【作者】 朱文忠；

【导师】 张宝仁； 徐美英；

【作者基本信息】 第二军医大学 ， 外科学（心胸外科）， 2001， 博士

【摘要】 腺苷作为内源性核苷，是一个重要的代谢性中间产物。腺苷通过和细胞表面 特殊腺苷受体的相互作用在整个机体起到广泛的调节和控制作用。文献报道腺苷 有显著的心脏保护作用，但这一作用的确切机制众说纷坛，没有定论。近年来， 腺苷在心脏能量代谢中的作用引起广泛关注。能量缺乏时容易生成腺苷的现象提 示腺苷的心血管作用似乎是调节氧供与能量需求之间的平衡，这意味着心脏代谢活性与腺苷释放之间存在敏感的联系。本课题通过建立离体鼠工作心脏灌注模 型，在灌注葡萄糖和脂肪酸的条件下研究腺苷对缺血和再灌注期间心脏的机械功 能、外源性葡萄糖酵解、糖氧化、代谢产物含量以及心脏超微结构的变化；同时， 在预先经短暂缺血(而非缺血预适应)的心脏中研究腺苷对随后长时间缺血和再 灌注心脏糖酵解、糖氧化、庄生成、代谢产物含量和心脏超微结构作用的变化， 并研究了α肾上腺素能受体阻滞剂──酚妥拉明在缓解腺苷于这类心脏中不良 反应中的作用，为腺苷心脏保护作用机制的进一步研究提供一条思路。主要研究 内容、方法和结果如下： 一、离体鼠工作心脏灌注模型的建立采用Neely等报道的方法，用雄性Sprague-Dawley大鼠灌注Krebs-Henseleit 缓冲液(37℃，pH7.4，95%O2和5%CO2充分饱和)建立非再循环式的离体鼠 工作心脏灌注模型，同时保留Langendorff灌注系统，使该灌注装置成为双灌流 系统。该模型较好地模拟了在体心脏做功时的工作状态，使左心房前负荷保持在 1.5kPa，主动脉后负荷保持在10.7kPa。非再循环式的灌注方法使离体心脏避免 了长时间灌注带来的代谢产物堆积和实验参数测定基点不同的缺点，便于对多种 干扰因素进行综合判断，使用该模型顺利地完成了后续实验。 二、腺苷对离体心脏缺血和再灌注期间糖代谢和机械功能的影响 在灌注改良Krebs-Henseleit缓冲液(含游离钙2.5mM、葡萄糖11mM、预先用3%牛血清白蛋白溶解结合的脂肪酸1.2mM、胰岛素500μU/ml)的离体鼠工 二 博士学位论文 中文摘要 作心脏中，缺血前或再灌注时使用腺昔O P M）显著改善了经 60ndn低流量 缺血（冠脉流量打）心脏机械功能的恢复（分别从1二．31士7．68o／增加至 7o．58士2一84％和6905士二28巴％）；缺血期间的糖氧化被抑制（从o．35士〕则到 0刀8士0刀2 n moVmin·g干重X这一抑制作用不受腺昔的影响，再灌注期间糖 氧化恢复至认29士0刀3 n moUmin·g干重，此时使用腺昔进一步增加了糖氧化 m．40士 0刀9 n moUmin·g千重入 缺血前使用腺昔抑制了不受缺血影响的糖酵 解的速率（从 4．77士0．66至 2．64士0．76 n moymin·g干重人缺血前或再灌注时 使用腺苦均使再灌注期间的糖酵解受到抑制（分别为2．77土1．03和L73土o．71 moUnun·g干重，对照组为 4刀7士045 u moUndnY干重X腺昔对糖代谢的作用 降低了缺血和再灌注期间来源于糖代谢的庄生成：腺苦增加了缺血后再灌注期 间的冠脉流量，这可能和心脏机械功能的增加有关，缺血期间冠脉流出液乳酸含c 量显著增高（从2．08土0．34到22．42士7．56mmo oo），再灌注后有所降低（8．65士 3，43mmow），缺血前和再灌注时使用腺昔进一步降低了缺血期间冠脉流出液的 乳酸含量（分别为5．gi士1．93和365土14lmmow），反映了这些心脏中糖氧化 作用的增强；腺苦保存了缺血期间的心肌糖原，降低了心脏摄取葡萄糖，但并不 增加缺血期间的糖原分解，再灌注期间腺昔抑制葡萄糖的摄取。 三、AIS心脏中腺昔对缺血后糖代谢和机械功能作用的转变 在经预先缺血应激（lS）——两段10min缺血继以sndn再灌注一一耗竭 糖原的心脏中，腺昔乃00 p M）刺激了糖酵解而非抑制糖酵解（从4．68士2．38，增至 5．92士 l．98 u moUmin·g干重），使庄的生成增加（从 8．64土 4．80到 10．70 士 4．18 n molimin·g干重L抑制了再灌注后心脏机械功能的恢复（恢复至缺血 前的16．46土2．96％，对照组为31．65土4．74％）。在灌注无底物（葡萄糖、脂肪酸） 灌注液以耗竭糖原（SFGD）的心脏中，药物治疗前的糖原含量与 lS心脏相似 （68．12士m．35和65．15士n65 pm叱干重），腺昔（500pM）抑制了有氧灌注 SFGD心脏中的糖酵解和庄生成。在SFGD心脏中，与未治疗心脏相比，长时 间缺血后的再灌注期间?更多还原

