【作者】 王永彬；

【导师】 范华骅；

【作者基本信息】 华东师范大学 ， 生物医学， 2009， 硕士

【摘要】 抗氧化酶交联的多聚血红蛋白（PolyHb-SOD-CAT）是红细胞代用品的一种,为了建立此代用品的制备方法,并对其理化特性、生物学安全性及抗氧化特性进行初步研究,我们从过期的人红细胞中提取血红蛋白（hemoglobin,Hb）,并利用阴离子交换层析方法进行纯化,采用戊二醛交联法制备抗氧化酶交联的多聚血红蛋白;利用中空纤维超滤仪纯化出300kD左右的多聚血红蛋白,浓缩、无菌过滤,冻干后-20℃长期保存。检测PolyHb-SOD-CAT的粒径,测定不同温度时间保存下制品高铁血红蛋白（MetHb%）含量的变化;流式细胞术检测制品与中性粒细胞孵育后细胞吞噬功能的变化以及制品对T淋巴细胞和血小板活化的影响;凝集试验检测血浆中凝血因子活力;Western Blot检测制品输注后在小鼠体内的清除速率;ELISA检测制品在小鼠体内的抗原性。实验结果显示,MetHb%为6.61±1.42%,粒径29nm;制品在4℃、24℃不同保存时间均呈自氧化稳定,37℃8h显示抗氧化酶交联的多聚血红蛋白比多聚血红蛋白有更强的自氧化稳定性;制品未明显影响中性粒细胞吞噬功能,对T淋巴细胞和血小板没有明显活化作用;对凝血因子活性也没有明显影响;制品输注后48h仍有检出;PolyHb-SOD-CAT的抗原性较弱。此外,为了研究PolyHb-SOD-CAT的抗氧化特性,我们利用过氧化氢（hydrogenperoxide,H₂O₂）对人脐静脉内皮细胞的氧化损伤建立了类似缺血再灌注损伤模型,化学比色法检测细胞内还原型谷胱甘肽（glutathione,GSH）、Ca²⁺浓度,免疫组化技术检测核转录因子NF-κB在细胞内的分布,RT-PCR技术检测血红素氧化酶（hemeoxygenase,HO-1）、内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）的mRNA表达情况,流式细胞术检测细胞死亡情况。结果表明,与对照组相比,单加H₂O₂组的GSH浓度为0.0064±0.0039mmol/L,Ca²⁺浓度为690.05±145.11nmol/ml,NF-kB大量位移入核,HO-1、eNOS的mRNA表达量上升,细胞死亡率高;而与单加H₂O₂组比,PolyHb-SOD-CAT+H₂O₂组的GSH浓度为0.0214±0.0098mmol/L,Ca²⁺浓度为172.43±24.09nmol/ml,NF-κB绝大部分仍位于胞浆内,HO-1、eNOS的mRNA表达量明显降低,细胞死亡率下降。PolyHb-SOD-CAT能够抑制H₂O₂引起的血管内皮细胞氧化损伤。上述实验结果表明,PolyHb-SOD-CAT是一种潜在的安全有效的红细胞代用品,在一定程度上体现了抗氧化的优越性,对于减轻缺血-再灌注损伤有重要作用。更多还原

【Abstract】 Antioxidant enzymes cross-linked polyhemoglobin（PolyHb-SOD-CAT）is a kind of red blood cell substitutes.To establish a method to prepare PolyHb-SOD-CAT and to analyse the biological characters,we isolated Hb from red blood cell and purified Hb using Anion Exchange Chromatography.Glutaraldehyde was used to crosslink hemogloibin and antioxidant enzymes.PolyHb-SOD-CAT（300kD）was prepared with hollow-fiber ultrafiltration,and was concentrated,frozen in-20℃.The diameter was detected using COULTER N4 Plus particle size analyzer. Drabkins method was used to measure the change of MetHb%of the product preserved in different time and temperature;change of neutrophil phagocytosis of the cells and activation of T lymphocytes and platelet incubated with products was estimated using flow cytometry;aggregation test was made to measure the function of serum accelerin incubated with products;rate of elimination of the products infused in the mouse was detected using Western Blot.Our results reveal that characters of the product were as follow:MetHb%6.61±1.42%,particle size 29nm;products preserved in different time and temperature were autoxidation stable,and PolyHb-SOD-CAT appeared to be more stable than PolyHb during the incubation（37℃8h）;no significant influence on the function of neutrophil phagocytosis and serum accelerin aggregation was measured;T lymphocytes and platelet stayed inactivated during the incubation;and the product could still be detected 48h after infused into the mouse.ELISA tests showed weak or no antigenicity.Besides,to investigate the antioxitant character of PolyHb-SOD-CAT,We examined the effect of PolyHb-SOD-CAT on human umbilical vein endothelial cells subjected to hydrogen peroxide（H₂O₂）.The level of glutathione（GSH）and Ca²⁺in cells were measured using the chemical colorimetry.Location of NF-kB was detected using the imrnunohistochemistry technique.Expression of the endothelial nitric oxide synthase（eNOS）mRNA and heme oxygenase-1（HO-1）mRNA were assessed by RT-PCR.Cellular death was evaluated by flow cytometry.Our results reveal that in H₂O₂ group,content of GSH in cells was 0.0064±0.0039 mmol/L,content of Ca²⁺in cells was 690.05±145.11 nmol/ml,and incubation with H₂O₂ alone produced greater activation of NF-kB,,greater expression of eNOS and HO-1 mRNA and increases in cellular death compared with control.However,comparing with H₂O₂ alone,content of GSH in cells was 0.0214±0.0098 mmol/L,content of Ca²⁺in cells was 172.43±24.09 nmol/ml in PolyHb-SOD-CAT+H₂O₂ group,most NF-kB was still in cytoplasm, expression of eNOS and HO-1 mRNA and cellular death decreased.PolyHb-SOD-CAT inhibited H₂O₂-induced oxidative damage to vascular endothelial cells.The above results showed that PolyHb-SOD-CAT could be a kind of potential safe and effective red blood cell substitutes with better antioxidant stability,and could alleviate ischemia-reperfusion injury.更多还原