人工红细胞及微囊包被血红蛋白的制备与鉴定

目的　确定有效制备及鉴定微囊包被血红蛋白和纳米级人工红细胞的方法。方法　首先通过低渗法制备高纯度、高浓度的血红蛋白 ,然后根据界面缩聚法的原理 ,利用匀速微囊制备喷头 ,将含有卵磷脂、聚乳酸的有机相 (丙酮、乙醇 )直接滴入含有 2 ,3 DPG和酶及抗氧化剂的血红蛋白水相中 ,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　低渗法制备的血红蛋白浓度为 1 4 .1 g/dl,纯度为 97.7%。微囊包被血红蛋白其微囊呈圆球状 ,表面光滑均匀 ,微囊直径 0 .2 5 4 μm(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2 1 0nm ,最小粒径为 1 90nm ;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 ,颗粒边缘有一窄带 ,电子密度低于中央为膜层 ,平均粒径为 2 37nm。人工红细胞的包埋率为 2 7.7% ,泄漏率在 4℃生理盐水中 30d仅释放 6 .9%。结论　界面缩聚法可制备纳米级人工红细胞 ,且具有携带和释放氧气及二氧化碳的生物活性

Preparation and assessment of artificial red blood cells and Henoglobin-vesicle(HbV)

Objective To determine the effective method to prepare and test hemoglobin vesicles and artificial red blood cells with diameters in nm. Methods Highly purified and concentrated hemoglobin was obtained by hypotonic hemolysis of human red cells. According to the principle of interface polymerize method, organic phase L α phosphatidylcholine, polylactic acid (PLA) was dropped into the hemoglobin water phase which contained 2,3-DPG, enzyme and antioxidant. 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 concentration was 14.1g/dl, purity was 97.7%. Hemoglobin vesicles were global, about 0.254μm 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 210nm and the smallest was 190nm. Scan electron microscope showed that the electronic density in the vesicle was high and heterogeneous, interlaced with fibre and granules, which were hemoglobin. There was a narrow edge around the vesicle, of which the electronic density was lower than that in the center. That was the membrane. The average diameters of the vesicles were 237nm. The envelop rate of artificial blood was 27.7%, leakage rate at 4℃ in 0.9% NaCl was 6.9% after 30 days. Conclusion Artificial red blood cells can be prepared with diameters around nanometers by interface polymerization method.