【作者】 冷玉芳；

【导师】 王建林；

【作者基本信息】 兰州大学 ， 动物学， 2008， 博士

【摘要】 背景和目的:心脏直视手术需要在体外循环（cardio-pulmonary bypass,CPB）状态下暂时阻断心脏冠状动脉血液循环。CPB可以导致心肌缺血再灌注损伤（myocardial ischemia/reperfusion injury,MI/RI）,再灌注损伤表现为心律不齐、可逆性收缩功能障碍——心肌顿抑、内皮功能障碍以及心肌超微结构的改变等,这些关系到心脏手术成败及患者预后。目前,MI/RI是阻碍缺血心肌从再灌注疗法中获得最佳疗效的主要难题,预防或减轻再灌注损伤已成为抗MI/RI的重要课题。针对I/R损伤的发生,很多研究结果表明药物对MI/RI具有防治作用,可减少心律失常、出血性坏死以及梗塞面积扩大等,但是由于药物本身的一些毒副作用及机体状态的影响,使其很难发挥真正的效能,因此仍然多限于基础研究阶段。为了进一步了解麻醉药、改善细胞能量代谢、NO合成的特异前体以及传统中药干预以后对心肌缺血再灌注药物的影响,本试验选择132例在CPB下行心内直视手术的心脏病患者,分为四大组,分别用异丙酚、氯胺酮、1.6-二磷酸果糖、左旋精氨酸及活血化瘀药物川芎嗪等不同药物干预,观察心肌组织热休克蛋白72、诱导型一氧化氮合酶和中性粒细胞NF-κB、Bcl-2的表达以及血清心肌肌钙蛋白T的变化。进一步认识不同药物干预对心肌缺血再灌注损伤的保护作用及其作用机理。方法:132例CPB下行心内直视手术的心脏病患者,根据试验时间分四大组。组一（G1）28例,随机分为对照组（C组）川芎嗪干预组（M组）,每组14例。M组麻醉诱导后经静内静脉滴入川芎嗪3 mg·kg^-1,30 min内滴完,转流后追加1 mg·kg^-1于氧合器中,C组于上述时间给予等量生理盐水。在主动脉阻断（ACC）前后切取右心房心肌标本,半定量逆转录聚合酶链式反应（RT-PCR）测定心肌细胞中HSP72mRNA的相对表达量;分别于转流前、主动脉开放后30min和术后24 h测定外周血天冬酸氨基转移酶（AST）、乳酸脱氢酶（LDH）、肌酸磷酸激酶（CK）和肌酸磷酸激酶同工酶（CK-MB）的变化;分别于转流前和主动脉开放后30min取右心耳心肌组织,透射电镜观察心肌超微结构变化,并采用Flameng线粒体半定量分析法对心肌线粒体进行计分。组二（G2）24例,随机分为对照组（C组）和氯胺酮干预组（K1组、K2组）,每组8例。在劈胸骨时、主动脉插管时和主动脉开放前5min,干预组分别静脉注射氯胺酮0.5 mg·kg^-1或1.0 mg·kg^-1,对照组注射等量生理盐水。于上腔静脉插管时（T1）、主动脉阻断后30min（T2）和主动脉开放后30min（T3）取适量右心耳心肌组织,一步法实时荧光定量RT-PCR技术（采用SYBR Green 1嵌合荧光法）检测心肌中iNOSmRNA和HSP72mRNA的表达量。并分别于T1、T2、T3和主动脉开放后2h（T4）取上腔静脉血4ml,检测血清中肌酸激酶（CK）和肌酸激酶同工酶（CK-MB）含量。组三（G3）40例,随机分为对照组（C组）、左旋精氨酸组（L组）、1,6-二磷酸果糖组（F组）和联合用药组（D组）,每组各10例。根据L-Arg和FDP作用机制的不同,L组于主动脉开放后即刻给予L-Arg200 mg·kg^-1,F组于主动脉阻断前给予FDP200 mg·kg^-1,D组联合应用两种药物。分别于主动脉插管时、主动脉开放30min、2h、6h检测血浆心肌肌钙蛋白T（cTnT）、乳酸脱氢酶（LDH）、肌酸激酶同工酶（CK-MB）、丙二醛（MDA）含量,以及超氧化物歧化酶（SOD）活力。组四（G4）40例,随机分为对照组（C组）和异丙酚组（P组）,每组20例。在麻醉诱导后,P组用TCI输注泵以2.5μg·ml^-1为初始血浆靶浓度持续输注异丙酚,根据麻醉深度调整异丙酚血浆靶浓度,最大可达3.4μg·ml^-1。分别于麻醉诱导后CPB前（T1）、主动脉阻断后30min（T2）、主动脉开放时（T3）、CPB停机后30min（T4）、停机后90min（T5）采集上腔静脉血检测血浆中肿瘤坏死因子-α（TNF-α）、白细胞介素-8（IL-8）、白细胞介素-10（IL-10）、血浆丙二醛（MDA）的浓度及超氧化物歧化酶（SOD）的活性,于T1、T4时间点采集上腔静脉血检测中性粒细胞NF-κB和Bcl-2的表达及中性粒细胞计数。结果:G1组:与ACC术前比较,ACC术后两组心肌组织HSP72 mRNA表达量、心肌酶含量及线粒体记分均显著增高（P＜0.05或P＜0.01）;与C组比较,ACC后M组心肌组织HSP72 mRNA表达量和线粒体记分显著增高,心肌酶AST、LDH、CK、CK-MB的升高幅度明显降低（P＜0.05）。心肌超微结构变化,CPB后对照组线粒体基质颗粒丢失,嵴断裂,肌浆网扩张,肌原纤维明显松弛,部分溶解、断裂。川芎组肌原纤维排列整齐,可见少数线粒体嵴肿胀模糊,嵴断裂,间隙增宽。局部内质网扩张。G2组:与T1比较,三组T2和T3心肌iNOSmRNA和HSP72mRNA表达量均显著升高（P＜0.05、P＜0.01）,CK、CK-MB含量在T2、T3、T4均显著增高（P＜0.01）;与T2比较,三组T3心肌iNOSmRNA和HSP72mRNA表达量均显著升高（P＜0.05、P＜0.01）,CK、CK-MB含量在T3、T4均显著增高（P＜0.01）;与T3比较,CK、CK-MB含量在T4均显著增高（P＜0.01）;与C组比较,K1组在T2而K2组在T2、T3心肌iNOSmRNA表达量及CK、CK-MB升高幅度在T2、T3、T4均显著降低（P＜0.05、P＜0.01）,K1、K2组HSP72mRNA表达量在T2、T3均显著增高（P＜0.01）;与K1组比较,K2组心肌iNOSmRNA表达量在T2、T3及CK、CK-MB升高幅度在T2、T3、T4均显著降低（P＜0.05、P＜0.01）,HSP72mRNA表达量差异无显著性（P＞0.05）。G3组:与T1比较,四组血浆cTnT、LDH、CK-MB、MDA和SOD在T2、T3、T4均显著增高（P＜0.05、P＜0.01）;与C组比较,血浆cTnT值L组和F组在T4而D组在T3、T4显著降低（P＜0.01或P＜0.05）,LDH升高幅度L组在T3而D组在T2、T3、T4显著降低（P＜0.01或P＜0.05）,CK-MB和MDA的升高幅度L、F、D组在T2、T3、T4均有显著降低（P＜0.01或P＜0.05）,三组SOD在T3显著升高（P＜0.05）;与L组比较,D组血浆cTnT值在T3时显著降低（P＜0.01）;与F组比较,D组血浆cTnT值在T3、T4时、LDH在T2时显著降低（P＜0.01或P＜0.05）G4组:与T1比较,CPB后（T4）两组中性粒细胞NF-κB、Bcl-2表达及中性粒细胞数均显著升高（P＜0.01）,T2～T5血清TNF-α、MDA和SOD、T3～T5血清IL-8和IL-10含量显著升高（P＜0.01、P＜0.05）;与C组比较,体外循环后P组中性粒细胞NF-κB、Bcl-2的表达及中性粒细胞数明显降低（P＜0.01）,P组T2～T5血清TNF-α和MDA、T3～T5血清IL-8含量显著降低（P＜0.05）,T3～T5血清IL-10含量、T2～T5血清SOD含量显著升高（P＜0.05）。