麻痹性贝毒毒素的制备和活性研究

IOCAS-IR > 海洋环流与波动重点实验室

	麻痹性贝毒毒素的制备和活性研究
	王云峰
学位类型	博士
	2001
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	麻痹性贝毒毒素膝沟藻毒素制备活性
摘要	麻痹性贝毒（Paralytic Shellfish Poisons, PSP）毒素主要是由海洋甲藻产生的一类活性物质，在赤潮毒素监测、神经与分子生物学基础研究以及新型药物开发方面都有潜在的应用价值。然而，纯化毒素的缺乏在很大程度上限制了相关工作的开展。本文通过大量培养塔玛亚历山大藻（Alexandrium tamarense）来提取PSP毒素，完善了毒素的分离纯化过程，并对纯化毒素的活性进行了研究和探讨。在实验室进行了塔玛亚历山大藻的规模培养，对其生长和培养条件进行了优化，探讨了有毒藻大量培养的采收方式，并获取了一批用于纯化毒素的有毒藻细胞。将藻细胞破碎后离心分离，提取出含PSP的粗毒素，再用大孔径凝胶过滤去除粗毒素中的大部分杂质，然后用小孔径凝胶和离子交换树脂将不同毒素逐级分离、纯化。结果表明，大孔径凝胶能有效地去除粗毒素中的杂质，小孔径凝胶过滤能将膝沟藻毒素（GTX）和C毒素分开，应用离子交换树脂能将GTX2,3和GTX1,4分开，最终得到GTX2,3和GTX1,4的纯化产品。分离纯化过程始终以小鼠法和荧光法进行毒素在线监测。以提取的毒素对小鼠进行毒性测试，小鼠呈现典型的PSP毒素中毒死亡症状；电生理分析的结果表明PSP毒素能够有效地阻断小鼠运动神经末梢钠电流，并可逆地阻断NG108-15全细胞的钠电流，与PSP毒素的作用机理一致；高效液相色谱分析结果表明，纯化毒素与标准毒素的保留时间完全一致；经质谱分析，纯化毒素与标准毒素具有相同的分子离子峰和碎片离子峰。应用运动模型研究了纯化毒素的药理活性。分别采用家兔椎管麻醉法、小白鼠热板法、小白鼠攀网法研究了GTX2,3对家兔的局麻作用、对小鼠的镇痛和肌松作用。结果表明，GTX2,3按剂量0.4μg STX equivalent/kg给予家兔椎管注射时，具有显著的局麻作用。GTX2,3按剂量5.99μg STX equivalent/kg给予小鼠肌肉注射时，具有显著的镇痛作用。GTX2,3按剂量6.49μg STX equivalent/kg给予小鼠皮下注射时，具有显著的肌松作用。
其他摘要	As a type of bioactive compounds, paralytic shellfish poisons (PSP) were mainly produced by dinoflagellate. PSP toxins have potential application in researches of harmful algal bloom toxicity, neurology and molecular biology, and development of new drug. However, the lackness of pufified PSP hampered the related researches. In this thesis, using the PSP-producing dinoflagellate Alexandrium tamarense, the technology on seperation and purification of PSP toxins have been studied and developed. Meanwhile, the activities of PSP were studied and discussed. The methods for culturing of Alexandrium tamarense were established and improved in the lab. The methods on concentrating and harvesting the cells were discussed. Crude PSP solution were obtained by sonicating Alexandrium tamarense cells and then centrifugating, After the impurities were excluded by large size gel filtration, Alexandrium tamarense were then seperated and purified by small size gel filtration and ion-exchange resin. The results showed that the impurities can be effectively excluded by large size gel, gonyautoxin (GTX) and C toxins could be seperated by small size gel filtration, GTX2,3 and GTX1,4 could be seperated by ion-exchange resin. GTX2,3 and GTX1,4 were the final products. Mouse assay and fluoresence detector were used in the whole preparation process to monitor the toxins. The mice used for test of toxicity showed typical symptoms of PSP poisoning. The extracted PSP could reversibly inhibit the voltage-gated sodium current of mouse motor nerve terminals and the whole NG108-15 cells, with the identical active mechanism of the standard PSP. The retention time of the purified GTX2,3 and GTX1,4 were the same as PSP standards by HPLC and the m/z of GTX2,3 and GTX1,4 were the same as PSP standards by MS. The pharmaceutical activities of the purified toxins were studied with animal models. The local anesthetic, analgesia, muscular relaxation of GTX2,3 were studied by rat sciatic anesthesia, mouse hot plate and mouse climbing web tests respectively. The results showed that the local anesthetic of rabbit was significant after percutaneous sciatic nerve injections with GTX2,3 equivalent to 0.4μg STX/kg. The analgesia of mouse was significant after muscular injections with GTX2,3 equivalent to 5.99μg STX/kg. The muscular relaxation of mouse was significant after subpercutaneous injections with GTX2,3 equivalent to 6.49μg STX/kg.
页数	93
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1325
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	王云峰. 麻痹性贝毒毒素的制备和活性研究[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,2001.

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