本文選題：心臟 + SR　； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:1.采用大鼠心臟冠脈結(jié)扎術(shù)制備心衰模型,觀察心臟的β3-AR對心肌結(jié)構(gòu)的影響。2.檢測SR 59230A對大鼠心臟MicroRNAs表達(dá)的影響,探討β3-AR對心臟MicroRNAs表達(dá)的可能作用機(jī)制。方法:1.心衰動(dòng)物模型的制備:將100只Sprague-Dawley(SD)大鼠(180-230g)隨機(jī)分為偽手術(shù)組(40只)和手術(shù)組(60只),手術(shù)組結(jié)扎心臟冠脈左前降支制備心衰模型,偽手術(shù)組實(shí)驗(yàn)大鼠心臟只穿線不結(jié)扎。術(shù)前記錄正常心電圖,術(shù)中實(shí)時(shí)監(jiān)測心電圖變化,術(shù)后八周對實(shí)驗(yàn)大鼠心臟做超聲心動(dòng)檢測,測定左心室收縮末期內(nèi)徑(LVIDS),左心室舒張末期內(nèi)徑(LVIDD),射血分?jǐn)?shù)(EF)和左心室短軸縮短率(%FS),確定心衰手術(shù)成功的大鼠為心衰組。2.動(dòng)物分組:將偽手術(shù)組大鼠隨機(jī)分為兩組,偽手術(shù)組(Sham control group,Sham組)和偽手術(shù)+SR 59230A組(Sham+SR group,Sham+SR組)。心衰組大鼠也隨機(jī)分為兩組:心衰組(CHF control group,CHF組)和心衰+SR 59230A組(CHF+SR group,CHF+SR組)。3.阻斷β3-AR對大鼠心臟MicroRNAs表達(dá)的影響:Sham+SR組和CHF+SR組大鼠腹腔注射1ml SR 59230A(濃度85mmol/l),Sham組和CHF組大鼠腹腔注射等量生理鹽水,每天兩次,連續(xù)注射7周后提取各組大鼠心臟RNA,采用NanoDrop2000C檢測RNA的濃度與質(zhì)量,通過microarray方法檢測β3-AR對大鼠心臟MicroRNAs表達(dá)的影響。4.心臟結(jié)構(gòu)的改變和可能的信號轉(zhuǎn)導(dǎo)途徑:取各組大鼠左室心肌組織于10%的福爾馬林溶液中固定,然后進(jìn)行石蠟包埋,組織切片,通過HE染色觀察心臟結(jié)構(gòu)和心肌細(xì)胞形態(tài)變化,免疫組織化學(xué)法檢測心肌β3-ar、nf-κbp65和p53等相關(guān)蛋白的表達(dá);同時(shí)取各組大鼠心臟冠脈結(jié)扎線下心肌組織做westernblot檢測,定量分析各組心肌組織β3-ar、nf-κbp65、p53和p53-phospho-serine15等蛋白的表達(dá),以確定β3-ar改變心臟結(jié)構(gòu)和功能的信號轉(zhuǎn)導(dǎo)途徑。結(jié)果:1.超聲心動(dòng)檢測結(jié)果:采用超高分辨率小動(dòng)物超聲實(shí)時(shí)影像系統(tǒng)測定sham組和chf組大鼠的lvidd、lvids及ef,并計(jì)算fs值,結(jié)果顯示模型成功的心衰組(chf)大鼠的心功能較偽手術(shù)組(sham)明顯下降。與sham組比較,chf組lvidd及l(fā)vids明顯增大(p0.05,p0.05),lvef及%fs則明顯降低(p0.01,p0.01),證明模型成功。2.sr59230a對大鼠心臟micrornas表達(dá)的影響:micrornamicroarray分析結(jié)果顯示大鼠在體給予sr59230a后,sham組與chf組有18種micrornas共同表達(dá)下調(diào),其中mir-125b-5p,mir-143-3p,mir-145-5p,mir-26a-5p,mir-30a-5p和mir-320-5p與nf-κb信號通路有關(guān)。3.he染色觀察各組心肌組織病理學(xué)改變:在sham組,心肌組織完好,未見損傷,心肌細(xì)胞排列整齊,核染色清晰;而在chf組,心肌組織大面積皺縮,心肌纖維腫脹,斷裂,間質(zhì)水腫。此外,心肌細(xì)胞排列紊亂,松散,經(jīng)sr59230a在體處理后,chf組大鼠心肌細(xì)胞排列紊亂減輕,心肌纖維斷裂減輕,炎性細(xì)胞減少,炎癥有逆轉(zhuǎn)趨勢。4.免疫組化結(jié)果:鏡下可見chf組大鼠心臟β3-ar較sham組表達(dá)增加(p0.01),nf-κbp65在胞核與胞質(zhì)均有表達(dá),在sham組均未見明顯表達(dá);chf組p53在胞核表達(dá)多于胞質(zhì)。免疫組化評分結(jié)果顯示,chf組nf-κbp65,p53表達(dá)較sham組增加(p0.01,p0.01);在體給予sr59230a后,chf組nf-κbp65,p53表達(dá)下降(p0.05,p0.05),而sham組nf-κbp65,p53表達(dá)增加(p0.01,p0.01)。5.westernblot結(jié)果:chf組β3-ar,nf-κbp65,p53-phospho-serine15蛋白表達(dá)明顯高于sham組(p0.05,p0.05,p0.05),chf組p53蛋白表達(dá)也高于sham組,但沒有顯著性差異。在體給予sr59230a后,chf組大鼠心臟nf-κbp65和p53-Phospho-Serine 15蛋白表達(dá)下降(P0.05,P0.01),但仍高于Sham組(P0.05,P0.05),而CHF+SR組與CHF組相比,p53蛋白表達(dá)沒有顯著性差異。Sham組大鼠在體給予SR 59230A后,NF-κB p65,p53和p53-Phospho-Serine 15蛋白表達(dá)均顯著增加(P0.05,P0.05,P0.05)。結(jié)論:1.在體阻斷β3-AR后,可緩解心衰大鼠心臟異常的形態(tài)結(jié)構(gòu)和炎癥傾向,提示β3-AR表達(dá)增加與心臟的損傷有關(guān)。2.給予SR 59230A可引起大鼠心臟MicroRNAs表達(dá)發(fā)生變化,其中miR-125b-5p,miR-143-3p,miR-145a-5p,miR-26a-5p,miR-30a-5p和miR-320-5p與NF-κB信號途徑有關(guān)。3.心衰大鼠心臟β3-AR,NF-κB p65和p53-Phospho-Serine 15表達(dá)增加,用SR 59230A阻斷β3-AR可以降低心衰大鼠心臟NF-κB p65和p53-Phospho-Serine 15的表達(dá),證明β3-AR在心臟的作用與p53磷酸化及NF-κB的表達(dá)有關(guān)。
[Abstract]:Objective: 1. rats with coronary artery ligation preparation to observe heart failure model, the effects of beta 3-AR on myocardial structure and.2. detection of SR 59230A expression of rat heart MicroRNAs, investigate the possible mechanism of beta 3-AR expression of cardiac MicroRNAs. Methods: 1. heart failure animal model preparation: 100 Sprague-Dawley (SD) rats (180-230g) were randomly divided into sham operation group (40 rats) and operation group (60 rats), operation group, ligation of the left anterior descending coronary artery in preparation of heart failure model, sham operation rats heart group without coronary artery ligation. The