p93基因在大鼠超负荷心肌肥厚过程中的表达变化
发布时间：2018-01-01 22:38
本文关键词：p93基因在大鼠超负荷心肌肥厚过程中的表达变化 出处：《河北医科大学》2007年硕士论文 论文类型：学位论文
更多相关文章： 压力超负荷心肌肥厚模型 p93 PrxIII 活性氧族 氧化应激
【摘要】： 心肌肥厚是心力衰竭时机体重要的代偿反应之一。其主要表现为心肌细胞的肥大和间质成分的改变(或称心脏重构),心脏顺应性和循环泵功能降低。心肌肥厚时,心肌细胞内收缩蛋白表型改变,同时伴有心肌细胞外基质改建,这是造成心肌收缩性降低的两个重要因素。心肌肥厚的不可逆性进展必然会导致心力衰竭的发生。因此,如果找到一条能改变或提高心肌收缩能力的途径必然会大大降低心力衰竭的发病率。 P93是近年来新发现的一个心肌特异性表达的蛋白激酶,分子量为93KD,可以进行自身磷酸化,它与心肌肌钙蛋白Ⅰ(Cardiac troponin, CTN1)、心肌肌动蛋白(a-action)等一系列与收缩有关的肌小节蛋白发生相互作用。推测p93可能通过一条未知的信号转导通路参与了对肌小节收缩蛋白的调控,进而调控心肌收缩能力,参与心肌肥厚的发生过程。 心肌收缩所需能量来自于线粒体的氧化磷酸化,但在氧化磷酸化过程中亦伴随着活性氧族(reactive oxygen species :ROS)的产生。PrxIII是P93的相关蛋白,其作用主要是清除线粒体内多余的ROS,从而保护细胞免受氧化应激损伤。因此,在心肌负荷发生变化时,心肌细胞的收缩力随之变化,其相关蛋白的表达亦可能发生变化。有研究证实:PrxIII在心肌梗塞后心室重构过程中表达增强,但其在心肌肥厚过程中的表达变化,及其与p93表达的相关性尚未见报道。 目的 为了探讨p93及相关蛋白PrxIII在超负荷心肌肥厚过程中的表达变化,及其与心肌细胞形态和功能变化的相关性,为进一步的p93功能研究奠定基础。 方法 1动物分组及压力超负荷大鼠心肌肥厚模型的制备选用190-200g的成年雄性SD大鼠,随机分为:①假手术对照组(Con): 24小时(Con_(24H))、2周(Con_(2W))、4周(Con_(4W))、6周(Con_(6W));②模型组(Mod): 24小时(Mod_(24H))、2周(Mod_(2W))、4周(Mod_(4W))、6周(Mod_(6W))。大鼠压力超负荷心肌肥厚模型的制备参考Anversa P的方法。假手术对照组只分离左肾动脉上方的腹主动脉,并不结扎。其他步骤与模型组相同。 2测定指标及方法 2.1血流动力学指标测定 各组大鼠做如下处理:称重,6%水合氯醛(5ml/kg体重)腹腔注射麻醉。固定大鼠,分离右侧颈总动脉,插入和泰盟BL-420E+生物机能实验分析系统相连的自制聚乙烯导管,稳定后记录右颈总收缩压(SBP)、舒张压(DBP),继续插入至左心室,记录左室收缩压(LVSP)、左室舒张末压(LVEDP)以及左心室收缩压最大上升速率(㧏dp/dt_(max))和左心室舒张压最大下降速率(-dp/dt_(max))。 2.2心脏重量指数HMI的测定 血流动力学指标测定完毕后,开胸迅速取出心脏,冰生理盐水洗掉余血,用滤纸吸干表面水分后称重并记录心脏重量,计算心脏重量(HW)与体重(BW)的比值,即为心脏重量指数(heate mass index:HMI)。 2.3 HE染色观察心肌的形态结构 心脏称重后,分离室间隔和左心室。将左心室分成两部分,一部分放入4%多聚甲醛固定液内固定。常规石蜡切片HE染色,于光学显微镜下观察形态变化,日本产Nikon光学显微镜下拍照。 2.4电镜观察左心室的超微结构变化 将另一部分左心室标本放入3%多聚甲醛和1%戊二醛混合固定液中固定。用于电镜观察左心室的超微结构变化 2.5大鼠室间隔p93和PrxIIImRNA的定量 将分离的室间隔迅速至于液氮内,-70℃保存,用于RNA提取。用Trizol法提取室间隔总RNA后,以GAPDH作为内参照,应用RT-PCR方法测定大鼠室间隔p93和Prx III mRNA的相对表达量。 