肥厚型心肌病不同品系小鼠模型筛选及双丹提取物的干预和机制研究

发布时间：2018-07-02 14:44

本文选题：心肌肥厚模型 + 肥厚标志基因　；参考：《安徽医科大学》2016年博士论文

【摘要】：背景肥厚型心肌病(hypertrophic cardiomyopathy, HCM)是以心肌肥厚为特征,心肌细胞肥大和肌纤维排列紊乱等是其典型表现。目前认为HCM的病因多与遗传有关,是一种常染色体显性遗传病。胚胎基因p-肌球蛋白重链(β-myosin heavy chain,β-MHC)、脑利尿钠肽(brain natriuretic peptide, BNP)和心房利尿钠肽(atrial natriuretic peptide, ANP)等基因重新表达被认为是心脏肥厚的分子标志,检测这些基因的表达不仅可以肯定心脏肥厚的存在,而且可以判断肥厚的程度及预后。当前基因芯片技术显示,不同心肌肥厚模型引起基因表达水平的方向、数目和种类各不相同,如利用动-静脉瘘和去甲肾上腺素制备的心肌肥厚模型中,其发生变化的34个基因中有14个调节方向相反。肾上腹主动脉缩窄(renal abdominal aortic coarctation, AAC)、动静脉瘘(arteriovenous fistula, AVF)和异丙肾上腺素(isoproterenol, ISO)法制备的心肌肥厚模型,都是经典的且被国内外研究者广泛认可的模型,但这三种不同心肌肥厚模型间肥厚性标志基因ANP、BNP和β-MHC表达有无差异尚不清楚。因此采用AAC、AVF和ISO法制备心肌肥厚小鼠模型,检测ANP、BNP和β-MHC的表达差异,以期优选出心肌肥厚标志基因表达明显的造模方法。动物造模,离不开动物品系的选择,不同品系动物,采用相同的方法造模亦存在差异,这些差异多数是由不同的遗传背景所造成的。遗传背景不同,导致特定的基因表达水平亦不同,但目前以与人类遗传密切相关的HCM肥厚分子标志(ANP、BNP和β-MHC)为靶标,对不同品系小鼠HCM心肌肥厚模型进行筛选将产生何种影响尚不明确,因此比较不同品系小鼠心肌肥厚模型间ANP、BNP和β-MHC的表达差异性具有一定的必要性。目前治疗HCM主要方法有药物和手术治疗等。2015年版药典记载的双丹口服液,功能主治为养心活血、化瘀止痛。本课题组前期研究结果显示,双丹提取物中两种主要有效成分(丹酚酸B和丹皮酚组合的新复方,简称SP)具有协同作用,并在心肌缺血模型中观察到SP具有明显的舒张血管功能和对动-静脉瘘心肌肥厚模型小鼠具有一定的防治作用,但其防治作用与ANP、BNP和P-MHC的表达是否相关及SP作用机制尚未明确。据报道心肌肥厚与纤维化密切相关且伴随着TGF-β1表达上调,通常Smad蛋白被认为是TGF-β1信号通路下游介质之一,其中Smad2、3可促进TGF-β1刺激信号从细胞质向细胞核传导,而当前认为TGF-β1信号转导可以被Smad7抑制,Smad7可阻断信号转导,抑制相关靶基因的转录和蛋白表达,这主要是由于TGF-β1型受体被配体激活后,Smad7与激活型Smad可与其竞争性结合。因此本研究亦将探索SP对心肌肥厚小鼠ANF、BNP和β-MHC的表达及TGF-β/Smad信号通路的影响。目的1.从三种造模方法AAC、AVF和ISO中优选心肌肥厚标志性基因ANP、BNP和β-MHC表达明显的造模方式；2.从四种不同品系BALB/c、C57BL/6、ICR和KM小鼠心肌肥厚模型中,优选心肌肥厚标志性基因ANP、BNP和β-MHC表达明显的小鼠品系；3.在优选、制备理想心肌肥厚模型的基础上,探索SP对其模型小鼠ANP、BNP和β-MHC表达及TGF-β/Smad信号通路的影响。方法1.将C57BL/6小鼠随机分为对照组和模型组,造模组分别采用AVF、AAC和ISO法制备心肌肥厚模型；再取KM、ICR、C57BL/6和BALB/c四个品系小鼠分别随机分为对照组和模型组,后者均采用前面实验结果制备心肌肥厚模型。上述小鼠均在4周后,称取体重(body weight, BW),颈椎脱臼处死,称量心脏重量(heartweight, HW)和左室重量(left ventricular weight, LVW),计算心脏指数(HW/BW)和左心室指数(LVW/BW).一部分心脏组织标本分离后快速冲洗、分装,并于液氮冻存,以备后续采用qRT-PCR检测；另一部分心脏组织用生理盐水漂洗,10%甲醛固定24h后,经冲洗、脱水、硬化、透明、浸渗等步骤后进行石蜡包埋、切片、苏木精-伊红(hematoxylin-eosin, HE)染色、摄片和组织芯片制作、免疫组织化学法观察ANP、BNP和β-MHC蛋白表达。2.将上述实验优选的小鼠品系,随机分为对照组和造模组,后者采用前面实验优选的方法制备,2周后将造模组随机分为：模型,阳性药和SP大、中、小剂量组,并开始灌胃给药至第4周末。阳性药组每天按35mg/kg给予卡托普利片,SP大、中、小剂量组分别每天按60,30和15mg/kg给予SP溶液,对照组和模型组给予等容量溶媒。末次给药后2小时,称取各小鼠BW,颈椎脱臼处死,称量HW和LVW,计算HW/BW和LVW/BW。一部心脏组织分装冻存,用于qRT-PCR和Western blot检测；另一部分心脏组织HE染色、摄片和组织芯片制作、免疫组织化学法观察ANP、BNP和β-MHC蛋白表达。结果1.不同方法诱导心肌肥厚模型小鼠间ANP、BNP和β-MHC的表达差异采用AAC、AVF和ISO法制备的心肌肥厚小鼠心脏肥厚指数HW/BW、 LVW/BW显著增加,在高倍镜下,可见心肌细胞肥大、排列稀疏和间质纤维化,提示采用AAC、AVF和ISO法诱导小鼠心肌肥厚模型是成功的；与AAC法比较,ISO组HW/BW、LVW/BW和AVF组HW/BW明显减轻,AVF和ISO组小鼠心肌室壁增厚、心室腔狭窄亦较轻,提示采用AAC法制备小鼠心肌肥厚模型产生的心肌肥厚程度要优于AVF和ISO法。采用AAC和AVF法制备的心肌肥厚小鼠心脏左心室ANP、BNP和β-MHC mRNA转录和蛋白表达水平均明显增加,同样ISO法小鼠ANP和BNP mRNA转录和蛋白表达水平亦显著增加,但β-MHC mRNA转录和蛋白表达水平仅有增加趋势；将三种方法AAC、AVF和ISO制备的心肌肥厚模型进行相互比较,显示AAC法小鼠左心室ANE、BNP和β-MHC mRNA转录和蛋白表达水平增加最明显,提示采用AAC法上调心肌肥厚模型小鼠左心室ANP、BNP和β-MHC mRNA转录和蛋白水平要优于AVF和ISO法。2.