【作者】 姜辉；

【导师】 田亚平；

【作者基本信息】 中国人民解放军军医进修学院 ， 临床检验诊断学， 2005， 硕士

【摘要】 研究发现,小分子一氧化氮（NO）在生物体内具有广泛的生理活性,参与诸多生理功能和病理生理过程。其兼有信号分子和自由基的双重性质。NO与肿瘤生长侵袭的关系也是近年来研究的热点,由于NO的双重特性决定其对于肿瘤的调控具有“两面性”一既存在抑制作用又存在促进作用。本课题通过细胞培养、动物模型等途径应用L精氨酸（L-Arg）、硝酸甘油（GTN）、氨基胍（AG）等三种调控NO生成的药物,观察对肿瘤细胞生长增殖的影响;应用GTN干预观察肿瘤细胞转移以及相关基因和表达蛋白质谱的改变。进一步探讨了NO调控肿瘤转移的机制,以及一氧化氮供体药物潜在的抗肿瘤转移功效。 一、不同一氧化氮调控药物对肿瘤细胞增殖抑制 在LLC肿瘤细胞培养体系中加入不同浓度L-Arg、GTN、AG后观察到,三种药物高浓度均抑制细胞增殖,随浓度降低抑制作用减弱,L-Arg和GTN在低浓度均产生对肿瘤增殖的促进作用。加入L-Arg和AG的细胞培养液中随药物浓度下降NO₂^-水平逐步升高,加入GTN的细胞培养液中NO₂^-水平与药物浓度正相关;观察不同药物组合对细胞增殖影响,L-Arg和AG在一定浓度出现拮抗作用,而硝酸甘油和氨基胍基本为协同作用; 二、GTN对肿瘤细胞转移相关基因和表达蛋白质谱的调控作用 根据前部试验在Hela细胞培养体系中加入较高浓度（40μg/ml）的GTN,形态学观察细胞生长受到抑制,通过基因芯片检测发现血管生成基因和转移相关基因的表达发生明显改变,而且变化趋势以下调较多;应用双向电泳技术观察到多个蛋白质点表达量有不同程度的改变,并出现新的差异表达蛋白点,运用RT-PCR和ELISA方法对基因芯片结果进一步确定,发现GTN干预后MMP-9基因表达和细胞外液蛋白表达水平均有下降,对TIMP-1无明显影响或轻微促进作用,二者比值明显改变; 三、高浓度GTN对肿瘤转移具有抑制作用并与调控MMP-9/TIMP-1表达有关通过小鼠LLC肿瘤转移模型观察到1.6g/L硝酸甘油溶液灌胃更多还原

【Abstract】 Many studies have confirmed that nitric oxide （NO）, as a small active molecule, participates in a variety of biological processes ranging from critical physiological functions to the pathophysiology of many diseases. NO acts as both signaling molecule and free radical in biological system. Recent years, the modulation effects of NO on tumor proliferation have been studied extensively and the results indicated that NO possesses both promotion and suppression effects on tumor. The goal of these studies is exploring the inhibitory effects of NO donors on tumor proliferation and metastasis by using cell culture and animal model. The mechanism was also studied by related gene expression and protein expression profile changing, are investigated. The mechanism of NO regulating tumor metastasis is further studied, as well as potential anti-tumor metastasis activities of NO donor. To used the contravesion properties of genotoxic and angiogenic of NO. In this study,1 Regulation of Proliferation inhibition on tumor Cell by Some Drugs influences the Nitric Oxide LevelDifferent concentrations of the drugs, including L-arginine（L-Arg）、 glyceryl trinitrate（GTN） and aminoguanidine（AG）, were added to Lewis Lung Carcinoma cell culture medium. It was found that all three drugs inhibited the cell proliferation in high concentrations and reduced inhibition of cell proliferation with concentration decreasing. As a result, cell proliferation was promoted in low concentration of L-Arg or GTN; NO₂^-level increased in L-Arg and AG treatment groups with concentration reducing, but there was a positive correlation between NO₂^- level and GTN concentration. In tests of regulations of drug-combination, there was anantagonistic joint action between L-Arg and AG treatment in certain concentrations, and had a synergistic joint action between GTN and AG treatment mostly.2 Regulation of GTN on genes related to metastasis and protein expression profile of tumor cellsHela cells treated with 40 μ g/ml GTN showed inhibition of the growth activity. Microarray test involved in angiogenesis and tumor metastasis showed that the gene expressions profiles changed greatly. Most of the genes were down-regulated, which were more obvious than genes up-regulated. In two-dimensional gel electrophoresis test, a few protein spots were changed obviously and some flew different expression spots were picked up. MMP-9 and TIMP-1 gene expressions were examined with RT-PCR. ELISA assay was applied to determine the protein concentrations of MMP-9 and TIMP-1 in culture medium. The expression of MMP-9 in GTN group was greatly lower than that in control group, but the expression of TIMP-1 was not obviously changed. MMP-9 concentration in culture medium reduced dramatically but TIMP-1 did not. And the ratio of MMP-9/TIMP-1 was altered statistically.3 GTN in high concentration inhibiting the tumor metastasis and concerned with regulating the expression of MMP-9/TIMP-1In the model of IXC-bearing C57BL/6 mice, GTN taken orally at the concentration of 1.6g/L in 40 days could inhibit the tumor metastasis. The inhibition ratio of tumor metastasis was 52% and significantly different with control group. While the MMP-9 expression was dramatically lower than that in Control group, the TIMP-1 expression was not different in each group. The production balance of MMP-9 and TIMP-1 was changed. Mice in GTN-treatment groups showed no obviously affected by the drug to bodies or weights, nor the life style.In conclusion, the series studies showed that NO could regulate tumor更多还原