BMSC在微泡及BiAb辅助下修复心肌纤维化的实验研究及PⅢNP对冠心病合并代谢综合征患者的预测价值
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摘要
病理性心肌纤维化是心血管疾病中的一个重要研究课题。近年来,干细胞移植在缺血性心脏病的治疗领域显示出独特的魅力。尽管在药物干预、介入治疗和外科手术方面取得了可喜的进展,使心肌纤维化的症状改善,闭塞的血管再通,但是心肌细胞缺乏增殖能力,对于已经梗死的心肌细胞和纤维化的心肌尚无办法使其恢复并发挥正常的舒缩功能。20世纪90年代以来,随着干细胞生物工程的蓬勃开展,具有多向分化潜能和自我复制能力的干细胞给心血管疾病的治疗带来了新的突破性的进展。
在细胞心肌成形术中,常用的干细胞有骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)、造血干细胞、内皮祖细胞、胚胎干细胞、骨骼肌成肌细胞等。BMSC取材方便、数量丰富、免疫排斥性低、不存在伦理争议、能分化为心肌细胞,这些优点使BMSC成为细胞心肌成形术治疗中的较优选择。
干细胞移植修复损伤心肌具有广阔的临床应用前景,但一些亟待解决的瓶颈问题制约着细胞心肌成形术的发展应用。随着研究不断深入,人们发现移植入的干细胞有效归巢到受损靶位的数量极少。这直接影响了干细胞的修复效果。此外,干细胞修复心肌纤维化的病理生理机制尚不完全清楚。
经静脉移植BMSC安全性高、易操作,但靶向性差、效率低。因此找寻一种既能从静脉移植,又能提高移植效率的方法极为重要。同时,这种方法若能增加BMSC的归巢率、优化归巢微环境,将使纤维化心肌的修复效果事半功倍。
近年来研究发现,超声微泡不仅是一种可以用于增强超声组织灰阶显像的造影剂,而且可作为一种新型的药物或基因运载工具。将特定的药物或基因与微泡结合在一起,通过外周血管注射,经体外超声定位辐照破坏与之结合的微泡,可使药物或基因在特定组织内定位释放,微泡破裂后引起机械效应和空化效应可使靶组织内毛细血管内皮间隙增宽或破裂,基因或药物可进入特定的组织内。前期研究显示超声微泡可促进肝细胞生长因子成功转移至大鼠缺血心肌,促进血管新生,微血管密度有明显的增加。亦有研究证实,超声破坏微泡可增加局部血管内皮细胞胞膜粘附分子的表达,从而促进骨髓单个核细胞粘附于内皮上,基于这些理论基础,超声破坏微泡技术是否可以作为一种新型的促进干细胞定向移植的有效方法有待进一步进行探讨。
CD29抗体是BMSC表面高表达的分子标志;抗肌凝蛋白轻链抗体(anti-myosin light chain antibody, AMLCA)是损伤心肌特异性抗体。AMLCA能与心室肌特异性地结合,而与肝、脾、肾等非肌性组织以及骨骼肌、血管和肠平滑肌无交叉反应。有研究发现,急性心肌损伤发生后AMLCA可通过破裂的肌细胞膜与坏死心肌结合,而不是与细胞膜完整的正常心肌细胞结合。本课题首先构建CD29×AMLCA这一双特异性抗体(bispecific antibody, BiAb),然后将BiAb和超声辐照微泡作为增效措施,辅助BMSC移植入心肌纤维化小鼠,检测BMSC归巢率的变化,同时探讨BMSC修复心肌纤维化的可能机制。
除了对BMSC在增效措施下干预心肌纤维化的效果和机制进行探讨外,本课题还开展了心肌纤维化血浆标志物PⅢNP预测冠心病合并代谢综合征患者近期心脏重塑及预后的探索性研究。在冠心病人群中,许多患者同时合并代谢综合征,如何客观衡量并预测这类患者的心血管重塑程度,并判断其预后,具有重要临床价值。间质胶原代谢失衡是心血管重塑的重要形态基础,血浆胶原标志物PⅢNP与慢性心衰、急性心梗相关。冠心病合并代谢综合征患者罹患慢性心衰的风险较大,这类人群的心血管重塑引起心功能恶化、动脉顺应性下降,活动耐力降低。血浆胶原标志物PⅢNP在冠心病合并代谢综合征患者中意义如何,与动脉粥样硬化疾病及其危险因素的相关性尚不完全清楚。此部分研究用血浆胶原标志物PⅢNP判断冠心病合并代谢综合征患者的近期心血管重塑程度,并预测其发展趋势,探寻心肌纤维化血浆标志物对预后的判断价值。
综上,本课题主要包括以下五部分内容:
第一部分心肌纤维化模型制备及其胶原沉积机制
目的建立理想的心肌纤维化小鼠模型并探讨其胶原沉积机制。方法将BALB/c小鼠随机分为实验组和对照组,每组10只。实验组予以腹部皮下注射异丙肾上腺素50mg/kg,每天2次,连续10天。对照组同法注射生理盐水。45天后处死小鼠,天狼猩红染色观察心脏Ⅰ、Ⅲ型胶原纤维含量,荧光定量PCR检测心脏基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)、基质金属蛋白酶组织抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)基因表达,免疫组化染色分析心、肝、肾组织层粘连蛋白(laminin,LN)的表达。结果实验组小鼠心脏胶原沉积较多,MMP-9、TIMP-1、LN表达上调(p<0.05),肝、肾组织LN无明显改变(p>0.05)。结论异丙肾上腺素能制备理想的心肌纤维化小鼠模型,其机制与心脏MMP-TIMP失衡有关。
第二部分小鼠骨髓间充质干细胞的分离、培养、鉴定
目的建立一种分离、培养小鼠骨髓间充质干细胞(BMSC)的稳定方法,并通过形态学、细胞表型、功能学指标进行鉴定。方法无菌分离小鼠股骨,以全骨髓贴壁法培养BMSC。稳定传代后,以光镜形态学观察,CD29、CD44、CD117流式细胞术,成骨、成脂诱导培养鉴定BMSC。结果全骨髓贴壁培养法能获得稳定的BMSC,呈平行排列或漩涡状生长。细胞呈梭形或多角型,CD29、CD44、CD117阳性表达率分别为99.94%、99.28%、1.35%,诱导成骨、成脂分化成功。结论全骨髓贴壁法能从小鼠骨髓中分离培养出均质性良好的BMSC。
第三部分超声微泡及双特异性抗体增效骨髓间充质干细胞归巢及干预心肌纤维化
目的探讨骨髓间充质干细胞(BMSC)在BiAb和超声辐射微泡(microbubble, MB)的辅助下归巢并干预心肌纤维化的效果。方法以CD29抗体(能识别间充质干细胞)和抗肌凝蛋白轻链抗体(AMLCA)(能特异性结合损伤心肌)制备BiAb(CD29×AMLCA)。将此BiAb与雄性小鼠BMSC输入异丙肾性心肌纤维化雌性小鼠,辅以超声辐照微泡(BMSC+BiAb+MB组)。另设单纯BMSC组、单纯BiAb组、单纯MB组、BiAb+BMSC治疗组、正常对照组、未治疗组。5周后处死小鼠,荧光定量PCR检测心肌Y染色体鉴别基因(sex-determining region of Y-chromosome,SRY)、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶组织抑制剂-1(TIMP-1)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达。天狼猩红染色观察胶原分布,western-blot观测心脏信号转导与转录激活因子1(signal transducer and activators of transcription-1,STAT1)和信号转导与转录激活因子3(signal transducer and activators of transcription-3,STAT3)的表达。结果BMSC归巢数最多的是BMSC+BiAb+MB组,其次为BMSC+BiAb组,BMSC组归巢数最少。与未治疗组相比,BMSC+BiAb+MB组、BMSC+BiAb组和单纯BMCS组的心肌MMP-9、TIMP-1和STAT1表达下调,STAT3表达升高,胶原沉积降低(p<0.05)。其中,与单纯BMSC组相比,BMSC+BiAb+MB组的心肌STAT3表达明显上调,TIMP-1明显下调(p<0.05)。结论CD29×AMLCA双特异抗体可促进小鼠BMSC的归巢,联合超声辐射微泡,可进一步增加干细胞的归巢率和修复效果。BMSC归巢后能改善缺血心肌MMP-TIMP表达,干预心肌纤维化,其机制可能与心肌STAT介导的信号通路有关。
第四部分骨髓间充质干细胞干预心肌纤维化的可能机制
目的探讨骨髓间充质干细胞(BMSC)移植干预心肌纤维化的可能机制。方法分离培养雄性小鼠BMSC,经尾静脉输入异丙肾性心肌纤维化雌性小鼠(治疗组)。另设未治疗组和正常对照组。5周后处死小鼠,免疫组织化学染色法观察心脏核转录因子-κb(nuclear factor-κb ,NF-κb)、核增殖抗原(proliferating cell nuclear antigen,PCNA)和细胞凋亡蛋白酶(caspase-3)的表达,荧光定量PCR检测心肌SRY、MMP-9、TIMP-1的表达,天狼猩红染色观察胶原分布。结果与未治疗组相比,治疗组心肌有雄性BMSC归巢,胶原沉积减少,NF-κb、caspase-3表达下调,PCNA表达增加(p<0.05)。结论BMSC移植到纤维化心肌,能抑制NF-κb过度活化,改善心肌增殖-凋亡状态。其干预机制与细胞因子网络介导有关。
第五部分PⅢNP预测冠心病合并代谢综合征患者近期心脏重塑程度及预后
目的探讨血浆Ⅲ型前胶原氨基末端肽(PⅢNP)对冠心病合并代谢综合征患者心血管重塑及预后的预测价值。方法连续入选108例冠心病合并代谢综合征患者(平均年龄63.29±3.32岁,女性56%),放免法测定PⅢNP水平,检测并随访1年后患者心脏、颈动脉彩超、6分钟步行距离(6MWT)。根据PⅢNP的中位数水平(3.5μg/L)将受试者分为高低水平两组,统计其各指标差异,多元逐步回归分析影响PⅢNP的因素,并分析PⅢNP与心血管重塑程度的相关性,COX比例风险模型分析心脑血管事件再入院的预测因子,ROC曲线分析PⅢNP判断再住院的阈值。结果血浆PⅢNP水平与冠心病合并代谢综合征患者年龄(β=0.686,p<0.001),高敏C反应蛋白(β=0.194,p=0.001),BMI(β=0.108,p=0.023)相关。与PⅢNP<3.5μg/L组相比,PⅢNP≥3.5μg/L组患者的心律不齐发生率、1年内再住院率和左心室心肌重量指数(LVMI)明显增加,6MWT缩短(p均<0.05)。随着Ln PⅢNP(r=0.271,p=0.007)升高,随访期间心脏重塑程度加重。COX比例风险模型显示,PⅢNP、LVEF、高敏C反应蛋白水平是冠心病合并代谢综合征患者随访期间心脑血管事件再住院的独立预测因子。PⅢNP4.0μg/L为衡量心脑血管事件再住院的最佳阈值。结论PⅢNP随冠心病合并代谢综合征患者的年龄、高敏C反应蛋白、BMI增加而上升,高水平的PⅢNP提示患者近期心脏重塑增加、活动耐力下降、预后不良。
Myocardial fibrosis is becoming one of the most important problems in cardiology. Stem cells have unique advantages in the treatment of myocardial damage. Medical, interventional and surgical intervention have made delectable advancement in revascularization, and improve the symptoms of myocardial fibrosis partly. But myocardial cell lack reproductive activity. How to repair the infarct myocardium and fibrotic heart is a hard problem. From 20th Century 90's, with the development of stem cell study[3], stem cell with multi-directional differentiation and self-duplication ability shine new light on the therapy of cardiovascular disease.
