发形霞水母触手提取物致红细胞与NRK-52E细胞损伤机制

发布时间：2018-06-29 03:27

本文选题：水母 + 触手提取物　；参考：《第二军医大学》2013年硕士论文

【摘要】：随着水母爆发日益频繁与人类涉海活动的逐渐增多，有毒水母蜇伤已成为最常见的海洋生物伤，水母蜇伤患者可出现多种局部或全身症状。研究表明水母毒素是一类结构新颖的肽类，具有溶血、心血管、肝肾、神经和皮肤坏死等多种生物活性，其中溶血活性，即损伤红细胞的效应，是水母毒素中普遍存在的一类生物活性。水母种类不同，其毒素的溶血机制亦有所区别，目前关于水母毒素的溶血机制主要有孔道形成和酶活性两种假说。我们推测，水母在亿万年的进化过程中还可能形成其他“高效、直接”发挥毒性的方式，本文第一部分旨在探索孔道形成之外，脂质过氧化损伤在发形霞水母（Cyanea capillata）触手提取物（tentacle extract, TE）产生溶血过程中的作用。水母蜇伤患者可分三种情况：极少数中毒非常严重，出现急性心衰而快速死亡；大多数患者中毒较轻，以蜇伤部位皮肤炎症为主，只需局部处理；另有一部分患者中毒程度介于前两者之间，可出现迟发的多器官功能损伤而需要住院治疗。本课题组前期研究表明，中等剂量TE可以引起动物延迟性的多器官损伤，而肾脏（尤其是肾小管）的急性损伤可能是TE致大鼠延迟死亡的主要原因。因此本文第二部分重点研究TE对肾小管上皮细胞（NRK-52E）的损伤机制。受溶血机制研究结果的启发，TE对NRK-52E细胞的损伤按照“脂质过氧化—活性氧—线粒体—细胞凋亡”的思路来展开。方法第一部分：TE致红细胞损伤（溶血活性）机制研究首先，以0.45%红细胞悬液为受试对象检测TE溶血活性的剂量效应关系，并检测Ca~(2+)和Ca~(2+)通道阻断剂地尔硫卓、维拉帕米、硝苯地平对TE溶血活性的影响。利用共聚焦显微镜观察TE作用后红细胞内Ca~(2+)浓度的变化，并观察Ca~(2+)通道阻断剂地尔硫卓的干预效应。通过系列PEG渗透保护实验，研究孔道形成在TE溶血活性机制中的作用。将抗氧化剂预先与红细胞孵育，检测Vc和GSH对TE溶血活性的影响。通过检测TE作用后红细胞MDA水平，分析TE对红细胞的脂质过氧化损伤效应，并在整体动物中，检测抗氧化剂Vc对TE引起的体内溶血和电解质紊乱的影响。第二部分：TE致NRK-52E细胞损伤机制初探利用MTT法检测不同浓度TE不同作用时间对NRK-52E细胞活力的影响，并在显微镜下观察细胞形态学变化。采用Annexin V-FITC/PI双染，流式细胞仪检测TE作用后细胞凋亡与坏死情况。利用荧光探针DCFH-DA标记技术观察TE作用后细胞内ROS水平，用TBA比色法检测细胞MDA水平，Rhodamine123染色检测线粒体膜电位的变化。结果第一部分：TE致红细胞损伤（溶血活性）机制研究 TE对0.45%红细胞悬液产生的溶血效应具有剂量依赖性，产生50%溶血时TE浓度约为150μg/ml。Ca~(2+)能增强TE的溶血活性，而三种Ca~(2+)通道阻断剂均能不同程度抑制TE的溶血活性。TE作用后，红细胞内Ca~(2+)明显升高，而Ca~(2+)通道阻断剂地尔硫卓可以部分拮抗TE作用后红细胞内Ca~(2+)升高的幅度。平均分子量超过4000的PEG可以抑制TE产生的溶血效应。TE作用后红细胞MDA水平随TE剂量依赖性升高，，且抗氧化剂Vc和GSH均可以显著拮抗TE的溶血活性；在体内，Vc也可以降低TE引起的溶血程度以及血清中K+和Lac升高的幅度。第二部分：TE致NRK-52E细胞损伤机制初探 TE对NRK-52E细胞活力的影响具有剂量和时间依赖性，TE作用后细胞形态发生改变，不再贴壁生长。随TE浓度增大，凋亡和坏死的细胞增多。TE作用后，NRK-52E细胞胞内ROS与MDA水平升高、线粒体膜电位降低。结论除了孔道形成之外，通过脂质过氧化损伤红细胞产生溶血也是TE可能的溶血机制；抗氧化剂Vc可以在体内和体外两个层面抑制TE的溶血活性，可以作为预防和治疗水母蜇伤的候选药物。通过损伤粒体，导致线粒体膜电位降低，胞内活性氧堆积，进而介导细胞凋亡与坏死是TE产生NRK-52E细胞毒性的机制之一。
[Abstract]:With the increasing frequency of jellyfish eruption and the increase of human involvement in the sea, toxic jellyfish stings have become the most common marine biological injuries. The jellyfish stings can occur in a variety of local or systemic symptoms. The study shows that the jellyfish toxin is a novel structure of peptides with a variety of organisms such as hemolysis, cardiovascular, liver and kidney, nerve and skin necrosis. The activity of hemolytic activity, that is, the effect of damage to red blood cells, is a kind of biological activity commonly found in the jellyfish toxin. The species of jellyfish are different, and the hemolysis mechanism of the toxin is also different. At present, the hemolysis mechanism of the jellyfish toxin mainly consists of two hypotheses of pore formation and enzyme activity. We speculate that the jellyfish is in the evolutionary process of hundreds of millions of years. Other "efficient, direct" forms of toxicity may also be formed. The first part of this article is to explore the role of lipid peroxidation damage in the process of hemolysis in the Cyanea capillata (tentacle extract, TE).
The jellyfish stings can be divided into three kinds of cases: very few intoxication is very serious, acute heart failure and rapid death; most patients are less toxic, with the main sting part of the skin inflammation, only local treatment, and another part of the patient's poisoning degree is between the former two, can be found late multiple organ function damage and need to be hospitalized. A preliminary study in our group showed that medium dose TE could cause delayed multiple organ damage in animals, and the acute injury of kidney (especially renal tubule) may be the main cause of delayed death in rats induced by TE. Therefore, the second part of this paper focuses on the damage mechanism of TE to renal tubular epithelial cells (NRK-52E). Inspired by the fruit, TE damages NRK-52E cells according to the idea of "lipid peroxidation - reactive oxygen - mitochondrion - cell apoptosis".
Method
