枯草芽孢杆菌LD-8547溶栓酶溶栓效果初步研究

发布时间：2018-08-14 09:36

【摘要】：随着物质生活水平的提高,血栓疾病也越来越威胁人类的健康和生命,并且会引发严重的后果。微生物溶栓酶是一类具有良好应用前景的溶栓酶,不仅能够大量生产,还能弥补传统溶栓剂的缺点。本实验室已从豆豉中分离出一株具有产高纤溶活性的溶栓酶的菌株,命名为枯草芽孢杆菌LD-8547。本实验经过发酵、盐析、透析、浓缩等步骤得到粗酶液,再用Sephadex G-100凝胶分子筛柱纯化豆豉溶栓酶,得到分子量约为31 kDa的单一条带,纯化后测得酶活为21750 U/mL。通过豆豉溶栓酶对优球蛋白凝块的溶解试验,表明溶栓酶(469 U)能显著溶解优球蛋白凝块(P0.05),并具有剂量依赖性。通过对豆豉溶栓酶急性毒性的初步研究,结果表明小鼠灌胃给药溶栓酶691586 U/Kg,发现给药小鼠与空白对照组情况相似,两周内均无死亡,小鼠解剖后,各脏体比均与对照组无显著差异(P0.05),说明溶栓酶的口服灌胃LD50远大于691586 U/Kg。小鼠连续灌胃给药豆豉溶栓酶一周,测定体内出血时间、体外凝血时间、全血块及全血浆凝块溶解作用。结果表明,溶栓酶(4412 U)对小鼠出血、凝血时间有一定延长作用(P0.05),溶栓酶(8200 U)能明显提高血块的溶解率,而溶栓酶(4100 U)能明显提高血浆凝块的溶解率,并随剂量的提高而作用加强。通过角叉菜胶诱发小鼠尾部血栓及兔颈动脉血栓的溶解试验,表明豆豉溶栓酶(2051 U)对小鼠尾静脉血栓形成平均长度有一定影响(P0.05),并随剂量增大抑制血栓形成的作用增强。溶栓酶(1035 U/kg)对兔颈动脉血栓有良好的溶解作用。小鼠腹腔注射豆豉溶栓酶(5137 U)后,发现血清中的豆豉溶栓酶活力先升高再下降,在20 min时酶活力最高,半衰期为32.75 min。通过实验,发现豆豉溶栓酶具有一定的抗栓及溶栓作用,且口服安全性好,是一种良好的潜在治疗血栓的药物。
[Abstract]:With the improvement of material standard of living, thrombus disease is more and more threatening human health and life, and will lead to serious consequences. Microbial thrombolytic enzyme is a kind of thrombolytic enzyme with good application prospect. It not only can be produced in large quantities, but also can make up for the shortcomings of traditional thrombolytic agents. A strain with high fibrinolytic activity was isolated from Douchi and named Bacillus subtilis LD-8547. After fermentation, salting out, dialysis and concentration, the crude enzyme solution was obtained, and then purified by Sephadex G-100 gel molecular sieve column, a single band with molecular weight of 31 kDa was obtained. After purification, the enzyme activity was measured to be 21750 U / mL. The results showed that the thrombolytic enzyme (469U) could significantly dissolve the euglobulin clot (P0.05) in a dose-dependent manner. The preliminary study on acute toxicity of thrombolytic enzyme in Douchi showed that mice were given 691586 U / kg thrombolytic enzyme by intragastric administration. The results showed that the situation of mice treated with the drug was similar to that of the blank control group, and there was no death within two weeks. After dissection, the mice were dissected. There was no significant difference between the visceral body ratio and the control group (P0.05), which indicated that the oral LD50 of thrombolytic enzyme was much greater than 691586 U / kg. Mice were administrated with Douchi thrombolytic enzyme continuously for one week. The bleeding time in vivo, the coagulation time in vitro, the whole blood clot and the whole plasma clot lysis were measured. The results showed that thrombolytic enzyme (4412 U) could prolong the blood coagulation time of mice (P0.05). Thrombolytic enzyme (8200 U) could significantly increase the dissolution rate of blood clots, while thrombolytic enzyme (4100 U) could significantly increase the dissolution rate of plasma clots. The effect was enhanced with the increase of dose. The experimental results of tail thrombosis and rabbit carotid thrombosis induced by carrageenan showed that the average length of thrombus formation in tail vein of mice was affected by Douchi thrombolytic enzyme (2051 U) (P0.05), and the effect of inhibiting thrombosis was enhanced with the increase of dosage. Thrombolytic enzyme (1035 U/kg) had a good dissolution effect on carotid thrombosis in rabbits. After intraperitoneal injection of Douchi thrombolytic enzyme (5137 U), it was found that the activity of Douchi thrombolytic enzyme in serum increased first and then decreased, and reached the highest level at 20 min, with a half-life of 32.75 min. It was found that Douchi thrombolytic enzyme had some antithrombotic and thrombolytic effects and had good oral safety. It was a potential drug for thrombus treatment.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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