本文選題：溶栓 + 超聲��； 參考：《新疆醫(yī)科大學》2010年博士論文

【摘要】：目的：研究制備攜帶尿激酶(UK)及精氨酸—甘氨酸—天冬氨酸—絲氨酸(RGDS)肽段靶向溶栓超聲微泡造影劑的可行性,并評價其理化性質及所攜帶尿激酶的活性。探討不同濃度的FeCl3和不同方法對制備兔股動脈內閉塞性、以血小板為主的混合性血栓的影響因素,為溶栓研究提供穩(wěn)定、方便的血栓模型。探索與血栓特異性靶向結合的超聲微泡攜帶藥物溶解血栓的靶向性和有效性。方法：尿激酶和RGDS進行熒光雙標記,根據(jù)尿激酶：RGDS的1：1、2：1和1：2不同比例實驗分為三組,以聲諾維(SonoVue)為載體,通過直接連接法,制備親血栓同時攜帶溶栓藥物的超聲微泡造影劑,并檢測三組微泡大小、形態(tài)、熒光亮度,微泡與尿激酶及RGDS的結合率以及所攜帶尿激酶的活性。30只新西蘭大白兔總60條股動脈,分別予以不同濃度的Fecl3 (10%FeCl3,10%FeCl3,10%FeCl3)、外敷法和內注射法以及股動脈遠端有無夾閉等影響因素將動物分為三組,觀察血栓形成情況,并對血管行HE染色和免疫組化血管假性血友病因子(von Willebrand factor,vWF)染色。42只新西蘭大白兔單側股動脈制作富含血小板的混合性血栓,分成7組,每組6只：1)單純超聲照射組(US)；2)超聲照射+造影劑注射組(US+M);3)單純尿激酶靜脈注射組(UK)；4)超聲照射+微泡造影劑+尿激酶靜脈注射組(US+M+UK);5)超聲照射+RGDS微泡造影劑組(US+R)；6)RGDS微泡造影劑+尿激酶靜脈注射組(R+UK)；7)超聲照射+RGDS微泡造影劑+尿激酶組(US+R+UK)。將RGDS、微泡造影劑(SonoVue)和尿激酶通過直接聯(lián)合法,按1：1：1配制6ml的溶液。3ml在5min內團注,3ml在20min內靜脈緩慢推注,超聲照射30min。120min后,通過超聲檢測、血流量測量和病理結果分析來對血栓的靶向性及溶栓效果進行評價。血流量持續(xù)監(jiān)測120min后對血管的再通率和溶栓過程中血流頻譜特點進行分析。結果：聲諾維、尿激酶及RGDS三者可以穩(wěn)定結合,以尿激酶和RGDS呈1：1比例配制的最佳。配制的造影劑在熒光顯微鏡下觀察,洗滌前、后微泡表面均可發(fā)出綠色及橙紅色熒光；流式細胞儀定量檢測結果顯示,洗滌前、后尿激酶攜帶率分別為73.4±11.0%、72.3±9.4%,RGDS攜帶率分別為67.1±10.9%、64.6±10.2%；體外血栓溶解實驗證實所制備造影劑中尿激酶具有活性(P0.01),血栓溶解率為18.4±3.2%。外敷10%FeCl3形成閉塞性血栓優(yōu)于其他濃度(P0.001),10%FeCl3外敷法形成血栓優(yōu)于內注射法(P0.001),10%FeCl3外敷法聯(lián)合遠端夾閉形成血栓優(yōu)于無夾閉的條件(P0.001)。在R+UK組和US+R+UK組,熒光顯微鏡下兔股動脈血栓部位同時顯示標記RGDS的橙紅色熒光和標記尿激酶的綠色熒光；UK、US、US+R、US+M組血流量基礎血流量15%,均未實現(xiàn)溶栓后血管再通；US+M+UK、R+UK組血流量在基礎血流量的15%～49%之間；US+R+UK組血流量75%基礎血流量。120min后US、UK、US+M、US+R及US+M+UK組均未實現(xiàn)再通(再通率15%),血流頻譜未出現(xiàn)變化為小、低幅雜波；R+UK組實現(xiàn)部分再通(再通率41.61%),US+R+UK完全再通(再通率85.81%),溶栓時血流頻譜出現(xiàn)持續(xù)高幅、雜亂的波(P0.001),在US+R+UK組出現(xiàn)超聲波與血流頻譜出現(xiàn)高幅、雜亂的共振變化血流量實現(xiàn)完全再通。結論：應用直接連接法可以制備同時攜帶尿激酶及RGDS的靶向溶栓超聲微泡造影劑。外敷10%FeCl3外敷聯(lián)合股動脈夾閉是成功形成股動脈內閉塞性、以血小板為主的混合性血栓的重要因素。應用直接聯(lián)合法制備RGDS微泡造影劑攜帶尿激酶對血栓具有靶向性,并能溶解兔股動脈在體血栓。
[Abstract]:Objective: To study the feasibility of preparation of urokinase (UK) and arginine - glycine - aspartate - serine (RGDS) peptide target thrombolytic ultrasound microbubble contrast agent, and evaluate its physicochemical properties and the activity of urokinase. Different concentrations of FeCl3 and different methods were used to prepare rabbit femoral artery occlusion, mainly platelets. The influencing factors of mixed thrombosis provide a stable and convenient thrombus model for thrombolytic study. Explore the targeting and effectiveness of thrombolytic microbubbles combined with thrombus specific targets. Methods: urokinase and RGDS were labeled with fluorescence, and the experiments were divided into different ratios of urokinase: RGDS 1:1,2:1 and 1:2. The three group, using SonoVue as the carrier, to prepare the ultrasound microbubble contrast agent with thrombolytic and thrombolytic drugs by direct connection, and detect the size, morphology, luminance, the binding rate of the three groups of microbubbles, the ratio of microbubbles to urokinase and RGDS, and the total 60 femoral arteries of the New Zealand white rabbit with active.30, respectively. The same concentration of Fecl3 (10%FeCl3,10%FeCl3,10%FeCl3), external application and internal injection, and the influence of the distal femoral artery were divided into three groups, the thrombosis was observed, and the blood vessel HE staining and the von Willebrand factor, vWF were used to stain the unilateral femoral of.42 New Zealand white rabbits. Arterial production of mixed thrombocytopenia rich in platelets was divided into 7 groups, which were divided into 7 groups, 6 in each group, 1) alone in ultrasound irradiation group (US); 2) ultrasound irradiation plus contrast agent injection group (US+M); 3) pure urokinase