Dextran-40对致命性水母蜇伤的保护作用及阳离子对水母毒素溶血作用的干预效应

发布时间：2018-09-11 14:53

【摘要】：水母是广泛分布在全球各海域的无脊椎浮游动物。近年来,海洋水温升高、海水富营养化等多种因素造成某些海域中特定种系水母暴发。多数种类水母无明显毒性作用,少数种类水母的刺丝囊中携带有毒性很强的毒液,其刺丝囊因化学或机械刺激发射刺丝释放毒素可以造成人员的严重蜇伤。全球每年被水母蜇伤的患者数以十万计,其中致死病例也并不罕见。不同种类水母蜇伤的症状和体征有所不同,有些水母蜇伤只造成一过性刺痛或轻微红肿,而有些水母蜇伤较严重,患者出现持续性疼痛,荨麻疹和水疱,表皮坏死,循环系统损伤,多器官功能障碍,甚至死亡。针对水母蜇伤预防和治疗药物的研究越来越受到关注,取得了一些进展,但尚没有达成救治措施方面的广泛共识。水母毒素成分复杂,易粘附和失活,毒作用靶点多样,毒素的这些理化特点增加了寻找特效抗毒药物的难度。本文第一部分在细胞水平和整体动物水平分析了低分子右旋糖酐(Dextran-40)对致命性水母蜇伤的保护作用。第二部分着重分析了5种金属阳离子对水母毒素溶血作用的干预效应。实验方法第一部分:Dextran-40对致命性水母蜇伤的保护作用本部分研究首先应用激光扫描共聚焦显微镜,在细胞水平研究Dextran-40对发形霞水母Cyanea capillata触手提取物(tentacle extract,TE)造成的小鼠心室肌细胞和红细胞损伤的保护作用。然后,应用本课题组建立的致命性水母蜇伤综合征(acute jellyfish envenomation syndrome,AJES和delayed jellyfish envenomation syndrome,DJES)模型,在整体动物水平分别研究Dextran-40对AJES小鼠模型生存率的影响、Dextran-40对AJES大鼠模型循环功能的影响以及Dextran-40对DJES大鼠模型血生化指标的影响。第二部分:金属阳离子对水母毒素溶血作用的干预效应本部分研究首先应用分光光度计法检测TE的溶血效应,观察5种据报道具有潜在抗溶血效应的阳离子(La3+,Mn2+,Zn2+,Cu2+和Fe2+)对TE溶血作用的影响,然后,分别测定阳离子溶液的吸光度值、阳离子对红细胞悬液的吸光度值影响、阳离子对血红蛋白溶液的吸光度值影响,并且在显微镜下对红细胞直接计数。综合分析La3+,Mn2+,Zn2+,Cu2+和Fe2+对水母毒素溶血作用的干预效应。实验结果第一部分:Dextran-40对致命性水母蜇伤的保护作用本部分实验结果发现,TE会引起心肌细胞和红细胞胞内Ca2+含量呈现剂量依赖性增加,心肌细胞和红细胞存活数量呈现剂量依赖性减少。Dextran-40可以对TE引起的心肌细胞胞内Ca2+含量升高和细胞存活率降低起到一定的保护作用;对TE引起的红细胞形态损伤和溶血作用没有明显保护作用,但是显著抑制TE引起的红细胞胞内钙超载。应用本课题组建立的致命性水母蜇伤综合征(AJES和DJES)模型,在整体动物水平研究结果发现,Dextran-40在0.8 g/kg时能明显降低TE所致AJES小鼠死亡率。Dextran-40在0.6 g/kg时能明显缓解TE所致AJES大鼠的血压下降,明显改善TE所致DJES大鼠的肝功能指标(ALT,AST,A/G)、心肌损伤相关酶指标(CK,LDH)。第二部分:金属阳离子对水母毒素溶血作用的干预效应本部分实验结果发现,有轻微颜色的Zn2+,Cu2+和Fe2+离子溶液导致红细胞悬液的吸光度值降低,而Mn2+溶液增加了红细胞悬液吸光度值,透明的La3+溶液对红细胞悬液的吸光度值几乎没有影响,这表明阳离子自身颜色可能干扰红细胞悬液中血红蛋白的吸收光谱。我们测试了5种阳离子对红细胞悬浮液释放的血红蛋白溶液的影响,表明结果透明La3+溶液不对血红蛋白溶液的吸光度值产生明显影响,Mn2+溶液导致血红蛋白溶液吸光度值轻微降低,有轻微颜色的Zn2+,Cu2+和Fe2+溶液则显著降低血红蛋白溶液的吸光度值。经校正分析,除La3+显示完全抑制TE的溶血作用之外,Zn2+,Cu2+,Fe2+和Mn2+使TE溶血曲线右移,即其实际抑制效应比之前报道的抑制效果偏弱。实验结论第一部分:Dextran-40对致命性水母蜇伤的保护作用我们推测Dextran-40一方面通过封堵水母毒素在细胞膜上形成的孔道,对细胞的钙超载起到了抑制作用,从而保护心肌细胞;另一方面可能通过提高血浆胶体渗透压,吸收血管外水分而增加血容量,稳定血压,以拮抗水母毒素引起冠脉痉挛导致的机体低血压症及其他心脏病变。第二部分:金属阳离子对水母毒素溶血作用的干预效应研究结果表明除La3+之外,其他4种金属阳离子(Mn2+,Zn2+,Cu2+和Fe2+)会干扰血红蛋白的吸光度,所以测试含金属离子类试剂对水母毒素溶血作用的影响时,应该进行校正分析。
[Abstract]:Jellyfish are invertebrate zooplankton widely distributed in various sea areas all over the world. In recent years, sea water temperature rises, eutrophication and other factors lead to the outbreak of certain species of jellyfish in certain sea areas. Most species of jellyfish have no obvious toxic effect. A few species of jellyfish carry highly toxic venom in their spiny sacs due to chemical factors. There are hundreds of thousands of jellyfish stings worldwide each year, and deaths are not uncommon. The symptoms and signs of different kinds of jellyfish stings vary. Some jellyfish stings cause only temporary stings or slight redness, while some jellyfish stings are more severe. Seriously, patients suffer from persistent pain, urticaria and blisters, epidermal necrosis, circulatory system damage, multiple organ dysfunction, and even death. Drug research for the prevention and treatment of jellyfish stings has attracted more and more attention, and some progress has been made, but there is no broad consensus on treatment measures. These physical and chemical characteristics of toxins make it difficult to find specific antitoxic drugs. In the first part of this paper, the protective effects of low molecular dextran (Dextran-40) on lethal jellyfish stings are analyzed at cellular and animal levels. In the second part, the solubility of five metal cations to jellyfish toxins is emphasized. Interventional effects of Dextran-40 on blood. Methods Part 1: Protective effects of Dextran-40 on lethal jellyfish stings. This part first studied the effects of Dextran-40 on ventricular myocytes and red and fine cells of mice induced by tentacle extract (TE) of Cyanea capillata at the cellular level by laser