【作者】 肖娟；

【导师】 肖颖彬；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 背景与目的心肌慢性缺氧是临床常见的病理生理过程,可导致心肌产生一系列代谢适应性改变,与线粒体的数量和功能密切相关的线粒体生物合成过程的适应性改变可能是其中重要的环节,但慢性缺氧对心肌线粒体生物合成影响的机制目前尚不完全清楚。一氧化氮(NO)可能参与了多种细胞线粒体生物合成的调控,其在慢性缺氧心肌线粒体生物合成中的作用尚不明了。深入研究NO与慢性缺氧心肌线粒体的适应性改变和调控之间的关系,将有望阐明缺氧状态下心肌的病理生理改变机制,为进一步增强缺氧状态下的心肌保护,提高临床治疗效果提供新的思路。本课题分别在先天性心脏病心肌组织标本和体外培养慢性缺氧心肌细胞模型上,对心肌细胞的线粒体生物合成和NO信号通路变化进行形态学和分子生物学研究,探讨NO在慢性缺氧状态下对心肌线粒体生物合成的调控作用机制。研究方法本研究共分为三部分:第一部分:选取紫绀型和非紫绀型先天性心脏病患者各10例,留取手术中切除的肥厚右室流出道组织,通过电镜观察分析标本心肌细胞超微结构,运用实时荧光PCR、RT-PCR及Western blot等方法比较心肌组织mtDNA拷贝数,细胞色素c氧化酶I (COX I)、过氧化物酶体增生物激活受体γ辅激活因子-1α(PGC-1α)、核呼吸因子1(NRF1)、线粒体转录因子A(Tfam)和三型NOS的mRNA或(和)蛋白水平,检测NOS酶活性,并对PGC-1α、eNOS蛋白水平与患者末梢血氧饱和度SaO2进行相关性分析。第二部分:体外培养乳鼠心肌细胞,根据不同干预因素将心肌细胞分为对照组,NO供体SNAP组,SNAP+NO清除剂氧合血红蛋白(OxyHb)组及SNAP+可溶性鸟苷酸环化酶(sGC)抑制剂(ODQ)组。应用线粒体荧光探针在激光共聚焦显微镜下观察各组细胞线粒体量的变化,应用实时荧光PCR、RT- PCR及Western blot等方法分别比较各组细胞mtDNA拷贝数,COX I、PGC-1α、NRF1和Tfam的mRNA水平以及COXI蛋白水平等。第三部分:通过1%O2浓度培养48小时建立体外培养乳鼠心肌细胞慢性缺氧模型,根据不同干预方式将心肌细胞分为对照组,慢性缺氧组,慢性缺氧+NOS抑制剂L-NAME组,慢性缺氧+OxyHb组以及慢性缺氧+ODQ组。检测细胞培养液上清NO浓度和心肌细胞NOS蛋白水平,应用线粒体荧光探针观察各组细胞线粒体量的变化,应用实时荧光PCR、RT- PCR及Western blot等方法分别比较各组细胞mtDNA拷贝数,COX I、PGC-1α、NRF1和Tfam的mRNA水平以及COX I蛋白水平等。研究结果本研究主要结果如下:1.紫绀型先天性心脏病右室流出道心肌组织中线粒体体密度Vv、数密度Nv明显高于非紫绀组(p<0.05, p<0.01),mtDNA拷贝数显著高于非紫绀组(p<0.01),COX I、PGC-1α、NRF1和Tfam的mRNA水平均显著高于非紫绀组(p<0.01),COX I、eNOS蛋白表达显著高于非紫绀组(p<0.05, p<0.05),心肌组织PGC-1αmRNA水平与SaO2呈显著的负相关(r=-0.75, p<0.01),eNOS蛋白水平与SaO2呈显著的负相关(r=-0.64, p<0.01),PGC-1αmRNA水平与eNOS蛋白水平呈显著的正相关(r=0.58, p<0.01);2. (1) NO供体SNAP干预组心肌细胞线粒体荧光强度、mtDNA拷贝数、COXI mRNA和蛋白水平均明显高于对照组(p<0.01, p<0.01, p<0.01, p<0.01),与线粒体生物合成相关的信号分子PGC-1α、NRF1和Tfam mRNA水平也高于对照组(p<0.01, p<0.01, p<0.05);(2)与SNAP组比较,SNAP+OxyHb组线粒体荧光强度、mtDNA拷贝数、COXI mRNA均降低(p<0.01, p<0.01, p<0.05),PGC-1α和NRF1 mRNA水平也降低(p<0.01, p<0.05);(3)与SNAP组比较,SNAP+ODQ组线粒体荧光强度、mtDNA拷贝数和COXI mRNA水平均降低(p<0.05, p<0.01, p<0.05),PGC-1αmRNA水平也降低(p<0.05);3. (1)慢性缺氧心肌细胞培养液上清NO浓度明显高于对照组(p<0.01),iNOS和eNOS蛋白水平明显高于对照组(p<0.05, p<0.05);慢性缺氧心肌细胞线粒体荧光强度、mtDNA拷贝数、COXI mRNA水平均明显高于对照组(p<0.01, p<0.01, p<0.01),与线粒体生物合成相关的信号分子PGC-1α、NRF1和Tfam mRNA水平也高于对照组(p<0.01, p<0.05, p<0.01);(2)与慢性缺氧组比较,慢性缺氧+ L-NAME组线粒体荧光强度、COXI mRNA和蛋白水平均降低(p<0.01, p<0.01, p<0.05),PGC-1α和Tfam mRNA水平也降低(p<0.05,p<0.01);(3)与慢性缺氧组比较,慢性缺氧+ OxyHb组PGC-1αmRNA水平降低(p<0.05),其它指标无明显变化;(4)与慢性缺氧组比较,慢性缺氧+ODQ组线粒体荧光强度、mtDNA拷贝数、COXI mRNA和蛋白水平均降低(p<0.01, p<0.05, p<0.01, p<0.05),PGC-1α和Tfam mRNA水平也降低(p<0.01, p<0.05)。结论本课题分别在先天性心脏病心肌组织标本和体外培养慢性缺氧心肌细胞模型上,对心肌细胞的线粒体生物合成和NO信号通路变化进行形态学和分子生物学研究,探讨NO在慢性缺氧状态下对心肌线粒体生物合成的调控作用机制。结果发现:1.紫绀型先天性心脏病右室流出道心肌组织中线粒体数量增多, mtDNA拷贝数增多,线粒体编码的COXI蛋白mRNA和蛋白水平升高。说明紫绀型先天性心脏病心肌线粒体生物合成明显增多;同时,紫绀型先天性心脏病右室流出道心肌组织中eNOS蛋白水平升高,并且与线粒体生物合成呈显著的正相关,提示其参与了线粒体生物合成的调节;2. NO供体在体外可以促进培养心肌细胞的线粒体生物合成,该过程可能通过sGC-cGMP途径介导;3.慢性缺氧的培养心肌细胞中iNOS和eNOS蛋白表达升高,NO合成增多,同时线粒体的生物合成上调;NO可能通过NOS-NO-sGC-cGMP途径参与慢性缺氧心肌细胞线粒体生物合成的调节。更多还原

