【作者】 吴东凯；

【导师】 陈胜喜；

【作者基本信息】 中南大学 ， 心胸外科， 2009， 博士

【摘要】 背景:随着移植技术的发展,供体的缺乏就成为研究人员所面临的一个全球性难题。因此有研究人员开始将研究目光转向无心跳供体/尸体供体（non-heart-beating donors,MHBD）,尝试使用临床死亡后的尸体停搏心脏（cadaver non-beating heart,CNBH）来进行心脏移植,以扩大供体的来源。目前研究认为临床有望开发利用的是院外急性失血性休克死亡的CNBH。由于在心脏停搏之前无法进行有效的预处理,因此只能期望在热缺血过程之后通过后处理的方法来减轻心脏因热缺血以及缺血/再灌注所带来的损伤、改善移植后供心的功能。目的:本研究在首先建立大鼠颈部异位心脏移植模型的基础上,尝试对热缺血死亡后大鼠不同时段的供心,进行缺血后处理以及腺苷后处理,检测受体术后24小时移植心脏的相关指标,来评价其心脏保护作用并探讨可能机制。方法:实验分为三大部分:第一部分为大鼠颈部异位心脏移植模型的建立。第二部分为缺血后处理对热缺血死亡大鼠供体心脏保护的研究。大鼠被随机分为7组:C组—对照组;I₅组、I₁₅组、I₃₀组—单纯热缺血组,供体大鼠失血性休克死亡后单纯热缺血5min、15min、30min取心移植组;P₅组、P₁₅组、P₃₀组—缺血后处理组,供体大鼠失血性休克死亡后热缺血5min、15min、30min取心移植并进行缺血后处理组。移植术后24小时取移植心脏标本,观察其病理改变,检测心甲橹疉TP、SOD、MDA、NF-κB mRNA、TNF-α、IL-6的含量,以及心肌细胞凋亡和Bcl-2、Bax蛋白的表达情况。第三部分为腺苷后处理对热缺血死亡大鼠供体心脏保护的研究。大鼠被随机分为7组:C组—对照组;I₅组、I₁₅组、I₃₀组—单纯热缺血组,供体大鼠失血性休克死亡后单纯热缺血5min、15min、30min取心移植组;A₅组、A₁₅组、A₃₀组—腺苷后处理组,供体大鼠失血性休克死亡后热缺血5min、15min、30min取心移植并进行腺苷后处理组。移植术后24小时取移植心脏标本,观察其病理改变,检测心肌组织ATP、SOD、MDA、NF-κB mRNA、TNF-α、IL-6的含量,以及心肌细胞凋亡和Bcl-2、Bax蛋白的表达情况。结果:1、热缺血死亡30分钟以内,冷缺血1小时左右的大鼠CNBH可以被成功移植。2、随着热缺血时间的延长,移植心脏的缺血/再灌注损伤越重,病理损害也越重;而在同一时间点与单纯热缺血组相比,缺血后处理组和腺苷后处理组心肌细胞损害被减轻。3、在同一时间点,缺血后处理以及腺苷后处理与单纯热缺血组相比,可以增加心肌组织的ATP、SOD含量,减少其MDA含量;减少心肌组织的NF-κB mRNA的表达及TNF-α、IL-6的含量;减轻心肌细胞的凋亡水平、上调Bcl-2蛋白表达而下调Bax蛋白表达。结论:1、热缺血死亡30分钟以内,冷缺血1小时左右的大鼠CNBH可以被成功应用于心脏移植;2、缺血后处理以及腺苷后处理对热缺血死亡大鼠心脏移植的供心缺血/再灌注损伤具有保护作用;3、缺血后处理以及腺苷后处理的心脏保护作用可能与其改善供心的能量代谢、减轻脂质过氧化损伤、抑制NF-κB表达所致炎性损伤以及减少心肌细胞的凋亡有关。创新点:1、本实验创新性的将后处理的概念引入心脏移植领域;2、本实验首次研究缺血后处理对热缺血死亡大鼠供体心脏保护的作用,国内外未见相关报道;3、本实验首次研究腺苷后处理对热缺血死亡大鼠供体心脏保护的作用,国内外未见相关报道。不足之处:由于经费、设备等多种限制,无法对移植后的供心进行功能上的检测、相关的检测指标尚不够全面、移植后的观察时间较短。更多还原