不同品系小鼠心肌肥厚模型ANP、BNP和P-MHC的表达差异采用四个品系小鼠C57BL/6、BALB/c、 KM和ICR制备心肌肥厚模型,其中C57BL/6、BALB/c、KM小鼠心脏肥厚指数HW/BW、LVW/BW显著增加,ICR小鼠HW/BW和LVW/BW仅有增加的趋势,HE染色显示四个品系小鼠心肌细胞肥大,部分肌纤维断裂,细胞间隙增加,提示采用C57BL/6、KM、BALB/c小鼠制备心肌肥厚模型是成功的,ICR小鼠不够理想；将四个品系小鼠C57BL/6、 BALB/c、KM和ICR制备的心肌肥厚模型进行相互比较,显示C57BL/6小鼠心脏肥厚指数HW/BW和LVW/BW增加最明显,其次为KM小鼠,提示采用C57BL/6小鼠制备小鼠心肌肥厚模型产生的心肌肥厚程度要优于BALB/c、KM和ICR。采用四个品系小鼠C57BL/6、BALB/c、KM和ICR制备心肌肥厚模型,其中C57BL/6、KM小鼠心脏左心室ANP、BNP和β-MHC mRNA转录和蛋白表达水平均明显增加,而BALB/c小鼠BNP和ICR小鼠ANP、BNP mRNA转录和蛋白水平仅有增加趋势；将四个品系小鼠C57BL/6、BALB/c、KM和ICR制备的心肌肥厚模型进行相互比较,显示C57BL/6小鼠左心室ANP、BNP和β-MHC mRNA转录和蛋白表达水平增加最明显,提示采用C57BL/6小鼠制备的心肌肥厚模型ANP. BNP和β-MHC mRNA转录和蛋白水平要优于BALB/c、KM和ICR小鼠。3.SP对心肌肥厚小鼠ANP、BNP和β-MHC表达及TGF-β/Smad信号通路影响采用AAC法制备C57BL/6小鼠心肌肥厚模型,显示造模小鼠心脏肥厚指数HW/BW和LVW/BW明显增加,心脏体积增加,心肌细胞肥大,部分肌纤维断裂,细胞间隙增加,提示本次实验的模型是成功的；CAP和SP组大、中剂量均可使模型小鼠肥厚指数HW/BW和LVW/BW显著降低,心脏体积减小,肌纤维断裂程度减轻,细胞间隙变小,且CAP和SP比较无显著差异,提示SP可明显降低AAC法致心肌肥厚模型C57BL/6小鼠心脏肥厚指数HW/BW、LVW/BW和心肌病理性损伤,其作用强度与卡托普利片相当。采用AAC法制备C57BL/6小鼠心肌肥厚模型,显示造模小鼠心脏左心室ANP、BNP和β-MHC mRNA转录和蛋白表达水平明显增强；CAP和SP组大、中剂量均可使造模小鼠左心室ANP、BNP和β-MHC mRNA转录和蛋白表达水平明显降低,且CAP和SP比较无显著差异,提示SP可明显降低AAC法致心肌肥厚模型C57BL/6小鼠左心室ANP、BNP和β-MHC mRNA转录和蛋白表达水平,其作用强度与卡托普利片相当。采用AAC法制备C57BL/6小鼠心肌肥厚模型,显示造模小鼠心脏左心室TGF-p1. Smad2和Smad3 mRNA转录水平显著增加,但Smad7 mRNA转录水平无明显差异；CAP和SP组大、中剂量均可显著下调造模小鼠左心室TGF-β1、Smad2、 Smad3 mRNA转录和Smad3蛋白表达水平,明显上调Smad7 mRNA转录和蛋白表达水平,提示SP对心肌肥厚的防治作用机制可能与调控TGF-β/Smad信号通路有关。结论1.AVF、AAC和ISO三种方法制备小鼠心肌肥厚模型,以心肌肥厚小鼠左心室ANP、BNP和β-MHC表达为筛选条件,结果表明以AAC法制备的小鼠心肌肥厚模型最理想；2.心肌肥厚小鼠左心室ANP、BNP和β-MHC表达为筛选条件,AAC法制备C57BL/6、BALB/c、KM和ICR四种不同品系小鼠心肌肥厚模型中,以C57BL/6品系小鼠模型最理想；3.SP明显抑制AAC法诱导的C57BL/6小鼠的心肌肥厚,抑制心肌肥厚小鼠的ANP、BNP和β-MHC的表达；4.SP抑制小鼠心肌肥厚的作用机制可能与调控TGF-β/Smad信号通路有关。
[Abstract]:Background hypertrophic cardiomyopathy (hypertrophic cardiomyopathy, HCM) is characterized by cardiac hypertrophy, cardiomyocyte hypertrophy and disorder of muscle fiber arrangement. It is believed that the etiology of HCM is mostly associated with heredity, and is an autosomal dominant genetic disease. The embryo gene p- myosin heavy chain (beta -myosin heavy chain, beta -MHC), brain benefit The reexpression of brain natriuretic peptide (BNP) and atrial natriuretic peptide (atrial natriuretic peptide, ANP) is considered as a molecular marker of cardiac hypertrophy. The expression of these genes can not only confirm the existence of cardiac hypertrophy, but also determine the degree and prognosis of hypertrophy. Current gene chip technology shows that The number and type of gene expression level in different cardiac hypertrophy models are different. For example, 14 of the 34 genes in the 34 genes that have changed by using arteriovenous fistula and norepinephrine have the opposite direction. The upper abdominal aorta coarctation (renal abdominal aortic coarctation, AAC), and arteriovenous fistula (a) The model of cardiac hypertrophy prepared by rteriovenous fistula, AVF) and isoproterenol (isoproterenol, ISO) method is classic and widely recognized by researchers at home and abroad. However, there is