生物信息学在控制SARS中的应用

目的 快速准确地对可疑病人进行筛选，早期诊断传染性疾病SARS。方法 运用生物信息学这一门数学、统计学、计算机学和生物医学相交融的学科作为研究工具。结果 破解SARS的病原一变异冠状病毒的DNA序列，从而获得SARS冠状病毒全基因组芯片。结论 生物信息学是新世纪跨专业的一门前沿学科。     

Cytokines as an adjuvant to tumor vaccines: Efficacy of local methods of delivery

Background: We examined alternative methods of delivering cytokines as an adjunct for priming lymph node (LN) cells draining sites of vaccine inoculation for the purpose of generating immune cells for adoptive immunotherapy. Methods: Using syngeneic murine tumors we examined the ability of IL-2, IL-4, or GM-CSF delivered locally to a site of tumor inoculum to induce antitumor reactive draining LN cells. Mice were inoculated subcutaneously with tumor cells transduced to secrete cytokine; tumor cells admixed with fibroblasts transduced to secrete cytokine; or intralesional inoculation of cytokine in established tumor to induce sensitized LN cells capable of mediating tumor regression in adoptive transfer. Results: Both IL-4 and GM-CSF cytokines were effective in enhancing the antitumor reactivity of vaccine-primed LN cells compared to IL-2, which was ineffective. The local delivery of GM-CSF by autocrine or paracrine secretion of genetically engineered cells, as well as direct intratumoral delivery was capable of upregulating LN sensitization compared to systemic administration, which did not. Conclusions: The local delivery of GM-CSF as an adjuvant for tumor vaccination can be accomplished by various methods, including direct injection, which avoids the need for gene transfer.     

心血管疾病基因治疗的基础研究Ⅵ．一种新的体内基因转移方法

将医用缝合线浸泡在质粒ＤＮＡ溶液中，然后缝合在大鼠股四头肌上，可以观察到外源基因在肌肉组织中高效、稳定地表达，其表达量高于磷酸钙共沉淀和ＤＮＡ沉淀块埋植法，较肌肉直接注射法表达量高１０倍左右。     

骨痹通天丸抗炎镇痛免疫作用研究

实验动物口服骨痹通天丸5．58g／kg．2．79g／kg，观察本品对炎症反应，局部疼痛和免疫功能的影响。结果表明，骨痹通天丸的两个剂量组对大鼠蛋清性关节肿具有不同程度的抑制作用（P＜0．01或0．05）；对二甲苯性耳廓肿仅大剂量组具有明显抑制作用（P＜0．01）；对小鼠热刺激所致的疼痛反应具有不同程度的抑制作用（P＜0．01或0．05）；对化学刺性疼痛两个剂量组均有明显抑制作用（P＜0．01）；并可抑制小鼠重要免疫器官的增长和巨噬细胞的吞噬功能（P＜0．05），对血清溶血素未显示其效应。     

P物质对肉芽组织成纤维细胞表皮生长因子的表达调控及其意义

目的 探讨感觉神经肽P物质(SP)对离体培养的肉芽组织成纤维细胞表皮生长因子(EGF)表达的调控作用及特点。方法 采用甲醛注射的方法造成Wistar大鼠局部无菌性炎性反应，提取肉芽组织进行成纤维细胞原代培养；采用逆转录—聚合酶链反应(RT—PCR)方法检测SP对肉芽组织成纤维细胞EGF基因表达的调控作用，观察时间及剂量—效应关系；采用Western-blotting方法检测EGF蛋白表达情况，观察时间及剂量—效应关系。结果 10^-7mol/L SP可诱导成纤维细胞EGF mRNA的表达，在作用后6h与对照组比，差异有显著性(P＜0．01)；SP在10^-8-10^-6mol/L范围内可以显著促进成纤维细胞EGF mRNA表达，在10^-7mol/L达到峰值，10^-5mol/L时与对照组差异无显著性(P＞0．05)。10^-7mol/L SP作用12h后可检测到EGF蛋白明显表达增强(P＜0．01)，24h达高峰，48h后逐渐有所回落。SP在在10^-8--10^-5mol/L范围可诱导EGF蛋白的表达，均在10^-7mol/L剂量点达到峰值(P＜0．01)。结论 SP可诱导肉芽组织成纤维细胞EGF基因和蛋白的表达，且呈现出一定的时间和剂量特点，在SP调控创伤愈合的作用中具有重要意义。     

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats

Objective: To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing. Methods: Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term =21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5 705 probes representing 5 705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin. Results: Among 5 705 rat genes, there were 53 genes (0.93%) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 ( FGF2 ) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method. Conclusions: High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.     

The effects of norepinephrine transporter inactivation on locomotor activity in mice.