结果 1血压 Con_(24H)的SBP为12.13±0.69,DBP为9.47±0.98, Mod_(24H)的SBP为12.51±1.37,DBP为10.04±1.01,与Con_(24H)相比,Mod_(24H)的SBP和DBP没有显著变化(P0.05); Con_(2W)的SBP为12.33±0.52,DBP为8.99±0.94,Mod_(2W)的SBP为14.55±1.04,DBP为12.03±2.08,与Con_(2W)相比,Mod_(2W)的SBP和DBP均上升(P0.05);Con_(4W)的SBP为12.40±0.74,DBP为9.32±0.78,Mod_(4W)的SBP为16.26±2.41,DBP为12.10±1.61。与Con_(4W)相比,Mod_(4W)的SBP和DBP也都有明显上升(P0.01);Con_(6W)的SBP为12.71±1.35,DBP为9.41±1.08,Mod_(6W)的SBP为16.04±2.32,DBP为13.40±1.94,与Con_(6W)相比,Mod_(6W)的SBP和DBP均上升且有显著性差异(P0.01)。 2左心室收缩和舒张功能 Con_(24H)的LVSP为12.37±0.74,LVEDP为0.66±0.07,㧏dp/dt_(max)为424±72,-dp/dt_(max)为406±58;Mod_(24H)的LVSP为12.58±1.01,LVEDP为0.71±0.10,㧏dp/dt_(max)为431±69,-dp/dt_(max)为419±63。与Con_(24H)相比,Mod_(24H)的LVSP、LVEDP及±dp/dt_(max)均没有显著变化(P0.05)。 Con_(2W)的LVSP为12.56±1.13,LVEDP为0.68±0.09,㧏dp/dt_(max)为416±77,-dp/dt_(max)为411±64;Mod_(2W)的LVSP为15.01±1.95,LVEDP为0.69±0.11,㧏dp/dt_(max)为491±85,-dp/dt_(max)为486±78。与Con_(2W)相比,Mod_(2W)的LVSP及±dp/dt_(max)增高,均有显著性差异(P0.05),但LVEDP没有显著变化(P0.05)。 Con_(4W)的LVSP为12.55±1.06,LVEDP为0.70±0.08,㧏dp/dt_(max)为407±81,-dp/dt_(max)为398±66;Mod_(4W)的LVSP为16.47±2.16,LVEDP为0.81±0.08,㧏dp/dt_(max)为421±80,-dp/dt_(max)为404±57。与Con_(4W)相比,Mod_(4W)的LVSP明显增高(P0.01),但LVEDP及±dp/dt_(max)均没有显著差异(P0.05)。 Con _(6W)的LVSP为12.77±1.87,LVEDP为0.75±0.09,㧏dp/dt_(max)为421±76, -dp/dt_(max)为410±65;Mod_(6W)的LVSP为16.16±1.54,LVEDP为1.83±0.22,㧏dp/dt_(max)为340±66, -dp/dt_(max)为338±52。与Con_(6W)相比,Mod_(6W)的LVSP及LVEDP显著升高(P0.01),±dp/dt_(max)均有明显下降(P0.05)。 3心脏重量指数HMI Con_(24H)的HMI为3.11±0.23,Mod_(24H)的HMI为3.15±0.19。Con_(2W)的HMI为3.07±0.49,Mod_(2W)的HMI为3.43±0.16,与各自的对照组相比,Mod_(24H)和Mod_(2W)的HMI均无明显变化(P0.05);Con_(4W)的HMI为3.10±0.37,Mod_(4W)的HMI为3.95±0.30,Con_(6W)的HMI为2.94±0.27,Mod_(6W)的HMI为4.35±0.74,与各自的对照组相比,Mod_(4W)和Mod_(6W)的HMI均升高,有显著性差异(P0.05)。 4大鼠左心室的光镜观察 各对照组心肌纤维排列整齐,胞核清晰。_(24H)模型组与对照组相比形态未见明显改变。