β3肾上腺素受体对大鼠心脏结构的影响及可能的机制
发布时间：2018-04-10 21:32
本文选题：心脏 + SR ； 参考：《山西医科大学》2017年硕士论文
【摘要】：目的:1.采用大鼠心脏冠脉结扎术制备心衰模型,观察心脏的β3-AR对心肌结构的影响。2.检测SR 59230A对大鼠心脏MicroRNAs表达的影响,探讨β3-AR对心脏MicroRNAs表达的可能作用机制。方法:1.心衰动物模型的制备:将100只Sprague-Dawley(SD)大鼠(180-230g)随机分为伪手术组(40只)和手术组(60只),手术组结扎心脏冠脉左前降支制备心衰模型,伪手术组实验大鼠心脏只穿线不结扎。术前记录正常心电图,术中实时监测心电图变化,术后八周对实验大鼠心脏做超声心动检测,测定左心室收缩末期内径(LVIDS),左心室舒张末期内径(LVIDD),射血分数(EF)和左心室短轴缩短率(%FS),确定心衰手术成功的大鼠为心衰组。2.动物分组:将伪手术组大鼠随机分为两组,伪手术组(Sham control group,Sham组)和伪手术+SR 59230A组(Sham+SR group,Sham+SR组)。心衰组大鼠也随机分为两组:心衰组(CHF control group,CHF组)和心衰+SR 59230A组(CHF+SR group,CHF+SR组)。3.阻断β3-AR对大鼠心脏MicroRNAs表达的影响:Sham+SR组和CHF+SR组大鼠腹腔注射1ml SR 59230A(浓度85mmol/l),Sham组和CHF组大鼠腹腔注射等量生理盐水,每天两次,连续注射7周后提取各组大鼠心脏RNA,采用NanoDrop2000C检测RNA的浓度与质量,通过microarray方法检测β3-AR对大鼠心脏MicroRNAs表达的影响。4.心脏结构的改变和可能的信号转导途径:取各组大鼠左室心肌组织于10%的福尔马林溶液中固定,然后进行石蜡包埋,组织切片,通过HE染色观察心脏结构和心肌细胞形态变化,免疫组织化学法检测心肌β3-ar、nf-κbp65和p53等相关蛋白的表达;同时取各组大鼠心脏冠脉结扎线下心肌组织做westernblot检测,定量分析各组心肌组织β3-ar、nf-κbp65、p53和p53-phospho-serine15等蛋白的表达,以确定β3-ar改变心脏结构和功能的信号转导途径。结果:1.超声心动检测结果:采用超高分辨率小动物超声实时影像系统测定sham组和chf组大鼠的lvidd、lvids及ef,并计算fs值,结果显示模型成功的心衰组(chf)大鼠的心功能较伪手术组(sham)明显下降。与sham组比较,chf组lvidd及lvids明显增大(p0.05,p0.05),lvef及%fs则明显降低(p0.01,p0.01),证明模型成功。2.sr59230a对大鼠心脏micrornas表达的影响:micrornamicroarray分析结果显示大鼠在体给予sr59230a后,sham组与chf组有18种micrornas共同表达下调,其中mir-125b-5p,mir-143-3p,mir-145-5p,mir-26a-5p,mir-30a-5p和mir-320-5p与nf-κb信号通路有关。3.he染色观察各组心肌组织病理学改变:在sham组,心肌组织完好,未见损伤,心肌细胞排列整齐,核染色清晰;而在chf组,心肌组织大面积皱缩,心肌纤维肿胀,断裂,间质水肿。此外,心肌细胞排列紊乱,松散,经sr59230a在体处理后,chf组大鼠心肌细胞排列紊乱减轻,心肌纤维断裂减轻,炎性细胞减少,炎症有逆转趋势。4.免疫组化结果:镜下可见chf组大鼠心脏β3-ar较sham组表达增加(p0.01),nf-κbp65在胞核与胞质均有表达,在sham组均未见明显表达;chf组p53在胞核表达多于胞质。免疫组化评分结果显示,chf组nf-κbp65,p53表达较sham组增加(p0.01,p0.01);在体给予sr59230a后,chf组nf-κbp65,p53表达下降(p0.05,p0.05),而sham组nf-κbp65,p53表达增加(p0.01,p0.01)。5.westernblot结果:chf组β3-ar,nf-κbp65,p53-phospho-serine15蛋白表达明显高于sham组(p0.05,p0.05,p0.05),chf组p53蛋白表达也高于sham组,但没有显著性差异。在体给予sr59230a后,chf组大鼠心脏nf-κbp65和p53-Phospho-Serine 15蛋白表达下降(P0.05,P0.01),但仍高于Sham组(P0.05,P0.05),而CHF+SR组与CHF组相比,p53蛋白表达没有显著性差异。Sham组大鼠在体给予SR 59230A后,NF-κB p65,p53和p53-Phospho-Serine 15蛋白表达均显著增加(P0.05,P0.05,P0.05)。结论:1.在体阻断β3-AR后,可缓解心衰大鼠心脏异常的形态结构和炎症倾向,提示β3-AR表达增加与心脏的损伤有关。2.给予SR 59230A可引起大鼠心脏MicroRNAs表达发生变化,其中miR-125b-5p,miR-143-3p,miR-145a-5p,miR-26a-5p,miR-30a-5p和miR-320-5p与NF-κB信号途径有关。3.心衰大鼠心脏β3-AR,NF-κB p65和p53-Phospho-Serine 15表达增加,用SR 59230A阻断β3-AR可以降低心衰大鼠心脏NF-κB p65和p53-Phospho-Serine 15的表达,证明β3-AR在心脏的作用与p53磷酸化及NF-κB的表达有关。
[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缁勮〃杈惧鍔，
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