no clear difference between these three different hypertrophy models of hypertrophy gene ANP, BNP and beta -MHC. Therefore, AAC, AVF and ISO are used. The difference of expression of ANP, BNP and beta -MHC was detected in the model of myocardial hypertrophy, in order to optimize the modeling method for the expression of cardiac hypertrophy marker gene. Animal model, the selection of animal strains, different strains of animals, and the same methods were also different, most of these differences were caused by different genetic background. The genetic background is different and the specific gene expression level is different, but at present, the HCM hypertrophy molecular markers (ANP, BNP and beta -MHC) which are closely related to human heredity are the targets. The effect of screening on the HCM myocardial hypertrophy model of different strains of mice is not clear. Therefore, ANP, BNP between different strains of murine myocardial hypertrophy model is compared. The difference between the expression of HCM and beta -MHC is necessary. At present, the main methods for the treatment of -MHC are double Dan oral liquid recorded in the Pharmacopoeia of medicine and operation, and the function is to nourish heart and promote blood circulation and relieve the pain and relieve pain. The new compound, called SP, has synergistic effect. In the myocardial ischemia model, SP has obvious vasodilatation function and has some preventive effect on the model mice with arteriovenous fistula myocardial hypertrophy, but its effect is related to the expression of ANP, BNP and P-MHC, and the mechanism of SP is not clear. It is reported that the myocardial hypertrophy and fibrinolysis is reported. It is closely related and up-regulated with the expression of TGF- beta 1. Generally, Smad protein is considered to be one of the downstream mediators of the TGF- beta 1 signaling pathway, in which Smad2,3 can promote the conduction of TGF- beta 1 stimulation signal from the cytoplasm to the nucleus. At present, it is believed that TGF- beta 1 signal transduction can be suppressed by Smad7, Smad7 can block signal transduction and inhibit the transfer of related target genes. The transcription and protein expression is mainly due to the competitive binding of Smad7 and activator Smad after the TGF- beta 1 receptor is activated by the ligand. Therefore, this study will also explore the effect of SP on the expression of ANF, BNP and beta -MHC and the TGF- beta /Smad signaling pathway in myocardial hypertrophy mice. Objective 1. the markers of cardiac hypertrophy in AAC, AVF and ISO were selected from three modeling methods. The expression of sex genes ANP, BNP and beta -MHC expressed a significant model. 