_(2W)、_(4W)、_(6W)模型组的心肌纤维逐渐较对照组宽大,肌纤维间隙逐渐增宽,间质内可见毛细血管扩张增生。 5大鼠左心室的电镜观察 各对照组大鼠心肌纤维排列整齐,明带、暗带及Z线清晰,线粒体结构正常。_(24H)模型组与对照组相比,结构未见明显改变。_(2W)、_(4W)、_(6W)模型组的心肌肌节清晰,线粒体逐渐增多,并出现大量线粒体集结现象,其中以_(4W)和_(6W)最为明显,线粒体嵴逐渐密集,_(6W)组的线粒体嵴较其它组明显增厚,并且在心肌细胞内,特别是在线粒体周围可观察到大量的高电子密度的分泌颗粒。 6 p93 mRNA的相对表达量的比较 Mod_(24H)的p93 mRNA相对表达量(0.47±0.07)与Con_(24H)(0.49±0.06)相比,无明显变化(P0.05);Mod_(2W)的p93 mRNA相对表达量(0.23±0.04)明显低于Con_(2W)(0.50±0.07)(P0.01);Mod4w的p93 mRNA相对表达量(0.54±0.09)与Con4w(0.51±0.08)相比,无明显变化(P0.05);Mod_(6W)的p93 mRNA相对表达量(1.12±0.16)明显高于Con_(6W)(0.41±0.07)(P0.01)。 7 PrxIII mRNA的相对表达量的比较 Mod_(24H)的PrxIII mRNA相对表达量(0.29±0.07)与Con_(24H)(0.65±0.10)相比,明显降低(P0.05);Mod_(2W)的PrxIIImRNA相对表达量(0.20±0.04)明显低于Con_(2W)(0.63±0.13)(P0.01);Mod4w的PrxIIImRNA相对表达量(0.34±0.02)与Con4w(0.61±0.08)相比,明显降低(P0.01);Mod_(6W)的PrxIII mRNA相对表达量(0.91±0.16)明显高于Con_(6W)(0.60±0.09)(P0.05)。 结论 1用7号针头结扎大鼠腹主动脉可成功制成压力超负荷心肌肥厚模型。 2血流动力学和心脏重量指数的变化与心肌肥厚的程度和心肌功能有关。 3随着心肌肥厚的出现及进展,心肌细胞的肌节逐渐延长,线粒体增多,线粒体嵴逐渐密集。 4随着超负荷心肌肥厚的发展,p93和PrxIII的表达逐渐增强,表明p93和PrxIII参与了超负荷心肌肥厚的发生,但其作用机制尚待研究。
[Abstract]:Heart failure myocardial hypertrophy is one of important compensation mechanism. Its main performance is the substance of cardiomyocyte hypertrophy and change (or cardiac remodeling), cardiac compliance and circulating pump function decreased. Myocardial hypertrophy, myocardial cell phenotype of contraction protein change accompanied by myocardial extracellular matrix remodeling, which is caused by two important factors to decrease of myocardial contractility. The irreversible progression of cardiac hypertrophy will inevitably lead to the occurrence of heart failure. Therefore, if you find a way to improve myocardial contractility approach will significantly reduce the incidence of heart failure.
