异氟烷预处理对心脏换瓣术病人心肌基因表达的影响
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摘要
目的：在确定异氟烷预处理能减轻心脏瓣膜置换术(cardiac valves replacement, CVR)病人术后心肌显微结构、降低血浆基质金属蛋白酶(metrix metalloproteinase, MMP)-9和血管假性血友病因子(von Willebrand factor, vWF)浓度的基础上,应用基因芯片技术,观察异氟烷预处理对CVR病人心肌基因表达的影响,构建相关基因表达改变框架,深入观察异氟烷预处理对CVR病人心肌保护机制,为CVR病人最适麻醉用药提供理论基础。
方法：择期体外循环(cardiopulmonary bypass, CPB)下心脏二尖瓣和主动脉瓣联合瓣膜置换术成年病人,随机分为对照组和试验组。试验组在麻醉诱导后吸入1.1%-1.2%(呼气末浓度)的异氟烷30min,洗脱15min。对照组不吸入异氟烷,其余麻醉用药同试验组。分别于麻醉诱导后吸入异氟烷前(T0)、主动脉开放30min (T1)、关胸后(T2)、主动脉开放后6h(T3)、12h(T4)及24h(T5)应用Swan-Gans漂浮导管测心功能；分别于T0、T1、T3、T4和T5经桡动脉取血测血浆MMP-9和vWF浓度；于上腔静脉插管时(主动脉阻断前)和上腔静脉退管时(主动脉开放后)取部分右心耳组织观察其显微结构和检测两组差异表达基因。最后,利用实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)技术对基因检测结果进行验证。
结果：和T0比较,试验组和对照组平均动脉压(mean arterial blood pressure, MABP)在T1-T4时降低(P<0.05或P<0.01),肺动脉阻塞压(pulmonary artery obstruction pressure, PAOP)在T2-T5时降低(P<0.05或P<0.01),体循环血管阻力(systemic vascular resistance,SVR)在T1-T5降低,左心室心搏做功指数(left ventricular-stroke work index, LVSWI)、心输出量(cardiac output, CO)和每博输出量(stroke volume, SV)在T1-T5时均升高(P<0.05或P<0.01)；血浆MMP-9浓度对照组在T1-T4升高(P<0.01)、试验组在T1升高(P<0.01)；血浆vWF浓度对照组在T1、T4和T5降低(P<0.05)、试验组在Tl-T5降低(P<0.05或P<0.01),以上差异均有统计学意义。和对照组比较,试验组PAOP在T5时降低(P<0.05),LVSWI和CO在T3-T5时升高(P<0.05或P<0.01),SV在T4-T5时升高(P<0.05),血浆MMP-9浓度在T3降低(P<0.01),vWF浓度在T1及T3降低(P<0.05),以上差异有统计学意义。光镜和电镜下可见试验组主动脉开放后心肌细胞显微结构损伤轻于对照组。基因芯片共检测出22条2倍及2倍以上差异表达基因。从基因编码的蛋白质细胞分布区域来看,有一半分布在细胞外区或细胞膜上。数量最多的是信号转导基因,共有9条,主要涉及蛋白激酶信号通路。将检测到的2倍及2倍以上差异表达基因从功能上分为以下几类：信号通路和信号转导、免疫功能、内环境稳态、细胞粘附、细胞外基质和细胞骨架、细胞凋亡及增加氧供。实时荧光定量PCR验证了NM 007350、NR 002196和BC042589三条基因。证实和对照组比较,实验组NM_007350表达下调,NR_002196和BC042589表达上调,与基因芯片检测结果一致。
结论：1.异氟烷预处理可改善心脏联合瓣膜置换术病人术后24h心功能；2.异氟烷预处理可降低心脏联合瓣膜置换术病人主动脉开放后6h内血浆MMP-9和vWF浓度；3.异氟烷预处理可减轻心脏联合瓣膜置换术病人术后心肌细胞显微结构损伤；4.异氟烷预处理对心脏联合瓣膜置换术病人术后心肌基因表达有影响,可能涉及对信号通路和信号转导、免疫功能、内环境稳态、细胞粘附、细胞外基质和细胞骨架、细胞凋亡及增加氧供等基因转录的改变。
Objective On the basis of confirmation that myocardial microstructure can be improved as well as plasma level of metrix metalloproteinase(MMP)-9 and von Willebrand factor(vWF) can be decreased by isoflurane preconditioning after surgery in patients undergoing cardiac valves replacement(CVR), myocardial gene expression profiles affected by isoflurane preconditioning were detected with gene microarray, aiming to constructing the framework of related gene expression profiles and revealing the mechanisms of myocardial protection with isoflurane preconditioning and to providing the proper anesthetics usage in CVR surgery patient.