2. from the four different strains of BALB/c, C57BL/6, ICR and KM mouse cardiac hypertrophy, the selection of cardiac hypertrophy marker gene ANP, BNP and beta -MHC expressed obviously in mice; 3. on the basis of optimal selection of ideal myocardial fat thickness model, SP on its model mice ANP The expression of beta -MHC and the effect of TGF- beta /Smad signaling pathway. Method 1. C57BL/6 mice were randomly divided into control group and model group. The model of myocardial hypertrophy was prepared by AVF, AAC and ISO, and KM, ICR, C57BL/6 and BALB/c were randomly divided into the control group and the model group, the latter were prepared by the previous experimental results. The model of muscle hypertrophy. After 4 weeks, the mice were weighed (body weight, BW), the cervical dislocations were killed, the weight of the heart (heartweight, HW) and the left ventricular weight (left ventricular weight, LVW) were weighed, and the cardiac index (HW/BW) and the left ventricular index (LVW/BW) were calculated. QRT-PCR test was used for follow-up; the other part of the heart tissue was rinsed with saline and 10% formaldehyde was fixed for 24h. After washing, dehydration, hardened, transparent, impregnated and other steps, paraffin was embedded, sliced, hematoxylin eosin (hematoxylin-eosin, HE) staining, film and tissue chips were made, and ANP, BNP and beta -MHC eggs were observed by immunohistochemistry. The white expression.2. was divided into the control group and the model group randomly. The latter was prepared by the preferred method of the previous experiment. After 2 weeks, the model was randomly divided into the model, the positive drug and the SP large, medium, small dose group, and began to fill the stomach for the fourth weekend. The positive drug group was given Captopril Tablets, SP, medium every day. The small dose groups were given SP solution by 60,30 and 15mg/kg each day respectively. The control group and the model group were given equal volume solvent. 2 hours after the last administration, the mice were given BW, the cervical dislocations were killed, the HW and LVW were weighed, and the HW/BW and LVW/BW. a cardiac tissue was frozen, used for qRT-PCR and Western blot detection; the other part of the heart tissue was stained HE, The expression of ANP, BNP and beta -MHC protein was observed by immunohistochemical method. Results 1. different methods induced ANP, BNP and -MHC expression in the model mice of myocardial hypertrophy using AAC, AVF and ISO method for cardiac hypertrophy of cardiac hypertrophy index HW/BW, LVW /BW increased significantly. Under high magnification, myocardial cell fertilizer was found. Large, arranged sparsely and interstitial fibrosis, suggesting that the AAC, AVF and ISO induced murine myocardial hypertrophy model was successful. Compared with the AAC method, HW/BW, LVW/BW and AVF groups in the ISO group were significantly reduced, the myocardium wall of the AVF and ISO group was thickened and the ventricular cavity