摘要
血红蛋白类氧载体(hemoglobin-based oxygen carriers，HBOCs)是尚处于不同新药研发阶段的一类新生物制剂药物，可用于代替红细胞的组织供氧作用。目前HBOCs制备所用的血红蛋白主要来源于血库过期人血和牛血，但是人源和牛源生物材料具有一些严重缺点，尤其是具有致命性病原体污染如艾滋病毒、肝病病毒、朊病毒等的威胁，因此探索新的血红蛋白来源对HBOCs产品开发具有十分重要的价值。近年来，我们对猪血红蛋白作为HBOCs基本原料的特点进行了较深入的研究，实验结果表明：猪血红蛋白的氧亲和力与人血红蛋白相似，明显高于牛血红蛋白，且具有较好的化学稳定性，是一种吸引力很强的HBOCs规模生产原料选择的对象。
目前，已有数种血液代用品进入临床研究，但是许多产品由于安全性问题而被提前终止。因此，血液代用品的潜在毒性已经成为许多血液代用品进入临床使用的最大障碍。免疫原性和免疫毒性是生物药品尤其是异源蛋白类生物药品研究开发的重要内容，是生物安全性研究的重要组成部分。
本文应用猪血为原料制备出高纯度的猪血红蛋白，以戊二醛为交联剂聚合猪血红蛋白制备聚合猪血红蛋白氧载体；通过在不同给药途径和给药条件下反复给动物免疫猪血红蛋白或戊二醛聚合猪血红蛋白，用间接ELISA法检测动物血清中的特异性IgG，研究了猪血红蛋白及其戊二醛聚合体的免疫原性；初步研究了戊二醛聚合猪血红蛋白免疫原性增强的机理；探讨了戊二醛聚合猪血红蛋白在静脉注射条件下无免疫原性的机理；此外本文还进一步研究了戊二醛聚合猪血红蛋白对小鼠抗菌免疫功能及其在体外对小鼠腹腔巨噬细胞系吞噬功能的影响。
实验结果表明：猪血红蛋白免疫原性很弱，在无佐剂条件下，经皮下或腹腔免疫途径，在小鼠中反复免疫注射都不能引起特异性抗体(IgG)生成反应。但是，在加福氏佐剂的条件下，猪血红蛋白反复免疫小鼠、兔子或大鼠均能刺激动物产生较高滴度的抗猪血红蛋白IgG。猪血红蛋白的戊二醛聚合产物免疫原性明显增强，在无佐剂条件下，戊二醛聚合猪血红蛋白皮下或腹腔免疫小鼠，均能刺激小鼠产生特异性IgG，戊二醛聚合猪血红蛋白皮下给药与腹腔给药相比对小鼠刺激强度大，抗体产生快，滴度高。但是，静脉注射不同治疗量的戊二醛聚合猪血红蛋白均不能刺激动物产生特异性IgG，进一步研究表明静脉注射治疗量戊二醛聚合猪血红蛋白可引起动物对聚合猪血红蛋白特异性的免疫耐受，动物对其它抗原(如牛血清白蛋白BSA)能够进行正常的免疫应答，而且，免疫耐受的程度与静脉注射的聚合猪血红蛋白呈现明显的剂量依赖关系，静脉注射的聚合猪血红蛋白剂量愈高，诱导的免疫耐受的程度愈强。
本文通过对戊二醛聚合猪血红蛋白免疫原性增强机理的初步探讨结果表明：戊二醛聚合兔血红蛋白在加福氏佐剂的条件下，反复皮下免疫兔子均不能刺激兔子产生抗兔血红蛋白特异性IgG。推测原因是：兔子对自身的血红蛋白具有特异性的免疫耐受，戊二醛聚合不能使血红蛋白形成新的抗原决定簇。通过不同分子量聚合猪血红蛋白免疫原性的研究发现：在平均分子量约为67.4KD-147.6kD之间，随着分子量的增大，聚合猪血红蛋白免疫原性增强；在平均分子量为147.6kD-1150KD之间，随着分子量的增大，聚合猪血红蛋白免疫原性逐渐减弱。通过抗人、牛或猪血红蛋白或其相应聚合产物抗体与人、牛或猪血红蛋白或其相应聚合产物抗原抗体交叉反应的比较，结果表明：戊二醛聚合／修饰没有使猪血红蛋白产生新的抗原决定簇；戊二醛不是引起血红蛋白免疫原性增强的原因，动物不能产生抗戊二醛的特异性抗体。
因此，我们通过一系列的实验推测：天然未经修饰的猪血红蛋白免疫原性很弱，只有在加佐剂条件下才会产生特异性的免疫反应的原因是：1)动物体包括人体主要通过吞噬细胞代谢体内老化红细胞释放的血红蛋白，血红蛋白被吞噬后大部分被水解，极少部分与MHCⅡ复合体结合，提呈给T细胞，这种代谢途径可能会存在某些漏洞，使极少量的肽段保持完整，与MHCⅡ复合体结合，在免疫系统形成的早期导致免疫系统对同种血红蛋白的免疫耐受；2)动物体的血红蛋白是高度保守的，不同种类的血红蛋白可能具有相同的内涵体或溶酶体蛋白酶裂解位点，因此，不同种类的血红蛋白在吞噬细胞内的裂解方式与动物自身的血红蛋白相同。即大部分的血红蛋白全部被水解。极少部分被提呈给T细胞，因此，只有加佐剂的条件下才能测出血红蛋白的免疫原性；3)佐剂诱导的局部炎症反应能够促进抗原提呈细胞对蛋白的吞噬作用，因此，较大量的异种血红蛋白泄露增加了抗原提呈细胞对异种血红蛋白水解片段的提呈作用，从而引起特异性的免疫反应。
戊二醛聚合猪血红蛋白免疫原性增强的原因主要是：1)聚合血红蛋白由于聚合发生局部环境的改变，蛋白酶裂解位点被覆盖而只能被部分水解，在抗原提呈细胞中，蛋白质抗原被部分水解，形成的肽段与MHCⅡ复合体结合从而提呈到细胞表面，从而引起特异性的免疫反应；2)聚合血红蛋白结构更加稳定，在体内代谢时间延长，对淋巴细胞的刺激作用时间延长，从而导致免疫原性增强；3)分子量增大也是聚合猪血红蛋白免疫原性增强的原因。4)如果血红蛋白聚合体太大，血红蛋白剧烈的构像变化会导致血红蛋白分子不能与淋巴细胞的抗原受体发生正常的吻合，或者使血红蛋白分子的特殊化学基团与淋巴细胞表面对应抗原受体相互接触的难度增大，从而使聚合猪血红蛋白的免疫刺激作用减弱。
许多研究者报道用HBOCs治疗的病人可能会增加细菌的易感性。本文通过研究在感染细菌的不同时间静脉注射不同剂量的聚合猪血红蛋白研究发现：小鼠在未感染绿脓杆菌之前4、8或20hrs尾静脉注射较高治疗量(130mg／kg)的聚合猪血红蛋白，并不能降低小鼠对细菌的抗感染能力，相反的却能够增强小鼠的抗感染能力。小鼠在感染绿脓杆菌之后或感染的同时静脉注射较高治疗量(130mg／kg体重)的聚合猪血红蛋白，小鼠对细菌的抗感染能力降低，因此，较高治疗量的聚合猪血红蛋白在感染的同时或感染之后使用能够增强细菌的毒性作用。但是，在感染绿脓杆菌的同时注射较低剂量(13或1.3mg／kg体重)的聚合猪血红蛋白不能降低小鼠的抗细菌感染能力，相反的却能够提高小鼠的抗细菌感染能力，其中，1.3mg／kg的促进作用最为显著。
病原微生物侵入机体后，在激发免疫应答以前即可被吞噬细胞吞噬并清除，这是机体非特异免疫防御机制的重要环节。本文通过聚合猪血红蛋白在体外对巨噬细胞吞噬绿脓杆菌的影响研究发现：在巨噬细胞和聚合猪血红蛋白共培养0min时感染绿脓杆菌，较高浓度的聚合猪血红蛋白(8或2mg／ml)对巨噬细胞吞噬绿脓杆菌有一定的抑制作用，而低剂量(200、20或2μg／ml)的聚合猪血红蛋白具有促进作用，其中以200μg／ml或20μg／ml的促进作用最为显著；在巨噬细胞和不同浓度的聚合猪血红蛋白(8mg／ml，2mg／ml、200μg／ml、20μg／ml或2μg／ml)共培养2或4小时后感染绿脓杆菌，不同浓度的聚合猪血红蛋白均能够促进巨噬细胞对绿脓杆菌的吞噬作用，其中以200μg／ml的促进作用最为显著。目前，聚合猪血红蛋白促进或抑制吞噬细胞吞噬功能的机理还在进一步的研究中。
Hemoglobin-based oxygen carriers(HBOCs) is a kind of novel therapeutic biological medicine which is in development at present. The solution can be used as oxygen carrier delivering oxygen. Hemoglobin used in HBOCs so far is of either human or bovine origin. But there are serious disadvantages of these two Hb resources, the most concern is the potential negative effects of the increasing incidence of new variant Creutzfeldt-Jacob disease,HIV and hepatitis virus. Therefore, other sources of hemoglobin may offer solutions to the problems encountered in using human and bovine hemoglobin. In the past few years we studied porcine hemoglobin as Hb resources of HBOCs ,the results indicated that Porcine hemoglobin has high oxygen affinity, similar to that of human hemoglobin and much higher than that of bovine hemoglobin. Rich supply, the unique properties of oxygen affinity and relative stability of porcine hemoglobin could make this type of protein a useful material in molecular design and large-scale production of HBOCs.