In cellular cardiomyoplasty, bone marrow mesenchymal stem cells (BMSC), hemopoietic stem cell, endothelial progenitor cell, embryonic stem cell, skeletal myoblast are the most commonly used stem cells. BMSC have many desirable characteristics, including low immunogenicity, liability for isolation and amplification, no- ethics debate[4], and multiple differentiation[5, 10], making these cells one of the most frequently used stem cells for the repairing and reconstruction of injured myocardium[6].
In investigations of cellular cardiomyoplasty, it was found that BMSC transplantation via the peripheral vein pathway reduced local trauma and had a high feasibility as compared to alternative treatments. However, the repairing efficacy is limited by the migratory number and colonization amount of targeted stem cells[7, 8]. And its repairing mechanism remains unclear.
BMSC transplantation via vein is safety and easy to approach, but low efficacy. So it is important to find a way to improve the efficiency. Meanwhile, the new way would be a better choice if it can increase the homing number of BMSC and promote the homing niche. Microbubble could improve the microenvironment in the target site for homing, which is helpful for the survival of stem cells. Microbubble act as cavitation nuclei or enhancer in myocardium. Once excited by a high peak pressure ultrasound pulse, the mechanical effects, like shock wave and microstream, released from cavitation could produce a series of bioeffects, contributing to sonoporation, microvascular rupture and hematoma[8]. Ultrasound-targeted microbubble destruction can effectively deliver hepatocyte growth factor into the infracted myocardium and facilitate angiogenesis, which provides a novel way in the gene therapy of myocardial infarction[9]. Targeted delivery of bone marrow mononuclearcell by ultrasound-targeted microbubble to the myocardium of the cardiomyopathic hamster increased the capillary densities and regional blood flow and inhibited cardiac remodeling.
CD29 is the molecular marker that shows a positive rate of more than 99% on the surface of BMSC, and AMLCA can specifically recognize injured myocardium[9]. Prepared CD29×AMLCA is the connecting bridge between injured myocardium and BMSC. Therefore, CD29×AMLCA can bind to both BMSC and injured myocardium, and it can act as the bridge between them. In order to promote the homing of BMSC and understand its repairing mechanism, we combined bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB) to guide BMSC to improve the homing number of BMSC and evaluate the therapeutic efficacy of this treatment on myocardial fibrosis.
Besides the animal study of BMSC preventing myocardial fibrosis, we also explored the role of plasma procollagenⅢN-terminal peptide (PⅢNP) in predicting the prognosis and cardiac remodeling in coronary heart disease (CHD) patients with metabolic syndrome (MS). In the CHD population, many patients also have MS[11] , resulting in an urgent need for objective parameters and predictors of the prognosis and the extent of cardiac remodeling in these patients. Recent studies have revealed that imbalanced metabolism of interstitial collagen provides a morphological foundation for cardiac remodeling, and the plasma collagen marker procollagenⅢN-terminal peptide (PⅢNP) is associated with chronic heart failure [12] and acute myocardial infarction [13]. CHD patients with MS often have a high risk of chronic heart failure, and cardiac remodeling may result in the deterioration of cardiac function, arterial compliance and exercise tolerance. However, it is still not known what role PⅢNP plays in these patients. Likewise, the association between PⅢNP and arterial atherosclerotic diseases and other risk factors remains unclear. The purpose of this cohort study was to explore the role of plasma PⅢNP in predicting the prognosis and cardiac remodeling in CHD patients with MS.
The study included the following five parts:
PARTⅠ: ESTABLISHMENT OF ISOPROTERENOL-INDUCED MYOCARDIAL FIBROSIS AND MECHANISM OF COLLAGEN DEPOSITION
Objective To establish a manipulation-easy animal model of myocardial fibrosis and investigate the mechanism of collagen deposition. Methods Twenty BALB/c mice were randomly divided into two groups: experiment group and control group. Mice in experiment group were subjected to hypodermic injection with isoproterenol at 50 mg/kg. The injection was carried out twice a day for continuous 10 days. Physiological saline was injected with the same method in control group. Forty-five days after injection, collagen distribution was observed using sirius red staining. Expression levels of matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1) in myocardium were detected by fluorescent qRT-PCR. The expression of laminin(LN) in heart, liver and kidney was detected by immunohistochemistry. Results Experiment group had increased levels of MMP-9, TIMP-1, LN and collagen deposition in myocardium than control group(p<0.05). The expression of LN in liver and kidney showed no difference(p>0.05). Conclusion Isoproterenol-induced collagen deposition in the experiment is a reproducible, handy and credible method to establish animal model of myocardial fibrosis. MMP-TIMP imbalance is involved in the mechanism.
PART II:ISOLATION, CULTURE AND IDENTIFICATION OF BONE MARROW MESENCHYMAL STEM CELLS FROM MOUSE
Objective To establish a stable method for isolation and culture of bone marrow mesenchymal stem cells(BMSC) from mouse, and morphological and functional indexes and cellular phenotype were used to identify the cultured BMSC. Methods Mouse limbs were sterilely isolated.The BMSC were enriched and expanded by using bone marrow adherent culture.Morphology of the BMSC was examined under light microscopy. The surface antigens CD29, CD44, CD117 phenotype was analyzed by flow cytometry. The ability of osteogenic and adipogenic diferentiation of the BMSC under diferent induction conditions was assessed.Results BMSC could obtain by the adherence separation method. The in vitro cultured BMSC grew in adherence and most of them grew in parallel arrays or a whirlpool-like pattern. The cells had a fusiform or polygonal shape. The expression of CD29,CD44,CD117 was 99.94%, 99.28%, 1.35%, and BMSC could be induced to differentiate into osteoblast and adipocytes cells. Conclusion BMSC isolated by the adherence separation method have a good homogeneity.
PARTⅢ: BISPECIFIC ANTIBODY AND ULTRASOUND-MEDIATED MICROBUBBLES PROMOTE BONE MARROW MESENCHYMAL STEM CELLS HOMING EFFICIENCY AND PREVENT MYOCARDIAL FIBROSIS
Objective To investigate the efficacy of myocardial fibrosis intervention using bone marrow mesenchymal stem cells (BMSC) with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). Methods BiAb (anti-CD29×anti-myosin light chain antibody (AMLCA)) was prepared and combined with isolated BMSC from male mice and transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein, followed by ultrasound-mediated microbubbles (BMSC+BiAb+MB). This study included seven groups: BMSC+BiAb+MB, BMSC, BiAb, MB, BMSC+BiAb, untreated, and control groups. Five weeks after treatment, expression levels of sex-determining region of Y-chromosome (SRY), matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent qRT-PCR. Collagen distribution was observed using sirius red staining. The protein expression of signal transducer and activators of transcription 1 (STAT1) and STAT3 was detected by Western blot. Results The highest homing number of BMSC was in the BMSC+BiAb+MB group, second highest in the BMSC+BiAb group, and lowest in BMSC alone. Compared to the untreated group, BMSC+BiAb+MB, BMSC+BiAb, and BMSC groups had decreased levels of MMP-9, TIMP-1, STAT1, and collagen deposition, and increased levels of STAT3. Up-regulated STAT3 and down-regulated TIMP-1 were significantly different in BMSC+BiAb+MB as compared to BMSC alone or BMSC+BiAb. Conclusion Homing rate and repairing efficacy of BMSC improved with treatment utilizing a combination of BiAb and MB. BMSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway.
PART IV: BONE MARROW MESENCHYMAL STEM CELLS PREVENT MYOCARDIAL DIBROSIS VIA THE POSSIBLE DIGNALING PATHWAY
Objective To investigate the potential mechanism of myocardial fibrosis intervention using BMSC. Methods Isolated BMSC from male mice were transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein (treated group). This study included three groups: treated, untreated, and control groups. Five weeks after transplantation, expression levels of nuclear factor-κb(NF-κb), heat shock protein-70(HSP-70), proliferating cell nuclear antigen(PCNA) and caspase-3 in myocardium were detected by immunohistochemistry. Results BMSC can home to fibroid heart. Compared to the untreated group, treated group had decreased levels of NF-κb, HSP-70, caspase-3 and increased levels of PCNA ( p<0.05 ) . Conclusion BMSC transplantation can improve collagen deposition in injured myocardium after homing, the mechanism that may be related to the NF-κb, HSP-70, caspase-3 and PCNA mediated signaling pathway.
PART V: PROCOLLAGENⅢN-TERMINAL PEPTIDE PREDICTS SHORT-TERM PROGNOSIS AND CARDIAC REMODELING IN CORONARY HEART DISEASE PATIENTS WITH METABOLIC SYNDROME
Objective To explore the role of plasma procollagenⅢN-terminal peptide (PⅢNP) in predicting the prognosis and cardiac remodeling in CHD patients with MS. Methods One hundred and eight patients were classified into high and low PⅢNP groups according to the median value of plasma PⅢNP. Cardiovascular examinations including echocardiogram, carotid color ultrasound examination, coronary angiography and 6-minute walking test (6MWT) were carried out before and after a one-year follow-up. Readmission for cardiac and cerebrovascular events was assessed during the follow-up period. Results Plasma PⅢNP level was significantly correlated with age(β=0.686,p<0.001), high-sensitivity C-reactive protein (hs-CRP)(β=0.194,p=0.001), and body mass index (BMI)(β=0.108,p=0.023) in a multiple stepwise regression model. There was a positive correlation between LnPⅢNP and a raised left ventricular mass index(LVMI) in partial correlation analysis. The COX proportional hazard model analysis indicated that the level of PIIINP, left ventricular ejection fraction, and hs-CRP were independent predictors of cardio-cerebral readmission during follow-up. A PIIINP value of 4.0μg/L was the best threshold value for determining the need for readmission. Conclusion PIIINP levels rise with increases in age, hs-CRP and BMI in CHD patients with MS, and a high level of PIIINP indicates recent deterioration of cardiac remodeling and exercise tolerance, as well as a poor prognosis.
