本文關(guān)鍵詞：蝮蛇毒血小板抑制因子對(duì)血小板信號(hào)通路影響及酶活性的研究　出處：《皖南醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蝮蛇毒 動(dòng)脈血栓 血小板抑制因子 纖溶酶原激活劑 蛋白激酶

【摘要】：目的:探討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機(jī)制。方法:實(shí)驗(yàn)新西蘭兔30只,隨機(jī)分為:空白實(shí)驗(yàn)組,頸動(dòng)脈血栓模型組,AHV-PI低劑量組(0.05mg/kg)、中劑量組(0.1mg/kg)、高劑量組(0.2mg/kg),陽性對(duì)照組(PI3K阻斷劑LY294002,20u M),共6個(gè)實(shí)驗(yàn)組,每組各5只。利用70%Fe Cl3通過化學(xué)損傷的方法制造家兔頸總動(dòng)脈血栓模型,AHV-PI各劑量組經(jīng)耳緣靜脈注入相應(yīng)體積的AHV-PI,空白實(shí)驗(yàn)組不復(fù)制模型,僅注入相同體積0.9%鹽水。1h后經(jīng)另一側(cè)頸總動(dòng)脈采血,離心取上清液,采用雙通道凝血儀測定家兔凝血功能,ELISA方法測定各組血漿中纖溶酶(plasmin)、5-核苷酸酶(5-NT)、血小板膜糖蛋白Ib(GPIb)的含量,XS-1000I血球計(jì)數(shù)儀進(jìn)行血小板計(jì)數(shù),流式細(xì)胞術(shù)(FCM)觀測AHV-PI干預(yù)下,熒光標(biāo)識(shí)的單克隆抗體CD61(FITC-CD61)與血小板膜糖蛋白IIb/IIIa(GPIIb/IIIa)結(jié)合率的變化,蛋白質(zhì)免疫印跡法(Western bloting)測定血小板內(nèi)Akt蛋白激酶磷酸化程度。結(jié)果:病理切片顯示AHV-PI中、高劑量組頸動(dòng)脈內(nèi)膜相對(duì)比較平滑,管腔內(nèi)未見血栓形成。陽性對(duì)照組、AHV-PI中劑量組和高劑量組中血小板數(shù)目和蛋白激酶Akt磷酸化水平與模型組比較均下降(P0.05),較空白實(shí)驗(yàn)組無明顯改變(P0.05),其中模型組與AHV-PI低劑量組之間、陽性對(duì)照組與AHV-PI中、高劑量組之間無明顯差異(P0.05)。與空白實(shí)驗(yàn)組比較,模型組和AHV-PI低劑量組中活化部分凝血酶(APTT)、凝血酶原時(shí)間(PT)與凝血酶時(shí)間(TT)都顯著縮短(P0.05),血漿中FIB含量增高(P0.05),纖溶酶水平下降(P0.05),5-NT含量無顯著變化(P0.05),GPIb水平升高(P0.05),中、高劑量組凝血功能指標(biāo)中除PT、TT外均無明顯改變(P0.05),纖溶酶水平進(jìn)一步下降(P0.05),5-NT含量升高(P0.05),GPIb含量降低(P0.05);與模型組相比,AHV-PI中、高劑量組APTT、TT延長(P0.05),PT無明顯改變(P0.05),血漿中FIB、纖溶酶和GPIb含量均下降(P0.05),5-NT含量升高(P0.05)。除PT外,模型組與AHV-PI低劑量組之間和AHV-PI中、高劑量組之間相比以上各項(xiàng)指標(biāo)差異無統(tǒng)計(jì)學(xué)意義(P0.05)。流式細(xì)胞術(shù)顯示AHV-PI對(duì)單克隆抗體CD61與血小板GPIIb/IIIa的結(jié)合率沒有顯著影響(P0.05)。結(jié)論:AHV-PI抑制血小板聚集的機(jī)制可能是降低了蛋白激酶Akt磷酸化水平,阻礙Akt通路下游的信號(hào)轉(zhuǎn)導(dǎo);也可能與自身各種酶的活性有關(guān),如纖溶酶原的激活、5-核苷酸酶能夠分解血漿中相應(yīng)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.

【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R54;R743

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