聚合猪血红蛋白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

发布时间：2018-05-24 02:48

本文选题：聚合猪血红蛋白 + 小肠保存　；参考：《西北大学》2013年博士论文

【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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)；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(P0.05)。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。
[Abstract]:Research background
Hemoglobin (Hb) is an important oxygen carrier in the blood red blood cells of vertebrates, and its content can reach more than 90% of the dry weight of blood red cells. For a long time, the Hemoglobin-Based Oxygen Carriers (HBOCs) has attracted wide attention because of its good oxygen carrying characteristics. The previous application research is mainly focused on the H. The application of BOCs to the use of red cell substitutes and the use of it as an oxygen carrier is still in its infancy. Small bowel transplantation is the most ideal treatment for short bowel syndrome and irreversible intestinal failure, but it is difficult to preserve in small intestine in vitro, which greatly restricts the clinical application of small intestine transplantation. The important reasons for the difficulty of small intestine in vitro preservation One of these is that small intestinal epithelial cells are very sensitive to ischemia, so pPolyHb with good oxygen carrying capacity is applied to the preservation of the small intestine in vitro or to optimize its preservation. The hemorrhagic shock is a disease caused by the body's loss of blood, the effective circulation of blood, the insufficiency of tissue perfusion, the disorder of cell metabolism, and the damage of multiple organ function. Fluid resuscitation is one of the important methods for the early treatment of shock. The common clinical resuscitation fluid, including crystal fluid, colloid fluid and blood products, has begun to be studied as an oxygen carrying resuscitation fluid in hemorrhagic shock. The results show that HBOCs is expected to be an important tool for patients with hemorrhagic shock in the clinic.
research objective
This study first selected pig blood as raw material and successfully developed Polymerized Porcine Hemoglobin (pPolyHb), which has high oxygen affinity and long cycle time, and has a stable four polymer structure. This study uses the pPolyHb as a Oxygen therapeutic agent (Oxygen therapeutic agent) to explore its transplantation in rats. Application of small intestine preservation and resuscitation of hemorrhagic shock.
research method
Polymerized porcine hemoglobin (pPolyHb) was prepared with glutaraldehyde, and its molecular weight and physicochemical properties were measured. PPolyHb was used as an oxygen carrying agent for hypertonic citrate adenine solution (Hypertonic Citrate Adenine Solution, HCA), to preserve oxygen supply in rat small intestine in vitro, and to optimize its preservation conditions. The rat small intestine was preserved in 2g/dl pPolyHb+HCA solution, 4g/dl pPolyHb+HCA solution and control group HCA solution, University of Wisconsin Solution (UW), and stored in 12h, 24h, and gradient time. Then histopathological analysis, histological analysis, blood gas analysis, etc. were used to improve the small intestine of the isolated small intestine. In order to detect the effect of pPolyHb on hemorrhagic shock and resuscitation, the rat model of hemorrhagic shock was established in this study. The time of hemorrhagic shock was 90min and the amount of blood loss was 60 + 5%., and SD rats were randomly divided into five groups, 8 rats in each group, combined with two times of pPoIyHb (2.0g/kg, 1.5g/ kg, 1.2g/kg, 1.0g/kg, 0.5g/kg). The blood pressure, heart rate, respiration, electrocardiogram and other basic vital signs and arterial blood gas indexes were monitored at the same time in rats of hemorrhagic shock. The pPolyHb concentration of the lowest effective dose was selected, and then randomly divided into six groups, 8 rats in each group, with two times of pPolyHb combined with saline, and two times of autologous blood (AB). Saline, red blood cells (RBC) combined with two times normal saline, hydroxyethyl starch (HES) combined with two times physiological saline, three times sodium lactate (LR) and three times physiological saline (SPSS), respectively, on the resuscitation experiment of rats with hemorrhagic shock, and the blood pressure, heart rate, respiration and electrocardiogram were monitored in each group. Pathological analysis of important visceral organs in rats.
Research results
The molecular weight and physicochemical properties of pPolyHb show that the average molecular weight is 550 to 800kD, the concentration is about 11g/dl, the content of MetHb is less than 5%, the pH value (7.4 + 0.3) is close to the physiological state, the colloid osmotic pressure is also within the physiological range, and the small molecule content is less than 3%. The P50 and Hill coefficient of the product from the Hemox blood oxygen analyzer can be measured. It was observed that the oxygen carrying capacity of hemoglobin was affected by chemical modification, and the synergy of the subunits also decreased.