结论:HSP72、iNOS、NF-κB和Bcl-2参与了心肌缺血/再灌注损伤的发生和发展。体外循环期间心内直视手术患者心肌组织中HSP72mRNA的表达增加;川芎嗪干预上调心肌细胞HSP72mRNA的表达,启动内源性心脏保护机制,使患者心肌超微结构损害减轻,使心肌线粒体结构和功能的完整性得到保护,在一定程度上减轻了心肌缺血再灌注损伤。麻醉(1.0mg·kg^-1)和亚麻醉(0.5mg·kg^-1)剂量的氯胺酮通过下调心肌iNOSmRNA的表达和上调心肌HSP72mRNA的表达,减少血清心肌酶的漏出,在一定程度上具有心肌保护作用,且麻醉优于亚麻醉。左旋精氨酸或1,6-二磷酸果糖由于其不同的作用机制对缺血再灌注损伤心肌有良好的保护作用,联合应用可发挥两种药物的协同作用,对心肌的保护作用更好。体外循环心内直视手术期间使用异丙酚不仅可以起到满意的麻醉镇静作用,还可通过影响中性粒细胞的效应作用、促/抗炎细胞因子水平、氧自由基等炎性反应的多个环节起到抑制炎症反应,减轻组织损伤的作用。更多还原

【Abstract】 Backgroud and objective Cardiopulmonary bypass(CPB)is applied in open heart operation to temporary block the blood circulation of the cardiac coronary artery. Myocardial ischemia/reperfusion injury(MI/RI)can be caused by CPB.The main presentations of MI/RI are arrhythmia,reversible contractile dysfunction(myocardial stunning),endothelium dysfunction and myocardial ultrastructure change,which play the important role to success in surgery.At present,MI/RI is a commonly occurred perioperative cardiac problem that hinders ischemic myocardium to acquire the best curative effect from the reperfusion therapy.The prevention or reduction of MI/RI has become an important subject. Recently,many research results show that MI/RI can be prevented by many drugs which suppressed the acute ischemic arrhythmia,hemorrhagic necrosis and the expansion of the infarcted area.But because of the adverse effects and the effects of drugs to the physiology,it is very difficult for the drugs to play a role.Therefore,most of the drugs are still in the stage of fundamental research.In order to get a better understanding of the effect of anesthetics,the improvement of energy metabolism of cells,the synthesis of the specific precursor of NO and the intervention of traditional Chinese herbs to the drugs of myocardial ischemia reperfusion(MIR),132 patients undergoing open heart surgery utilizing CPB were divided into four groups.Made intervention with Propofol,Ketamine,1, 6-diphosphate,L-arginine and tetramethylpyrazine and observed the expression of heat shock protein(HSP)72,myocardium induced nitric oxide synthase(iNOS), polymorphonuclear neutrophil(PMN)NF-κB,Bcl-2 and the change of serum cardiac troponin T(cTnT)for further understanding the protecting action and mechanism of different drugs to MI/RI.Methods A hundred and thirty-two patients undergoing open-heart-surgery by CPB were randomly divided into four groups.The first group(G1)has twenty-eight patients,which were randomly divided into two groups(n=14 each),control(C)and TMP groups(M).In group M,TMP 3 mg/kg were given for thirty minutes after anesthesia induction and 1 mg/kg were given in oxygenator during CPB.In group C,same volume 0.9%NaCl were given at same time.A piece