大鼠心肌梗死后差异蛋白质组学分析

目的:建立大鼠心肌梗死后蛋白表达变化谱,以进一步了解心肌梗死后心肌细胞重塑产生机制。方法:通过结扎大鼠冠状动脉左前降支建立急性心肌梗死模型,利用蛋白质双向凝胶电泳技术(two-dimensionalgelelectrophoresis,2-DE)分离心肌总蛋白,采用PDQuest7.3.1软件比较分析,获得差异表达蛋白总体变化趋势。进一步通过蛋白免疫印记技术(Western-blotting)检测碱性成纤维生长因子(Basefibroblastgrowthfactor,bFGF)在心肌梗死后的表达变化。结果:成功建立了大鼠急性心肌梗死模型;2-DE结果表明:以假结扎组为对照,梗死3天组有27个蛋白显著上调,18个蛋白显著下调,7个蛋白表达明显差异(ratio>5)。进一步研究发现梗死区心肌组织bFGF表达明显升高。结论:心肌梗死后蛋白表达变化趋势的探讨为心室重塑机制研究提供线索。     

microRNAs在大鼠急性心肌梗死过程的表达变化

目的研究分析microRNAs（miRNAs,miRs）在大鼠急性心肌梗死（acutemyocardialinfarction,AMI）心肌组织的梗死区与非梗死区的表达变化,为防治AMI提供基础数据。方法选择雄性sD大鼠为研究对象,建立结扎左冠状动脉造成的急性心肌梗死模型,取建模后6h的梗死区与非梗死区的心肌组织进行芯片检测．确定其中表达变化显著的miRNAs;最后进行定量逆转录聚合酶链反应（qRT—PCR）,定量分析梗死区与非梗死区心肌组织中miRNAs的表达。结果AMI大鼠心肌组织中,芯片筛选出在AMI前后发生显著波动的miRNAs,与非梗死区相比,梗死区心肌组织中有26个miRNAs表达发生了显著变化,其中19个miRNA表达下调,7个miRNA表达上调。结论在AMI后的心肌组织的梗死部位与非梗死部位miRNAs的表达是有显著差异的,这对AMI阶段心肌保护的救治具有重要意义。     

高血糖对大鼠心肌梗死后心肌组织SCF表达的影响

目的通过检测高血糖状态下正常及梗死后心肌组织中干细胞因子（SCF）的表达,探讨高血糖对心肌梗死后干细胞修复的影响。方法36只SD大鼠随机分为假结扎组、高血糖组、心肌梗死组和高血糖并心肌梗死组,每组9只,通过给予高糖饮食12周建立高糖血症模型,然后结扎冠状动脉左前降支建立心肌梗死模型。用RT-PCR检测各组大鼠心肌组织中SCFmRNA的表达水平;用免疫组织化学法检测心肌组织中SCF蛋白的表达。结果SCFmRNA和蛋白在正常心肌呈低水平表达,与假结扎组相比,心肌梗死组大鼠心肌梗死区周围组织表达明显增强（P〈0．05）;与心肌梗死组大鼠相比,高血糖并心肌梗死组大鼠心肌组织中SCF的表达明显减弱（P〈0．05）。结论心肌梗死后SCF的表达明显增强,而高血糖能抑制心肌梗死后心肌组织中SCF的表达,提示高血糖可能抑制了心肌梗死后干细胞的归巢及其梗死后心肌的修复。     

