抵抗素对自发性高血压大鼠肥厚心肌腺苷酸活化蛋白激酶信号通路的影响

发布时间：2018-06-18 23:19

本文选题：自发性高血压大鼠 + 心肌肥厚　；参考：《南方医科大学》2015年硕士论文

【摘要】：一、研究背景和立题依据心肌肥厚是导致心力衰竭、心律失常和猝死主要原因之一。本课题针对心肌肥厚的产生机制：抵抗素/脂旨联素/AMPK这一通路对心肌肥厚的影响提出。本课题对寻找新的治疗心肌肥厚及心衰的靶点有重要意义。实验由体内、体外实验完成,是在前人研究抵抗素、脂联素与心肌肥厚相关,激活腺苷酸活化蛋白激酶(AMPK)可抑制心肌肥厚基础上进一步深入探讨,通过注射载有抵抗素基因重组腺病毒注射液造模,并观察抵抗素对其血压收缩压(SBP)和脂联素(AN)及其下游蛋白酶AMPK信号转导子的影响。阐明抵抗素/脂联素/AMPK在心肌肥厚形成中的重要作用,为心肌肥厚的治疗提供新的靶点。二、研究目的探讨抵抗素对自发性高血压大鼠(spontaneous hypertensive rat, SHR)肥厚心肌腺苷酸活化蛋白激酶信号转导分子改变的影响。注射载有抵抗素基因重组腺病毒注射液大鼠造模,并观察抵抗素对其血压收缩压(SBP)和脂联素(AN)信号转导子的影响。三、材料与方法1.实验药品及主要试剂大鼠脂联素ELISA检测试剂盒,美国ADL公司。载有抵抗素基因重组腺病毒注射液1×1010PFU,3ml,上海吉凯基因化学技术有限公司。抗磷酸化AMPK抗体购自美国Cell Signaling Techno logy公司,非磷酸化AMPK抗体购自美国Santa Cruz公司,Trizo 1试剂购自美国Invitrogen公司,O ligo-dT、M-MLV逆转录酶、RNA酶抑制剂及Taq酶购自美国Promega公司,其他试剂均为分析纯产品。2.方法2.1实验动物8周龄Wistar大鼠及SHR大鼠,雄性,体重180-200 g,分别购自中山大学实验动物中心和北京维通利华实验动物技术有限公司提供,合格证号分别为SCXK(粤)2013-0001和SCXK(京)2013-0002。以8周龄Wistar大鼠作为正常对照,将同周龄SHR大鼠按体重随机分为4组, 即SHR组,高剂量组(载有抵抗素基因重组腺病毒注射液,剂量：1×109PFU/只),中剂量组(载有抵抗素基因重组腺病毒注射液,剂量：0.5×109PFU/只),低剂量组(载有抵抗素基因重组腺病毒注射液,剂量：0.25×109PFU/只)。2.3 ELISA检测血清脂联素水平经腹主动脉取血至离心管中, 室温静置1h,3000 r/m in离心10 m in后吸取血清,按试剂盒说明书测定血清脂联素水平。2.4 RT-PCR检测心肌AdipoRl的表达采用Trizo 1试剂盒提取心肌组织RNA,琼脂糖凝胶电泳鉴定RNA完整性,紫外分光光度法测定RNA的浓度和纯度。取RNA 5ug逆转录合成cDNA,进行半定量PCR反应。根据序列设计相应引物,引物序列见表1。PCR的扩增条件是：94℃预变性5 m in,94℃45 s-57℃ 45 s-72℃60s,30个循环后72℃充分延伸10 min。PCR产物经1.5%琼脂糖凝胶电泳, 凝胶扫描系统(DF-23B)英国UVP公司产品HZ-25。采用550 IW图像分析系统(LEICA公司)对PCR产物进行光密度扫描和分析,分别计算各指标的光密度比值。2.5 Western blot检测心肌AMPK的蛋白表达50 mg心肌组织研磨后放入20倍体积的裂解缓冲液(50mmol/L Tris-HCl, pH7.4,150mmol/L Nacl,40mmol/L NaF,5mmol/L EDTA,5mmol/L EGTA,2.175 mmol/L正钒酸钠,1% TritonX-100,0.1%脱氧胆酸钠,0.1%SDS,0.1%抑蛋白酶肽,1mmol/LPMSF)中1000×g离心10 min,收集上清,Lowry法测定蛋白浓度。配制SDS聚丙烯酰胺凝胶(4%浓缩胶,9%分离胶), 蛋白上样量为50ug,电泳(浓缩胶80V,分离胶150V)结束后,取出凝胶,将蛋白电转(200mA恒流冰浴转2h)至聚偏二氟乙烯膜(po lyvinylidene difuo ride, PVDF)上。PVDF膜用5%脱脂奶粉室温封闭2h,含0.05% Tween 20的PBS (PBST)冲洗后,用抗磷酸化AMPK(1：1000稀释)进行孵育,4℃过夜。经PBST冲洗后加入辣根过氧化物酶标记的二抗室温孵育2 h。PBST冲洗,用化学发光剂发光,在暗盒中胶片上曝光1-5 m in,显影、定影后观察结果。PVDF膜经洗膜液洗脱30min后加抗AMPK(1:1000稀释)孵育,其余操作同上。采用550IW图像分析系统对条带进行光密度扫描和分析。2.6统计学方法实验结果计量资料以均数±标准差表示,进行方差齐性检验,方差齐,多组组间比较采用方差分析,方差不齐采用秩和检验；计数资料采用计数(百分率)表达,率的比较采用贮检验。全部资料用采用SPSS 17.0统计软件进行统计分析。四、结果与Wistar大鼠比较,SHR大鼠血压收缩压、心肌肥大指数均显著升高(P0.05),血清脂联素(AN)、心肌组织AdipoR1、AMPK含量降低(P0.05)。与SHR大鼠比较, 给药高、中、低剂量组血压收缩压呈正相关；心肌肥大指数呈正相关；血清脂联素(AN)、心肌组织AdipoR1、AMPK含量呈负相关。五、结论SHR大鼠心肌肥厚与收缩压,脂联素及其下游激酶AMPK相关,抵抗素通过血压、脂联素发挥作用。血浆脂联素水平降低,心肌组织局部AdipoRl表达及AMPK活性下降,上述信号分子的变化可能是引起心肌肥厚及肥大心肌代谢改变的分子机制之一。
[Abstract]:First, the research background and the establishment of myocardial hypertrophy are one of the main causes of heart failure, arrhythmia and sudden death. This topic aims at the mechanism of cardiac hypertrophy: the effect of resistin / lipoid /AMPK on cardiac hypertrophy. This topic is important to find new targets for the treatment of cardiac hypertrophy and heart failure. In vivo and in vitro experiments were completed in the previous study of resistin, adiponectin was associated with myocardial hypertrophy, activated adenylate activated protein kinase (AMPK) can inhibit myocardial hypertrophy based on further in-depth study, by injection of