蝮蛇毒血小板抑制因子对血小板信号通路影响及酶活性的研究

发布时间：2017-12-30 21:51

本文关键词：蝮蛇毒血小板抑制因子对血小板信号通路影响及酶活性的研究　出处：《皖南医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的:探讨蝮蛇毒血小板抑制因子(platelet inhibitor from Askistrodon halys venom,AHV-PI)抑制血小板聚集的可能作用机制。方法:实验新西兰兔30只,随机分为:空白实验组,颈动脉血栓模型组,AHV-PI低剂量组(0.05mg/kg)、中剂量组(0.1mg/kg)、高剂量组(0.2mg/kg),阳性对照组(PI3K阻断剂LY294002,20u M),共6个实验组,每组各5只。利用70%Fe Cl3通过化学损伤的方法制造家兔颈总动脉血栓模型,AHV-PI各剂量组经耳缘静脉注入相应体积的AHV-PI,空白实验组不复制模型,仅注入相同体积0.9%盐水。1h后经另一侧颈总动脉采血,离心取上清液,采用双通道凝血仪测定家兔凝血功能,ELISA方法测定各组血浆中纤溶酶(plasmin)、5-核苷酸酶(5-NT)、血小板膜糖蛋白Ib(GPIb)的含量,XS-1000I血球计数仪进行血小板计数,流式细胞术(FCM)观测AHV-PI干预下,荧光标识的单克隆抗体CD61(FITC-CD61)与血小板膜糖蛋白IIb/IIIa(GPIIb/IIIa)结合率的变化,蛋白质免疫印迹法(Western bloting)测定血小板内Akt蛋白激酶磷酸化程度。结果:病理切片显示AHV-PI中、高剂量组颈动脉内膜相对比较平滑,管腔内未见血栓形成。阳性对照组、AHV-PI中剂量组和高剂量组中血小板数目和蛋白激酶Akt磷酸化水平与模型组比较均下降(P0.05),较空白实验组无明显改变(P0.05),其中模型组与AHV-PI低剂量组之间、阳性对照组与AHV-PI中、高剂量组之间无明显差异(P0.05)。与空白实验组比较,模型组和AHV-PI低剂量组中活化部分凝血酶(APTT)、凝血酶原时间(PT)与凝血酶时间(TT)都显著缩短(P0.05),血浆中FIB含量增高(P0.05),纤溶酶水平下降(P0.05),5-NT含量无显著变化(P0.05),GPIb水平升高(P0.05),中、高剂量组凝血功能指标中除PT、TT外均无明显改变(P0.05),纤溶酶水平进一步下降(P0.05),5-NT含量升高(P0.05),GPIb含量降低(P0.05);与模型组相比,AHV-PI中、高剂量组APTT、TT延长(P0.05),PT无明显改变(P0.05),血浆中FIB、纤溶酶和GPIb含量均下降(P0.05),5-NT含量升高(P0.05)。除PT外,模型组与AHV-PI低剂量组之间和AHV-PI中、高剂量组之间相比以上各项指标差异无统计学意义(P0.05)。流式细胞术显示AHV-PI对单克隆抗体CD61与血小板GPIIb/IIIa的结合率没有显著影响(P0.05)。结论:AHV-PI抑制血小板聚集的机制可能是降低了蛋白激酶Akt磷酸化水平,阻碍Akt通路下游的信号转导;也可能与自身各种酶的活性有关,如纤溶酶原的激活、5-核苷酸酶能够分解血浆中相应的致聚成分。
[Abstract]:Objective: To investigate the venom platelet inhibition factor (platelet inhibitor from Askistrodon halys venom, AHV-PI) could inhibit platelet aggregation mechanism. Methods: the 30 New Zealand rabbits were randomly divided into blank group, carotid artery thrombosis model group, AHV-PI low dose group (0.05mg/kg), middle dose group (0.1mg/kg), high dose group (0.2mg/kg), positive control group (PI3K inhibitor LY294002,20u M), a total of 6 experimental groups, 5 rats in each group. Making rabbit model of carotid artery thrombosis by method of chemical damage by 70%Fe Cl3, each AHV-PI group injected into the corresponding volume of AHV-PI by the ear vein, the experimental group does not copy the model of blank 0.9%, only the injection of the same volume saline.1h after the other side of the carotid artery blood, centrifugal supernatant, determination of blood coagulation in rabbits by double channel blood coagulation analyzer and ELISA method for the determination of plasma plasminogen activator (plasmin), 5- nucleotide The enzyme (5-NT), platelet membrane glycoprotein Ib (GPIb) content, platelet count XS-1000I blood cell count, flow cytometry (FCM) observation AHV-PI intervention, CD61 monoclonal antibody fluorescent labeling (FITC-CD61) IIb/IIIa and platelet membrane glycoprotein (GPIIb/IIIa) combined with the rate of change, Western blotting (Western bloting determination of platelet Akt) protein kinase phosphorylation. Results: histological examination showed that AHV-PI in high dose group, intima of carotid artery is relatively smooth, and there was no thrombosis of the lumen. The positive control group, the number of platelets in AHV-PI dose group and high dose group and protein kinase Akt phosphorylation levels were decreased (compared with the model group P0.05), compared with the experimental group had no significant change (P0.05), the model group and AHV-PI low dose group, positive control group and AHV-PI, there is no significant difference between the high dose group (P0.05). The experimental group and the blank Comparison of partial thrombin activated in the model group and AHV-PI low dose group (APTT), prothrombin time (PT) and thrombin time (TT) were significantly shortened (P0.05), increased the contents of plasma FIB (P0.05), plasminogen levels decreased (P0.05), the content of 5-NT had no significant change (P0.05), GPIb level (P0.05), and blood coagulation indexes in high dose group in addition to PT, TT showed no significant change (P0.05), plasminogen levels decline further increased the content of 5-NT (P0.05), (P0.05), decreased the content of GPIb (P0.05); compared with the model group, AHV-PI, APTT of high dose group (P0.05, TT extension), PT (P0.05), no significant changes in plasma FIB, plasmin and GPIb content decreased (P0.05), increased the content of 5-NT (P0.05). Except PT, between the model group and low dose group of AHV-PI and AHV-PI in high dose group compared the differences in the above indexes had no statistical significance (P0.05). AHV-PI of monoclonal flow cytometry Binding antibody CD61 and platelet GPIIb/IIIa rate had no significant effect (P0.05). Conclusion: the inhibition of platelet aggregation may be reduced AHV-PI protein kinase Akt phosphorylation of signal transduction pathways downstream of block Akt; also may be related to its activity of various enzymes, such as plasminogen activator, 5- nucleotidase can be decomposed by poly component corresponding in plasma.

【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R54;R743

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