人参皂甙Rb1改善转基因扩张型心肌病模型小鼠的心功能和心脏重构

目的利用cTnT^R141W转基因扩张型心肌病小鼠,研究人参皂甙Rb1对遗传性扩张型心肌病心功能及心脏重构的作用及其可能机制。方法将cTnT^R141W转基因小鼠随机分为模型组和人参皂甙Rb1治疗组（70 mg/kg/d）,连续给药7个月,取野生型小鼠作为对照组。心脏超声检测心脏功能及几何构型。HE染色观察心肌细胞变化。透射电镜分析心肌超微结构。RT-PCR检测心肌粘附蛋白的表达。免疫荧光激光共聚焦观察心肌粘附分子Itga8的表达与分布。结果Rb1长期给药能显著改善该模型的心脏功能及几何构型。光镜和透射电镜观察显示Rb1能减轻心肌细胞排列紊乱及超微结构的破坏。RT-PCR结果显示,在模型中Cx40表达降低,E-cad、itga8和itgb1bp3表达升高,但在Rb1组中接近正常水平。免疫荧光激光共聚焦结果显示Rb1可降低Itga8的表达量并调节其分布。结论Rb1可改善扩张型心肌病模型的心功能,抑制心脏重构,其作用可能部分通过调节粘附蛋白的表达而实现的。     

cTnT^（R141W）转基因小鼠扩张型心肌病模型的建立

目的建立cTnT^R141W扩张型心肌病的转基因小鼠模型。方法把cTnT^R141W基因插入-αMHC启动子下游,构建转基因表达载体,通过显微注射法建立cTnT^R141W转基因C57BL/6J小鼠。PCR鉴定cTnT^R141W转基因小鼠的基因表型,实时PCR检测基因的拷贝数,Northern blotting检测基因表达,光学显微镜和超声检测cTnT^R141W转基因小鼠心脏的病理改变。结果建立了3个系的cTnT^R141W转基因小鼠。3个系的基因拷贝数分别是15、20和59拷贝。cTnT^R141W基因在心脏组织的表达水平高于内源性cTnT。病理分析显示cTnT^R141W转基因小鼠心房心室明显大于野生型,心室壁明显变薄,心肌细胞不均匀肥大,心肌间质纤维增多。超声检查显示心室腔明显扩大,收缩期容积和舒张期容积显著增大,射血分数、短轴缩短率、室壁运动度明显降低。结论cTnT^R141W转基因小鼠的全心扩大,室壁变薄,心肌细胞肥大,间质纤维化以及心肌收缩力下降,说明成功建立了cTnT^R141W转基因小鼠扩张型心肌病模型,为研究扩张型心肌病发病机制和药物研发提供了有价值的动物模型。     

cTnT^R92Q转基因小鼠肥厚型心肌病模型的建立

目的建立cTnT^R92Q肥厚型心肌病的转基因小鼠模型。方法把cTnT^R92Q基因插入-αMHC启动子下游,构建转基因表达载体,通过显微注射法建立cTnT^R92Q转基因C57BL/6J小鼠。PCR鉴定cTnT^R92Q转基因小鼠的基因表型,RT-PCR检测基因表达,光学显微镜和超声检测cTnT^R92Q转基因小鼠心脏的病理改变。结果建立了3个不同表达水平的cTnT^R92Q转基因小鼠品系。转入的cTnT^R92Q基因在心脏组织的表达水平高于内源性cTnT。组织学分析显示cTnT^R92Q转基因小鼠心脏变大,心室壁肥厚,心腔变小,心肌细胞排列紊乱,心肌间质纤维增多。超声检查显示心室壁变厚,收缩期容积和舒张期容积显著缩小,射血分数、短轴缩短率明显增加。结论cTnT^R92Q转基因小鼠心脏变大,室壁变厚,心腔变小,心肌细胞排列紊乱,间质纤维化以及心肌舒张功能失调,说明成功建立了cTnT^R92Q转基因小鼠肥厚型心肌病模型,为研究肥厚型心肌病发病机制和药物研发提供了有价值的动物模型。     