At present time,many HBOCs have been brought into clinical trial,but several of them have been discontinued because of problems of safety evaluation.So potential toxicity of HBOCs become the main barrier of the clinical application of HBOCs,Immunogenity and immunological toxicity are important parts of development of biological protein medicine and safety evaluation of HBOCs.So it is important to assess the immunogicity and the immunotoxicity of glutaradehyde polymerized porcine hemoglobin.of immunological properties .
In this article, Using pig blood as the raw material, ultra-pure porcine hemoglobin was obtained through multiple purification steps; with glutaraldehyde as the cross-linker, polymerized porcine hemoglobin (pPolyHb) was then produced.The immunogenity of porcine hemoglobin and glutaradehyde polymerized porcine hemoglobin were investigated through detection of the serum specific IgG responses using indirect ELISA in animals after administration of the appropriated hemoglobin samples via different routes and under various conditions.In addition, the thesis was also concerned with the possible origin of enhanced immunogenicity of porcine glutaraldehyde-polymerized hemoglobin.The reasons why glutaraldehyde polymerized porcine hemoglobin were not antigenic when administrated via intravenous route repeatedly were also concerned in the thesis.
The results indicated that in the absence of an adjuvant, porcine hemoglobin did not elicit detectable specific serum IgG antibody response in mice after repeatedly subcutaneous or intraperitoneal administration of the protein samples. However, in the presence of Freuand's adjuvant, a significant anti-porcine hemoglobin antibody response was observed even after the first boosting injection through intraperitoneal or subcutaneous route administrated into mice,rats or rabbits. glutaraldehyde-polymerized porcine hemoglobin stimulated apparent production of specific serum IgG antibodies in the animals after repeated subcutaneous or intraperitoneal administration of the protein sample even in the absence of an adjuvant. Subcutaneous administration elicited stronger specific serum IgG responses than intraperitoneal delivery of the protein samples. However, repeatedly intravenous administration of therapeutic dosages of pPolyHb failed to induce detectable serum specific IgG responses in rats. Further experiments showed that in a clear dose-dependent manner, intravenous infusion of therapeutic amounts of the pPolyHb markedly inhibited the specific serum IgG responses elicited by subsequent repeated intraperitoneal administrations of pPolyHb in the presence of the Freund's adjuvant, but did not affect the IgG responses elicited by the co-administrated bovine serum albumin (BSA) in rats. These results indicated that intravenous administration of therapeutic dosages of glutaraldehyde-polymerized porcine hemoglobin led to significant, specific immune tolerance to glutaraldehyde-polymerized porcine hemoglobin, but did not affect humoral IgG responses to the stimulation of other unrelated antigens in tested animals.