narrower was lighter, suggesting that the AAC method was used to prepare the myocardial fertilizer produced by the murine myocardial hypertrophy model. The levels of ANP, BNP and beta -MHC mRNA in cardiac hypertrophy mice were significantly higher than those of AVF and ISO. The transcriptional and protein expression levels of ANP and BNP mRNA in ISO mice were also increased significantly. However, the level of the transcription and protein expression of the ANP and BNP mRNA was also increased, but the level of the transcription and protein expression was only increased. Three The comparison of the myocardial hypertrophy models prepared by AAC, AVF and ISO showed that the transcriptional and protein expression levels of ANE, BNP and beta -MHC mRNA in the AAC mice were most obvious, suggesting that the AAC method was used to increase the left ventricular ANP, BNP and beta -MHC transcription and protein levels were smaller than those of the AAC. The difference in expression of ANP, BNP and P-MHC in the rat cardiac hypertrophy model was made by four strains of mice, C57BL/6, BALB/c, KM and ICR to prepare the model of myocardial hypertrophy. In C57BL/6, BALB/c, KM mice, the cardiac hypertrophy index was HW/BW, LVW/BW significantly increased. Muscle fiber breakage and intercellular space increased, suggesting that the C57BL/6, KM, BALB/c mice were successful in preparing the myocardial hypertrophy model, and ICR mice were not ideal. The myocardial hypertrophy model of four strains of mice, C57BL/6, BALB/c, KM and ICR, was compared with each other, showing that the increase of the cardiac hypertrophy index HW/BW and LVW/BW was the most obvious in the C57BL/6 rat, followed by K. M mice showed that the degree of myocardial hypertrophy produced by C57BL/6 mice was better than that of BALB/c. KM and ICR. used four strains of mice C57BL/6, BALB/c, KM and ICR to prepare the model of myocardial hypertrophy. C57BL/6, KM mouse heart left ventricular ANP, and beta transcription and protein expression levels were significantly increased. The transcriptional and protein levels of ANP and BNP mRNA in mice BNP and ICR only increased, and the myocardial hypertrophy model of C57BL/6, BALB/c, KM and ICR in four strains of mice was compared, showing the ANP left ventricle of C57BL/6 mice, the most obvious increase in the transcription and protein expression level of BNP and beta, suggesting the use of the myocardium prepared by the mice. The transcriptional and protein levels of the hypertrophic model ANP. BNP and beta -MHC mRNA were superior to those of BALB/c, KM and ICR mouse.3.SP on the ANP, BNP and beta -MHC expression and the effect of TGF- beta signaling pathway on the myocardial hypertrophy model of mice. Myocardial hypertrophy, partial muscle fiber breakage, and intercellular space increased, suggesting that the model of this