腺苷预处理在非体外循环冠状动脉旁路移植术中心肌保护作用的初

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1、腺苷预处理在非体外循环冠状动脉旁路移植术中心肌保护作用的初步研究北京阜外心血管病医院麻醉科 ( 100037)杨静 杨大烜 叶珏 李立环【摘要 】目 的 探讨腺苷预处理在非体外循环冠状动脉旁路移植术(OPCAB)中的心肌保护作用。方法择期OPCAB患者40例，随机分为对 照组和腺苷组，每组 20 例。腺苷组在取乳内动脉时经颈内静脉输注腺苷 50 a g kg-1 min-1, 1min 后上调至 100 卩 g kg-1 min-1, 2min 后至 150 卩 g kg-1 min-1,维持此速度至10min,输注结束后5min开始血管吻合。对照 组给予生理盐水。 连续监测血流动力学变化，

2、并记录麻醉诱导后 10min( T0)、 血运重建后 30min(T1)、2h(T2)、6h(T3)、12h(T4)、24h(T5) 的 血流动力学参数,同时取静脉血测定血浆肌酸激酶同功酶( CK-MB )和心肌 肌钙蛋白I (cTnl)浓度。分别于给药前和血运重建后 15min取小块右心耳， 实时荧光定量聚合酶链反应(Real time-PCR)法检测TNF- a和ICAM-1mRNA 的相对表达量, 透射电镜观察超微结构的变化。 结果 两组术前一般资料比较 无显著性差异(P0.05)。血流动力学指标：与基础值比较，两组术中及 术后血压(BP)无显著变化，两组心率(HR)显著增快(PV0.0

3、1)，静脉压(CVP)、平均肺动脉压(MPAP)和肺动脉楔压(PCWP)有不同程度升高， 外周循环阻力(SVR)降低，肺循环阻力(PVR)无显著变化，两组心输出 量(CO)和心指数(CI)与基础值比较显著升高。两组每搏量(SV)、每搏 指数( SVI )和左室每搏功指数( LVSWI )降低,右室每搏功指数( RVSWI ) 和右室射血分数(RVEF)无明显变化。两组间比较，除血运重建后 2h腺苷 组CO和CI显著高于对照组(PV0.01)，余各循环动力学指标均无差异。两组 CK-MB 的变化：与基础值比较，两组各点 CK-MB 值均升高，对照组T1T5点都显著高于基础值（PvO.05）;腺苷

4、组仅T2点高于基础值（P 40%）, and with at least three vessel disease were selected for study. The 40 patients were allocated to two groups randomly （n=20 ADO, and n=20 control）. ADO group received infusion of ADO when the surgery dissociation left internal mammary artery, through a catheter via in ter nal jugu

5、lar vein. The in itial in fusi on rate was 50 ug kg-1 min-1, the rate of in fusi on was in creased every minute by 50 ug kg-1 min-1, un til the dose 150 ug kg-1 min-1, maintain this rate to 10min. The control group received 0.9% saline instead during the infusion period. 5min after the completion of

6、 adenosine or saline infusion protocol, revascularization began. Hemodynamic parameters were documented at following time points: T0 (10 minutes after anesthesia),T1 (30 minutes after revascularization), T2, T3, T4 and T5 (2 hours, 6 hours, 12 hours and 24 hours respectively afterwards). Blood sampl

7、es were collected simultaneously for MB isoenzyme of creatine kinase (CK-MB)and cardiac troponin I (cTnI) measurement. Right atrial myocardial were harvested before and 15 minutes after revascularization respectively, for analyzing the ultrastructure and TNF-a mRNA and ICAM-1mRNA expression.Results

8、Compared with the baseline, there were no significant differences in blood pressure postoperatively, but HR increased significantly ( P0.01)，pulmonary capillary wedge pressure (PCWP) , central venous pressure (CVP)and mean pulmonary artery pressure (MPAP) increased significantly (P0.01). Cardiac out