在细胞心肌成形术中,常用的干细胞有骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)、造血干细胞、内皮祖细胞、胚胎干细胞、骨骼肌成肌细胞等。BMSC取材方便、数量丰富、免疫排斥性低、不存在伦理争议、能分化为心肌细胞,这些优点使BMSC成为细胞心肌成形术治疗中的较优选择。
干细胞移植修复损伤心肌具有广阔的临床应用前景,但一些亟待解决的瓶颈问题制约着细胞心肌成形术的发展应用。随着研究不断深入,人们发现移植入的干细胞有效归巢到受损靶位的数量极少。这直接影响了干细胞的修复效果。此外,干细胞修复心肌纤维化的病理生理机制尚不完全清楚。
经静脉移植BMSC安全性高、易操作,但靶向性差、效率低。因此找寻一种既能从静脉移植,又能提高移植效率的方法极为重要。同时,这种方法若能增加BMSC的归巢率、优化归巢微环境,将使纤维化心肌的修复效果事半功倍。
近年来研究发现,超声微泡不仅是一种可以用于增强超声组织灰阶显像的造影剂,而且可作为一种新型的药物或基因运载工具。将特定的药物或基因与微泡结合在一起,通过外周血管注射,经体外超声定位辐照破坏与之结合的微泡,可使药物或基因在特定组织内定位释放,微泡破裂后引起机械效应和空化效应可使靶组织内毛细血管内皮间隙增宽或破裂,基因或药物可进入特定的组织内。前期研究显示超声微泡可促进肝细胞生长因子成功转移至大鼠缺血心肌,促进血管新生,微血管密度有明显的增加。亦有研究证实,超声破坏微泡可增加局部血管内皮细胞胞膜粘附分子的表达,从而促进骨髓单个核细胞粘附于内皮上,基于这些理论基础,超声破坏微泡技术是否可以作为一种新型的促进干细胞定向移植的有效方法有待进一步进行探讨。
CD29抗体是BMSC表面高表达的分子标志;抗肌凝蛋白轻链抗体(anti-myosin light chain antibody, AMLCA)是损伤心肌特异性抗体。AMLCA能与心室肌特异性地结合,而与肝、脾、肾等非肌性组织以及骨骼肌、血管和肠平滑肌无交叉反应。有研究发现,急性心肌损伤发生后AMLCA可通过破裂的肌细胞膜与坏死心肌结合,而不是与细胞膜完整的正常心肌细胞结合。本课题首先构建CD29×AMLCA这一双特异性抗体(bispecific antibody, BiAb),然后将BiAb和超声辐照微泡作为增效措施,辅助BMSC移植入心肌纤维化小鼠,检测BMSC归巢率的变化,同时探讨BMSC修复心肌纤维化的可能机制。
除了对BMSC在增效措施下干预心肌纤维化的效果和机制进行探讨外,本课题还开展了心肌纤维化血浆标志物PⅢNP预测冠心病合并代谢综合征患者近期心脏重塑及预后的探索性研究。在冠心病人群中,许多患者同时合并代谢综合征,如何客观衡量并预测这类患者的心血管重塑程度,并判断其预后,具有重要临床价值。间质胶原代谢失衡是心血管重塑的重要形态基础,血浆胶原标志物PⅢNP与慢性心衰、急性心梗相关。冠心病合并代谢综合征患者罹患慢性心衰的风险较大,这类人群的心血管重塑引起心功能恶化、动脉顺应性下降,活动耐力降低。血浆胶原标志物PⅢNP在冠心病合并代谢综合征患者中意义如何,与动脉粥样硬化疾病及其危险因素的相关性尚不完全清楚。此部分研究用血浆胶原标志物PⅢNP判断冠心病合并代谢综合征患者的近期心血管重塑程度,并预测其发展趋势,探寻心肌纤维化血浆标志物对预后的判断价值。
综上,本课题主要包括以下五部分内容:
第一部分心肌纤维化模型制备及其胶原沉积机制
目的建立理想的心肌纤维化小鼠模型并探讨其胶原沉积机制。方法将BALB/c小鼠随机分为实验组和对照组,每组10只。实验组予以腹部皮下注射异丙肾上腺素50mg/kg,每天2次,连续10天。对照组同法注射生理盐水。45天后处死小鼠,天狼猩红染色观察心脏Ⅰ、Ⅲ型胶原纤维含量,荧光定量PCR检测心脏基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)、基质金属蛋白酶组织抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)基因表达,免疫组化染色分析心、肝、肾组织层粘连蛋白(laminin,LN)的表达。结果实验组小鼠心脏胶原沉积较多,MMP-9、TIMP-1、LN表达上调(p<0.05),肝、肾组织LN无明显改变(p>0.05)。结论异丙肾上腺素能制备理想的心肌纤维化小鼠模型,其机制与心脏MMP-TIMP失衡有关。
第二部分小鼠骨髓间充质干细胞的分离、培养、鉴定
目的建立一种分离、培养小鼠骨髓间充质干细胞(BMSC)的稳定方法,并通过形态学、细胞表型、功能学指标进行鉴定。方法无菌分离小鼠股骨,以全骨髓贴壁法培养BMSC。稳定传代后,以光镜形态学观察,CD29、CD44、CD117流式细胞术,成骨、成脂诱导培养鉴定BMSC。结果全骨髓贴壁培养法能获得稳定的BMSC,呈平行排列或漩涡状生长。细胞呈梭形或多角型,CD29、CD44、CD117阳性表达率分别为99.94%、99.28%、1.35%,诱导成骨、成脂分化成功。结论全骨髓贴壁法能从小鼠骨髓中分离培养出均质性良好的BMSC。
第三部分超声微泡及双特异性抗体增效骨髓间充质干细胞归巢及干预心肌纤维化
目的探讨骨髓间充质干细胞(BMSC)在BiAb和超声辐射微泡(microbubble, MB)的辅助下归巢并干预心肌纤维化的效果。方法以CD29抗体(能识别间充质干细胞)和抗肌凝蛋白轻链抗体(AMLCA)(能特异性结合损伤心肌)制备BiAb(CD29×AMLCA)。将此BiAb与雄性小鼠BMSC输入异丙肾性心肌纤维化雌性小鼠,辅以超声辐照微泡(BMSC+BiAb+MB组)。另设单纯BMSC组、单纯BiAb组、单纯MB组、BiAb+BMSC治疗组、正常对照组、未治疗组。5周后处死小鼠,荧光定量PCR检测心肌Y染色体鉴别基因(sex-determining region of Y-chromosome,SRY)、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶组织抑制剂-1(TIMP-1)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达。天狼猩红染色观察胶原分布,western-blot观测心脏信号转导与转录激活因子1(signal transducer and activators of transcription-1,STAT1)和信号转导与转录激活因子3(signal transducer and activators of transcription-3,STAT3)的表达。结果BMSC归巢数最多的是BMSC+BiAb+MB组,其次为BMSC+BiAb组,BMSC组归巢数最少。与未治疗组相比,BMSC+BiAb+MB组、BMSC+BiAb组和单纯BMCS组的心肌MMP-9、TIMP-1和STAT1表达下调,STAT3表达升高,胶原沉积降低(p<0.05)。其中,与单纯BMSC组相比,BMSC+BiAb+MB组的心肌STAT3表达明显上调,TIMP-1明显下调(p<0.05)。结论CD29×AMLCA双特异抗体可促进小鼠BMSC的归巢,联合超声辐射微泡,可进一步增加干细胞的归巢率和修复效果。BMSC归巢后能改善缺血心肌MMP-TIMP表达,干预心肌纤维化,其机制可能与心肌STAT介导的信号通路有关。
第四部分骨髓间充质干细胞干预心肌纤维化的可能机制
目的探讨骨髓间充质干细胞(BMSC)移植干预心肌纤维化的可能机制。方法分离培养雄性小鼠BMSC,经尾静脉输入异丙肾性心肌纤维化雌性小鼠(治疗组)。另设未治疗组和正常对照组。5周后处死小鼠,免疫组织化学染色法观察心脏核转录因子-κb(nuclear factor-κb ,NF-κb)、核增殖抗原(proliferating cell nuclear antigen,PCNA)和细胞凋亡蛋白酶(caspase-3)的表达,荧光定量PCR检测心肌SRY、MMP-9、TIMP-1的表达,天狼猩红染色观察胶原分布。结果与未治疗组相比,治疗组心肌有雄性BMSC归巢,胶原沉积减少,NF-κb、caspase-3表达下调,PCNA表达增加(p<0.05)。结论BMSC移植到纤维化心肌,能抑制NF-κb过度活化,改善心肌增殖-凋亡状态。其干预机制与细胞因子网络介导有关。
第五部分PⅢNP预测冠心病合并代谢综合征患者近期心脏重塑程度及预后
目的探讨血浆Ⅲ型前胶原氨基末端肽(PⅢNP)对冠心病合并代谢综合征患者心血管重塑及预后的预测价值。方法连续入选108例冠心病合并代谢综合征患者(平均年龄63.29±3.32岁,女性56%),放免法测定PⅢNP水平,检测并随访1年后患者心脏、颈动脉彩超、6分钟步行距离(6MWT)。根据PⅢNP的中位数水平(3.5μg/L)将受试者分为高低水平两组,统计其各指标差异,多元逐步回归分析影响PⅢNP的因素,并分析PⅢNP与心血管重塑程度的相关性,COX比例风险模型分析心脑血管事件再入院的预测因子,ROC曲线分析PⅢNP判断再住院的阈值。结果血浆PⅢNP水平与冠心病合并代谢综合征患者年龄(β=0.686,p<0.001),高敏C反应蛋白(β=0.194,p=0.001),BMI(β=0.108,p=0.023)相关。与PⅢNP<3.5μg/L组相比,PⅢNP≥3.5μg/L组患者的心律不齐发生率、1年内再住院率和左心室心肌重量指数(LVMI)明显增加,6MWT缩短(p均<0.05)。随着Ln PⅢNP(r=0.271,p=0.007)升高,随访期间心脏重塑程度加重。COX比例风险模型显示,PⅢNP、LVEF、高敏C反应蛋白水平是冠心病合并代谢综合征患者随访期间心脑血管事件再住院的独立预测因子。PⅢNP4.0μg/L为衡量心脑血管事件再住院的最佳阈值。结论PⅢNP随冠心病合并代谢综合征患者的年龄、高敏C反应蛋白、BMI增加而上升,高水平的PⅢNP提示患者近期心脏重塑增加、活动耐力下降、预后不良。
Myocardial fibrosis is becoming one of the most important problems in cardiology. Stem cells have unique advantages in the treatment of myocardial damage. Medical, interventional and surgical intervention have made delectable advancement in revascularization, and improve the symptoms of myocardial fibrosis partly. But myocardial cell lack reproductive activity. How to repair the infarct myocardium and fibrotic heart is a hard problem. From 20th Century 90's, with the development of stem cell study[3], stem cell with multi-directional differentiation and self-duplication ability shine new light on the therapy of cardiovascular disease.
In cellular cardiomyoplasty, bone marrow mesenchymal stem cells (BMSC), hemopoietic stem cell, endothelial progenitor cell, embryonic stem cell, skeletal myoblast are the most commonly used stem cells. BMSC have many desirable characteristics, including low immunogenicity, liability for isolation and amplification, no- ethics debate[4], and multiple differentiation[5, 10], making these cells one of the most frequently used stem cells for the repairing and reconstruction of injured myocardium[6].
In investigations of cellular cardiomyoplasty, it was found that BMSC transplantation via the peripheral vein pathway reduced local trauma and had a high feasibility as compared to alternative treatments. However, the repairing efficacy is limited by the migratory number and colonization amount of targeted stem cells[7, 8]. And its repairing mechanism remains unclear.
BMSC transplantation via vein is safety and easy to approach, but low efficacy. So it is important to find a way to improve the efficiency. Meanwhile, the new way would be a better choice if it can increase the homing number of BMSC and promote the homing niche. Microbubble could improve the microenvironment in the target site for homing, which is helpful for the survival of stem cells. Microbubble act as cavitation nuclei or enhancer in myocardium. Once excited by a high peak pressure ultrasound pulse, the mechanical effects, like shock wave and microstream, released from cavitation could produce a series of bioeffects, contributing to sonoporation, microvascular rupture and hematoma[8]. Ultrasound-targeted microbubble destruction can effectively deliver hepatocyte growth factor into the infracted myocardium and facilitate angiogenesis, which provides a novel way in the gene therapy of myocardial infarction[9]. Targeted delivery of bone marrow mononuclearcell by ultrasound-targeted microbubble to the myocardium of the cardiomyopathic hamster increased the capillary densities and regional blood flow and inhibited cardiac remodeling.
CD29 is the molecular marker that shows a positive rate of more than 99% on the surface of BMSC, and AMLCA can specifically recognize injured myocardium[9]. Prepared CD29×AMLCA is the connecting bridge between injured myocardium and BMSC. Therefore, CD29×AMLCA can bind to both BMSC and injured myocardium, and it can act as the bridge between them. In order to promote the homing of BMSC and understand its repairing mechanism, we combined bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB) to guide BMSC to improve the homing number of BMSC and evaluate the therapeutic efficacy of this treatment on myocardial fibrosis.
