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Spirulina kinase anticoagulation and thrombolysis in rats

Author: LiXiaoHua
Tutor: PangHui
School: Guangxi Medical University
Course: Physiology
Keywords: SPK Venous thrombosis Arteriovenous loop thrombosis Anticoagulant Agarose - fibrin plate Thrombolysis D-dimer t-PA
CLC: R285.5
Type: Master's thesis
Year: 2009
Downloads: 122
Quote: 1
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Abstract

Objective To study the kinase Spirulina (Spirulina kinase, SPK) extract and crude enzyme in vitro and in vivo anticoagulant and thrombolytic effect, and to explore its possible mechanism. For the development of new anticoagulant and thrombolytic drugs and lay a good foundation. Method 1 In vitro experiments: (1) SPK Preparation of crude enzyme powder SPK soaked with saline (via Guangxi testing center activity of 7000 IU / g), stirred for 1 h at -4 ℃ placed in a refrigerator overnight, and then centrifuged for 10 min the pellet was discarded and the supernatant is SPK extract. Using ammonium sulfate salting SPK Break extract salting paragraphs product obtained by agarose - fibrin plate method to compare their activity, Coomassie blue protein determination, thrombolytic activity and protein content ratio of more than activity. Salting than and higher activity of frozen sections of the product is dried to give a crude enzyme SPK. (2) SPK extract anticoagulant effect in vitro test tube method, three test tubes were added to extract high concentrations of SPK, SPK low concentrations of extract and normal saline 0.5 ml, then add fresh rabbit blood 1ml, placed after mixing 37 ℃ water bath, start the stopwatch simultaneously recording blood clotting time. (3) SPK extracts in vitro thrombolytic effect of three test tubes were added fresh rabbit blood 1 ml, blood still solidified, discard serum obtained blood clots, check the weight. SPK were added to extract a high concentration and low concentration SPK extract and saline 0.5 ml, and placed in water bath at 37 ℃ was observed after 30 min, weighed residual thrombus weight, thrombolysis rates were calculated for each group. (4) SPK extract liquid outer rat PT, APTT and TT effects in rats after anesthesia, abdominal aortic blood, sodium citrate to obtain plasma, and the plasma is divided into three parts, each of 0.2 ml, respectively. addition of high concentrations of SPK extract, extract and low concentration saline SPK each 0.2 ml, according to kit instructions, measuring PT, APTT and TT values. And used to correct the fibrinogen solution experiments to explore the mechanism of its anticoagulant effect. 2 Animal experiments: (1) SPK crude enzyme impact of CT on mice were randomly divided into four groups, SPK group of low concentration and high concentration group, saline negative control group and the heparin positive control group (12.5 IU / ml) tail intravenous drug use, 20 min after the determination by capillary CT values ??in each group. (2) SPK crude enzyme solution of rat PT, APTT, TT SPK of rats were randomly divided into the low-dose group, SPK high dose group and negative control group, normal saline and heparin positive control group (12.5 IU / ml), shares intravenous administration, abdominal aortic blood, separated plasma, according to kit instructions, each group were measured PT, APTT and TT values. (3) SPK crude enzyme weight on the impact of venous thrombosis in rats were randomly divided into four groups, namely SPK low dose group and high dose group, saline negative control group and positive control group urokinase (2000 IU / ml). After intravenous administration of sublingual rats were anesthetized, the first push half the amount, 30 min after the preparation of venous thrombosis model, 1 h after intravenous injection of the other half by the sublingual amount. After 30 min re-open, remove the thrombus and weighed, placed in 70 ℃ oven for 1 h, let it dry weight was weighed after cooling were calculated thrombus wet weight and dry weight inhibition rate. (4) SPK crude enzyme in rat arteriovenous loop thrombus weight and plasma D-dimer and t-PA content in rats were randomly divided into four groups, namely crude enzyme SPK low dose group and high dose group, saline urokinase negative control group and positive control group (2000 IU / ml). After the rats were anesthetized intravenously stocks, production arteriovenous loop thrombosis model, remove the thrombus weight, ie thrombus wet weight. Oven at 70 ℃ for 1 h, let it dry weight was weighed after cooling, and find thrombus wet weight, dry weight inhibition rate. Abdominal aortic blood, citrated plasma was prepared, the First Affiliated Hospital of Guangxi Medical University, Department of Experimental Clinical detection of D-dimer levels, ELISA assay t-PA levels and to compare differences between groups. Results 1 in vitro (1) SPK crude enzyme preparation of ammonium sulfate salting SPK Break extract, 45% to 75% saturation highest specific activity obtained precipitate, after triple-distilled water, dialyzed frozen dried to SPK crude enzyme. (2) SPK extracts in vitro anticoagulant effect compared with the saline group, high and low dose SPK extract could significantly prolong rabbit blood clotting time (P lt; 0.01) (3) SPK extracts in vitro thrombolysis compared with the saline group, high and low dose SPK extract can significantly reduce the weight of the thrombus (P lt; 0.01) (4) SPK extract liquid outer pair PT, APTT and TT effects: compared with the saline group, high dose SPK rats can extract in vitro plasma PT, APTT was significantly prolonged (P lt; 0.01), high and low dose rats can SPK extract significantly prolonged plasma TT (low dose P lt; 0.05, high-dose P lt; 0.01). 2 animal experiments (1) SPK crude enzyme impact of CT on mice compared with the saline group, high dose SPK crude enzyme can prolong the CT (P lt; 0.01). (2) SPK crude enzyme solution on the rat PT, APTT, TT effects compared with the saline group, SPK crude enzyme allows the rat plasma PT, APTT and TT was significantly longer (low-dose P lt; 0.05, high-dose P lt; 0.01) (3) SPK crude enzyme solution on the weight of rat venous thrombosis compared with the saline group, high and low dose SPK vein thrombosis crude enzyme were so wet weight, dry weight was significantly reduced (P lt; 0.01) . (4) SPK crude enzyme in rat arteriovenous loop thrombus weight and plasma D-dimer and t-PA content in preparation arteriovenous loop thrombosis model, compared with the saline group, high and low dose SPK coarse enzymes in rat arteriovenous loop thrombus wet weight, dry weight was significantly reduced (P all lt; 0.05), high and low dose group, the plasma D-dimer levels were significantly increased (P all lt; 0.05), high-dose t-PA group was significantly higher (P lt; 0.05). Conclusion ammonium Break salting SPK extract, 45% to 75% saturation to obtain the highest specific activity of the crude enzyme SPK. SPK extract a significant in vitro anticoagulant effect and the direct dissolution of blood clots. SPK crude enzyme in vivo performance of a significant anticoagulant effect, on experimental thrombosis and venous thrombosis of arteriovenous loop significant dissolution. SPK crude fibrinolytic enzyme has a direct role in the plasma D-dimer levels were significantly increased, but also can promote the secretion of t-PA endothelial cells play a role in fibrinolysis.

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