抗氧化酶交联的多聚血红蛋白生物学特性的初步研究
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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,H_2O_2)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)、Ca~(2+)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H_2O_2组的GSH浓度为0.0064±0.0039mmol/L,Ca~(2+)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H_2O_2组比,PolyHb-SOD-CAT+H_2O_2组的GSH浓度为0.0214±0.0098mmol/L,Ca~(2+)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H_2O_2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。
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(H_2O_2).The level of glutathione(GSH)and Ca~(2+)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 H_2O_2 group,content of GSH in cells was 0.0064±0.0039 mmol/L,content of Ca~(2+)in cells was 690.05±145.11 nmol/ml,and incubation with H_2O_2 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 H_2O_2 alone,content of GSH in cells was 0.0214±0.0098 mmol/L,content of Ca~(2+)in cells was 172.43±24.09 nmol/ml in PolyHb-SOD-CAT+H_2O_2 group,most NF-kB was still in cytoplasm, expression of eNOS and HO-1 mRNA and cellular death decreased.PolyHb-SOD-CAT inhibited H_2O_2-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.
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,H_2O_2)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)、Ca~(2+)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H_2O_2组的GSH浓度为0.0064±0.0039mmol/L,Ca~(2+)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H_2O_2组比,PolyHb-SOD-CAT+H_2O_2组的GSH浓度为0.0214±0.0098mmol/L,Ca~(2+)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H_2O_2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。
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(H_2O_2).The level of glutathione(GSH)and Ca~(2+)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 H_2O_2 group,content of GSH in cells was 0.0064±0.0039 mmol/L,content of Ca~(2+)in cells was 690.05±145.11 nmol/ml,and incubation with H_2O_2 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 H_2O_2 alone,content of GSH in cells was 0.0214±0.0098 mmol/L,content of Ca~(2+)in cells was 172.43±24.09 nmol/ml in PolyHb-SOD-CAT+H_2O_2 group,most NF-kB was still in cytoplasm, expression of eNOS and HO-1 mRNA and cellular death decreased.PolyHb-SOD-CAT inhibited H_2O_2-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.
引文
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[2]王岱娟,牛津梁,张文博,菅晓娟,张文.血红蛋白氧载体研究进展及应用现状[J].中国生化药物杂志,2005,26(6):379-382.
[3]王红梅,王保龙,姚萍,苏虹.血液代用品的研究进展[J].临床输血与检验,2005,7(4):314-317.
[4]路秀玲.修饰血红蛋白作为红细胞代用品的研究进展和挑战[J].生物工程学报,2006,22(1):7-18.
[5]万英,周剑涛.血红蛋白氧载体研究进展[J].中国生化药物杂志,2004,25(3):178-180.
[6]王雁峰,潘继伦,俞耀庭.红细胞代用品的研究进展[J].生物医学工程学志,2004,21(3):490-494.
[7]吴红,吴道澄,李晓晔,等.血液代用品—血红蛋白修饰物的研究进展[J].中国输血杂志,2001,14(2):116-118.
[8]Thomas Ming Swi Chang Blood substitutes based on nanobiotechnology.TRENDS in Biotechnology,2006,24(8):372-377.
[9]王春玲,修瑞娟.人工血液代用品及其应用进展[J].国外医学输血及血液学分册,2005,28(3):260-263.
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[1]夏德宜,周剑涛.血液代用品及其临床应用[J].国外医学临床生物化学与检验学分册,2004,25(4):377-381.
[2]Buehler P W and Alayash A I.Oxidation of hemoglobin:mechanisms of control in vitro and in vivo[J].Transfusion Alternatives in Transfusion Medicine,2007,9:204-212.
[3]Reeder B J,Svistunenko D A,Cooper C E,et al.The radical and redox chem-istry of myoglobin and hemoglobin:from in vitro studies to human pathology[J].Antioxid Redox Signal,2004,6:954-966.
[4]Dunne J,Caron A,Menu P,et al.Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo[J].Biochemical Society, 2006,399:513-524.
[5]Vollaard N B J,Reeder B J,Shearman J P,et al.A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo[J].Free Radical Biology Medicine,2005,39:1216-1228.
[6]Chang E J,Lee S H,Mun K C,et al.Effect of Artificial Cells on Hepatic Function After Ischemia-Reperfusion Injury in Liver[J].Transplantation Proceedings,2004,36:1959-1961.
[7]Chang E J,Lee T H,Mun K C,et al.Effects of Polyhemoglobin-Antioxidant Enzyme Complex on Ischemia-Reperfusion in Kidney[J].Transplantation Proceedings,2004,36:1952-1954.
[8]Asanuma H,Nakai K,Sanada S,et al.S-nitrosylated and pegylated hemoglobin,a newly developed artificial oxygen carrier,exerts cardioprotection against ischemic hearts[J].Journal of Molecular and Cellular Cardiology,2007.
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[2]王岱娟,牛津梁,张文博,菅晓娟,张文.血红蛋白氧载体研究进展及应用现状[J].中国生化药物杂志,2005,26(6):379-382.
[3]王红梅,王保龙,姚萍,苏虹.血液代用品的研究进展[J].临床输血与检验,2005,7(4):314-317.
[4]路秀玲.修饰血红蛋白作为红细胞代用品的研究进展和挑战[J].生物工程学报,2006,22(1):7-18.
[5]万英,周剑涛.血红蛋白氧载体研究进展[J].中国生化药物杂志,2004,25(3):178-180.
[6]王雁峰,潘继伦,俞耀庭.红细胞代用品的研究进展[J].生物医学工程学志,2004,21(3):490-494.
[7]吴红,吴道澄,李晓晔,等.血液代用品—血红蛋白修饰物的研究进展[J].中国输血杂志,2001,14(2):116-118.
[8]Thomas Ming Swi Chang Blood substitutes based on nanobiotechnology.TRENDS in Biotechnology,2006,24(8):372-377.
[9]王春玲,修瑞娟.人工血液代用品及其应用进展[J].国外医学输血及血液学分册,2005,28(3):260-263.
[10]Hu T,Su ZG.A solid phase adsorption method for preparation of bovine serum albumin-bovine hemoglobin conjugate.J Biotechnol,2003,100:267-275.
[11]Hu T,Su ZG.Bovine serum albumin-bovine hemoglobin conjugate as a candidate blood substitute.Biotechnol Lett,2002,24(12):1027-1030.
[12]Lu XL,Zbeng CY,Shi XD et al.Conjugate of bovine hemoglobin and human serum albumin as a candidate for blood substitute:characteristics and effects on rats.Art Cells Blood Subs and Biotech,2005,33(2):83-99.
[13]Kobayashi K,Tsuchida,Horinouchi H.Artificial Oxygen Carrier.Tokyo:Springer-Verlag.2005.
[14]D' Agnillo F,Chang TMS.Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties.Nature Biotechnol,1998,16(7):667-671.
[15]Riess JG.Oxygen carriers(“blood substitutes”)—raison d'etre,chemistry,and some physiology.Chem Rev,2001,101(9):2797-2919.
[16]Workshop on Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes.Held under the joint auspices of the Food and Drug Administration,National Institute of Health and the Department of Defense.September,1999,27-28.Natcher Building,National Institute of Health,Bethesda,MD.http://www.fda.gov/cber/minutes/oxygen092799.doc
[17]Craston R,Koh M,Dermott A M et al.Temporal dynamics of CD69 expression on lymphoid cells.J Immuno Methods,1997,209:37.
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