Besides the animal study of BMSC preventing myocardial fibrosis, we also explored the role of plasma procollagenⅢN-terminal peptide (PⅢNP) in predicting the prognosis and cardiac remodeling in coronary heart disease (CHD) patients with metabolic syndrome (MS). In the CHD population, many patients also have MS[11] , resulting in an urgent need for objective parameters and predictors of the prognosis and the extent of cardiac remodeling in these patients. Recent studies have revealed that imbalanced metabolism of interstitial collagen provides a morphological foundation for cardiac remodeling, and the plasma collagen marker procollagenⅢN-terminal peptide (PⅢNP) is associated with chronic heart failure [12] and acute myocardial infarction [13]. CHD patients with MS often have a high risk of chronic heart failure, and cardiac remodeling may result in the deterioration of cardiac function, arterial compliance and exercise tolerance. However, it is still not known what role PⅢNP plays in these patients. Likewise, the association between PⅢNP and arterial atherosclerotic diseases and other risk factors remains unclear. The purpose of this cohort study was to explore the role of plasma PⅢNP in predicting the prognosis and cardiac remodeling in CHD patients with MS.
The study included the following five parts:
PARTⅠ: ESTABLISHMENT OF ISOPROTERENOL-INDUCED MYOCARDIAL FIBROSIS AND MECHANISM OF COLLAGEN DEPOSITION
Objective To establish a manipulation-easy animal model of myocardial fibrosis and investigate the mechanism of collagen deposition. Methods Twenty BALB/c mice were randomly divided into two groups: experiment group and control group. Mice in experiment group were subjected to hypodermic injection with isoproterenol at 50 mg/kg. The injection was carried out twice a day for continuous 10 days. Physiological saline was injected with the same method in control group. Forty-five days after injection, collagen distribution was observed using sirius red staining. Expression levels of matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1) in myocardium were detected by fluorescent qRT-PCR. The expression of laminin(LN) in heart, liver and kidney was detected by immunohistochemistry. Results Experiment group had increased levels of MMP-9, TIMP-1, LN and collagen deposition in myocardium than control group(p<0.05). The expression of LN in liver and kidney showed no difference(p>0.05). Conclusion Isoproterenol-induced collagen deposition in the experiment is a reproducible, handy and credible method to establish animal model of myocardial fibrosis. MMP-TIMP imbalance is involved in the mechanism.
PART II:ISOLATION, CULTURE AND IDENTIFICATION OF BONE MARROW MESENCHYMAL STEM CELLS FROM MOUSE
Objective To establish a stable method for isolation and culture of bone marrow mesenchymal stem cells(BMSC) from mouse, and morphological and functional indexes and cellular phenotype were used to identify the cultured BMSC. Methods Mouse limbs were sterilely isolated.The BMSC were enriched and expanded by using bone marrow adherent culture.Morphology of the BMSC was examined under light microscopy. The surface antigens CD29, CD44, CD117 phenotype was analyzed by flow cytometry. The ability of osteogenic and adipogenic diferentiation of the BMSC under diferent induction conditions was assessed.Results BMSC could obtain by the adherence separation method. The in vitro cultured BMSC grew in adherence and most of them grew in parallel arrays or a whirlpool-like pattern. The cells had a fusiform or polygonal shape. The expression of CD29,CD44,CD117 was 99.94%, 99.28%, 1.35%, and BMSC could be induced to differentiate into osteoblast and adipocytes cells. Conclusion BMSC isolated by the adherence separation method have a good homogeneity.
PARTⅢ: BISPECIFIC ANTIBODY AND ULTRASOUND-MEDIATED MICROBUBBLES PROMOTE BONE MARROW MESENCHYMAL STEM CELLS HOMING EFFICIENCY AND PREVENT MYOCARDIAL FIBROSIS
Objective To investigate the efficacy of myocardial fibrosis intervention using bone marrow mesenchymal stem cells (BMSC) with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). Methods BiAb (anti-CD29×anti-myosin light chain antibody (AMLCA)) was prepared and combined with isolated BMSC from male mice and transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein, followed by ultrasound-mediated microbubbles (BMSC+BiAb+MB). This study included seven groups: BMSC+BiAb+MB, BMSC, BiAb, MB, BMSC+BiAb, untreated, and control groups. Five weeks after treatment, expression levels of sex-determining region of Y-chromosome (SRY), matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent qRT-PCR. Collagen distribution was observed using sirius red staining. The protein expression of signal transducer and activators of transcription 1 (STAT1) and STAT3 was detected by Western blot. Results The highest homing number of BMSC was in the BMSC+BiAb+MB group, second highest in the BMSC+BiAb group, and lowest in BMSC alone. Compared to the untreated group, BMSC+BiAb+MB, BMSC+BiAb, and BMSC groups had decreased levels of MMP-9, TIMP-1, STAT1, and collagen deposition, and increased levels of STAT3. Up-regulated STAT3 and down-regulated TIMP-1 were significantly different in BMSC+BiAb+MB as compared to BMSC alone or BMSC+BiAb. Conclusion Homing rate and repairing efficacy of BMSC improved with treatment utilizing a combination of BiAb and MB. BMSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway.
PART IV: BONE MARROW MESENCHYMAL STEM CELLS PREVENT MYOCARDIAL DIBROSIS VIA THE POSSIBLE DIGNALING PATHWAY
Objective To investigate the potential mechanism of myocardial fibrosis intervention using BMSC. Methods Isolated BMSC from male mice were transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein (treated group). This study included three groups: treated, untreated, and control groups. Five weeks after transplantation, expression levels of nuclear factor-κb(NF-κb), heat shock protein-70(HSP-70), proliferating cell nuclear antigen(PCNA) and caspase-3 in myocardium were detected by immunohistochemistry. Results BMSC can home to fibroid heart. Compared to the untreated group, treated group had decreased levels of NF-κb, HSP-70, caspase-3 and increased levels of PCNA ( p<0.05 ) . Conclusion BMSC transplantation can improve collagen deposition in injured myocardium after homing, the mechanism that may be related to the NF-κb, HSP-70, caspase-3 and PCNA mediated signaling pathway.
PART V: PROCOLLAGENⅢN-TERMINAL PEPTIDE PREDICTS SHORT-TERM PROGNOSIS AND CARDIAC REMODELING IN CORONARY HEART DISEASE PATIENTS WITH METABOLIC SYNDROME
Objective To explore the role of plasma procollagenⅢN-terminal peptide (PⅢNP) in predicting the prognosis and cardiac remodeling in CHD patients with MS. Methods One hundred and eight patients were classified into high and low PⅢNP groups according to the median value of plasma PⅢNP. Cardiovascular examinations including echocardiogram, carotid color ultrasound examination, coronary angiography and 6-minute walking test (6MWT) were carried out before and after a one-year follow-up. Readmission for cardiac and cerebrovascular events was assessed during the follow-up period. Results Plasma PⅢNP level was significantly correlated with age(β=0.686,p<0.001), high-sensitivity C-reactive protein (hs-CRP)(β=0.194,p=0.001), and body mass index (BMI)(β=0.108,p=0.023) in a multiple stepwise regression model. There was a positive correlation between LnPⅢNP and a raised left ventricular mass index(LVMI) in partial correlation analysis. The COX proportional hazard model analysis indicated that the level of PIIINP, left ventricular ejection fraction, and hs-CRP were independent predictors of cardio-cerebral readmission during follow-up. A PIIINP value of 4.0μg/L was the best threshold value for determining the need for readmission. Conclusion PIIINP levels rise with increases in age, hs-CRP and BMI in CHD patients with MS, and a high level of PIIINP indicates recent deterioration of cardiac remodeling and exercise tolerance, as well as a poor prognosis.
引文
[1] Zimmermann S, Ruthrof S, Nowak K et al. Short-term prognosis of contemporary interventional therapy of ST-elevation myocardial infarction: does gender matter?[J]. Clin Res Cardiol, 2009;98(11):709-715.
[2]邓玮,陈庆伟.心脏纤维化的干预治疗[J].心血管病学进展, 2003(3):191-194.
[3] Abbott A. Stem-cell furore erupts[J]. Nature, 2010;466(7302):17.
[4] Vogel G. Stem cells. Reprogrammed cells come up short, for now[J]. Science, 2010;327(5970):1191.
[5] Wen Z, Zheng S, Zhou C et al. Repair mechanisms of bone marrow mesenchymal stem cells in myocardial infarction[J]. J Cell Mol Med, 2010.[Epub ahead of print].
[6] Joggerst SJ, Hatzopoulos AK. Stem cell therapy for cardiac repair: benefits and barriers[J]. Expert Rev Mol Med, 2009;11:e20.
[7] Jaiswal S, Jamieson CH, Pang WW et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis[J]. Cell, 2009;138(2):271-285.
[8] Yamashita YM. Cell adhesion in regulation of asymmetric stem cell division[J]. Curr Opin Cell Biol, 2010;22(5):605-610.
[9] Wang Z, Liao Y, Dong J et al. Clinical significance and pathogenic role of anti-cardiac myosin autoantibody in dilated cardiomyopathy[J]. Chin Med J (Engl), 2003;116(4):499-502.
[10] Battiwalla M, Hematti P. Mesenchymal stem cells in hematopoietic stem cell transplantation[J]. Cytotherapy, 2009;11(5):503-515.
[11] Chen Q, Liu Y, Yin Y et al. Relationship between metabolic syndrome (MS) and coronary heart disease (CHD) in an aged group[J]. Arch Gerontol Geriatr, 2008;46(1):107-115.
[12] Radauceanu A, Ducki C, Virion JM et al. Extracellular matrix turnover and inflammatory markers independently predict functional status and outcome inchronic heart failure[J]. J Card Fail, 2008;14(6):467-474.
[13] Poulsen SH, Host NB, Jensen SE et al. Relationship between serum amino-terminal propeptide of type III procollagen and changes of left ventricular function after acute myocardial infarction[J]. Circulation, 2000;101(13):1527-1532.
[14] Paul D, Samuel SM, Maulik N. Mesenchymal stem cell: present challenges and prospective cellular cardiomyoplasty approaches for myocardial regeneration[J]. Antioxid Redox Signal, 2009;11(8):1841-1855.
[15] Mias C, Lairez O, Trouche E et al. Mesenchymal stem cells promote matrix metalloproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction[J]. Stem Cells, 2009;27(11):2734-2743.
[16] Whittaker P, Kloner RA, Boughner DR et al. Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light[J]. Basic Res Cardiol, 1994;89(5):397-410.
[17] Brooks WW, Conrad CH. Isoproterenol-induced myocardial injury and diastolic dysfunction in mice: structural and functional correlates[J]. Comp Med, 2009;59(4):339-343.
[18] Sun Y, Weber KT. Animal models of cardiac fibrosis[J]. Methods Mol Med, 2005;117:273-290.
[19] Arteaga de Murphy C, Ferro-Flores G, Villanueva-Sanchez O et al. 99mTc-glucarate for detection of isoproterenol-induced myocardial infarction in rats[J]. Int J Pharm, 2002;233(1-2):29-34.
[20] Hynes RO. The extracellular matrix: not just pretty fibrils[J]. Science, 2009;326(5957):1216-1219.
[21] LaCroix C, Freeling J, Giles A et al. Deficiency of M2 muscarinic acetylcholine receptors increases susceptibility of ventricular function to chronic adrenergic stress[J]. Am J Physiol Heart Circ Physiol, 2008;294(2):H810-820.