of right atrium myocardium was obtained before and after aortic cross-cramping(ACC)during cardiopulmonary bypass to measure the levels of HSP72 mRNA by semi-quantitative RT-PCR.Peripheral levels of AST,LDH,CK and CK-MB were measured before the CPB and 30 minutes after opening of aorta clamp and 24 h after cardiosurgery as markers of heart damage.A piece of right auricle myocardium was obtained before the CPB and 30 min after opening aorta to observe myocardial ultrastructure and analyzing mitochondria semiquantatively according to Flameng method.The second group(G2)has thirty-six patients,which were randomly divided into three groups(n=12 each),control(C)and Ketamine interfered groups(K1 and K2). In group K1 and K2,Ketamine 0.5 mg/kg and 1.0 mg/kg were vein injected before sternum splitted at the time of aorta intubated and 5 minutes before aorta clamped off partly.A piece of right auricle myocardium was obtained at the time of right atria intubated(T1),30 minutes after aorta clamped on(T2)and 30 minutes after aorta clamped off(T3)to measured the levels of iNOSmRNA and HSP72mRNA by one step real time fluorescence quantitative RT-PCR(used SYBR Green 1 fluorescence inosculating method).Blood samples were drawn from superior vena at T1,T2,T3 and two hours after aorta clamped off(T4).The content of creatinekinase(CK)and creatinekinase isozyme(CK-MB)were detected.The third group(G3)has fourty patients,which were randomly divided into four groups(n=10 each),control(C),L-Arg(L),FDP(F)and L-Arg plus FDP groups(D). In group L,L-Arg 200mg/kg were infused after aorta clamped off.In group F,FDP 200mg/kg were infused before aorta clamped on.In group D,L-Arg 200mg/kg were infused after aorta clamped off and FDP 200mg/kg were infused before aorta clamped on.Blood samples were taken to measure the levels of cardiac Troponin T(cTnT), Lactate dehydrogenase(LDH),MB isoenzyme of creatine kinase(CK-MB), malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)before aortic-clamping on and 30 minutes,2 hours and 6 hours after aorta clamped off.The fourth group(G4)has fourty patients,which were randomly divided into two groups(n=20 each),control(C)and propofol groups(P).There were no differences between two groups about medication during operation except that the propofol was used for anesthesia maintenance in group P.In group P,the plasma concentration of propofol was targeted and started from 2.5-3.4μg/ml by target-controlled infusion, then adjusted plasma concentration to depend on blood pressure and heart rate.Blood samples were taken from superior vena cava after anesthesia induction and before CPB(T1),30 minutes after aorta clamped on(T2),while aorta clamped off(T3),30 minutes after CPB(T4)and 90 minutes after CPB(T5)to measure following data, NF-kB and Bcl-2 in neutrophil,and plasma levels of tumour necrosis factor-α(TNF-α), interleukin-8(IL-8),interleukin-10(IL-10),malondiadilyde(MDA)and