Part one: mechanism of erythrocyte injury (hemolytic activity) induced by TE
First, 0.45% erythrocyte suspension was used to detect the dose effect relationship of TE hemolytic activity, and the effects of Ca~ (2+) and Ca~ (2+) channel blocker diltiazem, Vera Pammy, nifedipine on the hemolytic activity of TE were detected. The changes of Ca~ (2+) concentration in erythrocytes after TE were observed by confocal microscopy, and the blockage of Ca~ (2+) channel was observed. The intervention effect of diltiazem. Through a series of PEG permeation protection experiments, the role of pore formation in the hemolytic activity of TE was studied. The effects of Vc and GSH on the hemolytic activity of TE were detected by incubating the antioxidants with red blood cells in advance. The lipid peroxidation damage effect of TE to erythrocytes was analyzed by detecting the MDA level of erythrocytes after TE action, and the effects of lipid peroxidation on erythrocytes were analyzed. In vivo, the effects of antioxidant Vc on hemolysis and electrolyte disturbances induced by TE in animals were examined.
The second part is about the mechanism of NRK-52E cell injury induced by TE.
The effects of different concentrations of TE on the activity of NRK-52E cells were detected by MTT, and the morphological changes of cells were observed under the microscope. Annexin V-FITC/PI double staining was used to detect the apoptosis and necrosis of the cells after the action of TE. The ROS level of the cells after the action of TE was observed by the fluorescence probe DCFH-DA labeling technique, and TBA was used for TBA. The level of MDA was detected by colorimetry, and the changes of mitochondrial membrane potential were detected by Rhodamine123 staining.
Result
Part one: mechanism of erythrocyte injury (hemolytic activity) induced by TE
The hemolysis effect of TE on 0.45% erythrocyte suspension was dose-dependent. When 50% hemolysis was produced, the concentration of TE was about 150 mu g/ml.Ca~ (2+) enhanced the hemolytic activity of TE, while the three Ca~ (2+) channel blockers could inhibit the hemolytic activity of TE in varying degrees, and the Ca~ (2+) in the erythrocytes was significantly increased, and the Ca~ channel blocker diltiazem The increase of Ca~ (2+) in erythrocytes after the action of TE can be partially antagonized. The average molecular weight of more than 4000 of PEG can inhibit the increase of erythrocyte MDA level with the dose dependence of TE, and the antioxidant Vc and GSH can significantly antagonize the TE hemolytic activity after the effect of.TE on TE. In the body, Vc also reduces the hemolytic process. The extent of the increase of K+ and Lac in the serum.
The second part is about the mechanism of NRK-52E cell injury induced by TE.
The effect of TE on the activity of NRK-52E cells was dependent on the dose and time. The cell morphology changed after the action of TE and no longer adhered to the wall. As the concentration of TE increased, the intracellular ROS and MDA level of NRK-52E cells increased and the mitochondrial membrane potential decreased after the apoptosis and necrotic cells increased.TE.
conclusion
In addition to the formation of the channels, the hemolysis of erythrocytes by lipid peroxidation is a possible hemolytic mechanism of TE, and the antioxidant Vc can inhibit the hemolysis activity of TE in two levels in vivo and in vitro, and can be used as a candidate for the prevention and treatment of jellyfish stings.
It is one of the mechanisms of TE to produce NRK-52E cytotoxicity by damaging the mitochondria, resulting in the decrease of mitochondrial membrane potential, the accumulation of intracellular reactive oxygen species, and cell apoptosis and necrosis.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R595.8

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