[22] Feng W, Li W, Liu W et al. IL-17 induces myocardial fibrosis and enhancesRANKL/OPG and MMP/TIMP signaling in isoproterenol-induced heart failure[J]. Exp Mol Pathol, 2009;87(3):212-218.
[23] Polyakova V, Loeffler I, Hein S et al. Fibrosis in endstage human heart failure: Severe changes in collagen metabolism and MMP/TIMP profiles[J]. Int J Cardiol, 2010.
[24] Truter SL, Catanzaro DF, Supino PG et al. Differential expression of matrix metalloproteinases and tissue inhibitors and extracellular matrix remodeling in aortic regurgitant hearts[J]. Cardiology, 2009;113(3):161-168.
[25] Pei Z, Meng R, Li G et al. Angiotensin-(1-7) ameliorates myocardial remodeling and interstitial fibrosis in spontaneous hypertension: Role of MMPs/TIMPs[J]. Toxicol Lett, 2010;199(2):173-181.
[26] Kandalam V, Basu R, Abraham T et al. Early activation of matrix metalloproteinases underlies the exacerbated systolic and diastolic dysfunction in mice lacking TIMP3 following myocardial infarction[J]. Am J Physiol Heart Circ Physiol, 2010;299(4):H1012-1023.
[27] Herpel E, Pritsch M, Koch A et al. Interstitial fibrosis in the heart: differences in extracellular matrix proteins and matrix metalloproteinases in end-stage dilated, ischaemic and valvular cardiomyopathy[J]. Histopathology, 2006;48(6):736-747.
[28] Bozkaya YT, Aydin HH, Celik HA et al. Increased myocardial collagen turnover in patients with progressive cardiac conduction disease[J]. Pacing Clin Electrophysiol, 2008;31(10):1284-1290.
[29] Pereira RF, Halford KW, O'Hara MD et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice[J]. Proc Natl Acad Sci U S A, 1995;92(11):4857-4861.
[30] Morikawa S, Mabuchi Y, Kubota Y et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow[J]. J Exp Med, 2009;206(11):2483-2496.
[31] Sung JH, Yang HM, Park JB et al. Isolation and characterization of mousemesenchymal stem cells[J]. Transplant Proc, 2008;40(8):2649-2654.
[32] Chamberlain G, Fox J, Ashton B et al. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing[J]. Stem Cells, 2007;25(11):2739-2749.
[33] Sordi V. Mesenchymal stem cell homing capacity[J]. Transplantation, 2009;87(9 Suppl):S42-45.
[34] Lee RJ, Fang Q, Davol PA et al. Antibody targeting of stem cells to infarcted myocardium[J]. Stem Cells, 2007;25(3):712-717.
[35] Sen M, Wankowski DM, Garlie NK et al. Use of anti-CD3 x anti-HER2/neu bispecific antibody for redirecting cytotoxicity of activated T cells toward HER2/neu+ tumors[J]. J Hematother Stem Cell Res, 2001;10(2):247-260.
[36] Lum LG, Fok H, Sievers R et al. Targeting of Lin-Sca+ hematopoietic stem cells with bispecific antibodies to injured myocardium[J]. Blood Cells Mol Dis, 2004;32(1):82-87.
[37] Zhao TC, Tseng A, Yano N et al. Targeting human CD34+ hematopoietic stem cells with anti-CD45 x anti-myosin light-chain bispecific antibody preserves cardiac function in myocardial infarction[J]. J Appl Physiol, 2008;104(6):1793-1800.
[38] Kraitchman DL, Tatsumi M, Gilson WD et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction[J]. Circulation, 2005;112(10):1451-1461.
[39] Cochlovius B, Kipriyanov SM, Stassar MJ et al. Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells[J]. J Immunol, 2000;165(2):888-895.
[40] Weiner LM, Holmes M, Adams GP et al. A human tumor xenograft model of therapy with a bispecific monoclonal antibody targeting c-erbB-2 and CD16[J]. Cancer Res, 1993;53(1):94-100.
[41] Rossi EA, Goldenberg DM, Cardillo TM et al. Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties ofanti-CD20/CD22 antibodies in lymphoma[J]. Blood, 2009;113(24):6161-6171.
[42] Skyba DM, Price RJ, Linka AZ et al. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue[J]. Circulation, 1998;98(4):290-293.
[43] Xu YL, Gao YH, Liu Z et al. Myocardium-targeted transplantation of mesenchymal stem cells by diagnostic ultrasound-mediated microbubble destruction improves cardiac function in myocardial infarction of New Zealand rabbits[J]. Int J Cardiol, 2009.
[44] Miyahara Y, Nagaya N, Kataoka M et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction[J]. Nat Med, 2006;12(4):459-465.
[45] Jiang W, Ma A, Wang T et al. Homing and differentiation of mesenchymal stem cells delivered intravenously to ischemic myocardium in vivo: a time-series study[J]. Pflugers Arch, 2006;453(1):43-52.
[46] Gnecchi M, He H, Liang OD et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells[J]. Nat Med, 2005;11(4):367-368.
[47] Li L, Zhang Y, Li Y et al. Mesenchymal stem cell transplantation attenuates cardiac fibrosis associated with isoproterenol-induced global heart failure[J]. Transpl Int, 2008;21(12):1181-1189.
[48] Molina EJ, Palma J, Gupta D et al. Reverse remodeling is associated with changes in extracellular matrix proteases and tissue inhibitors after mesenchymal stem cell (MSC) treatment of pressure overload hypertrophy[J]. J Tissue Eng Regen Med, 2009;3(2):85-91.
[49] Hsu CP, Huang CY, Wang JS et al. Extracellular matrix remodeling attenuated after experimental postinfarct left ventricular aneurysm repair[J]. Ann Thorac Surg, 2008;86(4):1243-1249.
[50] Dixon JA, Gorman RC, Stroud RE et al. Mesenchymal cell transplantation and myocardial remodeling after myocardial infarction[J]. Circulation, 2009;120(11Suppl):S220-229.
[51] Meirelles Lda S, Fontes AM, Covas DT et al. Mechanisms involved in the therapeutic properties of mesenchymal stem cells[J]. Cytokine Growth Factor Rev, 2009;20(5-6):419-427.
[52] Guo J, Lin GS, Bao CY et al. Anti-inflammation role for mesenchymal stem cells transplantation in myocardial infarction[J]. Inflammation, 2007;30(3-4):97-104.
[53] Wang TL, Yang YH, Chang H et al. Angiotensin II signals mechanical stretch-induced cardiac matrix metalloproteinase expression via JAK-STAT pathway[J]. J Mol Cell Cardiol, 2004;37(3):785-794.
[54] El-Adawi H, Deng L, Tramontano A et al. The functional role of the JAK-STAT pathway in post-infarction remodeling[J]. Cardiovasc Res, 2003;57(1):129-138.
[55] Wagner M, Siddiqui MA. Signaling networks regulating cardiac myocyte survival and death[J]. Curr Opin Investig Drugs, 2009;10(9):928-937.
[56] Scarabelli TM, Mariotto S, Abdel-Azeim S et al. Targeting STAT1 by myricetin and delphinidin provides efficient protection of the heart from ischemia/reperfusion-induced injury[J]. FEBS Lett, 2009;583(3):531-541.
[57] Boengler K, Hilfiker-Kleiner D, Drexler H et al. The myocardial JAK/STAT pathway: from protection to failure[J]. Pharmacol Ther, 2008;120(2):172-185.
[58] Barry SP, Townsend PA, McCormick J et al. STAT3 deletion sensitizes cells to oxidative stress[J]. Biochem Biophys Res Commun, 2009;385(3):324-329.
[59] Harada M, Qin Y, Takano H et al. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes[J]. Nat Med, 2005;11(3):305-311.
[60] Ng DC, Court NW, dos Remedios CG et al. Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts[J]. Cardiovasc Res, 2003;57(2):333-346.
[61] Furlani D, Li W, Pittermann E et al. A transformed cell population derived from cultured mesenchymal stem cells has no functional effect after transplantationinto the injured heart[J]. Cell Transplant, 2009;18(3):319-331.
[62] Lin YZ, Cai JM, Chen L et al. [Relationship between atrial fibroblast proliferation/fibrosis and atrial fibrillation in patients with rheumatic heart disease][J]. Zhonghua Xin Xue Guan Bing Za Zhi, 2009;37(9):813-817.
[63] Chavakis E, Urbich C, Dimmeler S. Homing and engraftment of progenitor cells: a prerequisite for cell therapy[J]. J Mol Cell Cardiol, 2008;45(4):514-522.
[64] Zavadzkas JA, Plyler RA, Bouges S et al. Cardiac-restricted overexpression of extracellular matrix metalloproteinase inducer causes myocardial remodeling and dysfunction in aging mice[J]. Am J Physiol Heart Circ Physiol, 2008;295(4):H1394-1402.
[65] Ries C, Egea V, Karow M et al. MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines[J]. Blood, 2007;109(9):4055-4063.
[66] Kolpakov MA, Seqqat R, Rafiq K et al. Pleiotropic effects of neutrophils on myocyte apoptosis and left ventricular remodeling during early volume overload[J]. J Mol Cell Cardiol, 2009;47(5):634-645.
[67] Mias C, Lairez O, Trouche E et al. Mesenchymal Stem Cells Promote Matrix Metalloproteinase Secretion By Cardiac Fibroblasts And Reduce Cardiac Ventricular Fibrosis After Myocardial Infarction[J]. Stem Cells, 2009.
[68] Chimenti I, Smith RR, Li TS et al. Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice[J]. Circ Res, 2010;106(5):971-980.
[69] Bujak M, Dobaczewski M, Chatila K et al. Interleukin-1 receptor type I signaling critically regulates infarct healing and cardiac remodeling[J]. Am J Pathol, 2008;173(1):57-67.
[70] Frangogiannis NG. The immune system and cardiac repair[J]. Pharmacol Res, 2008;58(2):88-111.
[71] Van der Heiden K, Cuhlmann S, Luong le A et al. Role of nuclear factor kappaB in cardiovascular health and disease[J]. Clin Sci (Lond),2010;118(10):593-605.
[72] Timmers L, van Keulen JK, Hoefer IE et al. Targeted deletion of nuclear factor kappaB p50 enhances cardiac remodeling and dysfunction following myocardial infarction[J]. Circ Res, 2009;104(5):699-706.
[73] Latanich CA, Toledo-Pereyra LH. Searching for NF-kappaB-based treatments of ischemia reperfusion injury[J]. J Invest Surg, 2009;22(4):301-315.
[74] Kim YS, Park HJ, Hong MH et al. TNF-alpha enhances engraftment of mesenchymal stem cells into infarcted myocardium[J]. Front Biosci, 2009;14:2845-2856.
[75] Hall G, Hasday JD, Rogers TB. Regulating the regulator: NF-kappaB signaling in heart[J]. J Mol Cell Cardiol, 2006;41(4):580-591.
[76] Xue Q, Luan XY, Gu YZ et al. The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity[J]. Stem Cells Dev, 2010;19(1):27-38.
[77] Chase AJ, Bond M, Crook MF et al. Role of nuclear factor-kappa B activation in metalloproteinase-1, -3, and -9 secretion by human macrophages in vitro and rabbit foam cells produced in vivo[J]. Arterioscler Thromb Vasc Biol, 2002;22(5):765-771.
[78] Potapova IA, Doronin SV, Kelly DJ et al. Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage[J]. Am J Physiol Heart Circ Physiol, 2008;295(6):H2257-2263.
[79] Jankowski M, Bissonauth V, Gao L et al. Anti-inflammatory effect of oxytocin in rat myocardial infarction[J]. Basic Res Cardiol, 2010;105(2):205-218.
