞研究

本论文由三章组成。第一章介绍了中药蜘蛛香的化学成分的研究成果，第二章为羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞的研究，第三章综述了人工细胞在生物医学领域的应用。 第一章报道了中药蜘蛛香（Valeriana wallichii）根部乙醇提取物的化学成分，采用正、反相硅胶层析等分离方法和MS、NMR等多种波谱手段，从中共分离鉴定出17个化合物，分别为缬草素(valtrate，1)，valechlorine（2），homobadrinal（3），baldrinal（4），乙酰缬草素(acevaltrate 5)，valeriotetrate C（6），valeriotetrate B（7），对羟基苯乙酮(4'-hydroxy-acetophenone 8)，7-hydroxy valtrate（9），8-methylvalepotriate（10），1,5-dihydroxy-3,8-epoxyvalechlorine A（11），二氢缬草素(didrovaltrate 12)，胡萝卜苷（13），橙皮苷 (hesperidin 14)，prinsepiol-4-O-β-D-glucopyranoside（15），longiflorone（16），乙基糖苷（17）。其中化合物6、7、10、和11为新化合物，化合物9、15、16为首次从该植物中得到。新化合物11为含有氯原子的刚性骨架环烯醚萜，并且确定了其绝对构型。 第二章报道了以羧甲基魔芋葡苷聚糖（CKGM）和壳聚糖（CS）为膜的固定化L-天冬酰胺酶人工细胞研究成果。利用羧甲基魔芋葡苷聚糖和壳聚糖两种生物相容性很好的天然多糖之间的静电吸引力，在非常温和的条件下制备成具有半透过性膜的人工细胞，将治疗儿童急性成淋巴细胞性白血病(ALL)的药物L-天冬酰胺酶包裹在内。通过考察温度和pH对人工细胞的影响,结果表明以CKGM- CS为膜的L-天冬酰胺酶人工细胞对温度和pH的稳定性和耐受性均高于自由酶，说明CKGM-CS对酶具有保护作用，而且小分子底物和产物可以自由进出膜内外，而包裹在膜内的生物大分子则不能泄露出来。 第三章综述了微囊化人工细胞的研究进展。 This dissertation consists of three parts. In the first part, the chemical constituents from the root of Valeriana wallichii were reported. In the second part, preparation and characteristics of L-Asparaginase Artificial cell were reported. The third part is a review on progress of microcapsule artificial cell. The first chapter is about the isolation and identification of the chemical constituents from the root of V. wallichii. Seventeen compounds were isolated from the ethanol extract of roots of V. wallichii through repeated column chromatography on normal and reversed phase silica gel. By the spectroscopic and chemical evidence, their structures were elucidated as valtrate (1), valechlorine (2), homobadrinal (3), baldrinal (4), acevaltrate (5), valeriotetrate C (6), valeriotetrate B (7), 4'-hydroxy-acetophenone (8), 7-hydroxy valtrate (9), 8-methylvalepotriate (10), 1,5-dihydroxy-3,8-epoxyvalechlorine A (11), didrovaltrate (12), daucosterol (13), hesperidin (14), prinsepiol-4-O-β-D-glucopyranoside (15), longiflorone (16), and ethyl glucoside (17). Among them, 6, 7, 10, and 11 are new compounds. 15, 16 and 9 were isolated from this plant for the first time. The absolute configuration of compound 11, an unusual iridoid bearing a C-10 chlor-group and an oxo-bridge connecting C-3 and C-8 resulting in a rigid skeleton, was confirmed. The second chapter is about the semi-permeable microcapsule of carboxymethyl konjac glucomannan-chitosan for L-asparaginase immobilization. Carboxymethyl konjac glucomannan-chitosan (CKGM-CS) microcapsules, which have good biocompatibility, prepared under very mild conditions via polyelectrostatic complexation, were used for immobilize L-asparaginase-a kind of drug for acute lymphoblastic leukemia (ALL). The activity and stability under different temperature and pH of the enzyme loaded-microcapsules were studied. The results indicated the immobilized enzyme has better stability and activity contrasting to the native enzyme. The study illustrates that the L-asparaginase could be protected in CKGM-CS microcapsules, the substrate and product could pass through the system freely.

Study on bioactivity of extracts from marine sponges in Chinese Sea

The bioactivity screening of fractions from two inter-tidal sponges collected from the north of China Yellow Sea and one sponge collected from the South Chinese Sea was reported in this study. In sponge Hymeniacidon perleve there were 9 fractions out of 15 from CHCl3 extract with anti Staphylococcus aureus activity, 9 fractions out of 19 from BuOH extract with anti Escherichia coli activity, and three fractions from CHCl3 extract which had moderate to strong activity in inhibiting Bacillus subtilis, Candida albicans, and Aspergilus niger. The fractions of Reniochalina sp. showed bioactivity against bacteria and fungi. The fractions of Acanthella acuta Schmidt showed bioactivity against S. aureus and fungi. One compound from H. perleve obtained by the bioactively directing isolation was tested for bioactivity against the human hepatoma cell line Qgy7701 (IC50 10.1 mug/ml), Burkitt's lymphoma cell line Raji (IC50 9.76 mug/ml) and chronic myelogenous leukemia K562 (IC50 1.90 mug/ml). (C) 2003 Elsevier B.V. All rights reserved.

Metabolic profiling studies on the toxicological effects of realgar in rats by H-1 NMR spectroscopy

The toxicological effects of realgar after intragastrical administration (1 g/kg body weight) were investigated over a 21 day period in male Wistar rats using metabonomic analysis of H-1 NMR spectra of urine, serum and liver tissue aqueous extracts. Liver and kidney histopathology examination and serum clinical chemistry analyses were also performed. H-1 NMR spectra and pattern recognition analyses from realgar treated animals showed increased excretion of urinary Kreb's cycle intermediates, increased levels of ketone bodies in urine and serum, and decreased levels of hepatic glucose and glycogen, as well as hypoglycemia and hyperlipoidemia, suggesting the Perturbation of energy metabolism. Elevated levels of choline containing metabolites and betaine in serum and liver tissue aqueous extracts and increased serum creatine indicated altered transmethylation. Decreased urinary levels of trimethylamine-N-oxide, phenylacetylglycine and hippurate suggested the effects on the gut microflora environment by realgar.