9、put (CO) and cardiac index (CI) increased markedly (P0.01). Systemic vascular resistance (SVR) , systemic vascular resistance index (SVRI) , stroke volume (SV) and stroke volume index (SVI) and left ventricular stroke work index (LVSWI) decreased significantly, while right ventricular stroke work in

10、dex (RVSWI) was decreased transiently (ADO group at T1 and control group at T2 , P0.01). Compared with the control group, CO andCI of ADO group increased markedly at T2 (P0.01). Compared with the baseline, CK-MB of control group increased from T1 to T5 (P0.05), that of adenosine group increased only

11、 at T2 (P0.05), patients of ADO group released significantly less CK-MB at T5(P0.01). Compared with the baseline, cTnI increased significantly from T1 to T5 (P0.05) in both groups, patients of ADO group released less cTnI than control group, especially at T4 and T5 point (P0.05). The myocardial ultr

12、astructure of control group after revascularization was damaged more seriously than that of ADO group.The expression of TNF- a mRNA in ADO group decreasedthan the control group, but not significant(P=0.07). ICAM-1mRNA expression decreased significantly inADO group (P=0.048). Conclusion The performan

13、ce of OPCAB result in ischemia/reperfusion injury and heart function inhibition. Adenosine preconditioning can reduce the release of CK-MB and cTnI, also can reduce the expression of TNF-aand ICAM-1mRNA, but can not improve cardiac function postoperatively.【 Keywords 】 Adenosine preconditioning, off

14、-pump, Coronary Artery Bypass Surgery缺血预处理对心肌的保护被认为是迄今为止最强的内源性保护。 腺苷 是内源性调节因子， 在缺血预处理的心肌保护中起着核心作用， 已在多种 动物实验中证实 1,2 。有研究表明在阻断主动脉前经中心静脉外源性输注腺苷3或给予腺苷受体激动剂同样可达到缺血预处理的效应。很多研究已证明腺苷在体外循环手术中能起到心肌保护作用，在非体外冠状动脉旁路移植术应用少有报道，本研究拟观察在非体外循环冠状动脉旁路移植术 中，腺苷预处理能否起到心肌保护作用。资料与方法1. 病例选择：选择择期初次非体外循环下冠状动脉旁路移植术患者，年龄 40%,多支冠

15、状动脉病变，收缩压90mmHg。 既往有糖尿病、哮喘或支气管痉挛病史、病态窦房结综合征或II、山度房室传导阻滞、合并瓣膜病、术前两月内心肌梗死病人或遗传性心脏病患者除外。 所有患者实验前两天不食用甲基黄嘌呤(咖啡、茶、茶碱)或双密达莫(潘 生丁)。所有患者随机分为腺苷组(n=20)或对照组(n=20)。2. 麻醉方法：视病人术前情况给予适当的心血管药物，术前2h 口服安 定10mg,术前30min肌肉注射吗啡10mg,入手术室后常规监测心电图和脉 搏血氧饱和度，行外周静脉和桡动脉穿刺。以 依托咪酯0.1 0.3mg / kg，哌 库溴铵0.1 0.15 mg / kg ,芬太尼10 20g /

16、 kg麻醉诱导。气管插管后以丙 泊酚持续静脉输注(TCI血浆靶浓度0.51 lg/ml)，间断注射芬太尼和阿端 维持麻醉。气管插管后行颈内静脉穿刺置漂浮导管，锁骨下静脉穿刺置三腔 静脉导管。3. 给药方法：取乳内动脉时腺苷组经颈内静脉导管输注腺苷50也g kg-1min-1,1min后上调至 100 卩 g kg-1min-1,2min后上调至150卩g kg-1 min-1,维持此速度至10min。输注过程中如遇收缩压低于 90mmHg, 加快补液速度和/或间断注射去甲肾上腺素 4-8卩g维持收缩压90mmHg以 上。对照组输注生理盐水。输注结束后 5min以上开始血管吻合。4. 检测指标：