[80] Semedo P, Palasio CG, Oliveira CD et al. Early modulation of inflammation by mesenchymal stem cell after acute kidney injury[J]. Int Immunopharmacol, 2009;9(6):677-682.
[81] Hu CH, Li ZM, Du ZM et al. Human umbilical cord-derived endothelial progenitor cells promote growth cytokines-mediated neorevascularization in ratmyocardial infarction[J]. Chin Med J (Engl), 2009;122(5):548-555.
[82] Singla DK, Singla RD, McDonald DE. Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway[J]. Am J Physiol Heart Circ Physiol, 2008;295(2):H907-913.
[83] Singla DK, Lyons GE, Kamp TJ. Transplanted embryonic stem cells following mouse myocardial infarction inhibit apoptosis and cardiac remodeling[J]. Am J Physiol Heart Circ Physiol, 2007;293(2):H1308-1314.
[84] Malfitano C, Alba Loureiro TC, Rodrigues B et al. Hyperglycaemia protects the heart after myocardial infarction: aspects of programmed cell survival and cell death[J]. Eur J Heart Fail, 2010;12(7):659-667.
[85] Crouse JR, Harpold GH, Kahl FR et al. Evaluation of a scoring system for extracranial carotid atherosclerosis extent with B-mode ultrasound[J]. Stroke, 1986;17(2):270-275.
[86] Devereux RB, Alonso DR, Lutas EM et al. Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings[J]. Am J Cardiol, 1986;57(6):450-458.
[87]陈志强.人体体表面积计算方法的比较研究[J].中国运动医学杂志, 2003;22(6):4.
[88] Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease[J]. Am J Cardiol, 1983;51(3):606.
[89] Timonen P, Magga J, Risteli J et al. Cytokines, interstitial collagen and ventricular remodelling in dilated cardiomyopathy[J]. Int J Cardiol, 2008;124(3):293-300.
[90] Zannad F, Radauceanu A. Effect of MR blockade on collagen formation and cardiovascular disease with a specific emphasis on heart failure[J]. Heart Fail Rev, 2005;10(1):71-78.
[91] Wang TJ, Larson MG, Benjamin EJ et al. Clinical and echocardiographic correlates of plasma procollagen type III amino-terminal peptide levels in the community[J]. Am Heart J, 2007;154(2):291-297.
[92] Sundstrom J, Vasan RS. Circulating biomarkers of extracellular matrix remodeling and risk of atherosclerotic events[J]. Curr Opin Lipidol, 2006;17(1):45-53.
[93] Huang Y, Hunyor SN, Jiang L et al. Remodeling of the chronic severely failing ischemic sheep heart after coronary microembolization: functional, energetic, structural, and cellular responses[J]. Am J Physiol Heart Circ Physiol, 2004;286(6):H2141-2150.
[94] Radauceanu A, Moulin F, Djaballah W et al. Residual stress ischaemia is associated with blood markers of myocardial structural remodelling[J]. Eur J Heart Fail, 2007;9(4):370-376.
[95] Alla F, Kearney-Schwartz A, Radauceanu A et al. Early changes in serum markers of cardiac extra-cellular matrix turnover in patients with uncomplicated hypertension and type II diabetes[J]. Eur J Heart Fail, 2006;8(2):147-153.
[96] McGavigan AD, Moncrieff J, Lindsay MM et al. Time course of plasma markers of collagen turnover in patients with acute myocardial infarction[J]. Heart, 2004;90(9):1053-1054.
[97] van den Borne SW, Isobe S, Zandbergen HR et al. Molecular imaging for efficacy of pharmacologic intervention in myocardial remodeling[J]. JACC Cardiovasc Imaging, 2009;2(2):187-198.
[98] McGavigan AD, Maxwell PR, Dunn FG. Serological evidence of early remodeling in high-risk non-ST elevation acute coronary syndromes[J]. Int J Cardiol, 2008;125(1):22-27.
[99] Iraqi W, Rossignol P, Angioi M et al. Extracellular cardiac matrix biomarkers in patients with acute myocardial infarction complicated by left ventricular dysfunction and heart failure: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) study[J]. Circulation, 2009;119(18):2471-2479.
[100] Lopez B, Gonzalez A, Querejeta R et al. The use of collagen-derived serumpeptides for the clinical assessment of hypertensive heart disease[J]. J Hypertens, 2005;23(8):1445-1451.
[101] Radovan J, Vaclav P, Petr W et al. Changes of collagen metabolism predict the left ventricular remodeling after myocardial infarction[J]. Mol Cell Biochem, 2006;293(1-2):71-78.
[102] Romero JR, Vasan RS, Beiser AS et al. Association of carotid artery atherosclerosis with circulating biomarkers of extracellular matrix remodeling: the Framingham Offspring Study[J]. J Stroke Cerebrovasc Dis, 2008;17(6):412-417.
[103] McNulty M, Mahmud A, Spiers P et al. Collagen type-I degradation is related to arterial stiffness in hypertensive and normotensive subjects[J]. J Hum Hypertens, 2006;20(11):867-873.
[104] Chatzikyriakou SV, Tziakas DN, Chalikias GK et al. Serum levels of collagen type-I degradation markers are associated with vascular stiffness in chronic heart failure patients[J]. Eur J Heart Fail, 2008;10(12):1181-1185.
[105] Ishikawa J, Kario K, Matsui Y et al. Collagen metabolism in extracellular matrix may be involved in arterial stiffness in older hypertensive patients with left ventricular hypertrophy[J]. Hypertens Res, 2005;28(12):995-1001.
[106] Martos R, Baugh J, Ledwidge M et al. Diagnosis of heart failure with preserved ejection fraction: improved accuracy with the use of markers of collagen turnover[J]. Eur J Heart Fail, 2009;11(2):191-197.
[107] Umar S, Bax JJ, Klok M et al. Myocardial collagen metabolism in failing hearts before and during cardiac resynchronization therapy[J]. Eur J Heart Fail, 2008;10(9):878-883.
[108] Garcia-Bolao I, Macias A, Lopez B et al. A biomarker of myocardial fibrosis predicts long-term response to cardiac resynchronization therapy[J]. J Am Coll Cardiol, 2006;47(11):2335-2337.
[109] Cicoira M, Rossi A, Bonapace S et al. Independent and additional prognostic value of aminoterminal propeptide of type III procollagen circulating levels inpatients with chronic heart failure[J]. J Card Fail, 2004;10(5):403-411.
[110] Picard F, Brehm M, Fassbach M et al. Increased cardiac mRNA expression of matrix metalloproteinase-1 (MMP-1) and its inhibitor (TIMP-1) in DCM patients[J]. Clin Res Cardiol, 2006;95(5):261-269.
[111] Hessel MH, Bleeker GB, Bax JJ et al. Reverse ventricular remodelling after cardiac resynchronization therapy is associated with a reduction in serum tenascin-C and plasma matrix metalloproteinase-9 levels[J]. Eur J Heart Fail, 2007;9(10):1058-1063.
[112] Fiotti N, Altamura N, Orlando C et al. Metalloproteinases-2, -9 and TIMP-1 expression in stable and unstable coronary plaques undergoing PCI[J]. Int J Cardiol, 2008;127(3):350-357.
[113] McGavigan AD, Maxwell PR, Dunn FG. Serological evidence of altered collagen homeostasis reflects early ventricular remodeling following acute myocardial infarction[J]. Int J Cardiol, 2006;111(2):267-274.
[114] Wagner DR, Delagardelle C, Ernens I et al. Matrix metalloproteinase-9 is a marker of heart failure after acute myocardial infarction[J]. J Card Fail, 2006;12(1):66-72.
[115] Hansson J, Lind L, Hulthe J et al. Relations of serum MMP-9 and TIMP-1 levels to left ventricular measures and cardiovascular risk factors: a population-based study[J]. Eur J Cardiovasc Prev Rehabil, 2009;16(3):297-303.
[116] Sugimoto M, Masutani S, Seki M et al. High serum levels of procollagen type III N-terminal amino peptide in patients with congenital heart disease[J]. Heart, 2009;95(24):2023-2028.
[117] Derosa G, Maffioli P, D'Angelo A et al. Evaluation of metalloproteinase 2 and 9 levels and their inhibitors in combined dyslipidemia[J]. Clin Invest Med, 2009;32(2):E124-132.
[118] Cicero AF, Derosa G, Manca M et al. Vascular remodeling and prothrombotic markers in subjects affected by familial combined hyperlipidemia and/or metabolic syndrome in primary prevention for cardiovascular disease[J].Endothelium, 2007;14(4-5):193-198.
[119] El Messal M, Beaudeux JL, Drissi A et al. Elevated serum levels of proinflammatory cytokines and biomarkers of matrix remodeling in never-treated patients with familial hypercholesterolemia[J]. Clin Chim Acta, 2006;366(1-2):185-189.
[120] Woodiwiss AJ, Libhaber CD, Majane OH et al. Obesity promotes left ventricular concentric rather than eccentric geometric remodeling and hypertrophy independent of blood pressure[J]. Am J Hypertens, 2008;21(10):1144-1151.
[121] Schram K, Sweeney G. Implications of myocardial matrix remodeling by adipokines in obesity-related heart failure[J]. Trends Cardiovasc Med, 2008;18(6):199-205.
[122] Goncalves FM, Jacob-Ferreira AL, Gomes VA et al. Increased circulating levels of matrix metalloproteinase (MMP)-8, MMP-9, and pro-inflammatory markers in patients with metabolic syndrome[J]. Clin Chim Acta, 2009;403(1-2):173-177.
[123] Krupinski J, Turu MM, Font MA et al. Increased tissue factor, MMP-8, and D-dimer expression in diabetic patients with unstable advanced carotid atherosclerosis[J]. Vasc Health Risk Manag, 2007;3(4):405-412.
[2]邓玮,陈庆伟.心脏纤维化的干预治疗[J].心血管病学进展, 2003(3):191-194.
[3] Abbott A. Stem-cell furore erupts[J]. Nature, 2010;466(7302):17.
[4] Vogel G. Stem cells. Reprogrammed cells come up short, for now[J]. Science, 2010;327(5970):1191.
[5] Wen Z, Zheng S, Zhou C et al. Repair mechanisms of bone marrow mesenchymal stem cells in myocardial infarction[J]. J Cell Mol Med, 2010.[Epub ahead of print].
[6] Joggerst SJ, Hatzopoulos AK. Stem cell therapy for cardiac repair: benefits and barriers[J]. Expert Rev Mol Med, 2009;11:e20.
[7] Jaiswal S, Jamieson CH, Pang WW et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis[J]. Cell, 2009;138(2):271-285.
[8] Yamashita YM. Cell adhesion in regulation of asymmetric stem cell division[J]. Curr Opin Cell Biol, 2010;22(5):605-610.
[9] Wang Z, Liao Y, Dong J et al. Clinical significance and pathogenic role of anti-cardiac myosin autoantibody in dilated cardiomyopathy[J]. Chin Med J (Engl), 2003;116(4):499-502.
[10] Battiwalla M, Hematti P. Mesenchymal stem cells in hematopoietic stem cell transplantation[J]. Cytotherapy, 2009;11(5):503-515.
[11] Chen Q, Liu Y, Yin Y et al. Relationship between metabolic syndrome (MS) and coronary heart disease (CHD) in an aged group[J]. Arch Gerontol Geriatr, 2008;46(1):107-115.
[12] Radauceanu A, Ducki C, Virion JM et al. Extracellular matrix turnover and inflammatory markers independently predict functional status and outcome inchronic heart failure[J]. J Card Fail, 2008;14(6):467-474.