【Abstract】 Background:With the development of transplantation,researchers must face a global problem—the shortage of donors.In order to enlarge the donor pool,some of them turn to non-heart-beating donors（NHBD） and attempt to use cadaver non-beating heart（CNBH）in the heart trans-plantation. Now they think the CNBH received from those who die from acute hemorrhagic shock may be useful.Because none preconditioning methods can be used before the heart stop beating.So we have to try some postconditioning methods to attenuate the warm ischemia and ischemia-reperfusion injury,and to improve the cardiac function after heart transplantation.Objective:Firstly we try to set up a cervical heterotopic heart trans-plantation in warm ischemic death rats.Then we use ischemic postcondi-tioning and adenosine postconditioning methods in the transplantation. 24 hours after transplantation we detect the change of the graft.Based on the data we evaluate the effects of these two methods and try to find the involving mechanisms.Methods:The experiment includes three parts.Part one:To set up a model of cervical heterotopic heart transplanta-tion in rats.Part two:Study on the cardioprotection of ischemic postconditioning in cadaver donor rats with warm ischemia.Rats were randomly divided into seven groups.C group—control group.I₅、I₁₅、I₃₀group—simple warm ischemia group,CNBH received from the cadaver rats which were die from acute hemorrhagic shock and the warm ischemic durations are 5 min、15min and 30min separately.Then the CNBH were used to heart transplantation.P₅、P₁₅、P₃₀group—ischemic postconditioning group, after the heart transplanted as the simple warm ischemia group,the donor hearts were treated with ischemic postconditioning.24 hours after transplantation we detect the change of the grafts including pathologic damages,ATP、SOD、MDA、NF-κB mRNA、TNF-αIL-6、apoptosis level、Bcl-2 and Bax proteins.Part three:Study on the cardioprotection of adenosine postconditioning in cadaver donor rats with warm ischemia.Rats were randomly divided into seven groups.C group—control group.I₅、I₁₅、I₃₀ group—simple warm ischemia group,CNBH received from the cadaver rats which were die from acute hemorrhagic shock and the warm ischemic durations are 5 min、15min and 30min separately.Then the CNBH were used to heart transplantation.A₅、A₁₅、A₃₀group—adenosine postconditioning group,the CNBH were received as mentioned and treated with adenosine postconditioning before heart transplantation.24 hours after transplantation we detect the change of the grafts including pathologic damages、ATP、SOD、MDA、NF-κB mRNA、 TNF-αIL-6、apoptosis level、Bcl-2 and Bax proteins.Result:1、The CNBH within 30min warm ischemia and 1h cold ischemia can be transplanted successfully.2、With the prolonged warm ischemia duration,the ischemia/reper-fusion injury became more severe and the pathologic damage became worse.But at the same time point,compared with the simple warm ischemia group,the myocardial injury of the ischemic postconditioning group and the adenosine postconditioning group were attenuated. 3、At the same time point,compared with the simple warm ischemia group,the ischemic postconditioning group and the adenosine post-conditioning group can increase ATP and SOD in myocardial tissue, decrease MDA,NF-κB mRNA,TNF-α,IL-6.In addition,the decrease of apoptosis level,up-regulation of Bcl-2 protein and down-regulation of Bax protein can be detected in these two groups.Conclusion:1、The CNBH within 30min warm ischemia and 1h cold ischemia can be transplanted successfully.2、Ischemic postconditioning and adenosine postconditioning can protect the graft from ischemia-reperfusion injury in cadaver donor rats with warm ischemia.3、The involving mechanisms may include that ischemic postcondi-tioning and adenosine postconditioning can improve myocardial energy metabolism,attenuate lipid peroxidation injury,suppress the expression of NF-κB mRNA,decrease the inflammatory damage of TNF-αand IL-6, up-regulation of Bcl-2 protein and down-regulation of Bax protein which lead to the shortage of myocardial cell apoptosis.New ideas:1、The study innovatively introduced the conception of postcondi-tioning into the area of heart transplantation.2、We study on the cardioprotection of ischemic postconditioning in cadaver donor rats with warm ischemia for the first time.The similar researches were not reported.3、We study on the cardioprotection of adenosine postconditioning in cadaver donor rats with warm ischemia for the first time.The similar researches were not reported.Limitation:For the restriction of funds and equipments,we cannot evaluate the cardiac function of the graft,the detected data were limited and the observing duration was not long.更多还原