The investigation of possible origin of glutaraldehyde polymerized porcine hemoglobin indicated that the repeated subcutaneous injections of glutaraldehyde polymerized rabbit hemoglobin back to rabbits in the presence of fruend's adjuvant didn't result in detective anti- glutaraldehyde polymerized rabbit hemoglobin IgG,.This indicated that there were no neo-antigens formed in polymerized hemoglobin,the rabbit's immune system was immunological torlerant to its own hemoglobin.
The data of immunogenicity of different molecular weight of pPolyHb indicated that the immunogenicity of pPolyHb enhanced along with the increase of molecular weight from 67.4KD to 147.6kD .the immunogenicity of pPolyHb decreased with the increase of of molecular weight from 147.6kD-1150KD .
The results of cross-binding reactions of mouse anti- hHb, bHb or pHb IgG or anti-hPolyHb, bPolyHb or pPolyHb IgG with hHb, bHb and pHb and their related polymers suggested that there were no neo-antigens induced in resultant glutaradehyde polymerized hb.The reason of enhanced immunogenicity of polymerized Hb was not glutaraldehyde,there was no production of specific anti-glutaraldehyde IgG.
The above series experimental results suggested that the reasons of weak immnogenicity of native/unmodified porcine hemoglobin are as follows .1)Animals including human metabolize a fairly large amount of hemoglobin constantly leased from aged red blood cells mainly through the phagocytical function of phagocytic cells.The fact that hemoglobin of various species are very weekly immunogenic indicates that the fate of hemoglobin after taken up by phagocytic cells , is being mostly hydrolyzed, leaving little or none bound to MHC II complex for presentation to T cells. This pathway would have some leakage, resulting in a very small amount of peptides remained intact and bound to MHC II complex. In the early stage of development of the immune system, this leakage pathway would result in tolerance of peptides derived from homologous hemoglobin. 2)Hemoglobins are highly conserved in animals. heterologous hemoglobin may share the same cleavage sites for endosomal/lysosomal proteases as the hemoglobin of the host so that hetero-hemoglobin would have the same fate inside endocytic/lysomal system of phagocytes as homo-hemoglobin of the host, i.g. most being directed to complete hydrolyzing pathway,This would explain why hetero-hemoglobin exhibits very week immunogenicity, which normally can only be detected when immunization is done in the presence of an adjuvant. Adjuvant-induced local inflammation would facilitate phagocytosis of the protein by DCs so that access amount of hetero-hemoglobin would result in a larger flux of the leakage leading to enhance production of peptides derived from the hetero-hemoglobin and increase opportunities for DC to present the peptides on MHC II complex and induce specific immuno-reactions to the protein.
The possible origin of enhanced immunogenicity of glutaraldehyde polymerized porcine hemoglobin is as followings.1) Polymerized hemoglobin may be able to remain partially hydrolyzed due to inaccessibility of some of protease cleavage sites because of local environmental change after polymerization. The final resultant peptides would be bound to MHC II complex and presented onto the cell surface to initiate subsequent specific T cell activation. 2)The polymerized Hb was more stable than native Hb,the T1/2 was prolonged in vivo,the stimulating time to lymphocytes was also prolonged,therefore, the immunogenicity was enhanced.3)The increase of molacular weight was also one of the reasons of the origin of enhanced immunogenicity of polymerized Hb. 4)To an extent, the larger the polymers, the more protease cleavage sites become inaccessible so that more peptides would remain intact in the endocytical pathway and be presented to the cell surface with MHC II.. Too large size of the protein polymers would reduce effectiveness of phagocysis of the protein, resulting in less specific immune response to the proteins.
Many investigators reported that patients treated with HBOCs may have increased susceptibility to bacterial infection.The data of intravenous infusion of different doses of pPolyHb at various time of infection indicated that the host defense of mice were not decreased but enhanced after intravenous infusion of 130mg/kg body weight of pPolyHb 4,8 0r 20 hrs before infection of p. aeruginosa.So pPolyHb may enhance host defense and accelerate the phagocytosis function of reticulation endothelium system.But the host defense was impaired when mice were infused 130mg/kg body weight of pPolyHb after 4 hrs or simutanously infection of p. aeruginosa.We found that when low doses of 13 or 1.3mg/kg of pPolyHb were infued simutanous with p. aeruginosa, the host defense of mice were enhanced,furthermore,the enhance of host defense of mice were enhanced signicantly at doses of 1.3mg/kg.
The microorganisms can be phagocytized by macrophages before the immune responses when invaded into the host,this is an important immunological defense.The data of effects of pPolyHb on phagocytosis of p. aeruginosa by TIB-67 indicated that at time of 0min TIB-67 were infected with p. aeruginosa when incubated with different concentrations of pPolyHb,the data indicated that high concentrations of pPolyHb (8 or 2mg/ml) can inhibit the phagocytosis of p. aeruginosa of TIB-67. On the contrary, the low concentration of pPolyHb (200、20μg/ml) can promote the phagocytosis function of TIB-67.Incubated with different concentrations of pPolyHb (8 mg/ml, 2mg/ml、200μg/ml、20μg/ml or 2μg/ml) 2 or 4 hrs later, the TIB-67 were infected with p. aeruginosa,the data showed that pPolyHb can promote the phagocytosis function of TIB-67,moreover, 200μg/ml pPolyHb could significantly enhance the phagocytosis function of TIB-67.At present,We are investigating the
引文
(1) Mary L.Nucci,Abraham Abuchowski.The threat of global shortages of blood and fears about comtamination have hastened attempts to find life-sustaining alternatives.Several of these compounds look promising.Scientic America. 1998,2:73-77
(2) Lawrence T Goodnough, Aryeh Shander, Mark E Brecher.Transfusion medicine: looking to the future. The Lancet. 2003,361:161-169.
(3) Hae Won Kim and A. Gerson Greenburg. Artificial Oxygen Carriers as Red Blood Cell Substitutes: A Selected Review and Current Status. Artificial Organs. 2004,28(9):813-828.
(4) A Gerson Greenburg and Hae Won Kim. Hemoglobin-based oxygen carriers. Critical Care. 2004,8(Suppl 2):S61-S64.