心脏特异表达人源FAM55A转基因小鼠的建立

目的建立心脏特异表达的人源FAM55A转基因小鼠,为研究该基因在心肌病发病中的作用提供模型。方法 Western blot检测FAM55A在野生型小鼠与cTnTR141W转基因小鼠心脏组织中的表达变化及其在野生小鼠的组织表达谱。克隆人源FAM55A基因入α-MHC启动子下游构建a-MHC-FAM55A表达载体,显微注射法建立FAM55A转基因小鼠。PCR鉴定转基因首建鼠的基因型。Western blot鉴定人源FAM55A在转基因小鼠心脏中的表达,超声检测转基因小鼠心脏的几何构型和功能。HE染色检测转基因小鼠心脏的病理改变。结果 FAM55A在野生型小鼠心脏中有少量表达,在扩张型心肌病小鼠的心脏中表达增加。建立了1个心脏组织特异表达人源FAM55A转基因小鼠品系。与野生型小鼠相比,FAM55A转基因小鼠的心脏收缩期和舒张期左室前壁从1月龄到5月龄持续增厚,3月龄转基因小鼠心脏射血分数和短轴缩短率稍有增强,1月龄和5月龄转基因小鼠心脏功能则与同龄野生型小鼠相比无变化。组织学检测显示,转基因小鼠心脏左室心肌细胞不均匀肥大,但不发生紊乱。结论 FAM55A在扩张型心肌病小鼠的心脏中表达上调,建立了心脏特异表达的人源FAM55A转基因小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。     

心脏特异表达NOL3转基因小鼠的建立

目的建立心脏特异表达NOL3转基因小鼠,用于研究该基因在心肌病发病中的作用。方法Western blot检测小鼠NOL3表达谱。构建aMHC-NOL3表达载体,显微注射法建立NOL3转基因小鼠。PCR鉴定转基因鼠的基因型,心脏超声检测转基因及野生型小鼠心脏功能及几何构型。结果NOL3在1月龄野生型鼠心脏、脑、骨骼肌中的高表达,在心脏中的表达不随年龄而改变。通过转基因小鼠的筛选,得到了3个NOL3转基因品系,其中1个品系心脏NOL3蛋白表达量与野生型鼠相比明显增加。单转NOL3基因的小鼠心脏功能及几何构型与野生型小鼠相比无显著变化。结论成功建立了心脏特异表达NOL3转基因小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。     

人参皂苷Rb1通过上调PGC-1α缓解糖尿病心肌病

目的:研究人参皂苷Rb1对糖尿病心肌病的治疗作用并阐明其分子机制。方法:采用腹腔注射链脲佐菌素的方法,建立糖尿病心肌病动物模型。将小鼠分为3组:WT组,DM组,DM+Rb1组。超声心动图分析小鼠心功能;Western blot分析PGC-1α、cleaved caspase-3、bcl-2等蛋白表达;MitoSOX染色分析线粒体ROS含量;透射电镜分析线粒体数目。结果:与WT组相比,DM组小鼠心功能显著下降(LVEF,P<0.01),PGC-1α表达下调(P<0.01),线粒体数目减少(P<0.01);而Rb1处理后,显著改善了DM小鼠心功能(LVEF,P<0.01),恢复了PGC-1α表达(P<0.05),增加了线粒体数目(P<0.05)。同时,Rb1处理后,减少了糖尿病小鼠心肌线粒体ROS产生(P<0.01),恢复了bcl-2蛋白表达(P<0.01),降低了cleaved caspase-3蛋白表达(P<0.01),从而减少了高糖引起的细胞凋亡(P<0.05)。而siPGC-1α处理后,阻断了Rb1的上述作用。结论:人参皂苷Rb1通过上调PGC-1α改善糖尿病小鼠心功能,缓解糖尿病心肌病。其机制可能与人参皂苷Rb1降低心肌线粒体ROS产生并减少心肌细胞凋亡有关。     