扶正化瘀胶囊对心肌梗死大鼠心肌纤维化的影响

目的：急性心肌梗死是危害人类健康的重大疾病之一,心肌梗死后心肌纤维化是造成心脏结构破坏、心功能下降、心律失常发生、心衰甚至猝死的微观病理机制。防治心肌纤维化是当前医学研究的重点和热点。本研究主要探讨扶正化瘀胶囊对心肌梗死大鼠心肌纤维化的干预作用。方法：大鼠随机分为假手术组、模型组、扶正化瘀胶囊组和卡托普利组,采用结扎冠状动脉前降支的方法建立心肌梗死模型,假手术组只穿线,不结扎。于造模成功后第10天开始给予相应药物治疗2个月。治疗结束后,检测左心室梗死范围和心肌胶原含量。结果：与假手术组比较,模型组、扶正化瘀胶囊组和卡托普利组的非梗死区面积显著减小（P〈0.01）。与模型组比较,扶正化瘀胶囊组和卡托普利组的梗死区面积和梗死百分比显著减小（P〈0.05,P〈0.01）。在心肌胶原表达上,与假手术组比较,模型组和扶正化瘀胶囊组胶原含量显著增加（P〈0.01）。与模型组比较,卡托普利组和扶正化瘀胶囊组胶原含量显著降低（P〈0.05,P〈0.01）。结论：扶正化瘀胶囊能够改善心肌缺血,缩小心肌梗死范围,抑制心肌胶原表达,除能用于肝纤维化的治疗外,还能用于防治心肌梗死后的心肌纤维化。     

过表达bFGF促进大鼠急性缺血心肌的毛细血管生成

研究过表达人碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）在大鼠急性心肌缺血模型中的表达及其对心肌血管 再生的影响. 将含人bFGF质粒pcDNA/b转染人脐带静脉内皮细胞（HUVEC） ,进行MTT检测. pcDNA/b转染人肾细胞293细胞系,进行G418稳定筛选, Western 印迹检测.建立大鼠急性心肌梗死模型,分别将生理盐水、空质粒 pcDNA3.1(+)和pcDNA/b分3点注射于梗死交界处心肌内4周后取材,做常规 HE 染色和Masson 染色,测量各组微血管数量和梗死面积. 经免疫组织化 学染色鉴定和电镜观察,过表达外源bFGF可以促进HUVEC（细胞）生长; pcDNA/b能在293细胞中高效表达外源bFGF基因. pcDNA/b注射组梗死交界处 可见大量新生血管,毛细血管总数明显大于对照组,梗死面积明显小于对 照组. pcDNA/b组在梗死交界区有bFGF阳性表达.电镜观察显示,在梗死交界 处心肌细胞间有毛细血管增生,分化成2个血管腔.本实验证明,过表达bFGF 具有促进大鼠急性缺血心肌的毛细血管生成的作用,为bFGF基因治疗缺血 性心肌病的研究提供实验基础.     

大鼠心肌发育相关基因在心肌梗死后的表达变化

探讨大鼠急性心肌梗死后与心肌发育相关基因的表达变化 .通过结扎大鼠冠状动脉左前降支建立急性心肌梗死模型 ,利用逆转录聚合酶链式反应 (RT PCR)和Northern印迹技术分别检测GATA4、Nkx2 5BMP2和Wnt11基因在心肌梗死后不同时间的表达变化趋势 .结果显示 ,在正常心肌中 ,与心肌发育相关的基因GATA4、Nkx2 5和BMP2有较强的表达 ,而Wnt11基础表达较低 .心肌梗死后 ,GATA4、Nkx2 5和BMP2表达下降 ,7d后有所恢复 ;Wnt11的表达在心肌梗死后呈上升趋势 ,至梗死 7d达到高峰 .结果提示 ,心肌发育相关基因的表达在心肌梗死后有明显改变 ,并发现启动心肌发育的关键信号分子———Wnt11基因表达上调 ,为心肌损伤后细胞分化机制的研究提供线索     

自体内皮前体细胞移植对急性心肌梗死大鼠微血管新生及心功能的影响

目的探讨外周血来源的内皮前体细胞自体移植,对大鼠急性心肌梗死后微血管新生与心功能的影响。方法抽取SD大鼠外周动脉血,应用Ficoll密度梯度离心法获取单个核细胞。应用含有VEGF和bFGF的特定培养基体外培养,得到内皮前体细胞;结扎SD大鼠冠状动脉左前降支,建立急性心肌梗死模型;然后将得到的自体内皮前体细胞植入缺血心肌局部区域。对照组动物注入细胞培养液。结果与对照组比较,细胞移植组大鼠心功能明显改善,心肌收缩力显著优于对照组;梗死心肌微血管新生更为明显。结论急性心肌梗死心肌局部移植外周血来源的自体内皮前体细胞,能够促进血管新生;对局部梗死心肌组织结构有一定的保护作用,并可在不同时点不同程度恢复心肌收缩力,显著改善心功能。     