resistin gene recombinant adenovirus injection model, and to observe its blood pressure systolic pressure (SBP) and lipoprotein The effect of AN and its downstream protease AMPK signal transduction. Elucidate the important role of resistin / Adiponectin /AMPK in the formation of myocardial hypertrophy and provide new targets for the treatment of myocardial hypertrophy. Two, the aim of this study was to explore the effect of resistin on the activation of adenylate activation protein in hypertrophic myocardium of spontaneously hypertensive rats (spontaneous hypertensive rat, SHR). Effect of enzyme signal transduction molecular changes. Injection of resistin gene recombinant adenovirus injection rat model, and the effect of resistin on its blood pressure systolic pressure (SBP) and adiponectin (AN) signal transduction. Three, materials and methods 1. experimental drugs and major reagents, lipoprotein ELISA detection kit, American ADL. Recombinant adenovirus injection 1 x 1010PFU, 3ml, Shanghai Jikai genetic Chemical Technology Co., Ltd.. Anti phosphorylated AMPK antibody purchased from American Cell Signaling Techno logy company. Non phosphorylated AMPK antibodies were purchased from American Santa Cruz company, Trizo 1 reagent purchased from American Invitrogen Gong, retroactive reverse transcriptase, inhibitor The Q enzyme was purchased from the Promega company of the United States. The other reagents were used to analyze the 8 week old Wistar rats and SHR rats of the pure product.2. method 2.1, the male and the weight 180-200 g, respectively purchased from the experimental animal center of Zhongshan University and the Beijing vitamin D experimental animal technology Co., Ltd., respectively, the joint certificate number was SCXK (Guangdong) 2013-0001 and SCXK (Beijing) 2013-0, respectively. 2 with 8 weeks old Wistar rats as normal control, the same week old SHR rats were randomly divided into 4 groups, namely, group SHR, high dose group (carrying resistin gene recombinant adenovirus injection, dosage: 1 x 109PFU/), medium dose group (carrying resistin gene recombinant adenovirus injection, dosage: 0.5 x 109PFU/), low dose group (carrying resistin) Recombinant adenovirus injection, dose: 0.25 x 109PFU/ only).2.3 ELISA detection of serum adiponectin level through the abdominal aorta to the centrifuge tube, at room temperature static 1H, 3000 r/m in centrifugation 10 m in after the absorption of serum, according to the reagent box instructions to determine the level of serum adiponectin level.2.4 RT-PCR detection of the AdipoRl expression of the myocardium using Trizo 1 Kit extract Myocardial tissue RNA, agarose gel electrophoresis identification of RNA integrity, ultraviolet spectrophotometry determination of the concentration and purity of RNA. RNA 5ug reverse transcriptase cDNA, semi quantitative PCR reaction. According to sequence design of the corresponding primers, primer sequence of the sequence of 1.PCR: 94 degrees C predenaturation of 5 m in, 94, 45 S-57, 45 S-72, 30 degrees, 30 evidence-based evidence-based After 72 