心脏特异表达Calponin 1转基因小鼠的心脏功能分析

目的建立心脏特异表达Calponin 1转基因小鼠,研究Calponin 1对心脏发育及心肌病的调节作用。方法利用心脏特异启动子α-MHC构建转基因表达载体,显微注射法建立Calponin 1转基因小鼠,PCR法鉴定转基因小鼠的基因型,Western Blot检测Calponin 1在心脏组织中的表达,心脏超声检测转基因小鼠的心脏结构和功能,HE染色和Masson染色检测转基因小鼠心脏的病理改变。结果 Calponin 1在野生型小鼠心脏中有表达,在扩张型心肌病小鼠的心脏组织表达降低。通过显微注射法,建立了2个心脏组织Calponin 1基因高表达的转基因小鼠系。与野生型小鼠相比,Calponin 1转基因小鼠收缩期左室内径（LVID,systolic）增加28%（P〈0.01,n=12）,舒张期左室内径（LVID,diastolic）增加16.2%（P〈0.01,n=12）,收缩期左室后壁厚度（LVPW,systolic）减小15.7%（P〈0.01,n=12）,舒张期左室后壁厚度（LVPW,diastolic）减小21%（P〈0.01,n=12）,射血分数（ejection fraction,EF）降低11.5%（P〈0.01,n=12）,短轴内径缩短率（fraction shortening,FS）降低14.6%（P〈0.05,n=12）。转基因小鼠心脏组织病理H＆E染色和Masson染色显示,转基因小鼠心室扩张,心肌细胞不均匀肥大,细胞间隙变大,心肌间质纤维增多。结论 Calponin 1在心脏特异过表达引起转基因小鼠心脏左室内径增加,收缩期容积和舒张期容积显著增大,心室壁变薄,射血分数及短轴缩短率降低等扩张性心肌病表型,推测Calponin 1是参与心肌病病理发生的基因之一。     

系统表达HB-EGF转基因小鼠的建立

目的建立系统表达肝素结合性表皮生长因子（HB-EGF）的转基因动物模型,利用转基因动物模型研究HB-EGF与组织纤维化的关系。方法RT-PCR法克隆小鼠HB-EGF基因,将其插入Chickenβ-actin启动子下游,构建Chickenβ-actin-HB-EGF表达载体,利用显微注射的技术把表达载体注射到受精卵的雄原核中,建立HB-EGF转基因小鼠。利用特异引物PCR的方法鉴定转基因的基因型,采用Western Blot方法鉴定HB-EGF基因在全身组织的表达。分别取HB-EGF转基因鼠与同窝阴性小鼠的肝、肾、肺及膀胱组织进行Massion染色。结果建立了系统表达HB-EGF转基因小鼠,Western Blot发现其HB-EGF在肝、肺、肾及膀胱的表达与同窝阴性对照小鼠相比明显增加。Massion染色结果表明转基因鼠肝、肺、肾及膀胱组织纤维化程度明显高于同窝阴性对照小鼠。结论成功建立了系统表达HB-EGF转基因小鼠,HB-EGF的过度表可显著加重组织纤维化程度。     

磁共振成像对扩张型心肌病转基因模型小鼠左右心室对比分析

目的对cTnTR141W扩张型心肌病转基因模型小鼠左、右心室进行对比分析,研究cTnTR141W转基因小鼠作为右心室心肌病的动物模型的可行性。方法利用7.0 T高场强磁共振成像(MRI)技术,定量分析了2、4、6和8月龄对照组及cTnTR141W转基因模型小鼠左、右心室的舒张末容积(EDV)、收缩末容积(ESV)和射血分数(EF)的变化情况,同时对6月龄对照组cTnTR141W转基因模型小鼠心肌组织进行组织学分析。结果转基因阴性对照小鼠相比,cTnTR141W转基因小鼠左、右心室的容积在2月龄时已有增大趋势,而射血分数有减小趋势。右心室射血分数减小出现最早也最显著(P<0.05)。随年龄增加,cTnTR141W转基因小鼠与转基因阴性对照小鼠相比,右心室的结构和功能的病理生理变化与左心室同时趋于严重。该小鼠左、右心室在4月龄后表现典型的扩张型心肌病表型。结论 cTnTR141W转基因模型小鼠左心室和右心室的扩张性心肌病表型同时出现,该小鼠可作为右室性心肌病等右心室功能下降相关疾病研究的动物模型。     