The expression of P93 was recently discovered a cardiac specific protein kinase, the molecular weight is 93KD, it can undergo autophosphorylation, and cardiac troponin I (Cardiac troponin, CTN1), myocardial actin (a-action) and a series of contraction of the sarcomere protein interaction. That signal transduction pathway p93 may be an unknown is involved in the regulation of sarcomeric contractile proteins, thereby regulating cardiac contractility, participate in the process of cardiac hypertrophy.
The energy required for myocardial contraction from mitochondrial oxidative phosphorylation, but in the process of oxidative phosphorylation is accompanied by reactive oxygen species (reactive oxygen, species: ROS) the.PrxIII is related to protein P93, its function is to eliminate the excess ROS in the mitochondria, thereby protecting cells from oxidative stress injury. Therefore, in the heart when the load changes, myocardial cell contraction changes, the expression of related protein may also change. Studies have shown that: PrxIII in ventricular remodeling after myocardial infarction in the process of expression, but in the process of cardiac hypertrophy expression and its correlation with the expression of p93 has not been reported.
objective
In order to explore the expression changes of p93 and related protein PrxIII in the process of overload cardiac hypertrophy and its correlation with the morphological and functional changes of myocardial cells, we will lay the foundation for further p93 function research.
Method
Adult male SD rats 1 animal grouping and pressure overload in rat cardiac hypertrophy model was prepared with 190-200g, were randomly divided into sham operation group (Con): 24 hours (Con_ (24H)), 2 weeks (Con_ (2W)), 4 weeks (Con_ (4W)). 6 weeks (Con_ (6W)); model group (Mod): 24 hours (Mod_ (24H), 2 weeks (Mod_) (2W) (Mod_), 4 weeks (4W), 6 weeks (Mod_) (6W)). Rat cardiac hypertrophy induced by pressure overload model for reference methods Anversa P. Sham operation group only isolated above the left renal artery abdominal aortic ligation. The same, not other steps with the model group.
2 measurement index and method
2.1 hemodynamic index determination
Rats were treated as follows: weighing 6% chloral hydrate (5ml/kg body weight) intraperitoneal injection of anesthesia. The rats were fixed right carotid artery, analysis of the self-made polyethylene catheter system is inserted and taimeng BL-420E+ biological function experiment, after recording the right carotid systolic blood pressure (SBP), diastolic blood pressure (DBP). To insert into the left ventricle, recording of left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular systolic pressure maximal rate of rise (? Dp/dt_ (max)) and left ventricular diastolic pressure decline rate (-dp/dt_ (max)).
Determination of 2.2 cardiac weight index HMI
After the hemodynamic parameters were measured, the heart was removed quickly, and the remaining blood was washed by ice saline. After drying the surface water with filter paper, the heart weight was recorded and the heart weight was recorded. The ratio of heart weight (HW) to body weight (BW) was calculated, which is the heate mass index: HMI.
Observation of the morphologic structure of myocardium by 2.3 HE staining
After the heart was weighed, the ventricular septum and left ventricle were separated. The left ventricle was divided into two parts. One part was fixed in 4% paraformaldehyde fixative. Routine paraffin section was stained with HE, and the morphological changes were observed under optical microscope. In Japan, Nikon was photographed under optical microscope.
2.4 ultrastructural changes of left ventricle by electron microscopy
Another part of the left ventricular specimen was placed in the mixed fixed solution of 3% polyformaldehyde and 1% glutaraldehyde. The ultrastructural changes of the left ventricle were observed by electron microscopy.
Quantitation of p93 and PrxIIImRNA in the interventricular septum of 2.5 rats
The separated interventricular septum was rapidly stored in liquid nitrogen, stored at -70 C for RNA extraction. After extracting the total RNA from ventricular septum by Trizol, GAPDH was used as an internal reference. RT-PCR was used to measure the relative expression of interventricular septal p93 and Prx III mRNA in rats.
Result
1 blood pressure
Con_(24H)鐨凷BP涓，
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