人脐带血浆纤维蛋白溶解酶原的纯化及其诱导玻璃体后脱离的研究

摘要

背景 近年来关于药物性玻璃体溶解的研究已陆续开展,主要包括软骨素酶、透明质酸酶、分散酶和纤维蛋白溶解酶等几大酶类,有些已应用于临床试验.然而对源于人脐带血浆的纤维蛋白溶解酶用于实验性诱导动物眼玻璃体后脱离(PVD)的研究尚未见报道.目的 从人脐带血血浆中实现有活性的纤维蛋白溶解酶原的提取及纯化,并探讨其在诱导PVD方面的初步作用.方法 利用低温醇沉液相分离法从人脐带血血浆中获得较纯的纤维蛋白溶解酶原(Plg)并进行活性测定.取新鲜离体猪眼25只,分为5组.第一组作为正常对照,第二组每只眼玻璃体腔注入0.1 ml平衡盐溶液(BSS),第三组每只眼注入0.1 ml含1000 μmol/(min·L)的重组链激酶(r-SK),第四组每只眼注入0.1 ml含1000 μmol/(min·L)的r-SK联合0.1 ml含3 μmol/(min·L)的Plg(r-SK+Plg),第五组每只眼注入0.1 ml 1000 μmol/(min·L)的r-SK联合0.1 ml 3 μmol/(min·L)的人纤维蛋白溶解酶原标准品(r-SK+Plg标准品),各组药物均经睫状体平坦部注射,37℃孵育60min后常规固定,在光学显微镜、扫描电子显微镜和透射电子显微镜下进行视网膜形态学和超微结构观察.结果 成功从人脐带血血浆中获得了纤维蛋白溶解酶原的分离及提取,初步纯化后获得有潜在纤溶活性的纤维蛋白溶解酶原.动物实验发现正常对照组、BSS组及r-SK组均未见PVD.除r-SK+Plg 标准品组外,r-SK+Plg组的猪眼后极部发生PVD,视网膜内界膜表面较光滑,光学显微镜及电镜下视网膜的组织结构和细胞结构均未见异常.结论 纤维蛋白溶解酶原能够通过低温醇沉联合液相分离方法从人脐带血血浆中获得并且具有潜在纤维蛋白溶解活性.1000 μmol/(min·L)r-SK与3 μmol/(min·L)Plg联合注入离体猪眼玻璃体腔60 min可以诱发后极部PVD,对视网膜未见毒性作用.%Background Many studies and clinical trials of pharmacologic vitreolysis are already under way to try to improve vitreo-retinal surgery and to liquefy and detach the vitreous from the retina ultimately, including chondroitinase,hyaluronidase,dispase and plasmin. However, there has not been any report on purification of human plasminogen from cord blood plasma and inducing posterior vitreous detachment of the animal eye at present.Objective This study was designed to isolate and purify the production of human plasminogen (Plg) from cord blood plasma with ethanol precipitation and evaluate the efficacy of Plg in inducing posterior vitreous detachment (PVD).Methods Human Plg was Separated and purified from cord blood plasma by ethanol precipitation method. The protein band corresponding to Plg with molecular mass of 92 000 was revealed in SDS-PAGE and confirmed by MALDI-TOF and Mascot database. Anion-exchange chromatography and plasminogen activity assay kit were used to obtain purified Plg with biological activity. Twenty-five fresh pig eyes were enucleated and assigned to 5 groups and 5 eyes for each group. The normal eyes were used as control group. Balanced salt solution(BSS)of 0.1 ml was intravitreally group and standard substance group. All of the eyes were then incubatedfor 60 minutes under the 37 ℃. Retinal histopathology and ultrastructure were examined under the light microscopy, scanning electron microscopy ( SEM ) and transmission electron microscopy (TEM). Results The Plg with potential fibrinolytic activity was successfully extracted and purified from cord blood plasma by ethanol precipitation method. No posterior vitreous detachment (PVD) was seen in normal control group, BSS group and r-SK group following the intravitreal injection under the sem. However,PVD was demonstrated in r-SK+ Plg group and standard substance group under the SEM. The inner limiting membrane ( ILM ) and the retina were well preserved in all of the experimental eyes. No retinal morphology and ultrastructural abnormality were found under the light and SEM and TEM. Conclusion Ethanol precipitation is a feasible way to isolate and purify Plg from human cord blood plasma. Extracted Plg shows potential fibrinolytic intravitreal injection of Plg.