Dkk3心脏特异表达转基因小鼠心脏功能分析

目的建立心脏特异表达Dkk3转基因模型小鼠,研究Dkk3对心脏发育及和心肌病的调节作用。方法把Dkk3基因插入心肌特异启动子-αMHC下游,构建转基因表达载体,显微注射法建立C57BL/6J Dkk3转基因小鼠,PCR鉴定转基因小鼠基因型,采用Northern blot检测Dkk3在心脏组织中的表达,HE染色和超声检查转基因小鼠心脏结构和功能。结果建立了3个不同表达水平的Dkk3转基因小鼠品系。转入的Dkk3基因在心脏组织的表达水平均高于同龄对照小鼠。组织学分析显示Dkk3小鼠室壁变厚,心腔减小,心肌细胞排列轻度紊乱。超声检查显示心室壁变厚,收缩期容积和舒张期容积显著减小,射血分数,短轴缩短率增加。结论Dkk3过表达导致转基因小鼠室壁变厚,心腔减小,心肌细胞排列轻度紊乱,心肌舒张功能轻度失调。     

Heparin-Binding EGF-Like Growth Factor Induces Heart Interstitial Fibrosis via an Akt/mTor/p70s6k Pathway

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is essential for maintaining normal function of the adult heart and is known to play an important role in myocardial remodeling. In the present study, we observed that heart-specific HB-EGF transgenic (TG) mice had systolic dysfunction with decreased fractional shortening (FS%), increased end-systolic diameter (LVIDs) at 5 months of age, increased heart fibrosis, and increased mRNA expression of Col1α1 and Col3α1 at 1, 3, 5 and 7 months of age compared to nontransgenic (NTG) littermates. However, the left ventricular anterior wall thickness at end-systole (LVAWs) of the TG mice was not different than the NTG mice. Phosphorylation levels of Akt, mTor and p70s6k were increased due to HB-EGF expression in TG mice compared with the NTG mice at 3 and 7 months of age. Additionally, activated Akt, mTor and p70s6k were co-localized with vimentin to cardiac fibroblasts isolated from TG mice. Furthermore, HB-EGF significantly increased phosphorylation levels of Akt, mTor and p70s6k and increased expression of type I collagen in cultured primary cardiac fibroblasts. Rapamycin (Rapa) and CRM197, inhibitors of mTor and HB-EGF respectively, could inhibit the expression of type I collagen in the cultured primary cardiac fibroblasts and Rapa suppressed interstitial fibrosis of the heart tissues in vivo. In addition, a BrdU assay showed that HB-EGF increased proliferation of cardiac fibroblasts by 30% compared with cells without HB-EGF treatment. HB-EGF-induced proliferation was completely diminished in the presence of Rapa. These results suggest that HB-EGF induced heart fibrosis and proliferation of cardiac fibroblasts occurs through activation of the Akt/mTor/p70s6k pathway.     

Expression of CYP2E1 increases oxidative stress and induces apoptosis of cardiomyocytes in transgenic mice

Cytochrome P450 2E1 (CYP2E1) is an effective generator of reactive oxygen species. Marked expression of CYP2E1 occurs in the heart and it is known to be regulated in the course of progression of myocardial ischemia and cardiomyopathy. We provide evidence that the expression of CYP2E1 is strongly up-regulated in cTnT(R141W) transgenic mice with dilated cardiomyopathy. Heart tissue-specific CYP2E1 transgenic mice were produced to study the effects of CYP2E1 overexpression on the heart. Increased mortality, chamber dilation and contractile dysfunction, as well as myocyte disarray, interstitial fibrosis, ultrastructural degeneration with myofibrillar disorganization and mitochondria damage, were observed in CYP2E1 transgenic mice and cTnT(R141W) transgenic mice. In addition, levels of H(2) O(2) and malondialdehyde were increased and levels of glutathione and total antioxidant capability were strongly reduced in CYP2E1 transgenic mice and cTnT(R141W) transgenic mice. Myocyte apoptosis was significantly increased by 19-fold in CYP2E1 transgenic mice and by 11-fold in cTnT(R141W) transgenic mice, respectively, compared to wild-type mice. Mitochondrial-dependent apoptotic signal transduction events, such as cytochrome?c release from mitochondria into the cytosol and the expression of cleaved (active) caspases 3 and 9, were significantly increased in CYP2E1 transgenic mice and cTnT(R141W) transgenic mice. These results demonstrate that CYP2E1 over-expression produces apoptosis and that the up-regulation of CYP2E1 in cTnT(R141W) transgenic mice also correlates with apoptosis in this model.     