superoxide dismutase(SOD)activity.Results In the first group(G1),there were no significant differences between the two groups in sex,age,weight,ACC time,CPB time and cardioplegic solution.The expression of HSP72mRNA and the serum concentration of AST,LDH,CK and CK-MB and the scores of myocardial mitochondria were significantly increased after ACC as compared to before ACC in the two groups(P＜0.05 or P＜0.01).The expression of HSP72 mRNA were significantly higher after ACC in the group M than in the control group(P＜0.05).The serum concentration of AST,LDH,CK and CK-MB and the scores of myocardial mitochondria were significantly lower in the group M than in the control group(P＜0.05)after CPB.The myocardial ultrastructure in the group had milder alterations than in the control group after CPB(P＜0.05).In the second group(G2),there were no significant differences among the three groups in sex,age,weight and time of aorta clamped on and cardiopulmonary bypass (P＞0.05).The expression of iNOSmRNA and HSP72mRNA were significantly increased at T2 and T3 and the serum concentration of CK,CK-MB were significantly increased at T2,T3 and T4 as compared to at T1 in the three groups(P＜0.05 or P＜0.01).The expression of iNOSmRNA and HSP72mRNA were significantly increased at T3 and the serum concentration of CK and CK-MB were significantly increased at T3 and T4 as compared to at T2 in the three groups(P＜0.05 or P＜0.01). The serum concentration of CK and CK-MB were significantly increased at T4 as compared to at T3 in the three groups(P＜0.01).Compared to the control group,the expression of iNOSmRNA significantly decreased in the group K1 at T2 and in the group K2 at T2 and T3(P＜0.01),the serum concentration of CK and CK-MB were significantly lower in the groups K1 and K2 at T2,T3 and T4(P＜0.01),the expression of HSP72mRNA were significantly increased in the group K1 and K2 at T2 and T3.Compared to the group K1,the expression of iNOSmRNA significantly decreased in the group K2 at T2 and T3(P＜0.01),the serum concentration of CK and CK-MB were significantly lower in the group K2 at T2,T3 and T4(P＜0.05 or P＜0.01),the expression of HSP72mRNA were no significant differences in the group K2(P＞0.05).In the third group(G3),the plasma concentration of cTnT,LDH,CK-MB,MDA and SOD were significantly increased at T2,T3 and T4 as compared to at T1 in the four groups(P＜0.05 or P＜0.01).Compared to the control group,the plasma concentration of cTnT were significantly lower in the group L and F at T4 and in the group D at T3 and T4(P＜0.05),the plasma concentration of LDH were significantly lower in the group L at T3 and in the group D at T2,T3 and T4(P＜0.05 or P＜0.01), the plasma concentration of CK-MB and MDA were significantly lower in the group L,F and D at T2,T3 and T4(P＜0.05 or P＜0.01),the plasma concentration of SOD were significantly higher in the group L,F and D at T3.The