17、循环指标：于麻醉诱导后10min(T0)、开侧壁钳后30min (T1)、开侧壁钳后 2h (T2)、6h (T3)、12h (T4)、24h (T5)记录收缩压（SBP）、舒张压（DBP）、中心静脉压（CVP）、肺动脉收缩压（SPAP）、 舒张压（DPAP）、肺动脉楔压（PCWP）、心输出量（CO）、心指数（CI）、 体循环阻力（SVR）、体循环阻力指数（SVRI）、肺循环阻力（PVR）、肺 循环阻力指数（PVRI）、每搏量（SV）、每搏指数（SVI）、左室每搏功指 数（LVSWI ）、右室每搏功指数（RVSWI）及混合静脉氧饱和度（SvO2 ）。生化指标：于上述六个时点取静脉血肝素抗凝化学

18、发光法（贝克曼化学发 光仪）检测血浆CK-MB和cTnl浓度。组织标本的制备：分别于输注腺苷 或生理盐水前和开侧壁钳后15min取右心耳，分成两份，一份快速液氮保存， 用于Real-time PCR反应测定心肌 TNF- a和ICAM-1mRNA 的相对表达量； 一份以3%戊二醛固定，透射电镜观察细胞超微结构的变化。5. TNF- a和ICAM-1mRNA表达测定以Oligo6.0软件进行引物设计（奥 科生物技术公司制作）。序列见表 1。表1 TNF- a ICAM-1和&actin引物序列及扩增片断长度引物名称序列扩增片断长度TNF- aF: 5 -GCCAGCTCCCTCTATTTATG

19、-3273bpR: 5-TGGTCACCAAATCAGCATTG -3ICAM-1F: 5 -TGCCCAGACATCTGTGTCCC -3336bpR: 5 -GCAGCGTAGGGTAAGGTTCTT- 3B -actinF: 5-CAGCACAATGAAGATCAAGATCA -3120bpR: 5-CGGACTCGTCATACTCCTGC -3PCR反应体系：10X Buffer （5卩,MgC （5） , SYBRGREEN I （0.5 ）1 , 引物（3gM, 5 ）1 , dNTP （3） , Taq DNA 聚合酶（0.4 ）1 , cDNA （20 ）1, dH2O （11

20、.1 ）1。PCR 反应程序为：95C变性 5min , 95C变性 30s, 55C退 火30s, 72C延伸30s,循环40次，72 C延伸5min。每对引物和模板均各以三个平行管反应。获得同一样品目的基因的Ct值与 阳Ct in的Ct值，计算出： Ct= Ct目的基因一Ct冏伽,目的基因的相对表达量=2CT( Ct= Ct未知Ct参照)。&统计学处理 采用SPSS10.0统计软件进行分析。计量资料以均数士标准差（X 士 S）表示，组间比较采用单因素方差分析，组内比较采用双因素方 差分析，PV0.05为差异有统计学意义。结果40例患者顺利完成实验。围术期无心肌梗死，IABP使用和死亡。1.

21、 两组术前一般资料和冠脉病变程度、搭桥数及阻断冠脉时间比较无显 著性差异（P0.05），见表1。表1两组一般资料组别年龄（岁）体重(Kg)性别比（男 /女）冠脉病变支数高血压史（有 / 无）陈旧心梗史（有 / 无）术前超声EF （%）搭桥数（支）阻断时间（min）对照组58 6.673.7士 7.217/32.9 0.411/96/1466 8.93.2 0.733.1 11.3腺苷组58.3 7.673.3 11.714/62.9 0.312/85/1562.3 5.83.5 0.634.6 12.92. 两组血流动力学变化及比较，见表 23. 两组各时点CK-MB变化及组内组间比较，见图1

