【作者】 黄鹤；

【导师】 陈超；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 研究背景血红蛋白(Hemoglobin, Hb)是脊椎动物血红细胞中重要的氧载体,其含量可达到血红细胞干重的90%以上。长期以来,血红蛋白类氧载体(Hemoglobin-Based Oxygen Carriers, HBOCs)由于其良好的载氧特性受到了广泛的关注。之前的应用研究主要集中在将HBOCs应用于红细胞代用品,将其用作携氧治疗剂的研究尚处于起步阶段。小肠移植是短肠综合征和不可逆肠功能衰竭的最理想治疗方法,但是小肠离体保存难度大,从而极大地限制了小肠移植的临床应用。小肠离体保存困难的重要原因之一是小肠上皮细胞对于缺血非常敏感,因此将具有良好携氧能力的pPolyHb应用于小肠的离体保存或可优化其保存效果。失血性休克是一种由于机体失血造成的有效循环血容量减少、组织灌注不足,细胞代谢紊乱和多器官功能受损的病理过程。液体复苏是休克早期治疗的重要手段之一,临床常用的复苏液包括晶体液、胶体液和血液制品等。HBOCs已经开始作为失血性休克的携氧复苏液进行研究,研究结果显示HBOCs有望成为临床中复苏失血性休克患者的重要工具。研究目的本研究首次选用猪血作为原料并成功研发了戊二醛聚合猪血红蛋白分子(Polymerized Porcine Hemoglobin, pPolyHb),该制品氧亲和力高,循环时间长,具有稳定四聚体结构。本研究以该pPolyHb作为携氧治疗剂(Oxygen therapeutic agent),探索其在大鼠移植小肠保存和失血性休克复苏中的应用。研究方法制备戊二醛聚合猪血红蛋白氧载体(Polymerized porcine hemoglobin, pPolyHb),并测量其分子量及理化性质指标。以pPolyHb作为高渗柠檬酸腺嘌呤溶液(Hypertonic Citrate Adenine Solution, HCA)的携氧治疗剂,为大鼠小肠的离体保存供氧,优化其保存条件。大鼠小肠分别保存于2g/dl pPolyHb+HCA液、4g/dl pPolyHb+HCA液和对照组HCA液、威斯康辛大学保存液(University of Wisconsin Solution, UW)中,以12h、24h和36h梯度时间保存,然后进行组织病理学分析、组织ATP含量检测、血气分析等,分析pPolyHb对于提高离体小肠保存的效果。为了检测pPolyHb在失血性休克复苏的作用,本研究建立了大鼠抗失血性休克模型,失血时间为90min,失血量为60±5%。将SD大鼠随机分为五组,每组8只,应用不同浓度的pPoIyHb(2.0g/kg、1.5g/kg、1.2g/kg、1.0g/kg、0.5g/kg)联合两倍生理盐水对失血性休克大鼠进行复苏实验,同时监测各组大鼠血压、心率、呼吸和心电图等基本生命体征和动脉的血气指标,最终筛选出最低有效剂量的pPolyHb浓度。然后随机分为六组,每组8只SD大鼠,采用pPolyHb联合两倍生理盐水、自体血(AB)联合两倍生理盐水、红细胞(RBC)联合两倍生理盐水、羟乙基淀粉(HES)联合两倍生理盐水、三倍乳酸钠林格液(LR)和三倍生理盐水(SPSS)分别对失血性休克大鼠进行复苏实验,同时监测各组大鼠血压、心率、呼吸和心电图等基本生命体征和动静脉的血气指标,并对大鼠重要内脏器官进行病理学分析。研究结果pPolyHb的分子量及理化性质指标结果显示其平均分子量在550～800kD,浓度在11g/dl左右,其中MetHb的含量小于5%;pH值(7.4±0.3)接近生理状态；胶体渗透压也在生理范围内；小分子含量小于3%；从Hemox血氧分析仪测定产品的P50和Hill系数可以看出,经化学修饰后,血红蛋白的载氧能力受到影响,亚基的协同性也有一定下降。在离体小肠保存实验中,病理学分析结果显示小肠在pPolyHb+HCA液中短期保存效果(12和24h)与UW液相近,无明显差异,但其长期保存效果(36h)则优于UW液,且无论长期与短期保存效果均优于单纯的HCA溶液(P<0.05)。组织ATP含量检测表明pPolyHb+HCA组中小肠组织的三磷酸腺苷(ATP)含量显著高于对照组UW组与HCA组(P<0.05)。血气分析显示pPolyHb+HCA保存液中的pH值始终维持在正常水平、HCO3-的变化值(ΔHCO3-)显著高于对照组,而Lac含量显著低于对照组(P<0.05)。这些研究结果表明pPolyHb能够改善离体小肠的保存效果,同时也提示提高离体小肠组织氧供应能够优化其离体保存。在大鼠失血性休克复苏实验中,生理监测指标显示：五组pPolyHb联合两倍生理盐水组复苏后大鼠的血压、心率、呼吸率等恢复平稳,较高浓度pPolyHb (2.0g/kg和1.5g/kg)联合两倍生理盐水对血流动力学的影响效果均优于其他三个较低浓度组(1.2g/kg、1.0g/kg、0.5g/kg)。血气指标显示：携氧能力方面,2.0g/kg pPolyHb与1.5g/kg pPolyHb组有更好的携氧能力,但是随着pPolyHb浓度的提高,高铁血红蛋白(MetHb)的含量有所增加；酸碱平衡方面,随着pPolyHb浓度的提高,可纠正休克期的代谢性酸中毒,但呼吸性酸中毒随着pPolyHb浓度的提高呈现下降的趋势；电解质平衡方面,五个浓度的pPolyHb均可有效平衡血液中的电解质；代谢物乳酸的生成量在不同浓度pPolyHb复苏后均有下降；存活率统计情况显示,1.5g/kg pPolyHb组的存活率和2.0g/kg pPolyHb组的存活率均为100%,故在接下来的不同溶液比较实验中选择1.5g/kg作为pPolyHb的最低有效剂量。生理监测指标显示：不同溶液复苏过程中,1.5g/kg pPolyHb+两倍生理盐水组复苏后血压恢复均优于红细胞+两倍生理盐水组、HES+两倍生理盐水组和三倍生理盐水组,心率和呼吸恢复平稳,说明有更好的扩容效果。血气指标分析结果显示：血氧方面,pPolyHb+两倍生理盐水组的携氧能力明显优于HES+两倍生理盐水组、乳酸钠林格液组和三倍生理盐水组(P<0.05)；酸碱平衡方面,pPolyHhH+两倍生理盐水组对于机体休克末的代谢性酸中毒缓解效果优于乳酸钠林格液组,与HES+两倍生理盐水组相似;无氧代谢方面：pPolyHb+两倍生理盐水组复苏后的乳酸含量明显低于HES+两倍生理盐水组、乳酸钠林格液组和三倍生理盐水组(P<0.05)。存活情况显示,pPolyHb+两倍生理盐水组大鼠存活时间与存活率均优于红细胞+两倍生理盐水组、HES+两倍生理盐水组和三倍生理盐水组,与自体血+两倍生理盐水无显著差异。失血性休克复苏大鼠病理检查结果显示,pPolyHb+两倍生理盐水组大鼠的心脏、肝脏、脾脏、肺脏和肾脏的病理性变化均优于对照组HES+两倍生理盐水组。结论(一)在12h和24h的短期保存中,pPolyHb+HCA组中的离体小肠保存效果与现行的临床标准UW液相近,但在36h长期保存中,其保存效果要优于UW溶液,pPolyHb可维持组织的有氧呼吸,阻止组织的无氧代谢,是用小肠离体保存良好的携氧治疗剂；(二)血液动力学、血气分析、病理分析的各项指标显示,与现行的临床复苏液相比,pPolyHb作为携氧治疗剂能够更为安全有效的复苏失血性休克大鼠；(三)pPolyHb在大鼠离体小肠保存与大鼠失血性休克复苏中的应用研究结果为聚合猪血红蛋白临床适应症的选择提供依据。更多还原

