直接连接法制备携尿激酶RGDS造影剂微泡靶向溶解在体血栓的实验研究

发布时间：2018-06-10 15:40

本文选题：溶栓 + 超声　；参考：《新疆医科大学》2010年博士论文

【摘要】：目的：研究制备携带尿激酶(UK)及精氨酸—甘氨酸—天冬氨酸—丝氨酸(RGDS)肽段靶向溶栓超声微泡造影剂的可行性,并评价其理化性质及所携带尿激酶的活性。探讨不同浓度的FeCl3和不同方法对制备兔股动脉内闭塞性、以血小板为主的混合性血栓的影响因素,为溶栓研究提供稳定、方便的血栓模型。探索与血栓特异性靶向结合的超声微泡携带药物溶解血栓的靶向性和有效性。方法：尿激酶和RGDS进行荧光双标记,根据尿激酶：RGDS的1：1、2：1和1：2不同比例实验分为三组,以声诺维(SonoVue)为载体,通过直接连接法,制备亲血栓同时携带溶栓药物的超声微泡造影剂,并检测三组微泡大小、形态、荧光亮度,微泡与尿激酶及RGDS的结合率以及所携带尿激酶的活性。30只新西兰大白兔总60条股动脉,分别予以不同浓度的Fecl3 (10%FeCl3,10%FeCl3,10%FeCl3)、外敷法和内注射法以及股动脉远端有无夹闭等影响因素将动物分为三组,观察血栓形成情况,并对血管行HE染色和免疫组化血管假性血友病因子(von Willebrand factor,vWF)染色。42只新西兰大白兔单侧股动脉制作富含血小板的混合性血栓,分成7组,每组6只：1)单纯超声照射组(US)；2)超声照射+造影剂注射组(US+M);3)单纯尿激酶静脉注射组(UK)；4)超声照射+微泡造影剂+尿激酶静脉注射组(US+M+UK);5)超声照射+RGDS微泡造影剂组(US+R)；6)RGDS微泡造影剂+尿激酶静脉注射组(R+UK)；7)超声照射+RGDS微泡造影剂+尿激酶组(US+R+UK)。将RGDS、微泡造影剂(SonoVue)和尿激酶通过直接联合法,按1：1：1配制6ml的溶液。3ml在5min内团注,3ml在20min内静脉缓慢推注,超声照射30min。120min后,通过超声检测、血流量测量和病理结果分析来对血栓的靶向性及溶栓效果进行评价。血流量持续监测120min后对血管的再通率和溶栓过程中血流频谱特点进行分析。结果：声诺维、尿激酶及RGDS三者可以稳定结合,以尿激酶和RGDS呈1：1比例配制的最佳。配制的造影剂在荧光显微镜下观察,洗涤前、后微泡表面均可发出绿色及橙红色荧光；流式细胞仪定量检测结果显示,洗涤前、后尿激酶携带率分别为73.4±11.0%、72.3±9.4%,RGDS携带率分别为67.1±10.9%、64.6±10.2%；体外血栓溶解实验证实所制备造影剂中尿激酶具有活性(P0.01),血栓溶解率为18.4±3.2%。外敷10%FeCl3形成闭塞性血栓优于其他浓度(P0.001),10%FeCl3外敷法形成血栓优于内注射法(P0.001),10%FeCl3外敷法联合远端夹闭形成血栓优于无夹闭的条件(P0.001)。在R+UK组和US+R+UK组,荧光显微镜下兔股动脉血栓部位同时显示标记RGDS的橙红色荧光和标记尿激酶的绿色荧光；UK、US、US+R、US+M组血流量基础血流量15%,均未实现溶栓后血管再通；US+M+UK、R+UK组血流量在基础血流量的15%～49%之间；US+R+UK组血流量75%基础血流量。120min后US、UK、US+M、US+R及US+M+UK组均未实现再通(再通率15%),血流频谱未出现变化为小、低幅杂波；R+UK组实现部分再通(再通率41.61%),US+R+UK完全再通(再通率85.81%),溶栓时血流频谱出现持续高幅、杂乱的波(P0.001),在US+R+UK组出现超声波与血流频谱出现高幅、杂乱的共振变化血流量实现完全再通。结论：应用直接连接法可以制备同时携带尿激酶及RGDS的靶向溶栓超声微泡造影剂。外敷10%FeCl3外敷联合股动脉夹闭是成功形成股动脉内闭塞性、以血小板为主的混合性血栓的重要因素。应用直接联合法制备RGDS微泡造影剂携带尿激酶对血栓具有靶向性,并能溶解兔股动脉在体血栓。
[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.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R543

【引证文献】