scanning confocal microscopy. Then, the effects of Dextran-40 on the survival rate of AJES mice model and the effects of Dextran-40 on AJES rat model were studied by using the acute jellyfish envenomation syndrome (AJES) and delayed jellyfish envenomation syndrome (DJES) models. The effect of Dextran-40 on blood biochemical parameters in DJES rats. Part 2: Intervention effect of metal cations on hemolysis of jellyfish toxin Part 2: The hemolysis effect of TE was detected by spectrophotometer, and five kinds of cations (La3+, Mn2+, Zn2+, Cu2+) with potential Anti-hemolysis effect were observed. Then, the absorbance of cationic solution, the absorbance of cationic solution to erythrocyte suspension, the absorbance of cationic solution to erythrocyte suspension, and the absorbance of cationic solution to erythrocyte were determined respectively. The erythrocyte count was also carried out under microscope. The hemolysis of jellyfish toxin by La3+, Mn2+, Zn2+, Cu2+ and Fe2+ was analyzed comprehensively. Intervention effect. Part I: Protective effect of Dextran-40 on lethal jellyfish sting. This part of the experiment found that TE could induce a dose-dependent increase in Ca2+ content in cardiomyocytes and erythrocytes, and a dose-dependent decrease in cardiomyocytes and erythrocytes survival. The increase of intracellular Ca2+ content and the decrease of cell survival rate play a certain protective role; they have no obvious protective effect on erythrocyte morphological damage and hemolysis induced by TE, but significantly inhibit intracellular calcium overload of erythrocyte induced by TE. Dextran-40 significantly reduced TE-induced mortality in AJES mice at 0.8 g/kg. Dextran-40 significantly alleviated TE-induced hypotension in AJES rats, significantly improved TE-induced liver function (ALT, AST, A/G) and myocardial injury-related enzymes (CK, LDH). Intervention effect of jellyfish toxin on hemolysis This part of the experimental results showed that slightly colored Zn2+, Cu2+ and Fe2+ ion solutions decreased the absorbance of erythrocyte suspension, while Mn2+ solution increased the absorbance of erythrocyte suspension, transparent La3+ solution had little effect on the absorbance of erythrocyte suspension, indicating that the cations were self-contained. Body color may interfere with the absorption spectrum of hemoglobin in erythrocyte suspensions. We tested the effects of five cations on the release of hemoglobin from erythrocyte suspensions. The results showed that transparent La3+ solution had no significant effect on the absorbance of hemoglobin solution. Mn2+ solution caused a slight decrease in the absorbance of hemoglobin solution. In addition to the complete inhibition of TE hemolysis by La3 +, Zn2 +, Cu2 +, Fe2 + and Mn2 +, the hemolysis curves of TE were shifted to the right by Zn2 +, Cu2 + and Mn2 +, i.e. the actual inhibition effect was weaker than previously reported. The protective effect of Dextran-40 on lethal jellyfish stings is hypothesized to protect cardiomyocytes by blocking the channels formed by jellyfish toxins on the cell membrane, inhibiting calcium overload, and on the other hand, increasing blood volume and stabilizing blood pressure by increasing plasma colloid osmotic pressure and absorbing extravascular water. Antagonizing hypotension and other heart diseases caused by jellyfish toxin-induced coronary spasm. Part II: Intervention effects of metal cations on the hemolysis of jellyfish toxin. The results showed that four other metal cations (Mn2+, Zn2+, Cu2+, and Fe2+) interfered with the absorbance of hemoglobin except La3+, so the metal ions were tested. The effect of reagents on hemolytic effects of jellyfish toxins should be corrected.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R646

【参考文献】