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.     

Hepatocyte growth factor prevents tissue fibrosis, remodeling, and dysfunction in cardiomyopathic hamster hearts

Structural remodeling of the myocardium, including myocyte hypertrophy, myocardial fibrosis, and dilatation, drives functional impairment in various forms of acquired and hereditary cardiomyopathy. Using cardiomyopathic Syrian hamsters with a genetic defect in delta-sarcoglycan, we investigated the potential involvement of hepatocyte growth factor (HGF) in the pathophysiology and therapeutics related to dilated cardiomyopathy, because HGF has previously been shown to be cytoprotective and to have benefits in acute heart injury. Late-stage TO-2 cardiomyopathic hamsters showed severe cardiac dysfunction and fibrosis, accompanied by increases in myocardial expression of transforming growth factor-beta1 (TGF-beta1), a growth factor responsible for tissue fibrosis. Conversely, HGF was downregulated in late-stage myopathic hearts. Treatment with recombinant human HGF for 3 wk suppressed cardiac fibrosis, accompanied by a decreased expression of TGF-beta1 and type I collagen. Suppression of TGF-beta1 and type I collagen by HGF was also shown in cultured cardiac myofibroblasts. Likewise, HGF suppressed myocardial hypertrophy, apoptosis in cardiomyocytes, and expression of atrial natriuretic polypeptide, a molecular marker of hypertrophy. Importantly, downregulation of the fibrogenic and hypertrophic genes by HGF treatment was associated with improved cardiac function. Thus the decrease in endogenous HGF levels may participate in the susceptibility of cardiac tissue to hypertrophy and fibrosis, and exogenous HGF led to therapeutic benefits in case of dilated cardiomyopathy in this model, even at the late-stage treatment.     

Cardioprotection by Hepc1 in cTnTR141W transgenic mice

Hepcidin 1 (Hepc1) is a peptide hormone secreted by the liver in response to iron loading. It is expressed in the heart and is thought to play a role in the regulation of iron homeostasis in an autocrine and paracrine fashion. We have shown that expression of Hepc1 is strongly down-regulated in the heart of the cTnT(R141W) transgenic mouse model of dilated cardiomyopathy (DCM) at 3?months of age. Transgenic mice with heart tissue-specific Hepc1 expression alone or in combination with the cTnT(R141W) mutation were produced to study the effects of Hepc1 on DCM. Transgenic expression of Hepc1 was found to be nonlethal and resulted in decreased mortality in cTnT(R141W) transgenic mice, from 29.6 to 7.4%(n =?27; P?相似文献   

Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression

Although cardiac myocyte apoptosis has been detected in explanted hearts from patients with end-stage dilated and ischemic cardiomyopathy, the relative contribution of apoptotic cell death to left ventricular (LV) remodeling and cardiac decompensation is not known. To determine whether progressive cardiac myocyte apoptosis contributes to the transition from a hypertrophic to a dilated cardiac phenotype that is observed in transgenic myosin heavy chain secreted TNF (MHCsTNF) mice with cardiac restricted overexpression of tumor necrosis factor (TNF), we assessed cardiac myocyte apoptosis (using a DNA ligase technique) in MHCsTNF mice and littermate control mice in relation to serial changes in LV structure, which was assessed using MRI. The prevalence of cardiac myocyte apoptosis increased progressively from 4 to 12 wk as the hearts of the MHCsTNF mice underwent the transition from a concentric hypertrophic to a dilated cardiac phenotype. Treatment of the MHCsTNF mice with the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino4-oxo-5-fluoropentanoic acid significantly decreased cardiac myocyte apoptosis and significantly attenuated LV wall thinning and adverse cardiac remodeling. Additional studies suggested that the TNF-induced decrease in Bcl-2 expression and activation of the intrinsic mitochondrial death pathway were responsible for the cardiac myocyte apoptosis observed in the MHCsTNF mice. These studies show that progressive cardiac myocyte apoptosis is sufficient to contribute to adverse cardiac remodeling in the adult mammalian heart through progressive LV wall thinning.