發(fā)布時(shí)間：2018-06-18 23:19

  本文選題：自發(fā)性高血壓大鼠 + 心肌肥厚��； 參考：《南方醫(yī)科大學(xué)》2015年碩士論文

【摘要】：一、研究背景和立題依據(jù)心肌肥厚是導(dǎo)致心力衰竭、心律失常和猝死主要原因之一。本課題針對心肌肥厚的產(chǎn)生機(jī)制：抵抗素/脂旨聯(lián)素/AMPK這一通路對心肌肥厚的影響提出。本課題對尋找新的治療心肌肥厚及心衰的靶點(diǎn)有重要意義。實(shí)驗(yàn)由體內(nèi)、體外實(shí)驗(yàn)完成,是在前人研究抵抗素、脂聯(lián)素與心肌肥厚相關(guān),激活腺苷酸活化蛋白激酶(AMPK)可抑制心肌肥厚基礎(chǔ)上進(jìn)一步深入探討,通過注射載有抵抗素基因重組腺病毒注射液造模,并觀察抵抗素對其血壓收縮壓(SBP)和脂聯(lián)素(AN)及其下游蛋白酶AMPK信號轉(zhuǎn)導(dǎo)子的影響。闡明抵抗素/脂聯(lián)素/AMPK在心肌肥厚形成中的重要作用,為心肌肥厚的治療提供新的靶點(diǎn)。二、研究目的探討抵抗素對自發(fā)性高血壓大鼠(spontaneous hypertensive rat, SHR)肥厚心肌腺苷酸活化蛋白激酶信號轉(zhuǎn)導(dǎo)分子改變的影響。注射載有抵抗素基因重組腺病毒注射液大鼠造模,并觀察抵抗素對其血壓收縮壓(SBP)和脂聯(lián)素(AN)信號轉(zhuǎn)導(dǎo)子的影響。三、材料與方法1.實(shí)驗(yàn)藥品及主要試劑大鼠脂聯(lián)素ELISA檢測試劑盒,美國ADL公司。載有抵抗素基因重組腺病毒注射液1×1010PFU,3ml,上海吉凱基因化學(xué)技術(shù)有限公司�？沽姿峄疉MPK抗體購自美國Cell Signaling Techno logy公司,非磷酸化AMPK抗體購自美國Santa Cruz公司,Trizo 1試劑購自美國Invitrogen公司,O ligo-dT、M-MLV逆轉(zhuǎn)錄酶、RNA酶抑制劑及Taq酶購自美國Promega公司,其他試劑均為分析純產(chǎn)品。2.方法2.1實(shí)驗(yàn)動物8周齡Wistar大鼠及SHR大鼠,雄性,體重180-200 g,分別購自中山大學(xué)實(shí)驗(yàn)動物中心和北京維通利華實(shí)驗(yàn)動物技術(shù)有限公司提供,合格證號分別為SCXK(粵)2013-0001和SCXK(京)2013-0002。以8周齡Wistar大鼠作為正常對照,將同周齡SHR大鼠按體重隨機(jī)分為4組, 即SHR組,高劑量組(載有抵抗素基因重組腺病毒注射液,劑量：1×109PFU/只),中劑量組(載有抵抗素基因重組腺病毒注射液,劑量：0.5×109PFU/只),低劑量組(載有抵抗素基因重組腺病毒注射液,劑量：0.25×109PFU/只)。2.3 ELISA檢測血清脂聯(lián)素水平經(jīng)腹主動脈取血至離心管中, 室溫靜置1h,3000 r/m in離心10 m in后吸取血清,按試劑盒說明書測定血清脂聯(lián)素水平。2.4 RT-PCR檢測心肌AdipoRl的表達(dá)采用Trizo 1試劑盒提取心肌組織RNA,瓊脂糖凝膠電泳鑒定RNA完整性,紫外分光光度法測定RNA的濃度和純度。取RNA 5ug逆轉(zhuǎn)錄合成cDNA,進(jìn)行半定量PCR反應(yīng)。根據(jù)序列設(shè)計(jì)相應(yīng)引物,引物序列見表1。PCR的擴(kuò)增條件是：94℃預(yù)變性5 m in,94℃45 s-57℃ 45 s-72℃60s,30個(gè)循環(huán)后72℃充分延伸10 min。