experiment was successful. CAP and SP groups were large, medium dose could reduce the hypertrophy index HW/BW and LVW/BW significantly, reduce the volume of heart, reduce the degree of muscle fiber rupture, and reduce the gap between the cells, and the CAP and SP have no significant difference, suggesting SP can be clear. The cardiac hypertrophy model of C57BL/6 mice was significantly reduced by the AAC method. The cardiac hypertrophy index (HW/BW, LVW/BW and myocardial pathological damage) were similar to that of Captopril Tablets. The AAC method was used to prepare the C57BL/6 mouse cardiac hypertrophy model, which showed that the transcriptional and protein expression levels of ANP, BNP and beta -MHC mRNA in the left ventricle of the left heart of the mice were obviously enhanced; CAP and SP. The level of ANP, BNP and beta -MHC mRNA in the left ventricle of the model mice decreased significantly, and there was no significant difference between CAP and SP, suggesting that SP could significantly reduce the level of ANP, BNP and beta -MHC transcription and egg white expression in the C57BL/6 mouse model C57BL/6 induced by AAC method, and the effect intensity was with the Captopril Tablets phase. The TGF-p1. Smad2 and Smad3 mRNA transcriptional levels in the left ventricle of the mice were significantly increased by AAC method, but there was no significant difference in the transcriptional level of the Smad7 mRNA in the left ventricle of the mice. The CAP and SP groups were large, and the middle dose of the mice could significantly reduce the TGF- beta 1 in the left ventricle of the mice. Level, obviously up regulation of Smad7 mRNA transcriptional and protein expression level, suggesting that the mechanism of SP's prevention and control of myocardial hypertrophy may be related to the regulation of TGF- beta /Smad signaling pathway. Conclusion 1.AVF, AAC and ISO have three methods of preparation of the model of myocardial hypertrophy in mice, and the expression of ANP, BNP and beta -MHC in the left ventricle of the cardiac hypertrophic mice is selected as the screening condition, and the results show that the AAC method is used. The model of ANP, BNP and beta -MHC in the left ventricle of the 2. hypertrophic mice was selected as the screening condition. The AAC method was used to prepare the C57BL/6, BALB/c, KM and ICR mice model of myocardial hypertrophy, and the C57BL/6 strain mouse model was the best. 3.SP obviously inhibited the cardiac hypertrophy and inhibition of the myocardium in C57BL/6 mice induced by AAC method. The expression of ANP, BNP and beta -MHC in muscle hypertrophic mice; the mechanism of 4.SP inhibiting myocardial hypertrophy may be related to the regulation of TGF- /Smad signaling pathway.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2;R-332

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