【摘要】：隨著物質(zhì)生活水平的提高,血栓疾病也越來(lái)越威脅人類的健康和生命,并且會(huì)引發(fā)嚴(yán)重的后果。微生物溶栓酶是一類具有良好應(yīng)用前景的溶栓酶,不僅能夠大量生產(chǎn),還能彌補(bǔ)傳統(tǒng)溶栓劑的缺點(diǎn)。本實(shí)驗(yàn)室已從豆豉中分離出一株具有產(chǎn)高纖溶活性的溶栓酶的菌株,命名為枯草芽孢桿菌LD-8547。 本實(shí)驗(yàn)經(jīng)過(guò)發(fā)酵、鹽析、透析、濃縮等步驟得到粗酶液,再用Sephadex G-100凝膠分子篩柱純化豆豉溶栓酶,得到分子量約為31 kDa的單一條帶,純化后測(cè)得酶活為21750 U/mL。 通過(guò)豆豉溶栓酶對(duì)優(yōu)球蛋白凝塊的溶解試驗(yàn),表明溶栓酶(469 U)能顯著溶解優(yōu)球蛋白凝塊(P0.05),并具有劑量依賴性。 通過(guò)對(duì)豆豉溶栓酶急性毒性的初步研究,結(jié)果表明小鼠灌胃給藥溶栓酶691586 U/Kg,發(fā)現(xiàn)給藥小鼠與空白對(duì)照組情況相似,兩周內(nèi)均無(wú)死亡,小鼠解剖后,各臟體比均與對(duì)照組無(wú)顯著差異(P0.05),說(shuō)明溶栓酶的口服灌胃LD50遠(yuǎn)大于691586 U/Kg。 小鼠連續(xù)灌胃給藥豆豉溶栓酶一周,測(cè)定體內(nèi)出血時(shí)間、體外凝血時(shí)間、全血塊及全血漿凝塊溶解作用。結(jié)果表明,溶栓酶(4412 U)對(duì)小鼠出血、凝血時(shí)間有一定延長(zhǎng)作用(P0.05),溶栓酶(8200 U)能明顯提高血塊的溶解率,而溶栓酶(4100 U)能明顯提高血漿凝塊的溶解率,并隨劑量的提高而作用加強(qiáng)。通過(guò)角叉菜膠誘發(fā)小鼠尾部血栓及兔頸動(dòng)脈血栓的溶解試驗(yàn),表明豆豉溶栓酶(2051 U)對(duì)小鼠尾靜脈血栓形成平均長(zhǎng)度有一定影響(P0.05),并隨劑量增大抑制血栓形成的作用增強(qiáng)。溶栓酶(1035 U/kg)對(duì)兔頸動(dòng)脈血栓有良好的溶解作用。 小鼠腹腔注射豆豉溶栓酶(5137 U)后,發(fā)現(xiàn)血清中的豆豉溶栓酶活力先升高再下降,在20 min時(shí)酶活力最高,半衰期為32.75 min。 通過(guò)實(shí)驗(yàn),發(fā)現(xiàn)豆豉溶栓酶具有一定的抗栓及溶栓作用,且口服安全性好,是一種良好的潛在治療血栓的藥物。
[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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378

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