(5) Thomas Ming Swi Chang. Hemoglobin-based Red Blood Cell Substitutes. Artificial Organs.2004,28(9):789-794.
(6) Lawrence T. Goodnough, MD. Risks of blood transfusion. Crit Care Med.2003,31(Suppl):S678-S686.
(7) Abdu I.Alayash.Oxygen therapeutics:Can we tame heamoglobin?Nature reviews.2004,3:152-159.
(8) M.F.Perutz,G.Fermi..Structure of Bovine Deoxyhaemoglobin,Absense of Specific Chloride-binding Sites and Origin of the Chloride-linked Bohr Effect in Bovine and Human Haemoglobin. J. Mol. Biol. 1993,233:536-545.
(9) Darin S. Katz,Steven P.White,Wen Huang,Ramesh Kumar and David W.Christianson. Structure Determination of Aquomet Porcine Hemoglobin At 2.8 A Resolution.J. Mol. Biol.1994,244:541-553.
(10) Kenneth S Kroeger and Craig E Kundrot. Structures of a hemoglobin-based blood substitute: insights into the function of allosteric proteins. Structure.1997, 5:227-237.
(11) Timothy C. Mueser, Paul H. Rogers, and Arthur Arnone. Interface Sliding As illustrated by the Multiple Quaternary Structures of Liganded Hemoglobin. Biochemistry. 2000,39:15353-15364.
(12) 吴保平．戊二醛聚合猪血红蛋白制备血红蛋白类氧载体的初步研究．西北大学硕士论文．2006．p47-55．
(13) M. F. Perutz. Regulation of affinity of hemoglobin: Influence of Structure of the Globin on the Heine Iron. Ann. Rev. Biochertt 1979,48:327-386.
(14) Clara Fronticelli, Enrico Bucci, and Charles Qrth. Solvent Regulation of Oxygen Affinity in hemoglobin.The Journal of Biological Chemistry.1984,259(17):10841-10844.
(15) Clara Fronticelli, Enrico Bucci and Anna Razynska. Modulation of Oxygen Affinity in Hemoglobin by Solvent Components Interaction of Bovine Hemoglobin with 2,3-Diphosphoglycerate and Monatomic Anions.J. Mol. Hid. 1988,202:343-348.
(16) Maria E. Clementil, Roberto Scatena, Alvaro Mordente Saverio G. Condo, Massimo Castagnola and Bruno Giardina. Oxygen Transport by Fetal Bovine Hemoglobin. J.Mol.Biol.1996,255:229-234.
(17) M. Marta, M. Patamia, A. Colella, S. Sacchi, M. Pomponi, K. M. Kovacs, C. Lydersen, and B. Giardina. Anionic Binding Site and 2,3-DPG Effect in Bovine Hemoglobin. Biochemistry.1998,37:14024-14029.
(18) Goodnough LH,Brecher ME,Kanter MH,Aubuchon JP.Transfusion medicine.First of two parts-blood transfusion.N Engl J Med.1999,340:438-47.
(19) Stowell CP,Levin J,Spiess BD,Winslow RM.Progress in the development of RBC substitutes,Transfusion.2001,41:287-99.
(20) Winslow RM.alphaalpha-crosslinked hemoglobin:was failure predicted by preclinical testing ?Vox Sang.2000,79:l-20.
(21) Keiji Shikama, and Ariki Matsuoka. Human haemoglobin: A new paradigm for oxygen binding involving two types of ab contacts. Eur. J. Biochem. 2003,270: 4041-4051.
(22) 吴保平．戊二醛聚合猪血红蛋白制备血红蛋白类氧载体的初步研究．西北 大学硕士论文．2006．p36-37．
(23) 董为人 综述 李进 审校．无基质血红蛋白及其血栓调节的一氧化氮基质。国外医学输血及血液学分册．1977，20(2)：91-94．
(24) R. M. Winslow.Current status of blood substitute research: towards a new paradigm.Journal of Internal Medicine.2003,253: 508-517.
(25) Savitsky J, Doczi J, Black J, Arnold J. A clinical safety trial of stroma-free hemoglobin. Clin Pharmacol Therap. 1978,23:73-80.
(26) 杨天楹 等编著．临床输血学．北京医科大学中国协和医科大学联合出版社．1993：P215-229。
(27) Moss GS,DeWoskin R,Rosen AL.Transport of oxygen and carbon dioxide by hemoglobin-saline solution in the red cell-free primate.Surg Gynecol Obstet.1976;142:357.
(28) Chang T.M.S. Semipermeable Microcapsules.Science. 1964,146: 524-525.
(29) Benesch R, Benesch R E, Yung S, Edalji R, Hemoglobin covalvently bridged across the polyphosphate binding site, Biochem. Biophs. Res. Commoun. 1975,63:1123.
(30) 胡远东，白坚石，焦克芳，卜凤荣，李松．PEG修饰牛血红蛋白的计算机模拟研究．生物物理学报．2000，6：315-321．
(31) Pmdyck J,Avorn J,Kuriyan M.Blood donation by the elderly:Clinical and Policy considerations.JAMA.1987,257:1186.
(32) Donat R.Spahn and Roman Kocian.Artificial O2 carriers:Status in 2005.Current pharmaceutical design.2005,11:4099-4114.
(33) Greenburg AG, Shooley M, Peskin GW. Improved retention of stroma free hemoglobin solution by chemical modification. J Trauma 1977,17:501-4.
(34) D'Agnillo F,Alayash A I.Site specific modification and toxicity of blood substitutes,the case of diaspirin cross-linked hemoglobin.Advanced drug delivery reviews.2000;40:199-212.
(35) Chang T M S. Stabilization of enzyme by microencapsulation with a concentrated Protein solution or by crosslinking with glutaraldehyde.Biochem.Biophys.Res. Com. 1971,44:1531—1533.
(36) Levy JH,Goodnough LT,Greilich PE,Parr GV,Stewart RW,Gratz I.Polymerized bovine hemoglobin solutions as a replacement for allogeneic red blood Cells transfusion after cardiac surgery:results of a randomized double-blind trial.J Thorac Cardiovasc Surg.2002,104:5-42.
