【作者】 任莉莉；

【导师】 王进义；

【作者基本信息】 西北农林科技大学 ， 中药学， 2010， 硕士

【摘要】 血栓栓塞性疾病是威胁人类健康的一大原因,且其发病率不断升高,对该疾病的研究也就刻不容缓。为了增加现有溶栓药物的溶栓作用及靶向性,本研究主要将溶解血栓的两种天然酶类蛋白(纳豆激酶和蚓激酶)连接于磁性纳米颗粒上,对其连接条件进行优化,产物性质进行表征,并进一步检测了其溶栓活性。此外为了实现体外条件下血栓形成及溶解的微型化、高效化模型检测系统,为血栓栓塞性疾病及治疗药物的研究提供依据和方法,本实验以微流控芯片技术为平台在体外逼真模拟体内血管血栓的形成,并用上述两种溶栓蛋白酶(Nattokinase and Lumbrukinase)进行溶栓行为研究。用EDC将两种重要的溶栓酶(NK和LK)固定于Fe3O4磁性纳米颗粒上,并研究了它们的溶栓活性。用透射电镜,傅立叶变换红外分光镜,振动探针式磁强计,X射线衍射和紫外可见分光光度计对Fe3O4磁性纳米粒子及NK和LK连接的磁性纳米粒子进行性能分析。在405nm和630nm处进行双波长吸光值测定,研究其溶栓活性。通过载药率分析,NK连接的最佳条件为pH6.00,MNPs : protein : EDC的值为2 : 1 : 1;LK连接的最佳条件为pH6.00,MNPs : protein : EDC的值为2 : 1 : 2。溶栓活性试验表明NK连接的磁性粒子溶栓活性可达91.89%,LK可达207.74%,甚至比纯的NK(82.86%)和LK (106.57%)还高。用PDMS制备不同管径的芯片,用注射泵以一定流速注入血小板丰富的血浆和凝血酶,在倒置荧光显微镜下实时观察血栓生成情况。芯片管径越小生成血栓越大且容易堵塞管道,溶解需较长时间;芯片管径越大则生成血栓较细小,溶解时所需时间也较少。在已生成血栓的管径注入不同浓度NK和LK使其溶解,在显微镜下观察其溶解过程,并用CCD照相机取图。通过溶解时间及血栓面积变化情况分析,管径内凝块在高浓度药物作用下溶解较快,2.5 mg/mL NK和LK溶解时间分别为21分钟和12分钟,且同等浓度下,LK的溶解效率比NK高(平均速率分别为23409.5 a.u./min和14208.4 a.u./min)。更多还原

【Abstract】 Thromboembolic diseases is the major cause of threatening human health, it’s of great urgency to study it as the morbidity is increasing every year. In order to strengthen the function and drug targeting of thrombolytic reagents, we conjugated two thrombolytic emzyms (nattokinase and lumbrukinase) to magnetic nanopartiles, optimized the conjugation conditions, characteristiced the products properties and tested the thrombolytic activity. In addition, a microfluidic chip was used to mimic the in vivo conditions of thrombosis in vessel, also the thrombolysis process was analyzed at the function of NK and LK, thus realized the foundation of micro and efficient detection system in vitro and provided groundwork and method for studying thromboembolic diseases and drugs.Two important thrombolytic enzymes, nattokinase (NK) and lumbrukinase (LK), were immobilized onto fine magnetic Fe3O4 nanoparticles using 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide (EDC) as the coupling reagent, and their thrombolytic activities were studied. The Fe3O4 nanoparticles and NK- and LK-conjugated magnetic nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, X-ray diffraction, and UV–vis absorption spectroscopy. Dual kinetic absorbance measurements at 405nm and 630 nm were employed to measure their thrombolytic activity. Analysis of protein amount showed that the optimum conditions for NK and LK binding to nanoparticles were respectively at a mass ratio of 2 : 1 : 1, 2 : 1 : 2 (magnetic nanoparticles : protein : EDC), and pH6.00. Thrombolytic activity assay showed that the best thrombolytic activity could reach 91.89% for NK–nanoparticle conjugates and 207.74% for LK–nanoparticle conjugates, which are much higher than the pure enzymes (NK, 82.86%; LK, 106.57%).PDMS was used as the material of making this microfluidic chip with different size, pletelet rich plasma and thrombin were injected into the channel by syringe pump under a certain flow rate, then observed the thrombosis with converted fluorescence microscope. With smaller channel the thrombus formed was big and easy to block, also needed longer time to lysis, whereas the thrombus was small and thin in the bigger channel and the thrombolysis time was shorter. Different concentrations of NK and LK were infused into the channel with formed thrombi, watched the thrombolytic process by microscope and got the pictures in real time by CCD camera. Analysis of thrombolysis time and thrombus area showed that the clot lysised faster in high concentration of the thrombolytic agents, the thrombolysis time were 21min and 12min respectively when the concentration was 2.5 mg/mL of NK and LK, the anverage thrombolysis rate of LK was higher than NK (23409.5 a.u./min and 14208.4 a.u./min respectively) at the same conditions.更多还原