Methods Patients with CVR surgery were randomly assigned to receive either isoflurane of 1.0 minimum alveolar concentration end-tidal(1.1%-1.2%) for 30 min followed by a 15-min washout period before the beginning of cardiopulmonary bypass(CPB)(study group) or not(control group). Cardiac function was assessed using a pulmonary artery catheter at the time after anesthetic induction as well as before inhalating isoflurane(T0), at 30 min after aortic clamp opening (T1), closion of thorax (T2), and at 6 h(T3),12 h(T4) and 24 h(T5) after aortic clamp opening, respectively. Blood samples were obtained at the time of To, T1, T3, T4, T5, respectively. Right atrial appendage was sampled for observation of myocardial microstructure and detection of gene expression profiles at the time when catheter of superior vena caval was inserted into precava and when the catheter was extracted respectively. At last, gene expression profiles that obtained in the study was verified with fluorescence quantitative real-time polymerase chain reaction(PCR).
Results Comparing with To, in both study and test group, a significant decrease of mean arterial blood pressure (MABP) was detected at T1-T4(P<0.05或P<0.01), a significant decrease of mean pulmonary arterial pressure(PAOP) was detected at T2-T5(P<0.05或P＜0.01), a significant decrease of systemic vascular resistance(SVR) was detected at T1-T5(P<0.05或P<0.01), a significant increase of left ventricular stroke work index(LVSWI), stroke volume(SV) and cardiac output(CO) were detected at T1-T5(P< 0.05 or P<0.01). Still comparing with To, plasma level of MMP-9 increased significantly at T1-T4 in control group and at T1 in study group, respectively. Plasma level of vWF decreased significantly at T1, T4 and T5 in control group and at T1-T5 in study group, respectively. Comparing with control group, PAOP decreased significantly at T5(P<0.05), LVSWI and CO increased significantly at T3-T5 (P<0.05 or P<0.01), SV increased significantly at T4-T5 in study group, and there was a significant decrease with MMP-9 level at T3 and vWF at T1 and T3 in study group(P<0.05) when comparing with control group. Myocardial microstructure was better in study group when comparing with control group. There were total 22 differential expression genes regulated up or down 2 fold or greater than 2 fold in study group when compared with control group detected with gene microarray. Half of these corresponding proteins locate extracellularly or in cell membrane. There were 9 genes that their functions involve in signaling pathways. The function of all these genes mainly involved in signaling pathway, immunity, homeostasis, cell adhesion, extracellular matrix and cell skeleton, apoptosis, oxygen delivery and so on, and the most amount of these genes was involved in signaling pathways. Then,3 of these 22 genes were detected with fluorescence quantitative real-time PCR and its results were coincident to that of gene microarray.
Conclusion 1. Cardiac function can be improved by isoflurane preconditioning in 24 h of CVR surgery patient.2. Plasma level of MMP-9 and vWF can be decreased by isoflurane preconditioning in 6 h of CVR surgery patient.3. Myocardial microstructure can be improved by isoflurane preconditioning in CVR surgery patient.4. Myocardial gene expression profiles can be altered by isoflurane preconditioning in CVR surgery patient which maybe principally involved in signaling pathway, immunity, homeostasis, cell adhesion, extracellular matrix and cell skeleton, apoptosis, oxygen delivery and so on.
方法：择期体外循环(cardiopulmonary bypass, CPB)下心脏二尖瓣和主动脉瓣联合瓣膜置换术成年病人,随机分为对照组和试验组。试验组在麻醉诱导后吸入1.1%-1.2%(呼气末浓度)的异氟烷30min,洗脱15min。对照组不吸入异氟烷,其余麻醉用药同试验组。分别于麻醉诱导后吸入异氟烷前(T0)、主动脉开放30min (T1)、关胸后(T2)、主动脉开放后6h(T3)、12h(T4)及24h(T5)应用Swan-Gans漂浮导管测心功能；分别于T0、T1、T3、T4和T5经桡动脉取血测血浆MMP-9和vWF浓度；于上腔静脉插管时(主动脉阻断前)和上腔静脉退管时(主动脉开放后)取部分右心耳组织观察其显微结构和检测两组差异表达基因。最后,利用实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)技术对基因检测结果进行验证。
结果：和T0比较,试验组和对照组平均动脉压(mean arterial blood pressure, MABP)在T1-T4时降低(P<0.05或P<0.01),肺动脉阻塞压(pulmonary artery obstruction pressure, PAOP)在T2-T5时降低(P<0.05或P<0.01),体循环血管阻力(systemic vascular resistance,SVR)在T1-T5降低,左心室心搏做功指数(left ventricular-stroke work index, LVSWI)、心输出量(cardiac output, CO)和每博输出量(stroke volume, SV)在T1-T5时均升高(P<0.05或P<0.01)；血浆MMP-9浓度对照组在T1-T4升高(P<0.01)、试验组在T1升高(P<0.01)；血浆vWF浓度对照组在T1、T4和T5降低(P<0.05)、试验组在Tl-T5降低(P<0.05或P<0.01),以上差异均有统计学意义。和对照组比较,试验组PAOP在T5时降低(P<0.05),LVSWI和CO在T3-T5时升高(P<0.05或P<0.01),SV在T4-T5时升高(P<0.05),血浆MMP-9浓度在T3降低(P<0.01),vWF浓度在T1及T3降低(P<0.05),以上差异有统计学意义。光镜和电镜下可见试验组主动脉开放后心肌细胞显微结构损伤轻于对照组。基因芯片共检测出22条2倍及2倍以上差异表达基因。从基因编码的蛋白质细胞分布区域来看,有一半分布在细胞外区或细胞膜上。数量最多的是信号转导基因,共有9条,主要涉及蛋白激酶信号通路。将检测到的2倍及2倍以上差异表达基因从功能上分为以下几类：信号通路和信号转导、免疫功能、内环境稳态、细胞粘附、细胞外基质和细胞骨架、细胞凋亡及增加氧供。实时荧光定量PCR验证了NM 007350、NR 002196和BC042589三条基因。证实和对照组比较,实验组NM_007350表达下调,NR_002196和BC042589表达上调,与基因芯片检测结果一致。