PCR產(chǎn)物經(jīng)1.5%瓊脂糖凝膠電泳, 凝膠掃描系統(tǒng)(DF-23B)英國UVP公司產(chǎn)品HZ-25。采用550 IW圖像分析系統(tǒng)(LEICA公司)對PCR產(chǎn)物進(jìn)行光密度掃描和分析,分別計(jì)算各指標(biāo)的光密度比值。2.5 Western blot檢測心肌AMPK的蛋白表達(dá)50 mg心肌組織研磨后放入20倍體積的裂解緩沖液(50mmol/L Tris-HCl, pH7.4,150mmol/L Nacl,40mmol/L NaF,5mmol/L EDTA,5mmol/L EGTA,2.175 mmol/L正釩酸鈉,1% TritonX-100,0.1%脫氧膽酸鈉,0.1%SDS,0.1%抑蛋白酶肽,1mmol/LPMSF)中1000×g離心10 min,收集上清,Lowry法測定蛋白濃度。配制SDS聚丙烯酰胺凝膠(4%濃縮膠,9%分離膠), 蛋白上樣量為50ug,電泳(濃縮膠80V,分離膠150V)結(jié)束后,取出凝膠,將蛋白電轉(zhuǎn)(200mA恒流冰浴轉(zhuǎn)2h)至聚偏二氟乙烯膜(po lyvinylidene difuo ride, PVDF)上。PVDF膜用5%脫脂奶粉室溫封閉2h,含0.05% Tween 20的PBS (PBST)沖洗后,用抗磷酸化AMPK(1：1000稀釋)進(jìn)行孵育,4℃過夜。經(jīng)PBST沖洗后加入辣根過氧化物酶標(biāo)記的二抗室溫孵育2 h。PBST沖洗,用化學(xué)發(fā)光劑發(fā)光,在暗盒中膠片上曝光1-5 m in,顯影、定影后觀察結(jié)果。PVDF膜經(jīng)洗膜液洗脫30min后加抗AMPK(1:1000稀釋)孵育,其余操作同上。采用550IW圖像分析系統(tǒng)對條帶進(jìn)行光密度掃描和分析。2.6統(tǒng)計(jì)學(xué)方法實(shí)驗(yàn)結(jié)果計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示,進(jìn)行方差齊性檢驗(yàn),方差齊,多組組間比較采用方差分析,方差不齊采用秩和檢驗(yàn)；計(jì)數(shù)資料采用計(jì)數(shù)(百分率)表達(dá),率的比較采用貯檢驗(yàn)。全部資料用采用SPSS 17.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。四、結(jié)果與Wistar大鼠比較,SHR大鼠血壓收縮壓、心肌肥大指數(shù)均顯著升高(P0.05),血清脂聯(lián)素(AN)、心肌組織AdipoR1、AMPK含量降低(P0.05)。與SHR大鼠比較, 給藥高、中、低劑量組血壓收縮壓呈正相關(guān)；心肌肥大指數(shù)呈正相關(guān)；血清脂聯(lián)素(AN)、心肌組織AdipoR1、AMPK含量呈負(fù)相關(guān)。五、結(jié)論SHR大鼠心肌肥厚與收縮壓,脂聯(lián)素及其下游激酶AMPK相關(guān),抵抗素通過血壓、脂聯(lián)素發(fā)揮作用。血漿脂聯(lián)素水平降低,心肌組織局部AdipoRl表達(dá)及AMPK活性下降,上述信號分子的變化可能是引起心肌肥厚及肥大心肌代謝改變的分子機(jī)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R544.1

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