22、图1 CK-MB的变化 （#与TO比较PvO.01, *组间比较P0.05）4. 两组各时点cTnl变化及组内组间比较，图2时点图2 cTnl的变化（#与TO比较P0.01, *组间比较P0.05）5. 心肌超微结构两组术前心肌纤维结构均清晰，排列整齐，线粒体形态结构正常（见图3）对照组术后部分肌节短缩、肌原纤维结构不清，溶解消失；部分线粒体、基质网中度肿胀扩张（见图 4）。而实验组术后心肌纤维排列整齐，肌节结构 清晰，肌原纤维结构排列清楚，仅部分肌质网轻度扩张，线粒体轻度肿胀（见 图5）。图3透射电镜下术前右心耳心肌组织形态（1X 10000倍）图4透射电镜下对照组血运重建后右心耳心肌组织形

23、态（1 X 10000倍）图5透射电镜下腺苷组血运重建后右心耳心肌组织形态（1X 10000倍）& 心肌 TNF- a和ICAM-1mRNA 的表达对照组血运重建后心肌细胞 TNF- a mRNA表达是术前的6.27倍，而腺 苷组为术前的3.08倍；对照组血运重建后心肌细胞ICAM-1mRNA表达是术 前的4.53倍，而腺苷组为1.62倍。两组比较，腺苷组ICAM-1mRNA表达低 于对照组，有统计学意义（P=0.048）, TNF- a mRNA表达未达到统计学意义（P=0.07）,但仍可看出腺苷组TNF- a mRNA表达弱于对照组，见图6。14.00数倍达表的前术于对相12.0010.0

24、08.006.004.002.000.00对照组I腺苷组图6血运重建后心肌细胞TNF- a和ICAM-1mRNA相对表达量（*组间比较P 40%), and with at least three vessel diseasewere selected for study. The 40 patients were allocated to two groups randomly (n=20 ADO, and n=20 control). ADO group received infusion of ADO when the surgeon dissociation left internal

25、 mammary artery, through a catheter via internal jugular vein. The initial in fusi on rate was 50 卩 g kg-1 mi n-1, the rate of in fusi on was in creased every minute by 50 卩 g kg-1 min-1, until the dose 150 卩 g kg-1 min-1, maintain this rate to 10min. The control group received 0.9% saline instead d

26、uring the infusion period. 5min after the completion of adenosine or saline infusion protocol, revascularization began. Hemodynamic parameters were documented at following time points: T0 (10 minutes after anesthesia), T1 (30 minutes after revascularization), T2, T3, T4 and T5 (2 hours, 6 hours, 12

27、hours and 24 hours respectively afterwards). Blood samples were collected simultaneously for MB isoenzyme of creatine kinase ( CK-MB ) and cardiac troponin I (cTnI) measurement. Right atrial myocardium were harvested before and 15 minutes after revascularization respectively, for analyzing the ultra

28、 structure and TNFa mRNA and ICAM-1mRNA expression.Results Compared with the baseline, there were no significant differences in blood pressure postoperatively, but HR increased significantly ( P0.01)， p u l m o n a ry capillary wedge pressure (PCWP) , central venous pressure (CVP)and mean pulmonary

29、artery pressure (MPAP) increased significantly (P0.01). Cardiac output (CO) and cardiac index (CI) increased markedly (P0.01). Systemic vascular resistance (SVR) , systemic vascular resistance index (SVRI) , stroke volume (SV) and stroke volume index (SVI) and left ventricular stroke work index (LVS

30、WI) decreased significantly, while right ventricular stroke work index (RVSWI) was decreased transiently (ADO group at T1 and control group at T2 , P0.01). Compared with the control group, CO and CI of ADO group increased markedly at T2 (P0.01). Compared with the baseline, CK-MB of control group inc

31、reased from T1 to T5 (P0.05), that of adenosine group increased only at T2 (P0.05), patients of ADO group released significantly less CK-MB at T5(P0.01). Compared with the baseline, cTnI increased significantly from T1 to T5 (P0.05) in both groups, patients of ADO group released less cTnI than contr

32、ol group, especially at T4 and T5 point (P0.05). The myocardial ultra structure of control group after revascularization was damaged more seriously than that of ADO group.The expression of TNF- a and ICAM-1mRNA decreased significantly in ADO group (P=0.03). Conclusion The performances of OPCAB result in ischemia/reperfusi