centigrade, 10 min.PCR products were fully extended by 1.5% agarose gel electrophoresis, gel scanning system (DF-23B), British UVP company product HZ-25. used 550 IW image analysis system (LEICA company) to carry out light density scanning and Analysis on PCR products, and calculated the light density ratio value.2.5 Western blot of each index, respectively, to detect the protein expression 50 m with 50 m. G 50mmol/L Tris-HCl, pH7.4150mmol/L Nacl, 40mmol/L NaF, 5mmol/L EDTA, 5mmol/L EGTA, 2.175 mmol/L sodium vanadate, 1% sodium deoxycholate, 0.1% anti protease peptide, 1000 x centrifugation 10 The SDS polyacrylamide gel (4% concentrated glue, 9% separation glue), the protein sample amount of 50ug, the gel electrophoresis (concentrated glue 80V, separation glue 150V), the gel was removed, the protein was converted (200mA constant flow ice bath to 2H) to the polyvinylidene fluoride membrane (PO lyvinylidene difuo ride, PVDF) on the.PVDF membrane with 5% skim milk powder at room temperature, containing 0.05% 20 After flushing BS (PBST), incubated with anti phosphorylation AMPK (1:1000 dilution), 4 C for overnight. After PBST flushing, two anti room temperature incubated with horseradish peroxidase, 2 h.PBST rinse, chemiluminescence, exposure to 1-5 m in on the film on the dark box, developing, and the results were observed, and the.PVDF film was washed away 30min after 30min plus AMPK (1:). 1000 dilution) incubation, the rest of the same operation. The 550IW image analysis system was used to carry out light density scanning and analysis of.2.6 statistical methods. The measurement data of the experimental results were expressed in the mean number of mean deviation, and the variance was tested and the variance was homogeneous. The variance was compared, the rank sum test was used for the variance of the variance, and the count data were adopted. SPSS 17 statistical software was used for statistical analysis. Four. Four. Compared with Wistar rats, the blood pressure systolic pressure of SHR rats increased significantly (P0.05), serum adiponectin (AN), myocardial tissue AdipoR1, AMPK content decreased (P0.05). Compared with SHR rats, the results were compared with that of SHR rats. The blood pressure systolic pressure was positively correlated with the low dose group and the low dose group, and the myocardial hypertrophy index was positively correlated; the serum adiponectin (AN), the myocardial tissue AdipoR1 and AMPK content showed negative correlation. Five, conclusion the myocardial hypertrophy and systolic pressure of the SHR rats, the adiponectin and its downstream kinase AMPK, the resistance of the adiponectin to the blood pressure, and the plasma adiponectin level drop. The expression of local AdipoRl and the activity of AMPK decreased in the myocardium, and the change of these signal molecules may be one of the molecular mechanisms that cause the changes of myocardial hypertrophy and hypertrophic myocardium.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R544.1

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