(37) Krieter H,Hagen G,Waschke KF,Kohler A,Wenneis B,Bruckner UB.Isovolemic hemodilution with a bovine Hemoglobin based oxygen carrier:effects on hemodynamics and oxygen transport in comparison with a nonoxygen carrying volume substitute.J Cardiothora Vasc Anesth.1997,11:3-9.
(38) Jahr JS,Varma N,Polyheme.IDrugs.2004;7:478-82.
(39) Gould SA,Moore EE,Hoyt DB,Ness PM,Norris EJ,Carson JL.The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable.J Am Coil Surg.2002,195:445-52.
(39) Cheng DC,Mazer CD,Martineau R,Ralph-Edwards A,Karski J,Robblee J.A phase Ⅱ dose-dependent study of hemoglobin raffimer(Hemolink)in elective coronary artery bypass surgery.J Thorac Cardiovasc.Surg.2004;127:79-86.
(41) Greenwald RB, Pendri A, Martinez A, et al. PEGthiazolidine-2-thione, a novel reagent for facile protein modification: conjugation of bovine hemoglobin.Bioconjug Chem,1996,7: 6638～6641.
(42) Abuchowski A,Van Es T,Palczuk NC.Alteration of immunological properties of bovine serum albumin by colvalent attachment of polyethylene glycol.J Bio Chem.1997,11:3578-3581.
(43) Spahn DR,Leone BJ,Reves JG,Pasch T.Cardiovascular and coronary physiology of acute isovolemic hemodilution:a review of nonoxygen-carrying and oxygen-carrying solution.Anesth Analg.1994,78:1000-21.
(44) Kasper SM,Walter M,Grune F,Bischoff A,Erasmi H,Buzello W.Effects of a hemoglobin-based oxygen carrier(Hboc-201) on hemodynamics and oxygen transport in patients undergoing preoperative hemodilution for elective abdominal aortic surgery.Anesth Analg.1996,83:921-7.
(45) Rohlfs RJ,Bruner E,Chiu A, Gonzales ML, Magde D,et al.Arterial blood pressure respomses to cell-free hemoglobin solutions and the reation with nitric oxide.J Biol Chem 1998;273:12128-34.
(46) Migita R,Gonzales ML, Vandegriff KD,Winslow RM.Blood volume and cardiac index in rats after exchange transfusion with hemoglobin-based oxygen carriers.J Appl Physiol 1997;82:1995-2002.
(47) 关口定美．人造血液的开发及其临床应用．日本医学介绍．1998,18(4)：151．
(48) Hoffman SJ,Looker DL, Roehrich JM,Cozart PE,Durfee SL, Tedesco JL, et al.Expression of fully functional tetrameric human hemoglobin in Escherichia Coli.Proc Natl Acad Sci USA 1990,87:8521-5.
(49) Looker D,Abbatt-Brown D, Cozart P, Durfee S, Hoffman S, Mathews AJ.Ahuman recombinant haemoglobin designed for use as a blood substitute.Nature 1992,356:258-60.
(50) Swanson ME,Martin MJ,O'Donnell J K, et al. Production of functional human hemoglobin in transgenic swine. Biotechnology.1992,10:557.
(51) M.J.Rao, K.Schneider, B.T,Chait, T.L.Chao, H.Keller, S.Anderson, B.N.Manjula, R.Kumar,and A.S.Acharya.Recombinant Hemoglobin a Produced in Transgenic Swine: Structural Equivalence with Human Hemoglobin A.Art.Cell,Blood Subs.,and Immob.Biotech.,1994,22(3):695-700
(52) Riess JG,Oxygen carriers(blood substitutes)Raison deter chemistry, and some physiology.Chem.Rev 2001,101:2797-920.
(53) Spence RK, McCoys,costabile J,Norcross ED,Pello MJ,Alevander JB,et al.Fluosol DA-20 in the treatment of severe anemia:randomized controlled study of 46 patients.Crit Care Med.1990,18:1227-30.
(54) Hess JR, Macdonald VW, Brinkley WW. Systemic and pulmonary hypertension after resuscitation with cell-free hemoglobin. Am J Physiol 1993;74:1769-78.
(55) Ning J, Peterson LMN, Anderson P J, Biro GP. Systemicoxygen carrier, does not affect activation and function of hemodynamics and renal effects of unmodified human SFH in dogs. Biomat Artif Cell Im.1992,20:723-7
(56) Dunlap E, Farrell L, Nigro C, Estep T, Marchand G, Burhop K. Resuscitation with diaspirin crosslinked hemoglobin in a pig model of hemorrhagic shock. Artif Cells Blood Substitesophagus.J Exp Ther 1995,273:762-7.
(57) Caron A, Menu P, Faivre-Fiorina B, Labrude P, Alayash AI, Vigneron C. Cardiovascular and hemorheological effects of three modified human hemoglobin solutions in hemodiluted rabbits. J Appl Physiol 1999;86:541-8.
(58) Lieberthal W, Fuhro R, Freedman JE, Toolan G, Loscalzo J, Valeri CR. O-raffinose cross-linking markedly reduces systemic and renal vasoconstrictor effects of unmodified human hemoglobin. J Pharmacol Exp Ther 1999,288:1278-87.
(59) Biro GP. Safety toxicology evaluation of o-raffinose cross linked hemoglobin solution by daily repeated infusions in rats and dogs. Crit Care Med 1999;27:A173.
(60) By Mehul J. Patel, Edwin J.Webb, Tina E. Shelbourn, Cynthia Mattia-Goldberg, Andrew J.T. George,Feng Zhang, Edwin G. Moore, and Deanna J. Nelson Absence of Immunogenicity of Diaspirin Cross-Linked Hemoglobin in Humans.Blood 1998,91:710-716.
(61) Chang TM, Lister C, Nishiya T, Varma R. Immunological effects of hemoglobin, encapsulated hemoglobin, and conju-gated hemglobin using different immunization schedules.Biomat Artif Cell Im 1992;20:611-8.
(62) Er SS, Wicks D, Wong LT. Pharmacokinetics of 3Hhemolink in rats. Artif Cells Blood Substit Immobil Biotechnol1996,24:435.