BACKGROUND: Acute administration of different classes of antidepressants can enhance or reduce spontaneous locomotor activity in a novel environment, but the effects of chronic antidepressant treatment on spontaneous locomotor activity in novel and familiar environments are less well characterized. Because norepinephrine is an important regulator of spontaneous locomotor activity, we speculated that norepinephrine transporter blockade contributes to the effects of some antidepressants on spontaneous locomotor activity. METHODS: Antidepressant drugs (reboxetine, desipramine, imipramine, venlafaxine, bupropion) were administered acutely (intraperitoneal) or chronically (via osmotic minipump) to control and norepinephrine transporter knockout mice, and spontaneous locomotor activity in novel or familiar environments was recorded. RESULTS: Acute treatment with most norepinephrine transporter-blocking antidepressants decreased spontaneous locomotor activity in a novel environment, whereas chronic treatment decreased spontaneous locomotor activity in both novel and familiar environments. The exception was bupropion, a dual norepinephrine transporter/dopamine transporter blocker, which tended to increase spontaneous locomotor activity. Coadministration of reboxetine and the dopamine transporter blocker GBR 12909 also increased spontaneous locomotor activity. Norepinephrine transporter knockout mice had low basal spontaneous locomotor activity, which was increased by bupropion, whereas reboxetine had no effect in norepinephrine transporter knockout mice. CONCLUSIONS: Acute or chronic inactivation of the norepinephrine transporter decreases spontaneous locomotor activity in novel and familiar environments unless coupled with dopamine transporter blockade.     

经皮局部隔离肝脏灌注化疗合并血液灌流方法学实验研究

目的　探讨经皮局部隔离肝脏灌注化疗的方法及可行性。方法　 12只实验用犬 ,对照组 4只 ,进行常规经肝动脉灌注化疗 ;实验组 8只 ,利用介入放射学方法进行经皮局部隔离肝脏灌注化疗结合血液灌流。化疗药物选用阿霉素 ,分别检测肝静脉及外周血液中的血药浓度。结果　实验组介入操作全部成功 ,1例死于血液灌流的血路管漏气 ,平均操作时间 ( 132 .3± 15 .3)min。血药浓度测定显示 ,对照组肝静脉血和外周静脉血浓度峰值分别为 ( 370 9.676± 385 .72 3)ng/ml、( 15 76.14 0± 2 2 6.933)ng/ml;实验组为( 465 3.42 0± 430 .2 0 4)ng/ml、( 433.612± 40 .5 0 1)ng/ml。统计学处理 ,两组相差非常显著。结论　经皮局部隔离肝脏灌注化疗结合血液灌流是一种可行的技术 ,具有简单、创伤小、可保护正常肝脏、便于重复治疗的优点。与常规经肝动脉灌注化疗相比 ,不仅可以增加局部血药浓度 ,更可以降低外周的血药浓度 ,降低毒副作用     

rhTPO在大肠杆菌中的表达、纯化及生物活性分析

目的：探讨人促血小板生成素(human thromlbopoietin，hTPO)在大肠杆菌中表达、分离纯化及生物学活性初步鉴定。方法：利用RT-PCR法从人胎肝细胞中扩增目的基因片段，将其定向插入pQE30表达质粒T5启动子下游的多克隆区，转化大肠杆菌M15，得到pQE30-TPO的工程菌，诱导目的蛋白表达、纯化。将表达产物给血小板减少模型小鼠尾静脉注射，观察注射后不同时间血小板量的改变。结果：经异丙基硫代-β-D-半乳糖苷(isopropylthio-β-D-galactoside IPTG)诱导培养，该工程菌可以产生单一特异性的高表达蛋白条带。将纯化后的表达蛋白注射小鼠，结果显示对实验性小鼠血小板减少症具有一定的治疗作用。结论：在大肠杆菌中成功地高效表达了重组hTPO，该产物具有促血小板生成的活性。     

重组逆转录病毒pLNCX2-GDNF转染许旺细胞及其表达

目的 利用基因转染技术将胶质细胞源性神经营养因子(GDNF)基因转入许旺细胞(SC)以提高其分泌该因子的水平。方法 应用逆转录聚合酶链反应(RT—PCR)方法合成GDNFcD—NA片段，以逆转录病毒pLNCX2为载体导入许旺细胞内，并运用RT—PCR、免疫细胞化学染色、ELISA及运动神经元联合培养法检测外源性基因在mRNA和蛋白水平的表达及其产物的活性。结果 RT—PCR检测结果示GDNF—SCs表达GDNFmRNA的水平明显优于SCs；免疫细胞化学检测发现GDNF—SCs抗GDNF蛋白染色呈强阳性，正常SCs呈弱阳性；ELISA法检测结果示转染后4周GDNF—SCs分泌GD—NF蛋白的水平升至正常SCs的5．1倍；运动神经元联合培养法结果显示GDNF—SCs分泌的外源性基因产物具有生物学活性。结论 GDNF基因转染SC在mRNA和蛋白水平表达GDNF明显优于正常SCs，外源性基因表达产物具有神经生物学活性。