【Abstract】 The development of HBOCs (Hemoglobin-Based Oxygen Carriers) as blood substitutes has reached an impasse due to clinically adverse outcomes, such as vasoconstriction caused by NO (Nitric Oxide) scavenging, oxidative damage and so on. Currently, the HBOCs can’t substitute for red blood cells completely in clinical blood transfusion, but its function as therapeutic agent can be applied in other clinical indications to carry oxygen. The FDA suggests for the categories of HBOCs indications:local perfusion, uncontrolled hemorrhagic shock, perioperative application. In this doctoral dissertation, a new type of HBOCs-pPolyHb (glutaraldehyde-polymerized porcine hemoglobin) was prepared and two types of clinical indications were chosen to test the effectiveness of pPolyHb as oxygen therapeutic agent.The SBTx (Small bowel transplantation) has became a standard clinical treatment for short bowel syndrome or irreversible intestinal function failure. The optimum preservation of the organ is essential for the success of transplantation. The aim of the present study is to investigate the time-related morphological changes and biochemical indicators of small bowel tissue in preservation solutions. Rat small bowels were preserved in HCA (hypertonic citrate adenine solution), HCA with pPolyHb or University of Wisconsin solution (UW), and light microscopy was used to evaluate the integrity of the small bowel. Small bowels were placed in different preservation solutions at4℃for12,24and36h. In the small intestine preservation experiment, pathology analysis results showed that the short-term (12and24h) preservation effect of pPolyHb+HCA solution approached the level of UW solution, there was no significant difference between these two groups. However, its long-term preservation effect (36h) was significantly better than the UW solution. Both long-term and short-term preservation effects were superior to the HCA solution. The ATP (Adenosine triphosphate) content test showed that the ATP content of pPolyHb+HCA group was significantly higher than the control group (UW group and HCA group). Blood gas analysis results indicated that the pH value of PolyHb+HCA solution maintained normal level all the time; the change of ΔHCO3-(HCO3-value) was significantly higher than the control groups; the content of Lac (Lactate) was significantly lower than the control groups. These results demonstrated that the pPolyHb could notably improve the preservation effects for small intestine, and also prompted to improve the oxygen supply of small intestine tissue as to optimize its preservation in vitro.Hemorrhagic shock is a pathologic state in which intravascular volume and tissue oxygen delivery are impaired, leading to circulatory collapse and cellular ischemia. Resuscitation by the HBOCs is appealing in both restore intravascular volume and tissue oxygenation, without