【作者】 宋华培；

【导师】 黄跃生；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 严重烧伤后,心肌局部血流量迅速减少,发生缺血缺氧损害和心功能减退,不仅引起心功能不全,还可诱发或加重休克,成为烧伤早期缺血缺氧的重要启动因素之一。根据上述认识,提出了烧伤后早期缺血缺氧损害的“休克心”假说。PI3K是细胞内重要的信号转导分子。PI3K激活后在质膜上产生第二信使PIP3,进一步引起信号蛋白Akt活化。活化的Akt通过磷酸化作用激活或抑制其下游靶蛋白Bad、Caspase9、NF-kB、GSK-3、FKHR、p21^Cip1和p27^Kip1等,进而调节细胞的增殖、分化、凋亡以及迁移等。PI3K/Akt信号途径在烧伤早期心肌细胞的表达、作用及调控机制尚不明确。我们假设:在烧伤早期心肌细胞遭受缺血缺氧损伤的同时,PI3K/Akt信号途径即被激活,并可能通过调控钙浓度、线粒体膜稳定性以及凋亡基因表达发挥抗凋亡作用,PI3K/Akt/HIF-1α则可能是这种抗凋亡作用的重要分子机制。本实验对这一假设进行了验证。一、材料方法:采用动物实验和体外实验相结合的方法。1.建立烫伤大鼠模型,应用RT-PCR检测心肌组织PI3K mRNA表达变化,Westernblot检测心肌组织PI3K及磷酸化Akt蛋白表达。2.建立体外培养的大鼠心肌细胞缺血缺氧模型,应用PI3K/Akt通路特异性抑制剂LY294002及激动剂IGF-1,并结合ELISA、激光共聚焦、TUNEL等方法等方法观察缺血缺氧条件下该信号通路对心肌细胞活力、LDH酶活性、线粒体膜电位、胞内钙浓度及凋亡率等指标的影响,明确PI3K/Akt信号途径在缺血缺氧心肌细胞中的作用。3.建立体外培养的大鼠心肌细胞缺血缺氧模型,应用RT-PCR观察PI3K/Akt对P53、C-myc、Bax、Bcl-2等凋亡相关基因以及HIF-1αmRNA的影响,并应用LY294002和Rapamycin,结合Western blot探讨可能存在的凋亡调控机制。二、主要结果与结论1.严重烧伤早期即有PI3K mRNA及蛋白的高表达,并伴有Akt磷酸化水平的显著增加,说明烧伤早期PI3K/Akt途径即被激活。2.缺血缺氧条件下,心肌细胞活力持续下降,PI3K/Akt抑制剂LY294002抑制PI3K/Akt激活后,心肌细胞活力下降更为明显。表明该途径可能具有维持细胞活性,拮抗缺血缺氧损害的作用。3.缺血缺氧刺激导致心肌细胞细胞膜结构破坏,LDH漏出增加;LY294002抑制PI3K/Akt激活后,缺血缺氧心肌细胞培养上清中LDH活性较单纯缺氧组明显升高。表明PI3K/Akt能够维持质膜稳定性,防止因膜结构的破坏导致细胞死亡。4.心肌细胞在缺血缺氧刺激下,细胞凋亡量明显增加。LY294002抑制PI3K/Akt激活后,缺血缺氧心肌细胞凋亡加剧;予以IGF-1激活PI3K/Akt,缺血缺氧心肌细胞凋亡量明显减少。说明PI3K/Akt的激活能够拮抗缺血缺氧心肌细胞凋亡,是细胞的内源性保护反应。5.缺血缺氧6h后,心肌细胞胞内钙浓度明显升高,LY294002抑制PI3K/Akt激活后,缺血缺氧心肌细胞胞内钙浓度升高更为显著;予以IGF-1激活PI3K/Akt,缺血缺氧心肌细胞胞浆钙离子浓度较单纯缺血缺氧组明显下降。说明PI3K/Akt的激活能够减轻胞内钙超载,避免钙超载引起的后续凋亡反应。6.缺血缺氧刺激6h、12h后,线粒体膜电位明显下降,提示线粒体膜通透性增加;LY294002抑制PI3K/Akt激活后,缺血缺氧心肌细胞线粒体膜电位下降更为显著;予以IGF-1激活PI3K/Akt,缺血缺氧心肌细胞线粒体膜电位下降幅度明显减小,表明PI3K/Akt能够维持线粒体膜稳定性,防止因线粒体MPTP的开放而导致后续凋亡反应的启动。7.缺血缺氧心肌细胞PI3K/Akt信号途径的活化可以上调HIF-1αmRNA转录活性,同时PI3K/Akt及mTOR蛋白磷酸化均可以在蛋白水平上调HIF-1α的表达,以发挥HIF-1α的抗凋亡作用,拮抗心肌细胞的缺血缺氧损害。8.缺血缺氧条件下,心肌细胞PI3K/Akt的活化可通过上调HIF-1α表达,进一步调控凋亡相关基因表达,如降低心肌细胞野生型P53的表达,提高Bcl-2/Bax比值,增加C-myc等表达,以减少凋亡,提高细胞存活率。更多还原

【Abstract】 Our previous studies demonstrated that myocardial damage occurs immediately following severe burns even before significant reduction in blood volume.Such myocardial damage leads to cardiac deficiency,and it is also a precipitating factor for burn shock and ischemic/hypoxic injury.Therefore,we called it ’shock heart’.PI3K is one of the most important intracellular signal transductuction molecules.After PI3K is activated,the second messenger PIP3 is produced at plasma menmbranes,which phosphorylates Akt at Ser308.The activated Akt further phosphorylates the target proteins, such as Bad,Caspase9,NF-kB,GSK-3,FKHR,p21^Cip1 and p27^Kip to modulate the cell proliferation,differentiation,apoptosis and immigration.The expression,function and potential mechanism of PI3K/Akt pathway in myocardium at early postburn stage remains unlear.We postulated that PI3K/Akt pathway is activated at the same time when the cardiocytes are inflicted with hypoxic/ischemic injury at early stage following severe burns,which may have a anti-apoptotic effect by modulation of calcium,mitochondrial membrane potential,HIF-1αexpression and apoptotic gene expression.The present study was designed to confirm this hypothesis.Material and MethodsMethods:Both in vivo and in vitro experiments were adopted in the present study.The animal model was established by scald in