preoperative normal ECG recording, real-time monitoring the changes of electrocardiogram during the operation, after eight weeks of experimental rats cardiac echocardiography, determination of left ventricular end systolic diameter (LVIDS), left ventricular end diastolic diameter (LVIDD), ejection fraction (EF) and left ventricular fractional shortening (%FS), ensure successful operation of rat centering ring for heart failure group.2 Animal grouping: sham group rats were randomly divided into two groups, sham operation group (Sham control, group, Sham group) and sham operation group (+SR 59230A Sham+SR group, Sham+SR group). Heart failure group rats were randomly divided into two groups: heart failure group (CHF control, group, CHF group) and heart failure +SR 59230A group (CHF+SR group, CHF+SR group).3. beta blocking effects of 3-AR on the expression of MicroRNAs in rat hearts: Sham+SR group and CHF+SR group rats by intraperitoneal injection of 1ml SR 59230A (85mmol/l, Sham concentration) group and CHF group rats were injected with saline, two times a day, continuous 7 weeks after injection to extract heart RNA rats, the concentration and quality of NanoDrop2000C for the detection of RNA by microarray method to detect the effects of 3-AR on the expression of beta MicroRNAs rat heart.4. the change of heart structure and the possible signal transduction pathways: formalin fixed solution of left ventricular myocardium were measured in the 10%, Then paraffin embedded tissue sections, observe the structure of heart and myocardial cell morphology by HE staining, detection of myocardial beta 3-Ar immunohistochemical method, the expression of nf- K bp65 and p53 and other related proteins; at the same time, coronary artery ligation in rats heart line of myocardial Westernblot detection, quantitative analysis of myocardial tissue were beta 3-Ar nf-, NF kappa bp65, expression of p53 and p53-phospho-serine15 protein, signal transduction of beta 3-Ar to determine the changes of cardiac structure and function approach. Results: 1. echocardiography results: Determination of sham group and CHF group rat ultrasonic real-time imaging system using ultra high resolution small animal lvidd, lvids and EF, and calculate the FS the value showed a successful model of heart failure group (CHF) on heart function in rats compared with sham operated group (sham) decreased significantly. Compared with sham group, CHF group, lvidd and lvids increased significantly (P0.05, P0.05), LVEF and%fs were significantly reduced (P0.01 , P0.01), proved the successful model effects of.2.sr59230a on the expression of microRNAs in rat hearts: micrornamicroarray analysis results showed that the rats given SR59230A in vivo, sham group and CHF group, there are 18 kinds of common microRNAs expression, including mir-125b-5p, mir-143-3p, mir-145-5p, mir-26a-5p, observe the pathological changes of myocardial tissue pathological mir-30a-5p and mir-320-5p and nf- k the B signaling pathway on.3.he staining: in group sham, the myocardial tissue intact, no injury, myocardial cells arranged in neat, clear nuclear staining; while in the CHF group, the myocardial tissue area shrinkage, myocardial fiber swelling, fracture, interstitial edema. Moreover, myocardial cell disorder, loose, by SR59230A in vivo after treatment the myocardial cells of rats in the CHF group arranged in disorder reduced, myocardial fiber rupture, inflammatory cells reduce inflammation, a reversal of the trend of.4. immunohistochemical results: under the microscope, the rats of group CHF beta 3-Ar Sha heart m緇勮〃杈懼鍔，

本文編號：1733001