血红蛋白的化学修饰与血液代用品

血液代用品系指具有携氧扩容功效的溶液,包括：氟碳化合物、脂质体包封血红蛋白（hemoglobin,Hb)、微囊化血红蛋白和无细胞基质血红蛋白（化学修饰Ｈb、基因重组Ｈb、化学合成Ｈb和转基因动物人Ｈb）。通过化学修饰稳定血红蛋白结构以避免其肾毒性,并延长循环半寿期是利用无基质血红蛋白制备血液代用品的主要研究内容,通常采用交联、聚合、偶联等方法完成。     

脂质体包埋猪血红蛋白的制备及其注入小鼠体内的初步试验

 为探索一条研制猪血红蛋白 (pHb)为基础的血液代用品新途径 ,开发了干膜超声法将猪血红蛋白和别构效应剂、超氧化物歧化酶等联合包埋于脂质体的技术 .考察了氢化大豆卵磷脂、二肉豆蔻酰磷脂酰胆碱、胆固醇、二硬脂酰磷脂酰乙醇胺 甲氧基聚乙二醇和维生素E等在制备脂质体包埋血红蛋白 (LEH)中的作用 .通过控制磷脂与其他成分的配比 ,制得包埋率为 10 3%,pHb浓度达 16 %的稳定的LEH ;进一步将pHb微囊通过小鼠尾静脉多次注入其体内 ,检测受试小鼠血液中红细胞和白细胞数、抗体滴度、血小板聚集率及肾脏组织学等方面的变化 .小鼠体内试验表明了所制备的LEH具有低免疫原性、对肾脏无明显损害等特点 .脂质体包埋pHb是一种极具开发前景的稳定的人工载氧系统     

辛二酸二琥珀酰亚胺酯交联血红蛋白制备红细胞代用品

探索了用辛二酸二琥珀酰亚胺酯(DSS)作为分子内交联剂交联血红蛋白的亚基, 以制备新型红细胞代用品. 采用多角度激光散射检测器(MALLS)与高效液相色谱(HPLC)连接进行检测, 辅之以变性聚丙烯酰胺凝胶电泳(SDS-PAGE)分析反应产物组成. 实验在局部百级的洁净室内进行, 低温4℃下反应1 h, 交联产物中83.8%为重均分子量(Mw)66.8 kD的分子内交联血红蛋白, 16.2%是Mw为146.6和306.4 kD的低聚血红蛋白. 通过流动相中添加1 mol/L MgCl₂的凝胶过滤色谱验证, 交联后的血红蛋白在生理环境下不会解聚. 联用反相高效液相色谱与质谱(RP-HPLC-MS)初步确定, 交联为非定点反应, 主要在a-a亚基间进行. 交联反应增强了血红蛋白亚基的疏水性, 影响了血红蛋白的载氧活性, P₅₀和Hill系数由交联前的21.8和2.22 mmHg分别降至14.3和1.41 mmHg, 仍具有一定的载氧能力. 等电聚焦(IEF)电泳表明, 制备的DSS交联血红蛋白的等电点降至4.6 ~ 5.2之间, 接近于不透过血管壁的血清白蛋白. 产品经小鼠异常毒性试验证实具有一定的生物安全性, 大鼠的换血试验表明DSS交联血红蛋白并未引起血压升高, 也未见其他明显的副作用. 还对DSS交联血红蛋白与文献已有的双阿司匹林交联血红蛋白的产物特性进行了比较.     

新型蛋白质修饰剂的合成及修饰牛血红蛋白的初步研究

以L-谷氨酸和己二酸为原料合成了一种新型的四官能团蛋白质修饰剂,并用核磁共振和红外光谱对其结构进行了表征。然后以其为修饰剂,对牛血红蛋白的化学修饰进行了初步的研究,并通过高效液相色谱、聚丙烯酰胺凝胶电泳和血氧分析仪对交联牛血红蛋白的分子量和携氧性能进行了表征。结果表明,该修饰剂可以使牛血红蛋白同时在分子内和分子间发生化学交联,并较好地保持携氧能力（P50:21.7mmHg,Hill系数:2.01）,因此在众多用于开发人工血液代用品的化学修饰剂中该修饰剂具有良好的应用前景。     

美研制脂类包衣血红蛋白代用品

美国体细胞遗传工程公司与美国海军研究署合作,正在研制一种脂类包衣的重组血红蛋白(rLEH)作为人血红蛋白的代用品。据称,人血红蛋白的代用品比人红血细胞产物有更多的优点。例如贮存期长,不需鉴定血     

