摘要
全世界范围内血源紧张、病毒污染等问题推动了血液代用品的研究。输血来源有限,且输注后可能引起严重的不良反应,因而国内外科学家对以携氧液作为输血替代品进行了长达一个多世纪的探索。迄今为止,几种第一代红细胞代用品已经有望获准上市。各种产品有其独自的生理特性、生物活性和副作用。本研究主要从脂质体包封血红蛋白(liposome encapsulated hemoglobin,LEH)的角度出发,作为氧治疗剂进行研发,可能成为今后红细胞代用品研制的发展方向。
关于本研究:
目的:确定有效制备及鉴定微囊包被血红蛋白和微米级人工红细胞的方法。
方法:首先通过低渗法制备高纯度、高浓度的膜蛋白,然后根据界面缩聚法的原理,利用匀速微囊制备喷头,将含有2 ,3-DPG、膜蛋白和血红蛋白水相直接滴入到含磷脂的植物油中,通过高速搅拌使囊心物(2 ,3-DPG与血红蛋白单体、二聚体构建的不稳定的四聚体)的周围形成单个球状膜壳型微囊。
结果:微囊包被血红蛋白其微囊呈球状,表面光滑均匀,微囊直径2-5μm(电子显微镜下估算值)。人工红细胞透射电镜:单个包囊颗粒呈圆球状,有的表面光滑,有的表面稍有凹凸,平均粒径为3μm,最小粒径为1μm;扫描电镜显示:颗粒内部电子密度较高、不均匀,呈纤维状和细颗粒状交错分布,是为血红蛋白。人工红细胞的包埋率为53%,泄漏率在4℃生理盐水中15d释放40.9%。
结论:双相乳化法可制备微米级人工红细胞,且具有携带和释放氧气及二氧化碳的生物活性。
各种修饰血红蛋白、氟碳乳剂、血红蛋白微囊等技术迅速发展,其中一些已经形成规模化制备工艺。但目前,血液代用品没有完整的评价体系,全面评估血液代用品产品仍较困难。因此,本文另一个重要部分是以课题研究的血液代用品为基础,结合世界范围内的最新进展,从血液代用品的评价角度出发,探讨血液代用品的评价参数,围绕血液代用品的理化性质、有效性与安全性、保存条件等方面进行了说明。
The shortage of healthy blood resource and the virus infection have promoted the study of blood substitute. Donor blood is a limited resource and its transfusion is possibly associated with significant adverse effects. An alternative to blood transfusion, based on oxygen-carrying solutions, has been sought by scientists for over a century. So far, several first generation red blood cell substitutes are approaching submission for licensing. While each formulation has its own physical characteristics, biological activities, and adverse reaction profile. In this study, we provide focus on the preparation processing of liposome encapsulated hemoglobin, and we presented some possible interests in the future artificial oxygen carriers’research.
About our study:
Objective:To determine the effective method to prepare and test hemoglobin vesicles and artificial red blood cells with diameters in micrometer.
Methods:Highly purified and concentrated membrane protein was obtained by hypotonic hemolysis of human red cells. According to the principle of interface polymerize method, the hemoglobin and membrane protein water which contained 2, 3–DPG and enzyme was dropped into organic phase with phospholipids. After mixing at high speed, the single global vesicle developed around the non - stable tetramers consisting 2, 3 - DPG and hemoglobin monomers or dimers.
Results:Hemoglobin vesicles were global, about 2-5μm in diameter (estimated under electron microscope), with smooth and homogenous surfaces. Transmission electron microscope revealed that single hemoglobin vesicles were global, some of which with smooth surfaces, some slightly unsmooth. The average diameter was 3μm and the smallest was 1μm. Scan electron microscope showed that the electronic density in the vesicle was high and heterogeneous, interlaced with fiber and granules, which hemoglobin was. The envelop rate of artificial blood was 53 % , leakage rate at 4℃in 0. 9 % NaCl was 40.9% after 15 days.
Conclusion:Artificial red blood cells can be prepared with diameters around micrometer by double Emulsification.
The technologies of modified Hemoglobin、Perfluorocarbons and Hb-Vesicles developed quickly, some of which have already formed into large-scale preparation and production. However, there is no completed evaluation system for the blood substitute at present, and it is still hard to estimate the function of blood substitute completely. Thus,The other significant part of the study is to take the evaluation of liposome encapsulated hemoglobin as a key point, discusses the evaluation parameters of blood substitute and presents the physical and chemical property, the availability, safety and the preservation condition of the blood substitute, which is based on the study in China and abroad and referred to the latest progress all over the world.
引文
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