本文選題：聚合豬血紅蛋白 + 小腸保存��； 參考：《西北大學(xué)》2013年博士論文

【摘要】：研究背景 血紅蛋白(Hemoglobin, Hb)是脊椎動(dòng)物血紅細(xì)胞中重要的氧載體,其含量可達(dá)到血紅細(xì)胞干重的90%以上。長(zhǎng)期以來(lái),血紅蛋白類氧載體(Hemoglobin-Based Oxygen Carriers, HBOCs)由于其良好的載氧特性受到了廣泛的關(guān)注。之前的應(yīng)用研究主要集中在將HBOCs應(yīng)用于紅細(xì)胞代用品,將其用作攜氧治療劑的研究尚處于起步階段。小腸移植是短腸綜合征和不可逆腸功能衰竭的最理想治療方法,但是小腸離體保存難度大,從而極大地限制了小腸移植的臨床應(yīng)用。小腸離體保存困難的重要原因之一是小腸上皮細(xì)胞對(duì)于缺血非常敏感,因此將具有良好攜氧能力的pPolyHb應(yīng)用于小腸的離體保存或可優(yōu)化其保存效果。失血性休克是一種由于機(jī)體失血造成的有效循環(huán)血容量減少、組織灌注不足,細(xì)胞代謝紊亂和多器官功能受損的病理過(guò)程。液體復(fù)蘇是休克早期治療的重要手段之一,臨床常用的復(fù)蘇液包括晶體液、膠體液和血液制品等。HBOCs已經(jīng)開(kāi)始作為失血性休克的攜氧復(fù)蘇液進(jìn)行研究,研究結(jié)果顯示HBOCs有望成為臨床中復(fù)蘇失血性休克患者的重要工具。 研究目的 本研究首次選用豬血作為原料并成功研發(fā)了戊二醛聚合豬血紅蛋白分子(Polymerized Porcine Hemoglobin, pPolyHb),該制品氧親和力高,循環(huán)時(shí)間長(zhǎng),具有穩(wěn)定四聚體結(jié)構(gòu)。本研究以該pPolyHb作為攜氧治療劑(Oxygen therapeutic agent),探索其在大鼠移植小腸保存和失血性休克復(fù)蘇中的應(yīng)用。 研究方法 制備戊二醛聚合豬血紅蛋白氧載體(Polymerized porcine hemoglobin, pPolyHb),并測(cè)量其分子量及理化性質(zhì)指標(biāo)。以pPolyHb作為高滲檸檬酸腺嘌呤溶液(Hypertonic Citrate Adenine Solution, HCA)的攜氧治療劑,為大鼠小腸的離體保存供氧,優(yōu)化其保存條件。大鼠小腸分別保存于2g/dl pPolyHb+HCA液、4g/dl pPolyHb+HCA液和對(duì)照組HCA液、威斯康辛大學(xué)保存液(University of Wisconsin Solution, UW)中,以12h、24h和36h梯度時(shí)間保存,然后進(jìn)行組織病理學(xué)分析、組織ATP含量檢測(cè)、血?dú)夥治龅?分析pPolyHb對(duì)于提高離體小腸保存的效果。為了檢測(cè)pPolyHb在失血性休克復(fù)蘇的作用,本研究建立了大鼠抗失血性休克模型,失血時(shí)間為90min,失血量為60±5%。將SD大鼠隨機(jī)分為五組,每組8只,應(yīng)用不同濃度的pPoIyHb(2.0g/kg、1.5g/kg、1.2g/kg、1.0g/kg、0.5g/kg)聯(lián)合兩倍生理鹽水對(duì)失血性休克大鼠進(jìn)行復(fù)蘇實(shí)驗(yàn),同時(shí)監(jiān)測(cè)各組大鼠血壓、心率、呼吸和心電圖等基本生命體征和動(dòng)脈的血?dú)庵笜?biāo),最終篩選出最低有效劑量的pPolyHb濃度。然后隨機(jī)分為六組,每組8只SD大鼠,采用pPolyHb聯(lián)合兩倍生理鹽水、自體血(AB)聯(lián)合兩倍生理鹽水、紅細(xì)胞(RBC)聯(lián)合兩倍生理鹽水、羥乙基淀粉(HES)聯(lián)合兩倍生理鹽水、三倍乳酸鈉林格液(LR)和三倍生理鹽水(SPSS)分別對(duì)失血性休克大鼠進(jìn)行復(fù)蘇實(shí)驗(yàn),同時(shí)監(jiān)測(cè)各組大鼠血壓、心率、呼吸和心電圖等基本生命體征和動(dòng)靜脈的血?dú)庵笜?biāo),并對(duì)大鼠重要內(nèi)臟器官進(jìn)行病理學(xué)分析。 研究結(jié)果 pPolyHb的分子量及理化性質(zhì)指標(biāo)結(jié)果顯示其平均分子量在550～800kD,濃度在11g/dl左右,其中MetHb的含量小于5%;pH值(7.4±0.3)接近生理狀態(tài)；膠體滲透壓也在生理范圍內(nèi)；小分子含量小于3%；從Hemox血氧分析儀測(cè)定產(chǎn)品的P50和Hill系數(shù)可以看出,經(jīng)化學(xué)修飾后,血紅蛋白的載氧能力受到影響,亞基的協(xié)同性也有一定下降。 在離體小腸保存實(shí)驗(yàn)中,病理學(xué)分析結(jié)果顯示小腸在pPolyHb+HCA液中短期保存效果(12和24h)與UW液相近,無(wú)明顯差異,但其長(zhǎng)期保存效果(36h)則優(yōu)于UW液,且無(wú)論長(zhǎng)期與短期保存效果均優(yōu)于單純的HCA溶液(P0.05)。