【Abstract】 BackgroundMyocardial chronic hypoxia is a common clinical pathophysiological process and leads to a series of myocardial metabolic adaptive changes. Mitochondrial biogenesis which controls the quantity and quality of mitochondria may plays an important role in myocardial metabolic adaptation. However, the effect of chronic hypoxia in myocardial mitochondrial biogenesis has not been fully understood. Nitric oxide (NO) may be involved in the regulation of mitochondria biogenesis in a variety of mammalian cell lines, but its role in cardiac mitochondrial biogenesis during chronic hypoxia remains unknown. Research on the relationship between NO and myocardial mitochondrial biogenetic adaptation during chronic hypoxia may help to clarify the mechanism of chronic hypoxic adaptation and provide some new ideas to enhance myocardial protection in health and patients. To better clarify the regulatory mechanisms of NO pathway in cardiac mitochondrial biogenesis during chronic hypoxia, we performed a detailed morphologic and molecular analysis on heart tissue samples from patients with congenital heart disease and cultured cardiomyocytes exposed to chronic hypoxia.MethodsIn the first part of this study, samples from the right ventricular outflow tract myocardium were taken from patients with cyanotic(n=10) and acyanotic(n=10) congenital heart diseases. The ultrastructure of samples was analyzed by transmission electron microscope. MtDNA copy number, cytochrome c oxidase I (COX I), peroxisome proliferators activated receptorγcoactivator-1α(PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (Tfam) and three nitric oxide synthase(NOS) isoforms transcript and(or) protein levels were detected by real-time PCR , RT-PCR and Western blot respectively. The activity levels of NOS were also analyzed.In the second part, cultured neonatal rat cardiomyocytes were treated with SNAP (NO donor), oxygenated hemoglobin (OxyHb, NO scavenger) or ODQ (soluble guanylate cyclase inhibitor). Mitochondrial specific fluorescence probe was used for detecting the mitochondrial mass, real-time PCR, RT-PCR and Western blot were used for detecting the expression of related gene and protein.In the last part, neonatal rat cardiomyocytes were cultured in 1%O2 for 48h and treated with L-NAME(NOS inhibitor),OxyHb or ODQ. The NO production in cardiomyocytes, mitochondrial mass, related gene and protein expression were also detected.Results1. Mitochondrial volume density (Vv) and numerical density (Nv) were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease (p<0.05, p<0.01). Elevated mtDNA and up-regulated COXI, PGC-1α, NRF1 and Tfam mRNA levels were observed in cyanotic patients (p<0.01). Levels of COXI and eNOS protein were significantly higher in the myocardium of cyanotic patients than those of acyanotic (p<0.05, p<0.05). Levels of PGC-1αtranscript and eNOS protein inversely correlated with SaO2(r=-0.75, p<0.01 and r=-0.64, p<0.01, respectively). Levels of PGC-1αtranscript correlated with the levels of eNOS(r=0.58, p<0.01).2. (1) In SNAP-treated cardiomyocytes, the mitochondria fluorescence intensity, copy number of mtDNA, levels of COXI mRNA and protein were significantly higher than those of control group (p <0.01, p <0.01, p <0.01, p <0.01). Mitochondrial biogenesis related signaling molecules PGC-1α, and NRF1 Tfam mRNA levels were also elevated (p <0.01, p <0.01, p <0.05).