the limitations in the supply and immunomodulatory effects of packed red blood cells. In this study, we established a rat model of uncontrolled hemorrhagic shock resuscitation to verify the effectiveness of pPolyHb. Firstly, we have chosen five gradient concentrations to determine the minimum effective dose of pPolyHb, and then used it as the final concentration of experimental group to compare with other five resuscitation solutions for the effects on changes of hemodynamic values and blood gas results to verify the validity of pPolyHb.Physiological indicators showed that the blood pressure, heart rate, respiratory rate were restored to the base value after resuscitation by2.0g/kg pPolyHb or1.5g/kg pPolyHb with twice saline. The results indicated that the hemodynamics effects of these two groups were better than the lower concentration groups. The blood gas results indicated that the oxygen-carrying capacity of2g/kg pPolyHb group and1.5g/kg pPolyHb group were better than that of low concentration group, and acid-base balance were also slightly better than the lower concentration groups. Meanwhile, the indicators of electrolyte balance and anaerobic metabolism had no significant difference among the five concentration groups. The survival rates of2.0g/kg and1.5g/kg pPolyHb group within72hours were100%, much higher than those of low concentration groups. Therefore,1.5g/kg pPolyHb was chosen as the minimum effective dose. Next, the1.5g/kg pPolyHb was compared with other five resuscitation fluids to verify the effectiveness. The physiological datum showed that the1.5g/kg pPolyHb and autologous blood group had better recovery effect than the other resuscitation fluids. The blood gas results notified that the1.5g/kg pPolyHb and autologous blood group showed a better capacity to carry oxygen and balance acid-base. Many of the blood gas indicators in1.5g/kg pPolyHb were significantly better than the negative control. The electrolyte balance datum were similar among each group, while the anaerobic metabolism index of the pPolyHb group, the AB (Autologous Blood) group and RBC (Red Blood Cell) group had shown obvious superiority.The survival rates of1.5g/kg pPolyHb and AB group were100%, higher than other liquid recovery groups. Rat pathological anatomic analysis results showed that the oxygen-carrying resuscitation liquid (1.5g/kg pPolyHb group, the AB group and RBC group) provided better tissue integrity than other three groups in heart, lung and liver.Conclusion:(1) The pPolyHb could maintain the aerobic respiration of organization and prevent anaerobic metabolism, which could be considered as an effective oxygen therapeutic agent in the small intestine preservation solution in vitro;(2) The pPolyHb, as an oxygen therapeutic agent could resuscitate the hemorrhagic shock rats effectively, which implies its possible clinical application in hemorrhagic shocktherapy.(3) The results of this study provided evidence for new selection of clinical indication for the pPolyHb.更多还原