大鼠红细胞作为SOD新型载体的细胞水平上的研究

用低渗透析 -等渗重封的方法制备了包埋超氧化物歧化酶 ( SOD)的大鼠载体红细胞 ,并从细胞水平上研究了透析条件对大鼠红细胞包埋 SOD的影响与载体红细胞的部分性质 .流式细胞计( FCM)研究表明 ,随透析时间延长和透析液渗透压降低 ,包埋 SOD的载体红细胞百分率升高 ,但载体细胞平均包埋 SOD的量无明显变化 ;SOD浓度对载体细胞百分率无明显影响 ,但与载体细胞平均包埋 SOD的量成线性关系 ;载体红细胞前向角散射 ( FLS)明显下降 ,但显微镜下观察到的载体细胞的大小无明显变化 ,当载体细胞反注射到大鼠体内后 FLS能迅速恢复 ;载体红细胞密度下降 ,其原因是低渗透析时红细胞膨胀未能完全恢复 ;载体红细胞未暴露与自身 Ig G结合的抗原位点 .激光扫描共聚焦显微镜 ( LSCM)分析表明 ,SOD在细胞内呈从细胞中心到细胞膜浓度逐步下降的辐射分布特征 .     

对地贫红细胞的显微激光散射和图象分析

应用显微准弹性激光散射(MQLS)技术与显微生物医学图象分析技术对地中海贫血红细胞及胞内血红蛋白动态特性进行了研究.在实验中,比较了正常人及地贫患者红细胞胞内血红蛋白聚集体的平均流体力学半径、平均平动扩散系数及红细胞膜的搏动频率等动态特性参数,以及细胞的截面积、规化形状因子、长径、短径、灰度等图象分析数据,发现地贫红细胞的血红蛋白聚合物平均流体力学半径远远大于正常人红细胞的,其大小变异亦较正常人大,且其膜搏动频率也较为缓慢,细胞的截面积也变小.这反映了地贫红细胞内有较大的蛋白质聚合物存在和红细胞变形能力差的特性.研究还表明,显微准弹性激光散射技术结合图象分析技术,可使测量的可比性和准确性大大提高,预期可广泛适用于各种活细胞动态特性的研究.     

功能红细胞研究的拉曼光谱技术与进展

红细胞在人体新陈代谢和物质运输方面起着非常重要的作用。作为一种无损、快速而灵敏的光谱学检测方法,拉曼光谱技术在生物医学领域的应用日渐广泛。本文综述了近年来拉曼光谱技术在红细胞及血红蛋白研究中的应用进展,详尽评述当前研究功能红细胞中较有优势的拉曼技术—显微拉曼光谱技术和光镊拉曼光谱技术,并对其技术的改进做出总结,以期为利用激光拉曼光谱研究红细胞提供较系统的参考。     

血液代用品的临床意义

红细胞代用品在临床试验方面发展很快,它是指在损伤和其他危急情况下使用的可保存且有携氧功能的溶液。这种最早提出的概念至今依然沿用。我们感兴趣的是红细胞代用品在外科和传统的自体输血领域的潜在应用。本文描述了其在外科领域的潜在应用价值。在外科病人中使用红细胞代用品要求很高,主要是由于安全性和某些干扰因素影响造成输血失败使其应用受到限制。尽管有越来越多的输血指南,但缺乏评价的指征。外科病人不仅仅担心传染病的传播,还有免疫系统、相关感染、肿瘤复发和恶性肿瘤的存活等问题。考虑到这些因素,出现了输血无效的新概念。…     

尺寸均一的壳聚糖微球的制备及其作为胰岛素控释载体的研究

采用新型微孔膜乳化技术制备了载胰岛素的壳聚糖微球。研究表明,要制备粒径均一的壳聚糖微球,必须将亲水性膜修饰成疏水性;制得的微球粒径和所采用的膜孔径之间存在很好的线性关系,使得微球粒径可控;以胰岛素为模型药物,主要考察了交联剂用量和交联时间对微球表面形态、药物包埋率和微球体外释药特性的影响。结果表明当氨基与醛基的摩尔比为1∶0.7、交联时间为1h时,所得载药微球的包埋率最高,随着戊二醛用量的增加和交联时间的延长,药物体外释放速率减慢。     

Future prospects for artificial blood

Concern about potential infective agents in donated blood has stimulated the recent development of blood substitutes. Chemically cross-linked hemoglobins are already undergoing clinical trials and might soon be ready for routine use. New generations of modified hemoglobin are being prepared to modulate the effects of nitric oxide and oxygen radicals, and artificial red blood cells are also under development.     

Liposome-Encapsulated Hemoglobin: A Red Blood Cell Substitute

Abstract

A safe and effective red blood cell (RBC) substitute would have broad implications in the practice of emergency medicine, trauma management, surgery, and several other areas of medicine. Several hemoglobin-based RBC substitutes have been developed that can deliver oxygen to peripheral tissues. However, although these RBC substitutes have desirable biophysical properties, their in vivo efficacy is limited by their significant toxicity. In view of the very high doses of blood substitute that are likely to be used clinically, toxicity as well as other safety issues that include hemostasis and thrombosis are critical considerations for the development and ultimate application of RBC surrogates.

