本文關鍵詞：p93基因在大鼠超負荷心肌肥厚過程中的表達變化　出處：《河北醫(yī)科大學》2007年碩士論文　論文類型：學位論文

  更多相關文章： 壓力超負荷心肌肥厚模型 p93 PrxIII 活性氧族 氧化應激

【摘要】： 心肌肥厚是心力衰竭時機體重要的代償反應之一。其主要表現(xiàn)為心肌細胞的肥大和間質(zhì)成分的改變(或稱心臟重構),心臟順應性和循環(huán)泵功能降低。心肌肥厚時,心肌細胞內(nèi)收縮蛋白表型改變,同時伴有心肌細胞外基質(zhì)改建,這是造成心肌收縮性降低的兩個重要因素。心肌肥厚的不可逆性進展必然會導致心力衰竭的發(fā)生。因此,如果找到一條能改變或提高心肌收縮能力的途徑必然會大大降低心力衰竭的發(fā)病率。 P93是近年來新發(fā)現(xiàn)的一個心肌特異性表達的蛋白激酶,分子量為93KD,可以進行自身磷酸化,它與心肌肌鈣蛋白Ⅰ(Cardiac troponin, CTN1)、心肌肌動蛋白(a-action)等一系列與收縮有關的肌小節(jié)蛋白發(fā)生相互作用。推測p93可能通過一條未知的信號轉(zhuǎn)導通路參與了對肌小節(jié)收縮蛋白的調(diào)控,進而調(diào)控心肌收縮能力,參與心肌肥厚的發(fā)生過程。 心肌收縮所需能量來自于線粒體的氧化磷酸化,但在氧化磷酸化過程中亦伴隨著活性氧族(reactive oxygen species :ROS)的產(chǎn)生。PrxIII是P93的相關蛋白,其作用主要是清除線粒體內(nèi)多余的ROS,從而保護細胞免受氧化應激損傷。因此,在心肌負荷發(fā)生變化時,心肌細胞的收縮力隨之變化,其相關蛋白的表達亦可能發(fā)生變化。有研究證實:PrxIII在心肌梗塞后心室重構過程中表達增強,但其在心肌肥厚過程中的表達變化,及其與p93表達的相關性尚未見報道。 目的 為了探討p93及相關蛋白PrxIII在超負荷心肌肥厚過程中的表達變化,及其與心肌細胞形態(tài)和功能變化的相關性,為進一步的p93功能研究奠定基礎。 方法 1動物分組及壓力超負荷大鼠心肌肥厚模型的制備選用190-200g的成年雄性SD大鼠,隨機分為:①假手術對照組(Con): 24小時(Con_(24H))、2周(Con_(2W))、4周(Con_(4W))、6周(Con_(6W));②模型組(Mod): 24小時(Mod_(24H))、2周(Mod_(2W))、4周(Mod_(4W))、6周(Mod_(6W))。大鼠壓力超負荷心肌肥厚模型的制備參考Anversa P的方法。假手術對照組只分離左腎動脈上方的腹主動脈,并不結扎。其他步驟與模型組相同。 2測定指標及方法 2.1血流動力學指標測定 各組大鼠做如下處理:稱重,6%水合氯醛(5ml/kg體重)腹腔注射麻醉。固定大鼠,分離右側頸總動脈,插入和泰盟BL-420E+生物機能實驗分析系統(tǒng)相連的自制聚乙烯導管,穩(wěn)定后記錄右頸總收縮壓(SBP)、舒張壓(DBP),繼續(xù)插入至左心室,記錄左室收縮壓(LVSP)、左室舒張末壓(LVEDP)以及左心室收縮壓最大上升速率(㧏dp/dt_(max))和左心室舒張壓最大下降速率(-dp/dt_(max))。 2.2心臟重量指數(shù)HMI的測定 血流動力學指標測定完畢后,開胸迅速取出心臟,冰生理鹽水洗掉余血,用濾紙吸干表面水分后稱重并記錄心臟重量,計算心臟重量(HW)與體重(BW)的比值,即為心臟重量指數(shù)(heate mass index:HMI)。 2.3 HE染色觀察心肌的形態(tài)結構 心臟稱重后,分離室間隔和左心室。將左心室分成兩部分,一部分放入4%多聚甲醛固定液內(nèi)固定。