原矛头蝮蛇毒及其组分对凝血功能的影响

发布时间：2018-09-06 13:15

【摘要】：目的研究原矛头蝮蛇毒(PMV)及其组分作用于血液循环系统的部分生物学活性,进一步探讨其毒性机制。方法采用Sephadex G-75凝胶层析方法分离不同分子质量范围的蛋白组分FⅠ,FⅡ和FⅢ。贫血小板血浆调节富血小板血浆使血小板为3×1011L-1,血小板悬液分别与PMV,FⅠ,FⅡ和FⅢ0.03 g·L-1预孵育5 min,血小板聚集仪测定PMV及其组分诱导血小板聚集活性;PMV,FⅠ,FⅡ和FⅢ0.05 g·L-1分别与纤溶酶原0.1 U·L-1预孵育10 min,采用单一时间点测定和酶动力学测定PMV及其组分酶切发色底物S-2251的作用;将大鼠血浆与PMV,FⅠ,FⅡ和FⅢ1.0 g·L-1分别孵育5和30 min,测定凝血酶时间(TT)、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和纤维蛋白原(FIB)水平;PMV,FⅠ,FⅡ和FⅢ10,50和250 mg·L-1处理微血管内皮细胞24 h,倒置相差显微镜观察细胞形态,MTT法检测细胞存活;PMV,FⅠ,FⅡ和FⅢ0.1 g·L-1与含卵磷脂和不含卵磷脂的豚鼠红细胞悬液分别孵育不同时间,计算溶血率。结果与对照组相比,PMV和高分子质量蛋白组分FⅠ(71 ku)诱导血小板聚集率显著升高〔(61.0±5.8)%和(56.9±5.9)%vs(12.4±4.1)%〕(P0.01)。酶切发色底物S-2251结果显示,PMV与中分子质量组分FⅡ(18~37 ku)具有酶切发色底物的作用(P0.01)。PMV和FⅠ可引起血浆凝固。与对照组相比,FⅡ组分和低分子质量蛋白组分FⅢ(10 ku)明显使TT,APTT和PT的时间延长(P0.01)。PMV,FⅠ及FⅡ导致内皮细胞解离悬浮,与对照组相比,细胞存活率下降,分别为(56.8±3.6)%,(71.6±3.8)%和(58.2±5.5)%。在无卵磷脂条件下,PMV和FⅡ可缓慢地引起红细胞轻度溶血;在卵磷脂参与下,PMV和FⅡ引起豚鼠红细胞急剧溶血,与正常对照组相比,在0.5 min内溶血率从(17.7±1.0)%分别升高至(81.0±4.0)%和(81.0±1.0)%(P0.01)。结论 PMV在体外表现出多方面的血循毒性质的活性,不同分子质量蛋白组分活性机制及作用不同。
[Abstract]:OBJECTIVE To study the biological activity of PMV and its components in the blood circulation system and to explore its toxicity mechanism. Methods Sephadex G-75 gel chromatography was used to isolate protein components F I, F II and F III with different molecular weight ranges. Anemic platelet plasma regulated platelet-rich plasma to make platelets 3 *1011. L-1, platelet suspension and PMV, FI, FII and FIII were pre-incubated for 5 min, respectively, and the platelet aggregation activity induced by PMV and its components was measured by platelet aggregation analyzer; PMV, FI, FII and FIII were pre-incubated with plasminogen 0.1 U.L-1 for 10 min, respectively, and the color base of PMV and its components was determined by single time point assay and enzyme kinetics. Rat plasma was incubated with PMV, FI, FII and FIII for 5 and 30 minutes, respectively, to determine thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FIB); PMV, FI, FII and FIII for 10, 50 and 250 mg L-1 for 24 hours, respectively. Cell morphology was observed and MTT assay was used to detect cell survival. PMV, FI, FII and FIII 0.1 g L-1 were incubated with lecithin-containing and non-lecithin-containing erythrocyte suspensions of guinea pigs for different periods to calculate the hemolysis rate. Enzyme-digested chromogenic substrate S-2251 showed that PMV and FII (18-37 ku) had the effect of Enzyme-digested chromogenic substrate (P 0.01). PMV and FI could induce plasma coagulation. Compared with the control group, FII and FIII (10 ku) significantly prolonged the time of TT, APTT and P T (P 0.01). MV, FI and FII led to the dissociation and suspension of endothelial cells. Compared with the control group, the cell survival rate decreased (56.8 (+) 3.6)%, (71.6 (+) 3.8)% and (58.2 (+) 5.5)%. In the absence of lecithin, PMV and FII could slowly cause mild hemolysis of erythrocytes; PMV and FII could cause acute hemolysis of erythrocytes in guinea pigs with the participation of lecithin. In 0.5 min, the hemolysis rate increased from (17.7 65507
【作者单位】：贵州省中国科学院天然产物化学重点实验室药理与活性筛选中心;
【基金】：国家自然科学基金项目(81260494)~~
【分类号】：R996.3

【参考文献】