白藜芦醇促进大鼠心脏低温保存后心功能恢复的研究

发布时间：2018-06-23 07:18

本文选题：心脏 + 低温保存　；参考：《浙江大学》2015年硕士论文

【摘要】：背景心脏移植手术是终末期心力衰竭和严重冠脉疾病患者首选的治疗手段。而如何减轻供体心脏低温保存后再灌注损伤是心脏移植成功的前提和关键。许多研究者对各种心脏低温保存液的成分进行了相应的调整,比如添加了氧自由基清除剂、钙离子拮抗剂等,虽然这些措施也确实不同程度地提高了供体心脏低温保存后再灌注期心功能的恢复,但当供体心脏的保存时间大于6h时,这些改进措施的效果明显下降。因此,如何通过改进心脏低温保存液的成分,延长心脏保存的时间,提高保存效果,减轻再灌注损伤,是我们现今迫切需要解决的问题。白藜芦醇(Resveratrol)是一种非类黄酮的多酚物质,1939年由日本科学家从植物白藜芦的根茎中提取并将其命名之。天然的白藜芦醇在葡萄、桑葚等多种植物中含量丰富。目前,大量的研究发现,白藜芦醇在哺乳动物的体内外均具有广泛的生物学效应,包括预防肿瘤、延长寿命、对抗代谢性疾病、保护心血管系统和神经系统等作用。白藜芦醇的这些作用被认为与其强大的抗氧化、抗炎特性有关。因此,我们推测,白藜芦醇可能具有延长心脏低温保存时间,提高供心低温保存效果的作用,从而使远距获取供心,扩大供心来源成为可能。目的利用大鼠心脏低温保存和Langendorff再灌注模型,观察在心脏低温保存液中添加白藜芦醇后,是否可延长心脏保存时间,减轻心脏低温保存后再灌注损伤；并初步探讨其可能的作用机制。方法(1)大鼠心脏低温保存模型的构建和心功能的测定离体SD大鼠心脏在Langendorff心脏灌注装置上平衡30min后,经主动脉根部逆行灌注心脏低温保存液(Celsior液),灌注温度为4℃,时间3min以内。待心脏停搏后分别置于4℃含不同浓度白藜芦醇(0、10、20、40μM)的Celsior液中保存9h,随后心脏重新置于Langendorff装置上,于37℃,用Krebs-Henseleit液体(KH液)再灌注60min。通过MedLab生物信号采集处理系统观察心脏血流动力学的改变,包括：左心室舒张末期压力(left ventricular end-diastolic pressure, LVEDP)、左心室发展压力(1eftventricular developed pressure, LVDP)、心室最大收缩速率(Maximal systolic velocity of left ventricular pressure,+dP/dtmax)、心室最大舒张速率(Maximal diastolic velocity of left ventricular pressure,-dP/dtmax)、心率(heart rate, HR)及冠脉流量(coronary flow, CF)等。(2) Western blot测定心脏解偶联蛋白2(uncoupling protein 2, UCP2)表达实验结束后取左心室肌,匀浆、离心后提取全细胞蛋白。每组取20μ g蛋白于10%SDS-PAGE后,转膜,封闭。硝酸纤维素膜用UCP2抗体(1：1000)于4℃孵育过夜,洗脱后用辣根过氧化物酶结合的二抗室温孵育2h。用增强化学发光法探测目标蛋白。结果(1)与空白对照组(即未于Celsior液中进行冷保存组)相比,大鼠离体心脏低温缺血保存9h后,在再灌注60 min时LVEDP明显抬高,LVDP、+dP/dtmax和-dP/dtmax恢复率均明显下降,心率和冠脉流量也显著下降。在Celsior心脏保存液中添加了不同浓度的白藜芦醇后,可浓度依赖性地对抗低温保存诱导的LVDP抬高,LVDP、+dP/dtmax和-dP/dtmax的下降,以及心率和冠脉流量的下降。(2)与空白对照组相比,大鼠离体心脏低温缺血保存9h后,心室肌组织中UCP2的表达明显增高,约为空白对照组的3.2倍。在Celsior心脏保存液中添加了中、高浓度白藜芦醇(20、40μM)后,对低温保存诱导的UCP2过表达有明显的抑制作用。(3)与单纯低温保存组(Celsior组)相比,在Celsior心脏保存液中添加了SIRT1 (Sirtuin type 1)抑制剂6-氯-2,3,4,9-四氢-1H-咔唑-1-甲酰胺((6-Chloro-2,3,4,9-tetrahydro-lH-Carbazole-l-carboxamide, EX-257)对心肌UCP2的表达,以及心功能指标均无明显影响。但EX-257可对抗高浓度白藜芦醇(40 μM)对低温保存诱导UCP2过表达的抑制作用,并取消白藜芦醇对低温保存心脏心功能的保护作用。结论在心脏低温保存液中添加了白藜芦醇后,可促进低温保存的大鼠心脏在再灌注期心功能的恢复。其机制可能与激活了SIRT1蛋白,并抑制低温保存诱导的UCP2过表达有关。该研究结果还有待于进一步在大鼠心脏同种移植模型上加以验证。
[Abstract]:Background heart transplantation is the first choice for patients with end-stage heart failure and severe coronary disease. And how to reduce reperfusion injury after cryopreservation is the prerequisite and key to the success of heart transplantation. Many researchers have adjusted the composition of various cardiac cryopreservation solutions, such as the addition of oxygen free. Basic scavengers, calcium antagonists, etc., although these measures do improve the cardiac function of the donor heart after cryopreservation in different degrees, but the effects of these improvements are obviously reduced when the donor heart's preservation time is greater than 6h. Therefore, how to improve the composition of the heart cryopreservation solution and prolong the heart Resveratrol is a non flavonoid substance that is extracted from the rhizomes of the plant resveratrol in 1939 and named by Japanese scientists. The natural resveratrol is in a variety of plants, such as grapes, mulberry and other plants. At present, a large number of studies have found that resveratrol has extensive biological effects in the body and body of mammals, including the prevention of cancer, prolongation of life, anti metabolic diseases, protection of the cardiovascular system and nervous system. The effects of resveratrol are considered to be related to its strong antioxidant and anti-inflammatory properties. Therefore, we speculate that resveratrol may have the effect of prolonging the cryopreservation time of the heart and improving the effect of the cryopreservation of the donor heart, thus making it possible to obtain the donor heart from the far distance and enlarge the source of donor heart. Prolonging heart preservation