(63) Lieberthal W, Fuhro R, Freedman JE, Toolan G, Loscalzo J,Valeri CR. O-raffinose cross-linking markedly reduces systemicand renal vasoconstrictor effects of unmodified human hemoglobin. J Pharmacol Exp Ther. 1999,288:1278-87.
(64) Baines AD, Christoff B, Wicks D, Wiffen D, Pliura D. Crosslinked hemoglobin increases fractional reabsorption and GFR in hyp0xic isolated perfused rat kidneys. Am J Physiol 1995,269(5 Part 2):F628-36.
(65) Leytin V, Mazer D, Mody M, Garvey B, Freedman J.Hemolink, an o-raffinose cross-linked hemoglobin-based oxygen carrier, does not affect activation and function of human platelets in whole blood in vitro. Br J Hematol 2003,120:535-41.
(66) Murray JA, Ledlow A, Lauspach J, Evans D, Loveday M,Conklin JL. The effects of recombinant human hemoglobin on esophageal motro function in humans. Gastroenterology. 1995.109:1241-8.
(67) Wong LT, Er SS, Ning J, Christoff B. Hemolink-induced effects on intestinal motor function and attenuation of these effects by selected agents. Artif Cells Blood Substit Immobil Biotechnol. 1998,26:529—48.
(68) C.P. Stowell. What happened to blood substitutes? Qu'est il arrive aux substituts du sang? Transfusion clinique et biologique. 2005, 12:374-379
(69) Saxena R, Wijnhoud AD, Carton H, et al. Controlled safety study of a hemoglobin-based oxygen carrier, DCL-Hb, in acute ischemic stroke. Stroke. 1999,30:993-6.
(70) Schubert A, Przybelski R J, Eidt JF, et al. Diaspirincrosslinked hemoglobin reduces blood transfusion in noncardiac surgery: a multicenter, randomized,controlled, double-blinded trial. Anesth Analg. 2003;97:323-32.
(71) Gould SA, Moore EE, Hoyt DB, et al. The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery. J Am Coil Surg. 1998,187:113-22.
(72) Gould SA, Moore EE, Hoyt DB, et al. The life sustaining capacity of human polymerized hemoglobin when red cells might be unavailable. J Am Coil Surg.2002,195:445-55.
(73) Carmichael FJL, Ali ACY, Campbell JA, et al. A phase I study of oxidized raffinose cross-linked human hemoglobin.Crit Care Med 2000,28:2283-92.
(74) Hill S, Gottschalk LI, Grichnik K. Safety and preliminaryefficacy of hemoglobin raffimer for patients undergoing coronary artery bypass surgery. J Cardiothorac Vasc Anesth. 2002,16:695-702.
(75) Levy JH, Goodnough LT, Greilich PE,Parr GVS, Stewart RW, Gratz I, Wahr J, Williams J, Comunale ME, Doblar D,Silvay G, Cohen M, Jahr JS, Vlahakes GJ.Polymerized bovine hemoglobin solution as a replacement for allogeneic red blood cell transfusion after cardiac surgery: results of a randomized,double-blind trial. J Thorac Cardiovasc Surg.2002,124:35-42
(76) Juraj Sprung,, James D. Kindscher,, Joyce A. Wahr, Jerrold H. Levy, Terri G. Monk, Mark W. Moritz, and Patrick J. O'Hara. The Use of Bovine Hemoglobin Glutamer-250 (Hemopure(?)) in Surgical Patients: Results of a Multicenter, Randomized,Single-Blinded Trial.Anesth Analg 2002,94:799-808.
(77) Burhop KE, Doyle M, Shick M, Matthews M. The development and preclinical testing of a novel second generation recombinant hemoglobin solution. Abstract S-I-6, Ninth International Symposium on Blood Substitutes, Tokyo,Japan, March 3-5,2003.
(78) Keipert PE. Clinical development of a perfluorochemical emulsion (Oxygent) as an intravascular oxygen therapeutic.Abstract S-I-3, Ninth International Symposium on Blood Substitutes, Tokyo, Japan, March 3-5, 2003.
(79) Nagababu, E. & Rifkind, J. M. Heme degradation during autoxidation of oxyhemoglobin. Biochem. Biophys. Res.Commun. 2000,273: 839-845.
(80) Nagababu, E. & Rifkind, J. M. Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation. Biochemistry 2000,39:12503-12511.
(81) Motterlini, R. et al. Oxidative-stress response in vascular endothelial cells exposed to acellular hemoglobin solutions.Am. J. Physiol. 1995, 269:H648-H655.
(82) Nagababu, E., Somasundaran, R., Rifkind, J. M., Jia, Y. & Alayash, A. I. Site-specific crosslinking of human and bovine hemoglobins differentially alters oxygen binding and redox side reaction producing rhombic heme and heme degradation. Biochemistry 2002,41:7407-7415.
(83) Gould, S. A. & Moss, G. S. Clinical development of human polymerized hemoglobin as blood substitute. World J. Surg.20,1200-1207 (1996).
(84) Liao J. C. et al. Intravascular flow decreases erythrocyte consumption of nitric oxide. Proc. Natl Acad. Sci. USA. 1999,96:8757-8761.
(85) Palaparthy, R., Saini, B. K. & Gulati, A. Modulation of diaspirin cross-linked hemoglobin induced systemic and reginal hemodynamic response by ethanol in rats. Life Sci. 2001,68:1383-1394.
(86) 王军志，生物技术药物研究开发和质量控制。科学出版社，P282-285。
(87) Rihova B,Riha I.Immunological problems of polymer-bound drugs.Crit Rev Ther Drug Carrier Syst.1985,1(4):311-74.
(88) 王军志，生物技术药物研究开发和质量控制。科学出版社，P721。
(89) Schimpff,S.C.,R.M.Miller,S.Polakayetz,and R.B.Hornick.Infection in the severely traumatized patient.Ann.Surg.1974,179:352-374.
(90) Rush, B.F., JR., A.J.Sori, T.F.Murphy, S.Smith, J.J.Flanagan, and G.W.Macheido.Endotoxemia and bacteremia during hemorrhagic shock.The link between trauma and sepsis?Ann.Surg.1988.207:549-554.)