plasma concentration of cTnT were significantly lower in the group D than in the group L at T3(P＜0.01)and in the group F at T3 and T4(P＜0.01).The plasma concentration of LDH were significantly lower in the group D than in the group F at T2(P＜0.05).In the fourth group(G4),there were no significant differences between two groups in sex,age,weight,ascending aorta clamping time,CPB time and operation time(P＞0.05).The expression of NF-kB and Bcl-2 and the count of neutrophil were significantly increased at T4 as compared to at Tl in the two groups(P＜0.01).The serum concentration of TNF-α,MDA and SOD were significantly increased at T2,T3, T4 and T5 as compared to at T1 in the two groups(P＜0.05 or P＜0.01).The serum concentration of IL-8 and L-10 were significantly increased at T3,T4 and T5 as compared to at T1 in the two groups(P＜0.05 or P＜0.01).Compared to the control group,the expression of NF-kB and Bcl-2 and the count of neutrophil were significantly decreased in the group P at T4(P＜0.01),the serum concentration of TNF-αand MDA were significantly lower in the group P at T2,T3,T4 and T5 (P＜0.05 or P＜0.01),the serum concentration of IL-8 were significantly lower in the group P at T3,T4 and T5(P＜0.05),the serum concentration of IL-10 were significantly higher in the group P at T3,T4 and T5(P＜0.05),the serum concentration of SOD were significantly higher in the group P at T2,T3,T4 and T5 (P＜0.05).Conclusion HSP72,iNOS,NF-kB and Bcl-2 take part in myocardial ischemia -reperfusion injury.HSP72 gene expression increase in myocardium of patients undergoing open heart surgery during CPB.TMP pre-conditioning can induce HSP72 gene expression and protect myocardial mitochondria from ischemia reperfusion injury and attenuates CPB-induced myocardial ischemia reperfusion injury through a self-protective mechanism.Ketamine in dose of anesthesia(1.0mg/kg)and subanaesthetic(0.5mg/kg)can decrease iNOSmRNA and increase HSP72mRNA expression in myocardium and reduce leakage of myocardium enzyme.Ketamine acts myocardial protection,and which in anesthesia dose is more effective than in subanaesthetic dose.The both of L-Arg and FDP can produce protective effects on myocardial ischemia reperfusion injury during open heart surgery with CPB through different protective mechanism.The combined use of L-Arg and FDP shows obvious synergistic effects on myocardial protection.Propofol is used to the patients undergoing open heart surgery during the CPB not only has the satisfactory actions of anesthesia and sedation but also has the actions of inhibiting Inflammatory reaction and abatement tissue injury by impact the function of neutrophil,the proinflammatory anti-inflammatory cytokines and oxygen free radicals etc.It has the prevention to MI/RI that was induced by infection or uninfection during cardiac surgery.So it has the clinic significance to use propofol in cardiac surgery anaesthesia.更多还原