The results of pathological analysis in isolated small intestine showed that the short term preservation effect of small intestine in pPolyHb+HCA solution (12 and 24h) was similar to that of UW solution, but the long-term preservation effect (36h) was superior to UW solution, and the long-term and short-term preservation effect was better than that of pure HCA solution (P0.05). Tissue ATP content detection showed that the pPolyHb+HCA group was in the pPolyHb+HCA group. The content of adenosine triphosphate (ATP) in small intestinal tissue was significantly higher than that of the control group UW and HCA group (P0.05). Blood gas analysis showed that the pH value in the pPolyHb+HCA preservation solution remained at the normal level, and the change value of HCO3- (delta HCO3-) was significantly higher than that of the control group, while the Lac content was significantly lower than that in the opposite group (P0.05). These results showed that pPolyHb could improve the separation. The preservation effect of the small intestine also suggests that increasing the oxygen supply of isolated small intestine tissue can optimize its preservation in vitro.
In the experiment of hemorrhagic shock and resuscitation in rats, the physiological monitoring indexes showed that the blood pressure, heart rate and respiratory rate of the five groups of pPolyHb combined with two times physiological saline group recovered smoothly. The effect of high concentration of pPolyHb (2.0g/kg and 1.5g/kg) combined with two times of normal saline on hemodynamics was better than that of the other three lower concentration groups (1. 2g/kg, 1.0g/kg, 0.5g/kg). Blood gas indicators showed that the oxygen carrying capacity, 2.0g/kg pPolyHb and 1.5g/kg pPolyHb group had better oxygen carrying capacity, but with the increase of pPolyHb concentration, the content of HB (MetHb) increased; with the increase of pPolyHb concentration, metabolic acidosis could be corrected with the increase of pPolyHb concentration, but the concentration of pPolyHb could correct metabolic acidosis in shock stage. Respiratory acidosis decreased with the increase of pPolyHb concentration; in the electrolyte balance, five concentrations of pPolyHb could effectively balance the electrolyte in the blood; the production of metabolite lactic acid decreased after the recovery of different concentrations of pPolyHb; the survival rate showed that the survival rate of 1.5g/kg pPolyHb group and 2.0g/kg p The survival rate of the PolyHb group was 100%, so 1.5g/kg was selected as the lowest effective dose of pPolyHb in the following different solution comparison experiments.
Physiological monitoring indexes showed that the blood pressure recovery of 1.5g/kg pPolyHb+ two times normal saline group was better than that of red blood cell + two times physiological saline group during the resuscitation of different solution, HES+ two times physiological saline group and three times physiological saline group, heart rate and respiration recovered smoothly, indicating better expansion effect. Blood gas index analysis results showed blood. In oxygen, the oxygen carrying capacity of pPolyHb+ two times normal saline group was better than that of HES+ two times normal saline group, sodium lactate Ringer solution group and three times physiological saline group (P0.05), and acid base balance, pPolyHhH+ two times physiological saline group had better effect on metabolic acidosis at the end of shock at the end of shock than the sodium lactate Ringer solution group and two times physiological HES+ physiology. The content of lactic acid in pPolyHb+ two times normal saline group was significantly lower than that of HES+ two times normal saline group, sodium lactate Ringer's solution group and three times normal saline group (P0.05). The survival time and survival rate of pPolyHb+ two times normal saline group were better than that of red blood cell + two times physiological saline group. HES+ two times normal saline group and three times physiological saline group had no significant difference with autologous blood + two times physiological saline. Pathological changes in rats with hemorrhagic shock and resuscitation showed that the pathological changes of heart, liver, spleen, lung and kidney of pPolyHb+ two times normal saline group were better than that of control group HES+ two times physiological saline group.
(1) in the short term preservation of 12h and 24h, the preservation effect of the isolated small intestine in group pPolyHb+HCA is similar to that of the current standard UW solution, but in the long-term preservation of 36h, the preservation effect is better than that of UW solution. PPolyHb can maintain aerobic respiration of the tissue and prevent the anaerobic metabolism of the tissue. It is a good oxygen carrying agent preserved in the small intestine in vitro; (two) blood. The indexes of liquid dynamics, blood gas analysis and pathological analysis show that pPolyHb can be more safe and effective in resuscitation of hemorrhagic shock in rats compared with the current clinical resuscitation fluid. (three) the results of pPolyHb in the preservation of small intestine in rats and the resuscitation of hemorrhagic shock in rats are the result of the polymerization of swine hemoglobin. The selection of bed indications provides the basis.
【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R459.7

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