intravenous group (UK); 4) ultrasound irradiation + microbubble contrast agent + urokinase intravenous injection group (US+M+UK); 5) +RGDS microbubble contrast agent group (US+R); 6) RGDS micro Vesicle contrast agent + urokinase intravenous injection group (R+UK); 7) ultrasound irradiation of +RGDS microbubble contrast agent + urokinase group (US+R+UK). RGDS, microbubble contrast agent (SonoVue) and urokinase were combined by direct combination method, 1:1:1 prepared 6ml solution.3ml in 5min internal injection, 3ml in 20min intravenous injection slowly, ultrasonic irradiation of 30min.120min, through ultrasonic testing. The blood flow measurement and pathological results were used to evaluate the target and thrombolytic effect of thrombus. After continuous monitoring of blood flow, the recanalization rate of blood vessels and the characteristics of blood flow spectrum during thrombolytic process were analyzed after 120min. Results: the combination of acoustic nove, urokinase and RGDS three was the best combination of urokinase and RGDS in the proportion of 1:1. The contrast agent was observed under the fluorescence microscope. Before washing, the surface of the posterior microbubble could be green and orange red fluorescence. The results of flow cytometry showed that the carrying rate of urokinase was 73.4 + 11% and 72.3 + 9.4% respectively before washing, and the carrying rate of RGDS was 67.1 + 10.9% and 64.6 + 10.2% respectively. In vitro thrombolytic experiment proved to be made by thrombolytic experiment in vitro Urokinase in contrast medium was active (P0.01), thrombolysis rate was 18.4 + 3.2%. external application and 10%FeCl3 was superior to other concentrations (P0.001), 10%FeCl3 external application was superior to internal injection (P0.001), 10%FeCl3 external application combined with distal occlusion to form blood thrombus was superior to non clipping condition (P0.001). In R+UK and US+R+UK groups, fluores The site of rabbit femoral artery thrombosis under light microscope showed the orange red fluorescence of RGDS and the green fluorescence labeling of urokinase; UK, US, US+R, US+M group blood flow basic blood flow was 15%, all did not realize blood vessel recanalization after thrombolysis; US+M+UK, R+UK group blood flow was between 15% and 49% of basic blood flow, US+R+UK group blood flow 75% basic blood flow.1 After 20min, the group of US, UK, US+M, US+R and US+M+UK did not realize recanalization (re pass rate 15%), the blood flow spectrum did not change to small, low amplitude clutter; the R+UK group realized partial recanalization (re pass rate 41.61%), US+R+UK complete recanalization (re pass rate 85.81%), the blood flow spectrum appeared sustained high amplitude, chaotic wave (P0.001) in the thrombolysis, in the US+R+UK group, the ultrasonic and blood frequency appeared in the US+R+UK group. Conclusion: the direct connection method can be used to prepare the target thrombolytic ultrasound microbubbles that carry urokinase and RGDS simultaneously. External application of 10%FeCl3 and femoral artery occlusion is an important cause of the successful formation of the femoral artery occlusion, and the main cause of the mixed thrombus with platelets. The preparation of RGDS microbubble contrast agent by direct combination method can carry urokinase to target thrombus and dissolve thrombus in rabbit femoral artery in vivo.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R543

【引證文獻】

相關碩士學位論文 前1條

1 李愛祥;4-甲氧基-1，3-苯二甲酰胺衍生物的合成及其體外抗血小板聚集活性測定[D];天津理工大學;2014年

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本文編號：2003734