(91) Saba, T.M.., F.A.Blumenstock, D.M.Shah, R.H.Landaburn, M.E.Hrinda, D.C.Deno, J.M.Holman,E.Cho, C.Dayton and P.M.Cardarelli. Reversal of opsonic deficiency in surgical,trauma and burn patients by infusion of purified plasma fibronectin.Correlation with experimental observation. AM.J.Med.1986, 80:229-240.
(92) Abraham, E., and Y.H.Chang. Cellular and humoral bases of hemorrhage induced depression of lymphocyte function.Crit.Care Med.1986, 4:81-86.
(93) Stephan, R.N., P.J.Conrad, c.a.Janeway, A.S.Geha, A.E.Baue,and I.H.Chaudry.Decreased interleukin-2 production following simple hemorrhage.Surg.Forum. 1986,37:73-75.
(94) Livingston, D.H., and M.A.Malangoni.An experimental study of susceptibility to infection after hemorrhagic shock.Surg.Gynecol.Obstet.1989,168:138-142.
(95) Abraham, E., a.a.Freitas, and A.A.Coutinho, Hemorrhage in mice produces alterations in B cell repertoires. Cell.Immunol, 1989, 122:208-217.
(96) Stephan,R.N.,C.A.Janeway,r.e.Dean,AND i.h.Chaudry.Decreased interleulin-2 production following simple hemorrhage and its restoration with ATP-MgCL2 treatment. Magnesium. 1987, 6:163.
(97) Stephan,R.N.,A.Ayala,J.M.Harkema,R.E.Dean,J.R.Border, and I.H.Chaudry.Me chenism of immunosuppression following hemorrhage:defective antigen pre sentation by macrophages.J.Surg.Res.1989,46:553-556.
(98) Ayala,A.,M.M.Perrin,M.A.Wagner,AND i.h.Chaudry.Enhanced suscepbility to sepsis following simple hemorrhage:depression of Fc and C3b receptor-mediated phagocytosis.Arch.Surg. 1990,125:70-75.
(99) Cuddy,B.G.,D.J.Loegering,F.A.Blumenstock,and D.J.Shan.Hepatic macrophage complement receptor clearance function following injury.J.Surg.Res.1986,40:216-224.
(100) Ernest E. Moore, Jeffrey L. Johnson, Aaron M. Cheng, Tomohiko Masuno, and Anirban Banerjee.Insights from studies of blood substitutes intrauma.Shock.2005,24:197-205.
(101) Feng Dong, Carrie H.Hall,Suzanne A.Golech,Nora B.Philbin,Jennifer P.Rice,Jennifer Gurney,Francoise G.Arnaud,Michael Hammett,Xia Ma,W.Shannon Flournoy,Jiang Hong,Lewis J.Kaplan,L.Bruce Pearce,kGerald Mc Gwin,Stephen Ahlers,Richard Mc Carron,and Daniel Freilich.Immune effects of resuscitation with HBOC-201,a hemoglobin-based oxygen carrier,in swine with moderately severe hemorrhagic shock from controlled hemorrhage.Shock.2006.25:50-55.
(102) Johnson JL, Moore E.E, Offner PJ.Resuscitation with a blood substitute abrogates pathologic postinjury neutrophil cytotoxic function. J.Trauma. 2001,50:449-456.
(103) Delio P.Ortegon,Patricia S.Dixon,Kathryn K.Crow,Muller DL and Jeffery D.Kerby.The effect of the bovine hemoglobin oxygen therapeutic HBOC-201 on human neutrophil activation in vitro.J.Trauma.2003,55:755-716.
(1) A Gerson Greenburg and Hae Won Kim. Hemoglobin-based oxygen carriers. Critical Care 2004, 8(Suppl 2):S61-S64.
(2) Darin S.Katz,Steven P.White,Wen Huang,Kamesh Kumar and David W.Christianson.Structure Determination of Aquomet Porcine Hemoglobin at 2.8AResolution.J.Mol.Biol.1994, 244: 541-553.
(3) Leblanc, A. La Cellule, 1901, 18:337.
(4) Ide, M. La Cellule 1902, 20:263.
(5) Thomsen,O.Z. Immunitatsforsch. Orig. 1909, 3:539
(6) Bradley, H.C and Sansum W.D.Some anaphylactic reactions. Journal of Biological Chemistry 1914, 18(3): 497-506.
(7) Bleeker WK, Zappeij LM, den Boer PJ, Agterberg JA, Rigter GM, Bakker JC. Evaluation of the immunogenicity of polymerized hemoglobin solutions in a rabbit model. Artif Cells Blood Substit Immobil Biotechnol. 1995,23(4):461-8.
(8) Estep TN, Gonder J, Bornstein I, Aono F.. Immunogenicity of diaspirin cross-linked human hemoglobin solutions. Biomater Artif Cells Immobilization Biotechnol. 1992,20(2-4):603-9.
(9) Chang TM, Lister C, Nishiya, Varma R.Immunological effects of hemoglobin, encapsulated hemoglobin, polyhemoglobin and conjugated hemoglobin using different immunization schedules. Biomater Artif Cells Immobilization Biotechnol.1992,20(2-4):611-8.
(10) By MehulJ.Patel,Edwin J.Webb,Tina E.Shelbourn,Cynthia Mattia Goldberg,Andrew J.T.George,FengZhang,Edwin G.Moore,AND Deanna J.Nelson.Absebse of immunogenicity of Diaspirin cross-linked hemoglobin in humans.Blood.1998,191:710-716.
(11) 陈琳，王鹤药，．免疫学杂志。2000，16：40-44．
(12) 王鹤药，郑文婕，陈琳，卜凤荣．人造血液基质聚合牛血红蛋白的抗原性研究．中华微生物学和免疫学杂志．2001，21：138-142．
(13) 但宁，陈超，吴宝平。一种血液代用品及制备方法。国防专利，申请号： 200310100233.5
(14) Dan, N., Middleton, R.B., Lehrman, M.A..Hamster UDP-N-acetylglucosamine:dolichol-P N-acetylglucosamine-1-P transferase has multiple transmembrane spans and a critical cytosolic loop. J Biol Chem. 1996,271(48):30717-30724.