time and reducing reperfusion injury after heart cryopreservation; and preliminary discussion on possible mechanism of action. Method (1) construction of heart cryopreservation model and cardiac function of rat heart of SD rat heart in Langendorff heart perfusion device balance 30min after retrograde perfusion of the aorta root to protect the heart Storage liquid (Celsior liquid), the perfusion temperature was 4 C, time 3min. After cardiac arrest, 9h was stored in Celsior solution with different concentration of resveratrol (0,10,20,40 mu M) at 4 degrees centigrade, then the heart was replaced on the Langendorff device. At 37, Krebs-Henseleit liquid (KH liquid) was used for 60min. through MedLab biological signal acquisition and processing system. Changes in cardiac hemodynamics were observed, including left ventricular end-diastolic pressure (LVEDP), left ventricular development pressure (1eftventricular developed pressure, LVDP), ventricular maximum systolic velocity (Maximal systolic velocity), and ventricular maximum diastolic velocity Rate (Maximal diastolic velocity of left ventricular pressure, -dP/dtmax), heart rate (heart rate, HR) and coronary flow (coronary), etc. (2) determine the left ventricular muscle after the end of the expression experiment of the cardiac uncoupling protein 2 (2), homogenate and extract the whole cell protein after centrifugation. Each group takes 20 micron protein. After 10%SDS-PAGE, the membrane was transferred and closed. The nitrocellulose membrane was incubated with UCP2 antibody (1:1000) at 4. After elution, the target protein was detected by two anti temperature incubating with horseradish peroxidase. The result (1) compared with the blank control group (that is, no cold preservation group in Celsior solution), the rat heart was low temperature. After ischemia and preservation of 9h, the LVEDP was significantly elevated at 60 min reperfusion, the recovery rate of LVDP, +dP/dtmax and -dP/dtmax decreased significantly, and the heart rate and coronary flow rate also decreased significantly. After adding different concentrations of resveratrol in the Celsior heart preservation solution, the concentration depended on the LVDP elevation induced by low temperature preservation, LVDP, +dP/dtmax and -dP/dtmax. The decrease of heart rate and coronary flow rate. (2) compared with the blank control group, the expression of UCP2 in the ventricular muscle tissue of the isolated rat heart was significantly higher than that in the blank control group, about 3.2 times that of the blank control group. In the Celsior heart preservation solution, the high concentration of resveratrol (20,40 mu M) was added to the Celsior heart preservation solution, and the UCP2 was induced by cryopreservation. (3) the expression of SIRT1 (Sirtuin type 1) inhibitor 6- chloro -2,3,4,9- four hydrogen -1H- carbazole -1- Methamide (6-Chloro-2,3,4,9-tetrahydro-lH-Carbazole-l-carboxamide, EX-257) was added to the heart preservation solution (group Celsior), and the cardiac function indexes were all in the heart preservation solution. But EX-257 can antagonize the inhibitory effect of high concentration resveratrol (40 u M) on the overexpression of UCP2 induced by cryopreservation, and cancels the protective effect of resveratrol on cardiac function at low temperature. Conclusion the addition of resveratrol in cryopreserved cardiopreservation solution can promote cardiac function in the reperfusion period of cryopreserved rat heart. The mechanism may be related to the activation of SIRT1 protein and the inhibition of UCP2 overexpression induced by cryopreservation. The results of this study are still to be further verified in rat heart allograft model.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2

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