Recent work conducted in our laboratories has demonstrated that administration of liposome-encapsulated hemoglobin (LEH) in rats was efficacious. Also our results have demonstrated that the replacement of more conventional lipids with the sterically-stabilizing lipid polyethyleneglycol distearoylphosphatidyl-ethanolamine in the LEH results in a significant decrease in LEH immunotoxicity, as measured by host resistance to infectious insult.     

Hemoglobin-based blood substitutes: oxygen carriers, pressor agents, or oxidants?

Hemoglobin-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and hypovolemic (low blood volume) shock. The ability of cell-free hemoglobin blood substitutes to affect vascular tone through the removal of nitric oxide has also prompted an evaluation of their usefulness for maintaining blood pressure in critically ill patients. Before the clinical potential of these substitutes can be fully realized, however, concerns remain as to the intrinsic toxicity of the hemoglobin molecule, particularly the interference of the heme prosthetic group with the tissue oxidant/antioxidant balance. This review provides some insights into the complex redox chemistry of hemoglobin and places an emphasis on how current knowledge may be exploited both to selectively enhance/suppress specific chemical reaction pathway(s) and to ultimately design safer hemoglobin-based therapeutics.     

Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P₅₀ than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.     

Ligand reactivity and allosteric regulation of hemoglobin-based oxygen carriers

Historically, exogenous administration of hemoglobin solutions to implement the oxygen transport capacity for clinical applications suffered from dramatic drawbacks, resulting in the failure of many attempts. In the last decades, the biochemical and physiological basis responsible for the therapeutic failures has been extensively investigated. It is now widely accepted that they mostly arise because, out of the confined and controlled environment of the red blood cell, hemoglobin exhibits tetramer instability, increased auto-oxidation rate, higher oxygen affinity, altered cooperativity and nitric oxide reactivity. Moreover, it became evident that the design of a hemoglobin-based oxygen carrier that exactly reproduces the "physiological" oxygen-binding curve is not only an overly ambitious task, but may also represent a wrong approach for many potential clinical applications. Under these premises, and given the complex chemical nature of blood, it is obvious that any strategy undertaken to modify the stability and function of the hemoglobin tetramer for clinical use should be driven by a detailed knowledge of its structure, dynamics and mechanism of allosteric regulation. We briefly review the most recent theories and experiments that increased our understanding of the mechanism of homo- and heterotropic effects in human hemoglobin, trying to interpret, on a biophysical basis, how diverse approaches like polymerization, cross-linking, site-directed mutagenesis, surface decoration and encapsulation may affect ligand affinity and allosteric regulation.     

Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Sickle cell disease and nitric oxide: a paradigm shift?

Traditionally the pathophysiology of sickle cell disease is thought to result from the polymerization of hemoglobin S in red cells, under hypoxic conditions, resulting in the occlusion of blood vessels. Adhesion of cells to the venular endothelium also appears to play a role. Recent studies have also suggested that in addition to the polymerization of hemoglobin S in the red blood cell, a deficiency of the endogenous vasodilator, nitric oxide may be involved. Hemoglobin released as a result of hemolysis rapidly consumes nitric oxide resulting in a whole program of events that inhibit blood flow. Therapies directed at decreasing the destruction of nitric oxide, increasing the production of nitric oxide, or amplifying the nitric oxide response may prove beneficial.     

Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.     

Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse.

Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human alpha-subunit and the bovine beta-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of approximately 3 Torr, and a cooperativity of n = 1.0. Anesthetized mice were transfused during 2-h occlusion of the middle cerebral artery. Compared with transfusion with 5% albumin, cerebral infarct volume was reduced by 41% with transfusion of a 3% solution of the high oxygen-affinity hemoglobin polymer and by 50% with transfusion of a 6% solution of the polymer. Transfusion of a 6% solution of a 500-kDa polymer possessing a P50 of 17 Torr and a cooperativity of n = 2.0 resulted in a 66% reduction of infarct volume. These results indicate that cell-free Hb polymers with P50 values much lower than that of red blood cell hemoglobin are highly capable of salvaging ischemic brain. The assumption that the P50 of blood substitutes should be similar to that of blood might not be warranted when used during ischemic conditions.     

Differential polarization imaging. III. Theory confirmation. Patterns of polymerization of hemoglobin S in red blood sickle cells.

In this paper we test the predictions of the differential polarization imaging theory developed in the previous two papers. A characterization of the patterns of polymerization of hemoglobin in red blood cells from patients with sickle cell anemia is presented. This system was chosen because it is relatively easy to handle and because previous studies have been done on it. A differential polarization microscope designed and built in our laboratory was used to carry out this study. This microscope uses an image dissector camera, a photoelastic modulator, and a phase-lock amplifier. This design represents a substantial modification with respect to the instrumentation used in the previous results communicated on this system. Therefore, the results presented here also permit us to confirm the validity of our conclusions. On the basis of the differential polarization images obtained, models of the patterns of polymerization of the hemoglobin S inside the sickle cells are proposed and their M12 and regular images are calculated by the theory. Good agreement between those models and the experimental systems is found, as well as with the results previously reported.