心肌细胞／胶原复合体移植心梗大鼠梗死周边区的电偶联网络变化

目的应用免疫组化技术和电生理技术记录心肌细胞／胶原复合体对心梗大鼠梗死周边区的有效不应期（ERP）及缝隙连接蛋白43（Cx43）改变,探讨梗死周边区的电偶联网络变化。方法将成年SD大鼠随机分组：假手术组、模型组、移植组。后2组制作心肌梗死动物模型,假手术组仅开胸,不结扎冠状动脉,移植组移植心肌细胞与胶原材料复合组织。结果①左室ERP变化：与假手术组相比,心梗组梗死周边区ERP显著延长（P〈0．01）;移植组梗死周边区ERP延长,但较心梗阻ERP缩短,差异无显著性（P〉0．01）。②Cx43免疫组化结果：移植组Cx43阳性蛋白表达高于心梗组。结论移植的心肌细胞／胶原复合体移植可改善大鼠心肌梗死周边区缝隙连接电偶联网络,进而调控心肌细胞／胶原复合体与宿主心肌同步收缩。     

1-磷酸鞘氨醇受体在心肌梗死后心室重塑过程中的变化差异

目的:探讨1-磷酸鞘氨醇(S1P)不同受体亚型(S1P1、S1P2、S1P3)在大鼠心肌梗死后心室重塑过程中的变化差异。方法:通过结扎大鼠左冠状动脉前降支,建立大鼠心肌梗死模型,在心肌梗死后1、4、8周,采用实时荧光定量PCR的方法,分别检测心脏非梗死区、梗死区、正常对照心肌组织S1P1、S1P2、S1P3的m RNA相对表达。结果:心梗后心室重塑阶段,S1P1 m RNA的表达水平在非梗死区与梗死区的下降程度不一致,1周组非梗死区与正常对照组比较无统计学差异,而在梗死区2组间差异有统计学意义(P0.05),在梗死区降低趋势更为明显。S1P2 m RNA在术后各组的非梗死区与正常对照心肌之间的表达比较,变化无统计学意义,且各时间点之间无差异,而在梗死区其表达变化较为显著(P0.01)。S1P3 m RNA在心梗后1周表达显著增强(P0.01),4周表达出现降低,这种表达抑制在梗死区更为明显(P0.01和P0.05)。结论:心室重塑阶段的S1P1、S1P2、S1P3 m RNA表达在非梗死区与梗死区变化不同步且各有特点,这种差异表达可能在心肌梗死后心室重塑过程中有重要意义。     

大鼠急性心肌梗死模型的制备及对肌钙蛋白T的影响

目的探讨建立大鼠急性心肌梗死（AMI）模型的方法,研究心肌梗死大鼠血浆中肌钙蛋白T（cTn-T）的动态变化。方法大鼠经结扎左冠状动脉前降支造成心肌梗死,建立稳定的心肌梗死模型;分别在结扎后31 h、48 h、69 h、168 h检测cTn-T含量和计算心肌梗死重量指数。结果成功制备心肌梗死大鼠模型,并对常规技术进行改进,降低动物死亡率。大鼠左冠状动脉结扎31 h、48 h模型组与假手术组cTn-T含量差异极显著（P〈0.001）;各时间点心肌梗死重量指数比较,差异极显著（P〈0.001）。cTn-T值与梗死重量指数呈显著性正相关（r=0.90,P〈0.01）。结论结合大鼠心肌梗死程度进一步佐证了模型制备较成功。cTn-T表现出特异性和敏感性,并在31 h最接近达峰时间,有早期诊断心肌梗死的价值,也可作为判断AMI时心肌梗死程度和预后的参考指标。     