常規(guī)石蠟切片HE染色,于光學顯微鏡下觀察形態(tài)變化,日本產(chǎn)Nikon光學顯微鏡下拍照。 2.4電鏡觀察左心室的超微結構變化 將另一部分左心室標本放入3%多聚甲醛和1%戊二醛混合固定液中固定。用于電鏡觀察左心室的超微結構變化 2.5大鼠室間隔p93和PrxIIImRNA的定量 將分離的室間隔迅速至于液氮內(nèi),-70℃保存,用于RNA提取。用Trizol法提取室間隔總RNA后,以GAPDH作為內(nèi)參照,應用RT-PCR方法測定大鼠室間隔p93和Prx III mRNA的相對表達量。 結果 1血壓 Con_(24H)的SBP為12.13±0.69,DBP為9.47±0.98, Mod_(24H)的SBP為12.51±1.37,DBP為10.04±1.01,與Con_(24H)相比,Mod_(24H)的SBP和DBP沒有顯著變化(P0.05); Con_(2W)的SBP為12.33±0.52,DBP為8.99±0.94,Mod_(2W)的SBP為14.55±1.04,DBP為12.03±2.08,與Con_(2W)相比,Mod_(2W)的SBP和DBP均上升(P0.05);Con_(4W)的SBP為12.40±0.74,DBP為9.32±0.78,Mod_(4W)的SBP為16.26±2.41,DBP為12.10±1.61。與Con_(4W)相比,Mod_(4W)的SBP和DBP也都有明顯上升(P0.01);Con_(6W)的SBP為12.71±1.35,DBP為9.41±1.08,Mod_(6W)的SBP為16.04±2.32,DBP為13.40±1.94,與Con_(6W)相比,Mod_(6W)的SBP和DBP均上升且有顯著性差異(P0.01)。 2左心室收縮和舒張功能 Con_(24H)的LVSP為12.37±0.74,LVEDP為0.66±0.07,㧏dp/dt_(max)為424±72,-dp/dt_(max)為406±58;Mod_(24H)的LVSP為12.58±1.01,LVEDP為0.71±0.10,㧏dp/dt_(max)為431±69,-dp/dt_(max)為419±63。與Con_(24H)相比,Mod_(24H)的LVSP、LVEDP及±dp/dt_(max)均沒有顯著變化(P0.05)。 Con_(2W)的LVSP為12.56±1.13,LVEDP為0.68±0.09,㧏dp/dt_(max)為416±77,-dp/dt_(max)為411±64;Mod_(2W)的LVSP為15.01±1.95,LVEDP為0.69±0.11,㧏dp/dt_(max)為491±85,-dp/dt_(max)為486±78。與Con_(2W)相比,Mod_(2W)的LVSP及±dp/dt_(max)增高,均有顯著性差異(P0.05),但LVEDP沒有顯著變化(P0.05)。 Con_(4W)的LVSP為12.55±1.06,LVEDP為0.70±0.08,㧏dp/dt_(max)為407±81,-dp/dt_(max)為398±66;Mod_(4W)的LVSP為16.47±2.16,LVEDP為0.81±0.08,㧏dp/dt_(max)為421±80,-dp/dt_(max)為404±57。與Con_(4W)相比,Mod_(4W)的LVSP明顯增高(P0.01),但LVEDP及±dp/dt_(max)均沒有顯著差異(P0.05)。 Con _(6W)的LVSP為12.77±1.87,LVEDP為0.75±0.09,㧏dp/dt_(max)為421±76, -dp/dt_(max)為410±65;Mod_(6W)的LVSP為16.16±1.54,LVEDP為1.83±0.22,㧏dp/dt_(max)為340±66, -dp/dt_(max)為338±52。