【Abstract】 Adenosine, an endogenous nucleoside, is an important metabolite intermediate and exerts widespread regulatoiy and control throughout the body by interacting with specific cell-surface adenosine receptors. It is said that adenosine possesses marked cardioprotective properties, but the mechanisms for this beneficial effect are unclear. The role of adenosine in cardiac energy metabolism as a homeostatic metabolite has recently attracted much attention. The cardiovascular effects of adenosine appear to regulate the balance between 02 supply and energy demand, and the formation of adenosine is facilitated during periods of energy deficit. This provides a sensitive link between cardiac metabolic activity and adenosine release. The objective of this study was to determine the effect of adenosine on mechanical function, exogenous glucose oxidation, glycolysis, metabolite levels, and ultrastructure changes of heart cells in isolated working rat hearts perfused with glucose and fatty acids. Meanwhile, glycolysis, oxidation, H+ production, metabolite levels, and ultrastructure changes in hearts subjected to antecedent ischemic stress (AIS) (not ischemic preconditioning) and subsequent ischemia and reperfusion was studied. The effect of phentolamine-an a -adrenoceptor antagonist in preventing adenosine-induced stimulation of glycolysis and reduced H+ production from glucose was also studied. The main work was described as foliows: 1. Establishment the model of isolated working rat hearts The model, reported by Neely et al, was established in Hearts from male Sprague-Dawley rats perfused with non-recirculating Krebs-Henseleit solusion (37℃, pH 7.4, gassed with a 95%O2-5%CO2 mixture). It is a biperfusing system because the Langendorff perfusion was attached. The model well mimicked the hearts in vivo. Perfusions were performed at a constant 1.5kPa left atrial preload and 1O.7kPa aortic 5 aflerload. The non-recirculating perfusion avoided the cumulation of metabolites and kept the the same baseline during the perfusion. It provide lots of help for the subsequent research work. 2. Adenosine altered glucose metabolism and mechanical function during ischemia and reperfusion in isolated rat heart In isolated working rat hearts perfused with modified Krebs-Henseleit solution containing 2. 5mM free Ca2~, 11mM glucose, 1.2mM palmitate pre-bound to 3% bovine serum albumin, 500 ju U/mi insulin, adenosine (100 ~.t M) pretreatment or adenosine (100 ~ M) at reperfusion markedly increased the recovery of mechanical function (from 12.31 ?7.68% to 70.58 ?24.84% and 69.05 ?12.88%, respectively) after 60 minutes of low-flow ischemia (coronary flow<lmllmin). Glucose oxidation was inhibited during ischemia (from 0.35 ?0.04 to 0.08 ?0.02 au mol/min g dry wt), and this was not altered by adenosine. During reperftision, glucose oxidation recovered to 0.29 ?0.03 ju md/mm g dry wt, and adenosine (100 ii M), given at reperflision, further increased glucose oxidation to 0.40 ?0.09 ~t mol/min g dry wt. The rate of glycolysis, which was unaffected by ischemia per se, was inhibited by adenosine pretreatment (from 4.77 ?0.66 to 2.64 ?0.76 ~t mollmin g dry wt). During reperfusion, glycolysis was also inhibited by adenosine relative to control (4.07 ?0.45 ~.i mol/min g dry wt) either when present during ischemia (2.77 ?1.03 ~t moVmin g dry wt) or during reperfusion (1.73 ?0.71 ji inol/min g dry wt). These e更多还原