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

IOCAS-IR > 海洋环流与波动重点实验室

	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
	张峰
学位类型	博士
	1999
学位授予单位	中国科学院海洋研究所
学位授予地点	中国科学院海洋研究所
学位专业	海洋生物学
关键词	皱纹盘鲍血细胞活性氧（ros）呼吸爆发农药重金属
摘要	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。
其他摘要	This study firstly reports that there is an oxygen-dependent against disease mechanism in the hemocytes of Haliotis discus hannai using reactive oxygen species (ROS). The results as follows: 1. The hemocytes of Haliotis discus hannai are stimulated in vitro with various particulate agents (yeast cells and zymosan) using a chemiluminescent method dependent on luminol (5'-amino-2,3-dihydro-1, 4-phtalazinedione) in order to determine whether Haliotis discus hannai hemocytes can show the chemiluminescence (CL) response of reactive oxygen species (ROS) production as a result of respiratory burst activities in phagocytosis. The results indicate the hemocytes of Haliotis discus hannai typically undergo a burst of respiratory activity and generate reactive oxygen species (ROS) in the phagocytosis. The hemocytes of Haliotis discus hannai to various stimulators show to generate defferent degree of chemiluminescence (CL), and to the same stimulators show to be dose-dependent response, and to opsonized and nonopsonized yeast cells show to generate defferent degree of chmeluminescence (CL). SOD and NaN_3 can inhibite the chemiluminescence production by the hemocytes of Haliotis discus hannai. All these prove that the hemocytes of Haliotis discus hannai can generate reactive oxygen species (ROS) to kill the pathogen in the host defense against microorganisms. 2. Effect of scavergers of reactive oxygen species and some reagents on chemiluminescence of Haliotis discus hannai hemocytes during phagocytosis was measured in vitro. In a manner resembling the respiratory burst of activated mammalian polymorphonuclear leukocytes, hemocytes of Haliotis discus hannai generated reactive oxygen species during in vitro phagocytosis of yeast cells. That Superoxide dismutase (SOD) can inhibit the chemiluminescence of Haliotis discus hannai hemocytes during phagocytosis (P < 0.01) showed Haliotis discus hannai hemocytes can generate superoxide anion (O_2~-), and NBT can decrease the chemiluminescence due to the intracellular blue formazan deposition was reduced by superoxide anion (O_2~-) that Haliotis discus hannai hemocytes released (P < 0.01). That Catalase (Cat.) can inhibit the chemiluminescence of Haliotis discus hannai hemocytes during phagocytosis (P < 0.01) showed that Haliotis discus hannai hemocytes can generate hydrogen peroxide (H_2O_2). The Sodium, benzoate can inhibit the chemiluminescence of Haliotis discus hannai hemocytes during phagocytosis (P < 0.01, P < 0.05) showed that Haliotis discus hannai hemocytes can generate hydoxyl radicals (OH~·). That 2,5-dimethyfuran (DMF)can inhibit the chemiluminescence of Haliotis discus hannai hemocytes during phagocytosis (P < 0.01) showed that the divalent cation Ca~(2+) are required to initiate phagocytic and oxidative activity in Haliotis discus hannai hemocytes. 3. Reactive oxygen species (ROS)-dependent bacterial killing mechanisms of Haliotis discus hannai hemocytes were quantified in vitro by luminol dependent Chemiluminescence (CL) assay to determine whether the mechanisms were affected by organophosphorous pesticides-Parathion, Dichlorovos, Dimethoate, 2,4-D butylester and Methamidophos. The results showed that in vitro soaking treatment of 1hr or 12hr. of Haliotis discus hannai hemocytes to defferent organophosphorous pesticides and defferent concentration of the same organophosphorous pesticide can inhibit ROS-dependent bacterial killing mechanisms of Haliotis discus hannai hemocytes and resulted in dose-dependent suppression of chemiluminescence (CL). 2,4-D butylester, Dichlorovos and Dimethoate have stronger effect to inhibit ROS production of Haliotis discus hannai hemocytes in phagocytosis than that of Parathion and Methamidophos have. The results shows that these pesticides can enhance the percent rate and index of phagocytosis of Haliotis discus hannai hemocytes in low concentration and can inhibit the percent rate and index of phagocytosis of Haliotis discus hannai hemocytes in high concentration. 4. In vitro, Reactive oxygen species (ROS)-dependent bacterial killing mechanisms of Haliotis discus hannai hemocytes were quantified by luminol dependent Chemiluminescence (CL) assay to determine whether the mechanisms were affected by different heavy metals and the different concentrations of the same heavy metal with 1hr treatments. These metals are Chromium (Cr), Cadmium (Cd), Hydrargyrum (Hg), Plumbum (Pb), Cuprum (Cu) and Zincum(Zn). The results showed that different species of metals and the different concentration of the same metal can inhibit ROS-dependent bacterial killing mechanisms of Haliotis discus hannai hemocytes and resulted in dose-dependent suppression of chemiluminescence (CL). Different metals have the different effect of inhibiting to the Haliotis discus hannai hemocytes and the order of inhibiting effects to the ROS production of Haliotis discus hannai hemocytes from strong to weak is Hg > Cd > Cu > Pb > Cr > Zn. The study indicates that Hg is strongest toxicity to Haliotis discus hannai hemocytes in the metals that were studied in the test.
页数	109
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/491
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张峰. 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[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,1999.

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