rats.The PI3K mRNA expression of myocardium was detected by RT-PCR.PI3K and phosphorylated Akt protein expression were detected by western blot.To elucidate the function of PI3K/Akt signal pathway in cardiomyocytes during hypoxia and ischemia,the in vitro model was established by cultured rat cardiomyocytes maintaining in hypoxia and ischemia environment.With treatment of specific PI3K/Akt inhibitor LY294002 and selective PI3K/Akt activator IGF-1,ELISA,laser scanning confocal fluorescence microscope,TUNEL technique were used to observe the influence of PI3K/Akt pathway on cardiomyocyte viability,LDH activity,mitochondrial membrane potential,inner Ca²⁺ concentration and apoptotic rate.LY294002 and Rapamycin treatment were used to explore the possible mechanism of apoptosis.Alteration of the apoptotic gene expression such as P53,C-myc,Bax,Bcl-2 and HIF-1αmRNA were detected by RT-PCR and/or Western blot.Results and Conclusion:1.PI3K mRNA and protein expression and Akt phospharylation were upregulated significantly at early stage of severe burn,which showed the PI3K/Akt pathway was activated.2.Hypoxia and ischemia induced progresive decrease of cardiocyte viability.The decrease of cardiocyte viability was more severe after PI3K/Akt inhibitor LY294002 was used,indicating that the PI3K/Akt pathway plays a role in the maintainance of cell viability and protection of cells from damage induced by hypoxia and ischemia.3.Hypoxia and ischemia caused the membrance damage of cardiocytes refleted by increase of LDH leakage.After pretreatment with LY294002,the LDH was obviously increased.It suggests that PI3K/Akt pathway can maintain the cardiocyte membrane stability.4.The apoptotic rate in cardiocytes cultured under hypoxia and ischemia environment was increased significantly,and was intensified by LY294002 pre-treatment.When PI3K/Akt activator IGF-1 was given,the apoptotic rate was decreaed.This result suggests that the PI3K/Akt pathway can protect cardiocytes from apoptosis as an endogenous cytoprotective factor.5.At 6h after hypoxia and ischemia,inner Ca²⁺ concentration increased obviously, which was intensified by LY294002 pre-treatment.When PI3K/Akt activator IGF-1 was given,concentration of inner Ca²⁺ was decreased,indicating that the activation of PI3K/Akt can relieve the Ca²⁺ overload and the consequent apoptotic reaction.6.At 6h and 12h after hypoxia and ischemia,mitochondrial membrane potential decreased obviously,which was also intensified by LY294002 pre-treatment.When PI3K/Akt activator IGF-1 was given,the decreasing of mitochondrial membrane potential was alleviated,suggesting that PI3K/Akt pathway can maintain the stability of mitochondrial membrane and prevent the apoptotic reaction induced by MPTP openning of mitochondria.7.PI3K/Akt/mTOR/p70S6K pathway may play a role in regulating HIF-1αexpression in mRNA and protein level to prevent cardiocyte apoptosis under hypoxia and ischemia condition.8.The activation of PI3K/Akt,through upregulation of HIF-1αexpression,further downregulated the wildtype P53 expression in cardiocytes,raised the Bcl-2/Bax ratio, upregulated C-myc expression so as to protect cells from apoptosis under hypoxia and ischemia condition.更多还原