[13] Poulsen SH, Host NB, Jensen SE et al. Relationship between serum amino-terminal propeptide of type III procollagen and changes of left ventricular function after acute myocardial infarction[J]. Circulation, 2000;101(13):1527-1532.
[14] Paul D, Samuel SM, Maulik N. Mesenchymal stem cell: present challenges and prospective cellular cardiomyoplasty approaches for myocardial regeneration[J]. Antioxid Redox Signal, 2009;11(8):1841-1855.
[15] Mias C, Lairez O, Trouche E et al. Mesenchymal stem cells promote matrix metalloproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction[J]. Stem Cells, 2009;27(11):2734-2743.
[16] Whittaker P, Kloner RA, Boughner DR et al. Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light[J]. Basic Res Cardiol, 1994;89(5):397-410.
[17] Brooks WW, Conrad CH. Isoproterenol-induced myocardial injury and diastolic dysfunction in mice: structural and functional correlates[J]. Comp Med, 2009;59(4):339-343.
[18] Sun Y, Weber KT. Animal models of cardiac fibrosis[J]. Methods Mol Med, 2005;117:273-290.
[19] Arteaga de Murphy C, Ferro-Flores G, Villanueva-Sanchez O et al. 99mTc-glucarate for detection of isoproterenol-induced myocardial infarction in rats[J]. Int J Pharm, 2002;233(1-2):29-34.
[20] Hynes RO. The extracellular matrix: not just pretty fibrils[J]. Science, 2009;326(5957):1216-1219.
[21] LaCroix C, Freeling J, Giles A et al. Deficiency of M2 muscarinic acetylcholine receptors increases susceptibility of ventricular function to chronic adrenergic stress[J]. Am J Physiol Heart Circ Physiol, 2008;294(2):H810-820.
[22] Feng W, Li W, Liu W et al. IL-17 induces myocardial fibrosis and enhancesRANKL/OPG and MMP/TIMP signaling in isoproterenol-induced heart failure[J]. Exp Mol Pathol, 2009;87(3):212-218.
[23] Polyakova V, Loeffler I, Hein S et al. Fibrosis in endstage human heart failure: Severe changes in collagen metabolism and MMP/TIMP profiles[J]. Int J Cardiol, 2010.
[24] Truter SL, Catanzaro DF, Supino PG et al. Differential expression of matrix metalloproteinases and tissue inhibitors and extracellular matrix remodeling in aortic regurgitant hearts[J]. Cardiology, 2009;113(3):161-168.
[25] Pei Z, Meng R, Li G et al. Angiotensin-(1-7) ameliorates myocardial remodeling and interstitial fibrosis in spontaneous hypertension: Role of MMPs/TIMPs[J]. Toxicol Lett, 2010;199(2):173-181.
[26] Kandalam V, Basu R, Abraham T et al. Early activation of matrix metalloproteinases underlies the exacerbated systolic and diastolic dysfunction in mice lacking TIMP3 following myocardial infarction[J]. Am J Physiol Heart Circ Physiol, 2010;299(4):H1012-1023.
[27] Herpel E, Pritsch M, Koch A et al. Interstitial fibrosis in the heart: differences in extracellular matrix proteins and matrix metalloproteinases in end-stage dilated, ischaemic and valvular cardiomyopathy[J]. Histopathology, 2006;48(6):736-747.
[28] Bozkaya YT, Aydin HH, Celik HA et al. Increased myocardial collagen turnover in patients with progressive cardiac conduction disease[J]. Pacing Clin Electrophysiol, 2008;31(10):1284-1290.
[29] Pereira RF, Halford KW, O'Hara MD et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice[J]. Proc Natl Acad Sci U S A, 1995;92(11):4857-4861.
[30] Morikawa S, Mabuchi Y, Kubota Y et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow[J]. J Exp Med, 2009;206(11):2483-2496.
[31] Sung JH, Yang HM, Park JB et al. Isolation and characterization of mousemesenchymal stem cells[J]. Transplant Proc, 2008;40(8):2649-2654.
[32] Chamberlain G, Fox J, Ashton B et al. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing[J]. Stem Cells, 2007;25(11):2739-2749.
[33] Sordi V. Mesenchymal stem cell homing capacity[J]. Transplantation, 2009;87(9 Suppl):S42-45.
[34] Lee RJ, Fang Q, Davol PA et al. Antibody targeting of stem cells to infarcted myocardium[J]. Stem Cells, 2007;25(3):712-717.
[35] Sen M, Wankowski DM, Garlie NK et al. Use of anti-CD3 x anti-HER2/neu bispecific antibody for redirecting cytotoxicity of activated T cells toward HER2/neu+ tumors[J]. J Hematother Stem Cell Res, 2001;10(2):247-260.
[36] Lum LG, Fok H, Sievers R et al. Targeting of Lin-Sca+ hematopoietic stem cells with bispecific antibodies to injured myocardium[J]. Blood Cells Mol Dis, 2004;32(1):82-87.
[37] Zhao TC, Tseng A, Yano N et al. Targeting human CD34+ hematopoietic stem cells with anti-CD45 x anti-myosin light-chain bispecific antibody preserves cardiac function in myocardial infarction[J]. J Appl Physiol, 2008;104(6):1793-1800.
[38] Kraitchman DL, Tatsumi M, Gilson WD et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction[J]. Circulation, 2005;112(10):1451-1461.
[39] Cochlovius B, Kipriyanov SM, Stassar MJ et al. Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells[J]. J Immunol, 2000;165(2):888-895.
[40] Weiner LM, Holmes M, Adams GP et al. A human tumor xenograft model of therapy with a bispecific monoclonal antibody targeting c-erbB-2 and CD16[J]. Cancer Res, 1993;53(1):94-100.
[41] Rossi EA, Goldenberg DM, Cardillo TM et al. Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties ofanti-CD20/CD22 antibodies in lymphoma[J]. Blood, 2009;113(24):6161-6171.
[42] Skyba DM, Price RJ, Linka AZ et al. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue[J]. Circulation, 1998;98(4):290-293.
[43] Xu YL, Gao YH, Liu Z et al. Myocardium-targeted transplantation of mesenchymal stem cells by diagnostic ultrasound-mediated microbubble destruction improves cardiac function in myocardial infarction of New Zealand rabbits[J]. Int J Cardiol, 2009.
[44] Miyahara Y, Nagaya N, Kataoka M et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction[J]. Nat Med, 2006;12(4):459-465.
[45] Jiang W, Ma A, Wang T et al. Homing and differentiation of mesenchymal stem cells delivered intravenously to ischemic myocardium in vivo: a time-series study[J]. Pflugers Arch, 2006;453(1):43-52.
[46] Gnecchi M, He H, Liang OD et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells[J]. Nat Med, 2005;11(4):367-368.
[47] Li L, Zhang Y, Li Y et al. Mesenchymal stem cell transplantation attenuates cardiac fibrosis associated with isoproterenol-induced global heart failure[J]. Transpl Int, 2008;21(12):1181-1189.
[48] Molina EJ, Palma J, Gupta D et al. Reverse remodeling is associated with changes in extracellular matrix proteases and tissue inhibitors after mesenchymal stem cell (MSC) treatment of pressure overload hypertrophy[J]. J Tissue Eng Regen Med, 2009;3(2):85-91.
[49] Hsu CP, Huang CY, Wang JS et al. Extracellular matrix remodeling attenuated after experimental postinfarct left ventricular aneurysm repair[J]. Ann Thorac Surg, 2008;86(4):1243-1249.
[50] Dixon JA, Gorman RC, Stroud RE et al. Mesenchymal cell transplantation and myocardial remodeling after myocardial infarction[J]. Circulation, 2009;120(11Suppl):S220-229.
[51] Meirelles Lda S, Fontes AM, Covas DT et al. Mechanisms involved in the therapeutic properties of mesenchymal stem cells[J]. Cytokine Growth Factor Rev, 2009;20(5-6):419-427.
[52] Guo J, Lin GS, Bao CY et al. Anti-inflammation role for mesenchymal stem cells transplantation in myocardial infarction[J]. Inflammation, 2007;30(3-4):97-104.
[53] Wang TL, Yang YH, Chang H et al. Angiotensin II signals mechanical stretch-induced cardiac matrix metalloproteinase expression via JAK-STAT pathway[J]. J Mol Cell Cardiol, 2004;37(3):785-794.
[54] El-Adawi H, Deng L, Tramontano A et al. The functional role of the JAK-STAT pathway in post-infarction remodeling[J]. Cardiovasc Res, 2003;57(1):129-138.
[55] Wagner M, Siddiqui MA. Signaling networks regulating cardiac myocyte survival and death[J]. Curr Opin Investig Drugs, 2009;10(9):928-937.
[56] Scarabelli TM, Mariotto S, Abdel-Azeim S et al. Targeting STAT1 by myricetin and delphinidin provides efficient protection of the heart from ischemia/reperfusion-induced injury[J]. FEBS Lett, 2009;583(3):531-541.
[57] Boengler K, Hilfiker-Kleiner D, Drexler H et al. The myocardial JAK/STAT pathway: from protection to failure[J]. Pharmacol Ther, 2008;120(2):172-185.
[58] Barry SP, Townsend PA, McCormick J et al. STAT3 deletion sensitizes cells to oxidative stress[J]. Biochem Biophys Res Commun, 2009;385(3):324-329.
[59] Harada M, Qin Y, Takano H et al. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes[J]. Nat Med, 2005;11(3):305-311.
[60] Ng DC, Court NW, dos Remedios CG et al. Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts[J]. Cardiovasc Res, 2003;57(2):333-346.
[61] Furlani D, Li W, Pittermann E et al. A transformed cell population derived from cultured mesenchymal stem cells has no functional effect after transplantationinto the injured heart[J]. Cell Transplant, 2009;18(3):319-331.
[62] Lin YZ, Cai JM, Chen L et al. [Relationship between atrial fibroblast proliferation/fibrosis and atrial fibrillation in patients with rheumatic heart disease][J]. Zhonghua Xin Xue Guan Bing Za Zhi, 2009;37(9):813-817.
[63] Chavakis E, Urbich C, Dimmeler S. Homing and engraftment of progenitor cells: a prerequisite for cell therapy[J]. J Mol Cell Cardiol, 2008;45(4):514-522.
[64] Zavadzkas JA, Plyler RA, Bouges S et al. Cardiac-restricted overexpression of extracellular matrix metalloproteinase inducer causes myocardial remodeling and dysfunction in aging mice[J]. Am J Physiol Heart Circ Physiol, 2008;295(4):H1394-1402.
[65] Ries C, Egea V, Karow M et al. MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines[J]. Blood, 2007;109(9):4055-4063.
[66] Kolpakov MA, Seqqat R, Rafiq K et al. Pleiotropic effects of neutrophils on myocyte apoptosis and left ventricular remodeling during early volume overload[J]. J Mol Cell Cardiol, 2009;47(5):634-645.
[67] Mias C, Lairez O, Trouche E et al. Mesenchymal Stem Cells Promote Matrix Metalloproteinase Secretion By Cardiac Fibroblasts And Reduce Cardiac Ventricular Fibrosis After Myocardial Infarction[J]. Stem Cells, 2009.
[68] Chimenti I, Smith RR, Li TS et al. Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice[J]. Circ Res, 2010;106(5):971-980.
[69] Bujak M, Dobaczewski M, Chatila K et al. Interleukin-1 receptor type I signaling critically regulates infarct healing and cardiac remodeling[J]. Am J Pathol, 2008;173(1):57-67.
[70] Frangogiannis NG. The immune system and cardiac repair[J]. Pharmacol Res, 2008;58(2):88-111.