(15) Harris, E.L.V.Protein purification methods.a practical approach. IRL Press, Oxford University Press., 1989, pp. 24-45
(16) X.L.Zhu,Wang T.W., Chen C,Dan N,and Daidi Fan.Immunological studies of Porcine Hemoglobin and Its Glutaraldehyde-polymerized/modified Derivatives: Immunogenicity and basic immunological properties. To be published.
(1) Bleeker WK, Zappeij LM, den Boer PJ, Agterberg JA, Rigter GM, Bakker JC.Evaluation of the immunogenicity of polymerized hemoglobin solutions in a rabbit model. Artif Cells Blood Substit Immobil Biotechnol. 1995;23(4):461-8.
(2) By MehulJ.Patel,Edwin J.Webb,Tina E.Shelbourn,Cynthia Mattia Goldberg,Andrew J.T.George,FengZhang,Edwin G.Moore,AND Deanna J.Nelson.Absebse of immunogenicity of Diaspirin cross-linked hemoglobin in humans.Blood.1998,191:710-716.
(3) HayGlass KT, Strejan GH. Suppression of the IgE antibody response by glutaraldehyde-modified ovalbumin: dissociation between loss of antigenic reactivity and ability to induce suppression.Int Arch Allergy Appl Immunol. 1984;74(4):332-40.
(4) Onica D, Dobre MA, Lenkei R. Antibodies against glutaraldehyde-modified albumin in normal mouse sera.Mol Immunol. 1982 Aug;19(8):1021-7.
(5) Hertzman CM, Keipert PE, Chang TM.Serum antibody titers in rats receiving repeated small subcutaneous injections of hemoglobin or polyhemoglobin: a preliminary report. Int J Artif Organs. 1986 May;9(3): 179-82.
(6) Xiaoli Zhu,Wei Chu, Tongwen Wang,Fan Wang,Daidi Fan. Variations in dominant antigen determinants of glutaraldehyde polymerized human, bovine and porcinehemoglobin.Artificial cells blood substitutes and biotechnology. 已接收。
(7) Chang TM, Lister C, Nishiya, Varma R.Immunological effects of hemoglobin, encapsulated hemoglobin, polyhemoglobin and conjugated hemoglobin using different immunization schedules. Biomater Artif Cells Immobilization Biotechnol. 1992;20(2-4):611-8.
(8) Dominik J. Schaer, Christian A. Schaer, Paul W. Buehler, Robert A. Boykins, Gabriele Schoedon, Abdu I. Alayash, and Andreas Schaffner. CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin. Blood. 2006,107:373-380.
(1) C.P. Stowell. What happened to blood substitutes? Qu'est il arrive aux substituts du sang? Transfusion clinique et biologique. 2005, 12:374-379.
(2) Hae Won Kim and A. Gerson Greenburg. Artificial Oxygen Carriers as Red Blood Cell Substitutes: A Selected Review and Current Status. Artificial Organs. 2004,28(9):813-828.
(3) Rabiner SF, Helbert JR, Lopas H, Friedman LH. Evaluation of stroma-free hemoglobin solution for use as plasma ex-pander. J Exp Med. 1967,126:1127-42.
(4) Bleeker WK, van der Plas J, Feitsma RIJ, et al. In vivo distribution and elimination of hemoglobin modified by intramolecular cross-linking with 2-nor-2-formylpyridoxal 5'-phosphate. J Lab Clin Med. 1989,113:151-61.
(5) Lee JT, Ahrenholz DH, Nelson RD, Simmons RL. Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis. V. The significance of the coordinated iron component. Surgery. 1979,86:41-8.
(6) Hau T, Simmons RL. Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis. Ⅲ. The influence of hemoglobin on phagocytosis and intracellular killing by human granulocytes. Surgery.1980.87:588-92.
(7) Eaton JW, Brandt P, Mahoney JR, Lee JT. Haptoglobin—a natural bacteriostat. Science. 1982,215:691-2,
(8) Otto BR, Verweij-van, Vught AMJJ,MacLaren DM. Blood substitutes and infection. Nature.1992, 358: 23-24.
(9) Bullen JJ, Griffiths E. Iron and infection: molecular, physiological and clinical aspects. Chichester: John Wiley &Sons, 1987.
(10) Lee JT,Arenholz DH, Nelson RD, Simmons RL. Mechanisms of the adjuvant effect of haemoglobin in experimental peritonitis V: the significance of the co-ordinated iron component. Surgery1979, 86: 41-47.
(11) Kim YM, Yamazaki I,Piette LH. The effect of haemoglobin, hematin and iron on inactivation in neutrophil superoxide generating systems.Arch Biochem Biophys.1994, 309: 308-14.
(12) Hoyt DB, Greenburg AG, Peskin GW, Forbes S,Reese H.Resuscitation with pyridoxalated stroma free haemoglobin: tolerance to sepsis. J Trauma. 1981, 21: 938-42.
(13) Crowley JP, Metzger J, Gray A, Plvacek LE, Cassidy G, Valeri CR.Infusion of stroma-free cross-linked Gram hemoglobin during acute negative bacteremia. Circ Shock. 1993,41: 144-49.
(14) Elwyn Griffiths,Angeles Cortes,Nicholas Gilbert,Pauline Stevenson,Shirley Macdonald,Duncan Pepper.Hemoglobin-based blood substitutes and sepsis.Lancet. 1995,345:158-60.
(15) Jan A M Langersman,Wim K Bleeker. Haemoglobin-based blood substitutes and infection.Lancet. 1995,345:863-864.
(16) Sherwood RL, McCormick DL, Zheng S, Beissinger RL.Influence of steric stabilization of liposome-encapsulated hemoglobin on Listeria monocytogenes host defense. Artif Cells Blood Substit Immobil Biotechnol. 1995,23(6):665-79.
(17) David P.Speert and Siamon Gordon.Phagocytosis of unopsionized Pseudomonas aeruginosa by murine macrophages is a two-step process requiring Glucose.J.Clin.Invest.1992,(90):1085-1092.
(18) Donghui Su,Robert I.Roth,Minoru Yoshida,Jack Levin.Hemoglobin increases mortality from bacteria endotoxin.Infection and immunity. 1997,65:1258-1266.