組織ATP含量檢測(cè)表明pPolyHb+HCA組中小腸組織的三磷酸腺苷(ATP)含量顯著高于對(duì)照組UW組與HCA組(P0.05)。血?dú)夥治鲲@示pPolyHb+HCA保存液中的pH值始終維持在正常水平、HCO3-的變化值(ΔHCO3-)顯著高于對(duì)照組,而Lac含量顯著低于對(duì)照組(P0.05)。這些研究結(jié)果表明pPolyHb能夠改善離體小腸的保存效果,同時(shí)也提示提高離體小腸組織氧供應(yīng)能夠優(yōu)化其離體保存。 在大鼠失血性休克復(fù)蘇實(shí)驗(yàn)中,生理監(jiān)測(cè)指標(biāo)顯示：五組pPolyHb聯(lián)合兩倍生理鹽水組復(fù)蘇后大鼠的血壓、心率、呼吸率等恢復(fù)平穩(wěn),較高濃度pPolyHb (2.0g/kg和1.5g/kg)聯(lián)合兩倍生理鹽水對(duì)血流動(dòng)力學(xué)的影響效果均優(yōu)于其他三個(gè)較低濃度組(1.2g/kg、1.0g/kg、0.5g/kg)。血?dú)庵笜?biāo)顯示：攜氧能力方面,2.0g/kg pPolyHb與1.5g/kg pPolyHb組有更好的攜氧能力,但是隨著pPolyHb濃度的提高,高鐵血紅蛋白(MetHb)的含量有所增加；酸堿平衡方面,隨著pPolyHb濃度的提高,可糾正休克期的代謝性酸中毒,但呼吸性酸中毒隨著pPolyHb濃度的提高呈現(xiàn)下降的趨勢(shì)；電解質(zhì)平衡方面,五個(gè)濃度的pPolyHb均可有效平衡血液中的電解質(zhì)；代謝物乳酸的生成量在不同濃度pPolyHb復(fù)蘇后均有下降；存活率統(tǒng)計(jì)情況顯示,1.5g/kg pPolyHb組的存活率和2.0g/kg pPolyHb組的存活率均為100%,故在接下來(lái)的不同溶液比較實(shí)驗(yàn)中選擇1.5g/kg作為pPolyHb的最低有效劑量。 生理監(jiān)測(cè)指標(biāo)顯示：不同溶液復(fù)蘇過(guò)程中,1.5g/kg pPolyHb+兩倍生理鹽水組復(fù)蘇后血壓恢復(fù)均優(yōu)于紅細(xì)胞+兩倍生理鹽水組、HES+兩倍生理鹽水組和三倍生理鹽水組,心率和呼吸恢復(fù)平穩(wěn),說(shuō)明有更好的擴(kuò)容效果。血?dú)庵笜?biāo)分析結(jié)果顯示：血氧方面,pPolyHb+兩倍生理鹽水組的攜氧能力明顯優(yōu)于HES+兩倍生理鹽水組、乳酸鈉林格液組和三倍生理鹽水組(P0.05)；酸堿平衡方面,pPolyHhH+兩倍生理鹽水組對(duì)于機(jī)體休克末的代謝性酸中毒緩解效果優(yōu)于乳酸鈉林格液組,與HES+兩倍生理鹽水組相似;無(wú)氧代謝方面：pPolyHb+兩倍生理鹽水組復(fù)蘇后的乳酸含量明顯低于HES+兩倍生理鹽水組、乳酸鈉林格液組和三倍生理鹽水組(P0.05)。存活情況顯示,pPolyHb+兩倍生理鹽水組大鼠存活時(shí)間與存活率均優(yōu)于紅細(xì)胞+兩倍生理鹽水組、HES+兩倍生理鹽水組和三倍生理鹽水組,與自體血+兩倍生理鹽水無(wú)顯著差異。失血性休克復(fù)蘇大鼠病理檢查結(jié)果顯示,pPolyHb+兩倍生理鹽水組大鼠的心臟、肝臟、脾臟、肺臟和腎臟的病理性變化均優(yōu)于對(duì)照組HES+兩倍生理鹽水組。結(jié)論 (一)在12h和24h的短期保存中,pPolyHb+HCA組中的離體小腸保存效果與現(xiàn)行的臨床標(biāo)準(zhǔn)UW液相近,但在36h長(zhǎng)期保存中,其保存效果要優(yōu)于UW溶液,pPolyHb可維持組織的有氧呼吸,阻止組織的無(wú)氧代謝,是用小腸離體保存良好的攜氧治療劑；(二)血液動(dòng)力學(xué)、血?dú)夥治�、病理分析的各�?xiàng)指標(biāo)顯示,與現(xiàn)行的臨床復(fù)蘇液相比,pPolyHb作為攜氧治療劑能夠更為安全有效的復(fù)蘇失血性休克大鼠；(三)pPolyHb在大鼠離體小腸保存與大鼠失血性休克復(fù)蘇中的應(yīng)用研究結(jié)果為聚合豬血紅蛋白臨床適應(yīng)癥的選擇提供依據(jù)。
[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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R459.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 張浩,毛秉智,王全立;提高血紅蛋白四聚體穩(wěn)定性的研究進(jìn)展[J];中國(guó)輸血雜志;2000年01期

2 單闖;失血性休克的病理生理及治療[J];現(xiàn)代實(shí)用醫(yī)學(xué);2002年11期

3 ;低血容量休克復(fù)蘇指南(2007)[J];中國(guó)實(shí)用外科雜志;2007年08期

，

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