不同年龄鼠骨髓干细胞移植治疗心梗后心衰的实验研究

目的：通过对比观察不同年龄鼠骨髓干细胞在体外的生长状态和移入受损心肌后对心肌梗死大鼠心功能的影响,说明年龄对骨髓干细胞的增殖能力和对移植效果的影响。方法：分别分离培养3日龄,1月龄,6月龄,12月龄Wister雄鼠骨髓干细胞,观察细胞生长状态,描记生长曲线。将60只大鼠用结扎冠状动脉前降支的方法制成心肌梗死模型后两周,随机分为三组：Ⅰ组：3日龄组（n=20只）：给予3日龄的5-溴脱氧尿嘧啶（Brdu）标记的鼠骨髓干细胞;Ⅱ组：6月龄组（n=20只）：给予6月龄的Brdu标记的鼠骨髓干细胞;Ⅲ组：对照组（n=20只）：给予培养液。各组均于治疗前及治疗后4周进行心脏超声检查评价心功能改善情况。处死动物取出心脏,进行直接测量后取左心室作石蜡切片,行苏木素-伊红（HE）染色及免疫组化检测,鉴定植入的细胞和心肌、毛细血管再生情况。结果：不同年龄鼠骨髓干细胞培养结果示3日龄及1月龄组增殖能力均高于6月龄及12月龄组,但3日龄与1月龄组之间无统计学差异,12月龄组在原代培养后期即死亡。治疗前超声心动图显示Ⅰ、Ⅱ、Ⅲ组心功能无显著差异（P〉0.05）,治疗之后四周超声心动图检查结果示：Ⅰ、Ⅱ组LVEF、FS、IVST、LVPW和LVESD较治疗前均有明显提高,且显著高于Ⅲ组;Ⅰ组LVEF、FS、IVST、LVPWs和LVESD又明显高于Ⅱ组;Ⅰ、Ⅱ、Ⅲ组LVEDD与治疗前比较无统计学意义。心脏直测结果显示Ⅰ、Ⅱ组的Wh、Wh、Wb、L、I度均较第Ⅲ组有所增加,其中Ⅰ组又明显高于Ⅱ组。免疫组化结果显示：Ⅰ、Ⅱ组于梗死周边均发现Brdu（＋）细胞存在,心肌特异性抗体阳性,且Ⅰ组阳性率明显高于Ⅱ组。毛细血管密度检测结果显示Ⅰ组毛细血管增生情况明显优于Ⅱ组。结论：不同年龄鼠骨髓干细胞的体外增殖能力不同,年龄越小,增殖能力越强。骨髓干细胞移植可以改善心肌梗死后大鼠的心脏功能,增加梗死区毛细血管密度,抑制心室重构,且年龄与移植效果成反比。     

Alteration of collagenous protein profile in congestive heart failure secondary to myocardial infarction

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure.     

Retracted: IRAK3 gene silencing prevents cardiac rupture and ventricular remodeling through negative regulation of the NF-κB signaling pathway in a mouse model of acute myocardial infarction

Cardiac rupture and ventricular remodeling are recognized as the severe complications and major risk factors of acute myocardial infarction (AMI). This study aims to evaluate the regulatory roles of interleukin-1 receptor-associated kinase 3 (IRAK3) and nuclear factor-κB (NF-κB) signaling pathway in cardiac rupture and ventricular remodeling. Microarray analysis was performed to screen AMI-related differentially expressed genes and IRAK3 was identified. The models of AMI were established in male C57BL/6 mice to investigate the functional role of IRAK3. Afterwards, lentivirus recombinant plasmid si-IRAK3 was constructed for IRAK3 silencing. Next, cardiac function parameters were measured in response to IRAK3 silencing. The regulatory effects that IRAK3 had on myocardial infarct size and the content of myocardial interstitial collagen were analyzed. The regulation of IRAK3 silencing on the NF-κB signaling pathway was further assayed. The obtained results indicated that highly expressed IRAK3 and activated NF-κB signaling pathway were observed in myocardial tissues of mouse models of AMI, accompanied by increased expression of matrix metalloproteinase (MMP)-2/9 and tissue inhibitor of metalloproteinase 2 (TIMP-2). Notably, IRAK3 gene silencing inhibited the activation of NF-κB signaling pathway. Furthermore, IRAK3 gene silencing led to the decreased thickness of infarct area and collagen content of myocardial interstitium, alleviated diastolic, and systolic dysfunctions, as well as, facilitated cardiac functions in mice with AMI, corresponding to decreased expression of MMP-2/9 expression and increased expression of TIMP-2. Taken together, silencing of IRAK3 inactivates the NF-κB signaling pathway, and thereby impeding the cardiac rupture and ventricular remodeling, which eventually prevents AMI progression.     