與Con_(6W)相比,Mod_(6W)的LVSP及LVEDP顯著升高(P0.01),±dp/dt_(max)均有明顯下降(P0.05)。 3心臟重量指數(shù)HMI Con_(24H)的HMI為3.11±0.23,Mod_(24H)的HMI為3.15±0.19。Con_(2W)的HMI為3.07±0.49,Mod_(2W)的HMI為3.43±0.16,與各自的對照組相比,Mod_(24H)和Mod_(2W)的HMI均無明顯變化(P0.05);Con_(4W)的HMI為3.10±0.37,Mod_(4W)的HMI為3.95±0.30,Con_(6W)的HMI為2.94±0.27,Mod_(6W)的HMI為4.35±0.74,與各自的對照組相比,Mod_(4W)和Mod_(6W)的HMI均升高,有顯著性差異(P0.05)。 4大鼠左心室的光鏡觀察 各對照組心肌纖維排列整齊,胞核清晰。_(24H)模型組與對照組相比形態(tài)未見明顯改變。_(2W)、_(4W)、_(6W)模型組的心肌纖維逐漸較對照組寬大,肌纖維間隙逐漸增寬,間質(zhì)內(nèi)可見毛細血管擴張增生。 5大鼠左心室的電鏡觀察 各對照組大鼠心肌纖維排列整齊,明帶、暗帶及Z線清晰,線粒體結構正常。_(24H)模型組與對照組相比,結構未見明顯改變。_(2W)、_(4W)、_(6W)模型組的心肌肌節(jié)清晰,線粒體逐漸增多,并出現(xiàn)大量線粒體集結現(xiàn)象,其中以_(4W)和_(6W)最為明顯,線粒體嵴逐漸密集,_(6W)組的線粒體嵴較其它組明顯增厚,并且在心肌細胞內(nèi),特別是在線粒體周圍可觀察到大量的高電子密度的分泌顆粒。 6 p93 mRNA的相對表達量的比較 Mod_(24H)的p93 mRNA相對表達量(0.47±0.07)與Con_(24H)(0.49±0.06)相比,無明顯變化(P0.05);Mod_(2W)的p93 mRNA相對表達量(0.23±0.04)明顯低于Con_(2W)(0.50±0.07)(P0.01);Mod4w的p93 mRNA相對表達量(0.54±0.09)與Con4w(0.51±0.08)相比,無明顯變化(P0.05);Mod_(6W)的p93 mRNA相對表達量(1.12±0.16)明顯高于Con_(6W)(0.41±0.07)(P0.01)。 7 PrxIII mRNA的相對表達量的比較 Mod_(24H)的PrxIII mRNA相對表達量(0.29±0.07)與Con_(24H)(0.65±0.10)相比,明顯降低(P0.05);Mod_(2W)的PrxIIImRNA相對表達量(0.20±0.04)明顯低于Con_(2W)(0.63±0.13)(P0.01);Mod4w的PrxIIImRNA相對表達量(0.34±0.02)與Con4w(0.61±0.08)相比,明顯降低(P0.01);Mod_(6W)的PrxIII mRNA相對表達量(0.91±0.16)明顯高于Con_(6W)(0.60±0.09)(P0.05)。 結論 1用7號針頭結扎大鼠腹主動脈可成功制成壓力超負荷心肌肥厚模型。 2血流動力學和心臟重量指數(shù)的變化與心肌肥厚的程度和心肌功能有關。 3隨著心肌肥厚的出現(xiàn)及進展,心肌細胞的肌節(jié)逐漸延長,線粒體增多,線粒體嵴逐漸密集。 4隨著超負荷心肌肥厚的發(fā)展,p93和PrxIII的表達逐漸增強,表明p93和PrxIII參與了超負荷心肌肥厚的發(fā)生,但其作用機制尚待研究。
[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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