结论：1.异氟烷预处理可改善心脏联合瓣膜置换术病人术后24h心功能；2.异氟烷预处理可降低心脏联合瓣膜置换术病人主动脉开放后6h内血浆MMP-9和vWF浓度；3.异氟烷预处理可减轻心脏联合瓣膜置换术病人术后心肌细胞显微结构损伤；4.异氟烷预处理对心脏联合瓣膜置换术病人术后心肌基因表达有影响,可能涉及对信号通路和信号转导、免疫功能、内环境稳态、细胞粘附、细胞外基质和细胞骨架、细胞凋亡及增加氧供等基因转录的改变。
Objective On the basis of confirmation that myocardial microstructure can be improved as well as plasma level of metrix metalloproteinase(MMP)-9 and von Willebrand factor(vWF) can be decreased by isoflurane preconditioning after surgery in patients undergoing cardiac valves replacement(CVR), myocardial gene expression profiles affected by isoflurane preconditioning were detected with gene microarray, aiming to constructing the framework of related gene expression profiles and revealing the mechanisms of myocardial protection with isoflurane preconditioning and to providing the proper anesthetics usage in CVR surgery patient.
Methods Patients with CVR surgery were randomly assigned to receive either isoflurane of 1.0 minimum alveolar concentration end-tidal(1.1%-1.2%) for 30 min followed by a 15-min washout period before the beginning of cardiopulmonary bypass(CPB)(study group) or not(control group). Cardiac function was assessed using a pulmonary artery catheter at the time after anesthetic induction as well as before inhalating isoflurane(T0), at 30 min after aortic clamp opening (T1), closion of thorax (T2), and at 6 h(T3),12 h(T4) and 24 h(T5) after aortic clamp opening, respectively. Blood samples were obtained at the time of To, T1, T3, T4, T5, respectively. Right atrial appendage was sampled for observation of myocardial microstructure and detection of gene expression profiles at the time when catheter of superior vena caval was inserted into precava and when the catheter was extracted respectively. At last, gene expression profiles that obtained in the study was verified with fluorescence quantitative real-time polymerase chain reaction(PCR).
Results Comparing with To, in both study and test group, a significant decrease of mean arterial blood pressure (MABP) was detected at T1-T4(P<0.05或P<0.01), a significant decrease of mean pulmonary arterial pressure(PAOP) was detected at T2-T5(P<0.05或P＜0.01), a significant decrease of systemic vascular resistance(SVR) was detected at T1-T5(P<0.05或P<0.01), a significant increase of left ventricular stroke work index(LVSWI), stroke volume(SV) and cardiac output(CO) were detected at T1-T5(P< 0.05 or P<0.01). Still comparing with To, plasma level of MMP-9 increased significantly at T1-T4 in control group and at T1 in study group, respectively. Plasma level of vWF decreased significantly at T1, T4 and T5 in control group and at T1-T5 in study group, respectively. Comparing with control group, PAOP decreased significantly at T5(P<0.05), LVSWI and CO increased significantly at T3-T5 (P<0.05 or P<0.01), SV increased significantly at T4-T5 in study group, and there was a significant decrease with MMP-9 level at T3 and vWF at T1 and T3 in study group(P<0.05) when comparing with control group. Myocardial microstructure was better in study group when comparing with control group. There were total 22 differential expression genes regulated up or down 2 fold or greater than 2 fold in study group when compared with control group detected with gene microarray. Half of these corresponding proteins locate extracellularly or in cell membrane. There were 9 genes that their functions involve in signaling pathways. The function of all these genes mainly involved in signaling pathway, immunity, homeostasis, cell adhesion, extracellular matrix and cell skeleton, apoptosis, oxygen delivery and so on, and the most amount of these genes was involved in signaling pathways. Then,3 of these 22 genes were detected with fluorescence quantitative real-time PCR and its results were coincident to that of gene microarray.
Conclusion 1. Cardiac function can be improved by isoflurane preconditioning in 24 h of CVR surgery patient.2. Plasma level of MMP-9 and vWF can be decreased by isoflurane preconditioning in 6 h of CVR surgery patient.3. Myocardial microstructure can be improved by isoflurane preconditioning in CVR surgery patient.4. Myocardial gene expression profiles can be altered by isoflurane preconditioning in CVR surgery patient which maybe principally involved in signaling pathway, immunity, homeostasis, cell adhesion, extracellular matrix and cell skeleton, apoptosis, oxygen delivery and so on.
引文
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