【作者】 郭文怡；

【导师】 贾国良；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 博士

【摘要】 研究背景缺血性心脏病严重威胁人们的健康。阐明缺血性心脏病的发病机理,寻求合理的治疗措施,是当今医学界亟待攻克的重要课题。已有研究证实干细胞移植和基因治疗可能促进心梗后心肌的再生及促进新生血管生成,在一定程度上改善心功能。骨髓基质细胞(BMSCs)自体移植技术以及血管内皮生长因子(VEGF)的“基因搭桥技术”已被许多研究证实各自的安全性和有效性,但VEGF并不能挽救瘢痕区的心肌,对于已经凋亡和变性的心肌细胞无法弥补或者逆转,而BMSCs移植后,虽然可以增加瘢痕区的新生心肌细胞,但缺血心肌内缺血缺氧环境使得移植细胞的存活率较低。同时BMSCs和VEGF都具有促进血管新生的功能,将两种治疗方法联合应用是否可以弥补两者各自的不足,达到促进血管新生和心肌细胞再生,进一步改善心功能的目的,尚需要进一步的研究。而且,VEGF在缺血性心肌病治疗研究中的作用表现,仅仅用促进血管新生来解释是不够的,是否存在额外的心肌保护作用,需要进行进一步的机制方面的研究。实验目的本研究第一部分采用猪慢性心肌梗死模型,应用左室心内膜电机械标测系统及NOGA注射导管,利用不同的检测指标,观察BMSCs结合VEGF165心内膜心肌内注射对心肌梗死的疗效。研究第二部分针对VEGF的表现,观察VEGF是否存在心肌细胞的直接保护作用,并探讨VEGF是否在心肌中作为SDF-1α/CXCR4轴和VEGF/VEGFR通路中的一种存活因子,通过PI3K/Akt/SDF-1α通路参与了对H2O2诱导的心肌细胞死亡的保护作用。实验方法第一部分:通过建立猪心肌梗死模型,胸骨穿刺抽取骨髓,分离培养BMSCs,部分细胞进行VEGF165基因转染。模型建立成功4周后,进行左室心内膜电机械标测并行心肌膜注射等操作,按照注射成分不同分为3组,联合组(n=8)注射转染VEGF165基因的BMSCs,细胞组(n=8)注射BMSCs,对照组(n=6)注射生理盐水。于细胞移植前及移植后4周分别进行左室心内膜电机械标测、选择性冠脉造影及超声心动图检查。第二部分:细胞水平研究,分离新生鼠的心室肌细胞,H2O2诱导心肌细胞损伤,培养上清中分别加入VEGF和SDF-1α分子,收集细胞样品,提取蛋白或mRNA,免疫印迹电泳方法检测培养细胞的SDF-1α、CXCR4和Akt表达水平以及Akt磷酸化水平。利用实时定量PCR方法和ELISA方法检测和VEGF的蛋白及mRNA表达水平,细胞活性和损伤检测通过胎盘蓝染色及LDH活性检测进行。实验结果:第一部分研究结果显示:术后4周时联合组较细胞组及对照组的心梗面积明显缩小,侧支循环、心脏泵血功能以及心肌收缩力改善更加明显。第二部分研究结果显示:H2O2可诱导分离培养的心肌细胞损伤,胎盘蓝染色及LDH活性测定显示细胞培养上清中加入VEGF可显著增加细胞活力,促进细胞存活。WB结果显示Akt磷酸化水平增加,SDF-1α和CXCR4表达上调,此作用可被PI3K特异性抑制剂消除。培养上清中加入SDF-1α,可显著增加心肌细胞VEGF的表达,此作用与CXCR4-PI3K/Akt的激活相关。结论上述结果表明,自体BMSCs结合VEGF165基因治疗心肌梗死效果显著。VEGF可减少H2O2诱导的细胞死亡,保护心肌细胞,其保护作用的机制可能与PI3K/Akt信号和SDF-1α/CXCR4轴和VEGF/VEGFR通路在心肌中的正反馈相关。更多还原

【Abstract】 Background: Ischemic heart disease is the major cause of morbility and mortality in the world. Although myocardial salvage due to early reperfusion therapy has significantly reduced early mortality rates, postinfarction heart failure resulting from ventricular remodeling remains a problem.One possible approach to reversing postinfarction heart failure is enhancement of the regeneration of cardiac myocytes as well as stimulation of neovascularization within the infarcted area. The purpose of this study is to evaluate the efficacy of t ransplantation of vascular endothelial growth factor (VEGF) - expressing bone marrow st romal cells (BMSCs) into ischemic swine myocardium .The cardiomyocyte protection effect of VEGF and the mechanism involved will also be studied.Methods and results: A swine model of chronic myocardial ischemia was made in this study. BMSCs were obtained f rom swines′breastbone marrow and cultured in vitro, then BMSCs were t ransfected with the human VEGF165 gene. Four weeks later, VEGF - expressing BMSCs (Combo group, n = 8), BMSCs (BMSCs cont rol group, n = 8) or saline water only (Control group, n = 6) was injected into swine heart s using lef t vent ricular elect romechanical (NOGA) mapping system and injection catheter. NOGA mapping, coronary angiography and echocardiography were conducted before and 4 weeks after cell transplantation. Rat neonatal ventricular myocytes were isolated and cultured in DMEM with 10% fetal bovine serum. Cardiomyocytes death was induced with H2O2 and VEGF was added to the culture medium. Expression of CXCR4c-20 and Akt and Akt phosphoratliation were was detected with Western blotting. Protein level and mRNA expression of SDF-1αand VEGF were detected with qRT-PCR and ELISA respectively. Cytoactivity and cell injury were measured with trypan blue dying assay and LDH activity detection. This study show that after four weeks after cell transplantation, combination therapy resulted in superior improvement in all indexes of perfusion and function compared with other two treatment groups (P < 0.05). Treatment of neonatal rat ventricular myocytes with VEGF stimulated phosphorylation of Akt in dose- and Flk-1- dependent manner. VEGF attenuated H2O2-induced cardiomyocytes death. LY294002, PI3K/Akt inhibitor and Flk-1 antibody abolished the beneficial effect of VEGF. Concomitantly, SDF-1αand its receptor, CXCR4, were up-regulated by VEGF through the PI3K-Akt signaling pathway, contributing to the protective effect of VEGF on H2O2-induced cell death. Furthermore, through the PI3K-Akt pathway, SDF-1αalso stimulated in vitro VEGF production, and prevented H2O2-induced cardiomyocyte death. In conclusion, these results suggest an Akt-centered loop, with VEGF and SDF-1αplaying critical roles in cardiomyocytes survival.Conclusion: These results provide the first evidence that that combined strategy of bone marrow stromal cell transplantation with VEGF gene therapy could be of importance for the treatment of myocardial infarction. And a crosstalk between VEGF and SDF-1αthrough PI3K-Akt serves a survival role in cardiomyocytes in vitro.更多还原