钠离子通道类毒素的细胞毒性和检测方法的研究

IOCAS-IR > 海洋环流与波动重点实验室

	钠离子通道类毒素的细胞毒性和检测方法的研究
其他题名	Study on the cytotoxicities and detecting method of sodium ion-channel toxins
	张艺
学位类型	博士
	2005-06-05
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	钠离子通道类毒素 Neuro-2a 细胞毒性检测方法
摘要	赤潮毒素广泛存在于各种赤潮藻和各类海洋生物中，不仅对渔业、养殖业危害甚大，而且还直接威胁着人类的生存健康。其中，离子通道类毒素是一类毒性较高的毒素。除一些赤潮藻可以产生此类毒素之外，海洋中还存在某些生物也能够产生离子通道类毒素。为进一步阐明钠离子通道类毒素对细胞的毒性效应机制，本文选取一株小鼠神经母细胞瘤（Neuro-2a）作为受试对象，研究了四种钠离子通道类毒素STX、GTX1,4、GTX2,3、TTX对Neuro-2a细胞的毒性影响机制，并利用STX和TTX，建立了钠离子通道类毒素的细胞毒性检测方法，且应用此方法检测了贝体内、藻体内的毒素含量，进一步与小鼠法和HPLC法进行了比较。研究表明：STX、GTX1,4、GTX2,3、TTX四种钠离子通道类毒素在长时间内均会对Neuro-2a细胞的增殖产生不利影响。在短时间（24h）内，以上各毒素均没有抑制Neuro-2a细胞的增殖，但是48h后，以上各毒素对Neuro-2a细胞的增殖均产生了抑制作用，且随着各毒素剂量的增加，细胞增殖受抑制程度也表现出一定程度的增高，二者呈剂量-反应关系。STX、GTX1,4、GTX2,3、TTX对Neuro-2a细胞的48h半数抑制浓度（IC50）分别为：250ng/ml、1000ng/ml、1300ng/ml、700ng/ml。本论文还首次研究了STX、GTX1,4、GTX2,3、TTX四种钠离子通道类毒素对Neuro-2a细胞内酶活性的影响。研究发现，STX、GTX1,4、GTX2,3、TTX四种钠离子通道阻断剂类毒素均能够影响Neuro-2a细胞内Na+-K+-ATP酶和乙酰胆碱酯酶TChE的活性。当各毒素作用24h后，Neuro-2a细胞内Na+-K+-ATP酶和乙酰胆碱酯酶TChE的活力均会受到抑制，并且随着各毒素剂量的增加，两种酶的活性也逐渐降低。可见，钠离子通道阻断剂类毒素能对细胞内酶的功能产生一定的影响，此影响连同阻断细胞膜钠离子通道，造成离子流的失衡作用，进一步对细胞产生毒性效应。在对细胞膜通透性的研究中发现，上述四种钠离子通道阻断剂类毒素各剂量组细胞培养液乳酸脱氢酶LDH的漏出率与对照组相比均无显著差异，它们均未引起Neuro-2a细胞膜内LDH的改变，看来钠离子通道阻断剂类毒素不会通过影响细胞膜的通透性而对细胞引起毒性效应。本研究还利用STX和TTX两种钠离子通道标准毒素以及乌苯苷、藜芦定两种生物毒素，参照Jellett（1992）方法，建立了STX和TTX两种钠离子通道类毒素的细胞毒性检测的标准曲线，分别为：Y=0.266X+51.184和 Y=1.6068X+47.186。检出限分别为5ng/ml和0.8ng/ml。并且利用已建立的细胞毒性检测方法检测了来自浙江舟山和连云港赣榆市的19个织纹螺样品和5株实验室培养的亚历山大藻，得到的实验结果与小鼠生物测试和HPLC检测的结果存在较好的相关关系。鉴于该方法具有高通量、省时、检出限低等优点，因此更具有在沿海环境检测中推广应用的潜力。
其他摘要	Algal toxins widely exist in a variety of red-tide algae and other marine organisms. They not only endanger fishery and aquaculture industry, but also cause direct menace to the heath of human beings. Ion-channel toxin, produced by red-tide algae and also some marine organisms is one of the most poisonous algal toxins. In order to elucidate the cytotoxic mechanisms of ion-channel toxins, the toxic effect of four sorts of sodium ion-channel toxin—STX、GTX1,4、GTX2,3、TTX were determined by utilizing a cell strain from Neuro-2a. As a result, an approach for detecting the cytotoxicities of sodium ion-channel toxins was established by taking advantage of STX and TTX. The established method was utilized to detect toxin level of the shellfish and algae. The results were then compared with that obtained by mouse bioassay and HPLC method. Our studies showed that STX、GTX1,4、GTX2,3、TTX all caused negative effect on the growth of Neuro-2a cells in the long term. Within 24h, no significant inhibition of cell growth was observed. However, after 48h, cells growth were inhibited in the presence of either the four toxins and the extent of the inhibition was shown to be in a dose-dependent manner. The half inhibition concentrations (IC50) were 250ng/ml、1000ng/ml、1300ng/ml、700ng/ml for STX、GTX1,4、GTX2,3、TTX, respectively. The desmoenzyme active effect of four sorts of sodium ion-channel toxin—STX、GTX1,4、GTX2,3、TTX to Neuro-2a cells also studied in this paper for the first time. The studies showed that four toxins were also found to influence the Na+-K+-ATPase and TChE activity of Neuro-2a. After 24h, Na+-K+-ATPase and TChE activity were inhibited and decreased with the increased dose of toxins. Obviously, the sodium ion-channel toxins have certain influence to the desmoenzyme function, with blocking the sodium ion channel of cell membrane and resulting the ion stream imbalanced, thus have the poisonous effect to the cells. A later investigation on cell membrane permeability showed that regardless of their dosage， the LDH leakage rate of four toxin groups exhibited no significant difference compared with control group, which suggested the cytotoxicity of four toxins were not through their effect on cell membrane permeability. Additionally, based on method reported by Jellett (1992) and using the two standard toxins of STX、TTX and two biological toxins of Ouabain、Veritridine, we successfully established the standard curves for determining the cytotoxicities of STX and TTX,Y=0.266X+51.184 and Y=1.6068X+47.186 with a detection limit of 5ng/ml and 0.8ng/ml ,respectively. Moreover, the toxin level of 19 samples of Nassarius from Zhoushan and Ganyu and 5 samples of A.tamarense kept in our lab were tested using our established methods. Results showed good correlation to those obtained by mouse bioassay and HPLC. Accordingly, based this detecting method was proved to be high-throughput, time-saving, and low detection limit, this method has the potential of application in coastal environment.
页数	75
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1165
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张艺. 钠离子通道类毒素的细胞毒性和检测方法的研究[D]. 海洋研究所. 中国科学院海洋研究所,2005.

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