[71] Van der Heiden K, Cuhlmann S, Luong le A et al. Role of nuclear factor kappaB in cardiovascular health and disease[J]. Clin Sci (Lond),2010;118(10):593-605.
[72] Timmers L, van Keulen JK, Hoefer IE et al. Targeted deletion of nuclear factor kappaB p50 enhances cardiac remodeling and dysfunction following myocardial infarction[J]. Circ Res, 2009;104(5):699-706.
[73] Latanich CA, Toledo-Pereyra LH. Searching for NF-kappaB-based treatments of ischemia reperfusion injury[J]. J Invest Surg, 2009;22(4):301-315.
[74] Kim YS, Park HJ, Hong MH et al. TNF-alpha enhances engraftment of mesenchymal stem cells into infarcted myocardium[J]. Front Biosci, 2009;14:2845-2856.
[75] Hall G, Hasday JD, Rogers TB. Regulating the regulator: NF-kappaB signaling in heart[J]. J Mol Cell Cardiol, 2006;41(4):580-591.
[76] Xue Q, Luan XY, Gu YZ et al. The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity[J]. Stem Cells Dev, 2010;19(1):27-38.
[77] Chase AJ, Bond M, Crook MF et al. Role of nuclear factor-kappa B activation in metalloproteinase-1, -3, and -9 secretion by human macrophages in vitro and rabbit foam cells produced in vivo[J]. Arterioscler Thromb Vasc Biol, 2002;22(5):765-771.
[78] Potapova IA, Doronin SV, Kelly DJ et al. Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage[J]. Am J Physiol Heart Circ Physiol, 2008;295(6):H2257-2263.
[79] Jankowski M, Bissonauth V, Gao L et al. Anti-inflammatory effect of oxytocin in rat myocardial infarction[J]. Basic Res Cardiol, 2010;105(2):205-218.
[80] Semedo P, Palasio CG, Oliveira CD et al. Early modulation of inflammation by mesenchymal stem cell after acute kidney injury[J]. Int Immunopharmacol, 2009;9(6):677-682.
[81] Hu CH, Li ZM, Du ZM et al. Human umbilical cord-derived endothelial progenitor cells promote growth cytokines-mediated neorevascularization in ratmyocardial infarction[J]. Chin Med J (Engl), 2009;122(5):548-555.
[82] Singla DK, Singla RD, McDonald DE. Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway[J]. Am J Physiol Heart Circ Physiol, 2008;295(2):H907-913.
[83] Singla DK, Lyons GE, Kamp TJ. Transplanted embryonic stem cells following mouse myocardial infarction inhibit apoptosis and cardiac remodeling[J]. Am J Physiol Heart Circ Physiol, 2007;293(2):H1308-1314.
[84] Malfitano C, Alba Loureiro TC, Rodrigues B et al. Hyperglycaemia protects the heart after myocardial infarction: aspects of programmed cell survival and cell death[J]. Eur J Heart Fail, 2010;12(7):659-667.
[85] Crouse JR, Harpold GH, Kahl FR et al. Evaluation of a scoring system for extracranial carotid atherosclerosis extent with B-mode ultrasound[J]. Stroke, 1986;17(2):270-275.
[86] Devereux RB, Alonso DR, Lutas EM et al. Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings[J]. Am J Cardiol, 1986;57(6):450-458.
[87]陈志强.人体体表面积计算方法的比较研究[J].中国运动医学杂志, 2003;22(6):4.
[88] Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease[J]. Am J Cardiol, 1983;51(3):606.
[89] Timonen P, Magga J, Risteli J et al. Cytokines, interstitial collagen and ventricular remodelling in dilated cardiomyopathy[J]. Int J Cardiol, 2008;124(3):293-300.
[90] Zannad F, Radauceanu A. Effect of MR blockade on collagen formation and cardiovascular disease with a specific emphasis on heart failure[J]. Heart Fail Rev, 2005;10(1):71-78.
[91] Wang TJ, Larson MG, Benjamin EJ et al. Clinical and echocardiographic correlates of plasma procollagen type III amino-terminal peptide levels in the community[J]. Am Heart J, 2007;154(2):291-297.
[92] Sundstrom J, Vasan RS. Circulating biomarkers of extracellular matrix remodeling and risk of atherosclerotic events[J]. Curr Opin Lipidol, 2006;17(1):45-53.
[93] Huang Y, Hunyor SN, Jiang L et al. Remodeling of the chronic severely failing ischemic sheep heart after coronary microembolization: functional, energetic, structural, and cellular responses[J]. Am J Physiol Heart Circ Physiol, 2004;286(6):H2141-2150.
[94] Radauceanu A, Moulin F, Djaballah W et al. Residual stress ischaemia is associated with blood markers of myocardial structural remodelling[J]. Eur J Heart Fail, 2007;9(4):370-376.
[95] Alla F, Kearney-Schwartz A, Radauceanu A et al. Early changes in serum markers of cardiac extra-cellular matrix turnover in patients with uncomplicated hypertension and type II diabetes[J]. Eur J Heart Fail, 2006;8(2):147-153.
[96] McGavigan AD, Moncrieff J, Lindsay MM et al. Time course of plasma markers of collagen turnover in patients with acute myocardial infarction[J]. Heart, 2004;90(9):1053-1054.
[97] van den Borne SW, Isobe S, Zandbergen HR et al. Molecular imaging for efficacy of pharmacologic intervention in myocardial remodeling[J]. JACC Cardiovasc Imaging, 2009;2(2):187-198.
[98] McGavigan AD, Maxwell PR, Dunn FG. Serological evidence of early remodeling in high-risk non-ST elevation acute coronary syndromes[J]. Int J Cardiol, 2008;125(1):22-27.
[99] Iraqi W, Rossignol P, Angioi M et al. Extracellular cardiac matrix biomarkers in patients with acute myocardial infarction complicated by left ventricular dysfunction and heart failure: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) study[J]. Circulation, 2009;119(18):2471-2479.
[100] Lopez B, Gonzalez A, Querejeta R et al. The use of collagen-derived serumpeptides for the clinical assessment of hypertensive heart disease[J]. J Hypertens, 2005;23(8):1445-1451.
[101] Radovan J, Vaclav P, Petr W et al. Changes of collagen metabolism predict the left ventricular remodeling after myocardial infarction[J]. Mol Cell Biochem, 2006;293(1-2):71-78.
[102] Romero JR, Vasan RS, Beiser AS et al. Association of carotid artery atherosclerosis with circulating biomarkers of extracellular matrix remodeling: the Framingham Offspring Study[J]. J Stroke Cerebrovasc Dis, 2008;17(6):412-417.
[103] McNulty M, Mahmud A, Spiers P et al. Collagen type-I degradation is related to arterial stiffness in hypertensive and normotensive subjects[J]. J Hum Hypertens, 2006;20(11):867-873.
[104] Chatzikyriakou SV, Tziakas DN, Chalikias GK et al. Serum levels of collagen type-I degradation markers are associated with vascular stiffness in chronic heart failure patients[J]. Eur J Heart Fail, 2008;10(12):1181-1185.
[105] Ishikawa J, Kario K, Matsui Y et al. Collagen metabolism in extracellular matrix may be involved in arterial stiffness in older hypertensive patients with left ventricular hypertrophy[J]. Hypertens Res, 2005;28(12):995-1001.
[106] Martos R, Baugh J, Ledwidge M et al. Diagnosis of heart failure with preserved ejection fraction: improved accuracy with the use of markers of collagen turnover[J]. Eur J Heart Fail, 2009;11(2):191-197.
[107] Umar S, Bax JJ, Klok M et al. Myocardial collagen metabolism in failing hearts before and during cardiac resynchronization therapy[J]. Eur J Heart Fail, 2008;10(9):878-883.
[108] Garcia-Bolao I, Macias A, Lopez B et al. A biomarker of myocardial fibrosis predicts long-term response to cardiac resynchronization therapy[J]. J Am Coll Cardiol, 2006;47(11):2335-2337.
[109] Cicoira M, Rossi A, Bonapace S et al. Independent and additional prognostic value of aminoterminal propeptide of type III procollagen circulating levels inpatients with chronic heart failure[J]. J Card Fail, 2004;10(5):403-411.
[110] Picard F, Brehm M, Fassbach M et al. Increased cardiac mRNA expression of matrix metalloproteinase-1 (MMP-1) and its inhibitor (TIMP-1) in DCM patients[J]. Clin Res Cardiol, 2006;95(5):261-269.
[111] Hessel MH, Bleeker GB, Bax JJ et al. Reverse ventricular remodelling after cardiac resynchronization therapy is associated with a reduction in serum tenascin-C and plasma matrix metalloproteinase-9 levels[J]. Eur J Heart Fail, 2007;9(10):1058-1063.
[112] Fiotti N, Altamura N, Orlando C et al. Metalloproteinases-2, -9 and TIMP-1 expression in stable and unstable coronary plaques undergoing PCI[J]. Int J Cardiol, 2008;127(3):350-357.
[113] McGavigan AD, Maxwell PR, Dunn FG. Serological evidence of altered collagen homeostasis reflects early ventricular remodeling following acute myocardial infarction[J]. Int J Cardiol, 2006;111(2):267-274.
[114] Wagner DR, Delagardelle C, Ernens I et al. Matrix metalloproteinase-9 is a marker of heart failure after acute myocardial infarction[J]. J Card Fail, 2006;12(1):66-72.
[115] Hansson J, Lind L, Hulthe J et al. Relations of serum MMP-9 and TIMP-1 levels to left ventricular measures and cardiovascular risk factors: a population-based study[J]. Eur J Cardiovasc Prev Rehabil, 2009;16(3):297-303.
[116] Sugimoto M, Masutani S, Seki M et al. High serum levels of procollagen type III N-terminal amino peptide in patients with congenital heart disease[J]. Heart, 2009;95(24):2023-2028.
[117] Derosa G, Maffioli P, D'Angelo A et al. Evaluation of metalloproteinase 2 and 9 levels and their inhibitors in combined dyslipidemia[J]. Clin Invest Med, 2009;32(2):E124-132.
[118] Cicero AF, Derosa G, Manca M et al. Vascular remodeling and prothrombotic markers in subjects affected by familial combined hyperlipidemia and/or metabolic syndrome in primary prevention for cardiovascular disease[J].Endothelium, 2007;14(4-5):193-198.
[119] El Messal M, Beaudeux JL, Drissi A et al. Elevated serum levels of proinflammatory cytokines and biomarkers of matrix remodeling in never-treated patients with familial hypercholesterolemia[J]. Clin Chim Acta, 2006;366(1-2):185-189.
[120] Woodiwiss AJ, Libhaber CD, Majane OH et al. Obesity promotes left ventricular concentric rather than eccentric geometric remodeling and hypertrophy independent of blood pressure[J]. Am J Hypertens, 2008;21(10):1144-1151.
[121] Schram K, Sweeney G. Implications of myocardial matrix remodeling by adipokines in obesity-related heart failure[J]. Trends Cardiovasc Med, 2008;18(6):199-205.
[122] Goncalves FM, Jacob-Ferreira AL, Gomes VA et al. Increased circulating levels of matrix metalloproteinase (MMP)-8, MMP-9, and pro-inflammatory markers in patients with metabolic syndrome[J]. Clin Chim Acta, 2009;403(1-2):173-177.
[123] Krupinski J, Turu MM, Font MA et al. Increased tissue factor, MMP-8, and D-dimer expression in diabetic patients with unstable advanced carotid atherosclerosis[J]. Vasc Health Risk Manag, 2007;3(4):405-412.
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