Proteomic analysis of left ventricular remodeling in an experimental model of heart failure

The development of chronic heart failure (CHF) following myocardial infarction is characterized by progressive alterations of left ventricle (LV) structure and function called left ventricular remodeling (LVR), but the mechanism of LVR remains still unclear. Moreover, information concerning the global alteration protein pattern during the LVR will be helpful for a better understanding of the process. We performed differential proteomic analysis of whole LV proteins using an experimental model of CHF in which myocardial infarction was induced in adult male rats by left coronary ligation. Among 1000 protein spots detected in 2D-gels, 49 were differentially expressed in LV of 2-month-old CHF-rats, corresponding to 27 different identified proteins (8 spots remained unidentified), classified in different functional groups as being heat shock proteins, reticulum endoplasmic stress proteins, oxidative stress proteins, glycolytic enzymes, fatty acid metabolism enzymes, tricarboxylic acid cycle proteins and respiratory chain proteins. We validated modulation of selected proteins using Western blot analysis. Our data showed that proteins involved in cardiac metabolism and oxidative stress are modulated during LVR. Interestingly, proteins of stress response showed different adaptation pathways in the early and late phase of LVR. Expression of four proteins, glyceraldehyde-3-phosphate dehydrogenase, alphaB-crystallin, peroxiredoxin 2, and isocitrate dehydrogenase, was linked to echographic parameters according to heart failure severity.     

大鼠心肌梗塞后心室重构中miRNA的差异表达

目的：筛选大鼠急性心梗后的心室重构过程中差异表达的微小RNA（microRNA,miRNA）,为miRNA调控心室重构的机制研究提供靶标。方法：20只成年雄性Wistar大鼠,分组如下：心肌梗死组（MI组,n=10）和假手术组（Sham组,n=10）。通过结扎大鼠左冠状动脉前降支构建急性心梗模型建模。4周后对大鼠进行超声心动图检查和梗死边缘区心肌HE染色观察心室重构程度。利用miRNA芯片对心梗边缘区的miRNA进行差异表达检测,采用实时定量PCR验证芯片结果的可靠性。结果：心脏超声显示MI组大鼠左室重构明显,心梗边缘区心肌HE染色可见细胞间质大量胶原纤维沉积。miRNA芯片结果显示15个miRNA在心梗4周后呈差异表达,其中11个miRNA（miR-21、miR-23a、miR-125b、miR-132、miR-146b、miR-181b、miR-199a、miR-320、miR-324、miR-328和miR-499）表达上调,4个miRNA（miR-29、miR-30c、miR-133a和miR-208）表达下调。实时定量PCR验证结果与芯片结果一致。结论：这些差异表达的miRNA可能与心梗后心室重构相关,进一步深入研究特定miRNA的调控机制有望为基因治疗提供新靶点。     

TGF-beta(1) and prepro-ANP mRNAs are differentially regulated in exercise-induced cardiac hypertrophy.

The induction of transforming growth factor (TGF)-beta and prepro-atrial natriuretic peptide (ANP) mRNAs represent hallmark features of pathological cardiac hypertrophy. The present study examined whether this pattern of mRNA expression was conserved in a physiological model of cardiac hypertrophy. To address this thesis, female Sprague-Dawley rats were individually housed and permitted to run freely. Voluntary exercise for 3 and 6 wk resulted in biventricular hypertrophy and increased cytochrome c oxidase activity in the triceps muscle. In the hypertrophied left ventricle, the steady-state mRNA level of the cardiac fetal gene prepro-ANP and the extracellular matrix proteins preprocollagen-alpha(1) and fibronectin were similar in exercise-trained and sedentary rats. By contrast, an increased expression of TGF-beta(1) mRNA was observed, whereas TGF-beta(3) mRNA level was unchanged in the hypertrophied left ventricle of exercise-trained compared with sedentary rats. These data highlight a heterogeneity in the regulation of TGF-beta isoforms, and the increased expression of ventricular TGF-beta(1) mRNA in physiological cardiac hypertrophy may contribute to myocardial remodeling.