沙蜇毒素致红细胞损伤的机制初步探究

发布时间：2018-03-26 19:25

本文选题：沙蜇　切入点：溶血活性　出处：《青岛大学》2017年硕士论文

【摘要】：研究目的研究沙蜇毒素致红细胞损伤(溶血活性)的机制,评估不同浓度的维生素C对沙蜇毒素溶血活性的影响。研究方法采用沙蜇自溶法,从沙蜇中分离刺丝囊细胞,利用超声波细胞破碎法提取刺丝囊细胞中的毒素,以牛血清白蛋白为标准,测得毒素蛋白的浓度。选取健康自愿者,男女各8名,各抽取新鲜血3~4ml,重复洗涤红细胞2~3次后,配制成20%(V/V)人血红细胞悬浮液和0.45%(V/V)人血红细胞悬浮液。取不同体积的沙蜇毒素加入到1ml的20%人血红细胞悬浮液中,加0.9%的氯化钠溶液稀释至5ml,使得沙蜇毒素终浓度为0、2、4、8、16μg/ml,应用硫代巴比妥(TBA)法,检测不同浓度的沙蜇毒素作用后,溶血反应体系当中的脂质过氧化产物丙二醛(MDA)的水平。取一定体积的沙蜇毒素,加入到1ml的0.45%人血红细胞悬浮液中,后加入不同体积的维生素C,再加入0.9%的氯化钠溶液稀释至5ml,使维生素C终浓度分别0、0.5、1、1.5、2、2.5mmol/L,测定不同浓度的维生素C对沙蜇毒素溶血活性的影响。结果实验数据所得,本研究中沙蜇毒素蛋白浓度为395.45μg/ml。不同浓度的沙蜇毒素作用后,红细胞溶血体系中的MDA水平随毒素剂量增大呈依赖性升高,实验中沙蜇毒素浓度为16μg/ml时,MDA浓度由基础浓度(0.79±0.07μmol/L)升高至(19.90±1.22μmol/L),采用单因素方差分析及LSD-t检验,实验各组与对照组比较,差异有统计学意义(p0.05);实验各组互相比较,差异有统计学意义(p0.05)。所研究浓度范围内的维生素C可以拮抗沙蜇毒素的溶血活性,随着维生素C浓度的升高,溶血活性不断下降。当维生素C浓度在0~1mmol/L范围内,毒素的溶血活性下降明显,由75.79%(未添加维生素C)显著下降至31.95%;当维生素C浓度在1~2.5mmol/L范围内,溶血活性由31.95%下降至26.66%,下降趋势减慢。采用单因素方差分析及LSD-t检验,实验各组与对照组相比,差异有统计学意义(p0.05);实验各组互相比较,当实验中维生素C浓度为1.5、2、2.5mmol/L时,差异无统计学意义(p0.05),其余各组差异有统计学意义(p0.05)。结论脂质过氧化损伤是沙蜇毒素溶血活性的机制之一,有其他机制参与了溶血过程。维生素C能够一定程度上抑制沙蜇毒素的溶血活性,可以作为预防和治疗沙蜇蜇伤的候选药物。沙蜇毒素中存在对红细胞产生脂质过氧化损伤的成分,为沙蜇毒素中溶血蛋白的提纯提供了实验帮助。意义了解沙蜇毒素致红细胞损伤的机理,为沙蜇蜇伤的预防和治疗提供实验依据,为深入研究沙蜇毒素中各类生物活性物质提供基础资料。
[Abstract]:Objective to study the mechanism of erythrocyte damage (hemolytic activity) induced by jellyfish toxin and to evaluate the effect of different concentrations of vitamin C on the hemolytic activity of jellyfish toxin. The toxin was extracted by ultrasonic cell fragmentation method. The concentration of toxin protein was determined by using bovine serum albumin as the standard. Healthy volunteers, eight men and eight men, were selected to extract 3ml of fresh blood and washed red blood cells 23 times. We prepared 20 V / V human erythrocyte suspensions and 0.45 V / V human erythrocyte suspensions. Different volumes of jellyfish toxin were added to the 20% human erythrocyte suspensions of 1ml. When 0.9% sodium chloride solution was added to 5 ml, the final concentration of jellyfish toxin was 816 渭 g / ml, the final concentration of jellyfish toxin was 816 渭 g / ml. The method of thiobarbital TBA was used to detect the effect of different concentrations of jellyfish toxin. The level of malondialdehyde (MDA), a product of lipid peroxidation in hemolytic reaction system. A certain volume of jellyfish toxin was added to 0.45% human erythrocyte suspension of 1ml. Different volumes of vitamin C were added, then 0.9% sodium chloride solution was added to dilute to 5 ml. The final concentration of vitamin C was 0 ~ (0.5) ~ (1) ~ (-1) ~ (1.5) ~ (2.5) mmol / L, respectively. The effects of different concentrations of vitamin C on the hemolytic activity of jellyfish stings were determined. In this study, the concentration of jellyfish toxin protein was 395.45 渭 g / ml. The level of MDA in erythrocyte hemolysis system increased in a dependent manner with the increase of toxin dose after different concentrations of jellyfish toxin. When the concentration of jellyfish toxin was 16 渭 g/ml, the concentration of malondialdehyde increased from 0.79 卤0.07 渭 mol / L to 19.90 卤1.22 渭 mol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). The results of single factor variance analysis and LSD-t test showed that there was a significant difference between the experimental groups and the control group (P 0.05). The difference was statistically significant (P 0.05). Vitamin C in the studied concentration range could antagonize the hemolytic activity of jellyfish toxin. With the increase of vitamin C concentration, the hemolytic activity decreased. When the concentration of vitamin C was in the range of 0~1mmol/L, The hemolytic activity of the toxin decreased significantly from 75.79 (without vitamin C) to 31.95. When the concentration of vitamin C was in the range of 1~2.5mmol/L, the hemolytic activity decreased from 31.95% to 26.66%, and the downward trend slowed down. Univariate analysis of variance and LSD-t test were used. Compared with the control group, there was a significant difference between the experimental groups and the control group (p 0.05), and when the concentration of vitamin C in the experiment was 2.5 mmol / L, the concentration of vitamin C in the experiment was 2.5 mmol / L. There was no significant difference between the two groups (P 0.05). Conclusion the damage of lipid peroxidation is one of the mechanisms of the hemolytic activity of jellyfish toxin. There are other mechanisms involved in the hemolysis process. Vitamin C can inhibit the hemolytic activity of jellyfish toxin to some extent. It can be used as a candidate drug for the prevention and treatment of jellyfish stings. It provides experimental help for the purification of hemolytic protein from jellyfish toxin. The significance of this study is to understand the mechanism of erythrocyte damage induced by jellyfish toxin, and to provide experimental basis for the prevention and treatment of jellyfish stings. To provide basic data for further study of various bioactive substances in jellyfish toxin.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R751

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