(2) Comparing with the SNAP group, the mitochondria fluorescence intensity, copy number of mtDNA and COXI mRNA decreased in SNAP + OxyHb group (p <0.01, p <0.01, p <0.05), and levels of PGC-1αand NRF1 mRNA had also decreased (p <0.01, p <0.05).(3) Comparing with the SNAP group, the mitochondria fluorescence intensity, mtDNA copy number and COXI mRNA levels decreased in the SNAP + ODQ group (p <0.05, p <0.01, p <0.05), and PGC-1αmRNA levels have also decreased (p < 0.05).3. (1) Comparing with the control group, NO production, eNOS and iNOS protein levels were significantly higher in cardiomyocytes exposed to chronic hypoxia (p <0.01, p <0.05, p <0.05). The mitochondria fluorescence intensity, mtDNA copy number and COXI mRNA levels were significantly higher (p <0.01, p <0.01, p <0.01). Mitochondrial biogenesis related signaling molecules PGC-1α, and NRF1 Tfam mRNA levels were also elevated in cardiomyocytes exposed to chronic hypoxia (p <0.01, p <0.05, p <0.01).(2) Comparing with the chronic hypoxia group, the mitochondria fluorescence intensity, COXI mRNA and protein levels decreased (p <0.01, p <0.01, p <0.05), and PGC-1αand Tfam mRNA levels have also decreased (p <0.05, p <0.01) in chronic hypoxia + L-NAME group.(3) Comparing with the chronic hypoxia group, levels of PGC-1αmRNA decreased in chronic hypoxia + OxyHb group (p <0.05). No significant changes were observed in other parameters.(4) Comparing with the chronic hypoxia group, the mitochondria fluorescence intensity, mtDNA copy number, COXI mRNA and protein levels decreased (p <0.01, p <0.05, p <0.01, p <0.05), levels of PGC-1αand Tfam mRNA were also decreased (p <0.01, p <0.05) in chronic hypoxia + ODQ group.ConclusionsThis study based on a detailed morphologic and molecular analysis on heart tissue samples from congenital heart disease patients and cultured cardiomyocytes exposed to chronic hypoxia. The results indicated that:1. The mitochondrial mass, mtDNA copy number, mitochondrial encoded COXI mRNA and protein levels were increased in the right ventricular outflow tract myocardium of congenital heart disease with cyanosis, suggesting that mitochondrial biogenesis is activated in myocardium in congenital heart disease with cyanosis. Meanwhile, levels of eNOS protein were also elevated in cyanotic group, and were significantly positive correlated with mitochondrial biogenesis, which implied that eNOS may be involved in the regulation of mitochondrial biogenesis.2. NO donor can promote the mitochondrial biogenesis in cultured cardiomyocytes, possibly mediated by sGC-cGMP pathway.3. NO production, eNOS and iNOS protein expression as well as mitochondrial biogenesis were up-regulated in cardiomyocytes exposed to chronic hypoxia. NO may regulate mitochondrial biogenesis through NOS-NO-sGC-cGMP pathway in cardiomyocytes exposed to chronic hypoxia.更多还原