【作者】 李峰；

【导师】 胡建国；

【作者基本信息】 中南大学 ， 外科学， 2008， 博士

【摘要】 缺血性心脏病（IHD）是由于冠状动脉循环改变引起冠状血流和心肌需求之间不平衡而导致的心肌损害,其发病率高,死亡率高,严重危害着人类的身体健康。IHD的主要病理改变是不可逆性功能心肌细胞丢失,梗死区被纤维疤痕替代,以及残余心肌发生心室重构,进而发展为充血性心力衰竭。目前的药物治疗、介入治疗及冠脉旁路手术能够改善心肌缺血,但仍然不能逆转已经坏死的心肌,更不能促进心肌细胞的再生。治疗性血管发生（therapeutic angiogenesis）根据血管生成的机制,用人工干预的手段刺激心肌缺血区小血管生长和侧支循环形成,从而修复与再生坏死心肌细胞,阻止或延缓心室重构和心功能衰竭,是当前治疗缺血性心肌病的研究热点。目前治疗性血管发生可通过三种方法得以实现:生长因子蛋白治疗,细胞移植以及基因治疗。缺氧诱导因子-1α（Hypoxia-inducible factor-1α,HIF-1α）是细胞在缺氧条件下产生的具有转录活性的核蛋白,不仅可以结合多种促血管因子如血管内皮生长因子（VEGF）、转化生长因子β（TGF-β）等及其受体基因启动子上的缺氧反应元件,从而增强相应蛋白的表达,启动血管新生过程,提高血流灌注,挽救濒死缺氧的心肌细胞,还可以提高缺血心肌对缺氧环境的适应性,增强酵解过程,为心肌提供更多的能量代谢,可以设想,HIF-1α用于基因治疗具有两大优势:1.作为上游调控基因将比VEGF、ANG-2等单纯基因治疗更具效率;2.HIF-1α在常氧条件下迅速降解,具有可控性,减低了VEGF持续高表达诱发血管瘤的危险性。骨髓间充质干细胞（MSCs）具有分化为心肌细胞、内皮细胞的多向分化潜能,且分离方便、增殖容易、遗传稳定、无免疫排斥和伦理障碍等优势,是干细胞治疗的理想供体细胞。由此,假设将转染HIF-1α基因的MSCs移植到心梗区,以求基因和细胞治疗相互协同、相互促进,能同时、更好地实现心肌再生和血管发生,是一种极具潜能的替代疗法。因此,本研究拟在大鼠的心肌缺血的模型上,通过移植体外转染HIF-1α基因的MSCs,探求这种方法对IHD的治疗效果,为进一步的临床应用提供铺垫性实验依据。本实验共分为三部分:第一部分HIF-1α基因克隆及HIF-1α-pcDNA3.1载体的构建目的:构建并鉴定HIF-1α真核表达载体HIF-1α-pcDNA3.1方法:抽提大鼠心肌细胞mRNA,以mRNA为模版,逆转录得到cDNA,PCR扩增HIF-1α目的基因,连接到pGEM-T载体中,酶切,测序证实,双酶切将HIF-1α转至pcDNA3.1中。结果:琼脂糖凝胶电泳结果显示RT-PCR扩增出的特异片段约2480bp,基因测序结果与GenBank报道的完全一致,成功构建了HIF-1α-pcDNA3.1真核表达载体。第二部分HIF-1α基因转染骨髓间充质干细胞及其表达目的:研究HIF-1α真核表达载体HIF-1α-pcDNA3.1体外转染MSCs及HIF-1α基因在MSCs中的表达情况。方法:采用密度梯度离心—贴壁培养法获Wistar大鼠MSCs。对其进行表型及生长曲线测定,并进行向成骨细胞及脂肪细胞诱导分化的研究。采用脂质体介导技术将HIF-1α-pcDNA3.1转染至MSCs,在倒置显微镜下观察转染后细胞形态和生长情况的变化,通过RT-PCR,Western和ELISA鉴定HIF-1α在细胞中的表达情况。结果:1.采用Percoll密度梯度离心-贴壁培养法易获取大鼠MSCs,形态学观察呈纤维样细胞外观;免疫组化检测结果为CD44、SH3（CD73）阳性,CD34、CD45阴性,符合MSCs表面标记;流式细胞仪分析,CD44、SH3（CD73）阳性细胞分别占94.7%、97.3%,表明分离培养的MSCs纯度较高;经诱导后,所培养的MSCs可成功向成骨细胞及脂肪细胞分化。2.RT-PCR证实采用阳离子脂质体Lipofectamine2000转染的MSCs表达HIF-1αmRNA明显增加,Western和ELISA检测证实转基因MSCs表达HIF-1α蛋白明显增加。第三部分HIF-1α转染骨髓间充质干细胞移植对大鼠心功能的影响目的:利用冠脉结扎法构建Wistar大鼠心肌缺血动物模型,探讨HIF-1α基因转染MSCs后心肌移植对缺血性心脏病心功能及心室重构的影响,并比较联合治疗与基因治疗、细胞治疗的疗效差别。方法:80只Wistar近交系大鼠随机分为5组（各组16只）,首先结扎前降支,2周后形成慢性心肌缺血动物模型。A组于心梗区移植转HIF-1α的MSCs（联合组）,B组单纯移植等量的MSCs（细胞组）,C组单纯注射HIF-1α-pcDNA3.1复合物（基因组）,D组注射无血清培养液（对照组）,E组仅用于模型评估（评估组）。另取16只未结扎冠脉的Wistar近交系大鼠为假手术组,亦未作任何治疗,设为F组。移植4周后以Buxco系统检测心功能,测量心梗面积,免疫组化检测Brdu、肌钙蛋白T双染确定移植细胞的存活与分化,通过Ⅷ因子染色检测血管新生,通过RT-PCR检测HIF-1α基因的体内表达。结果:冠脉结扎法制作的大鼠心梗模型,其有效使用率为78.4%（80/102）。移植治疗4周后,联合组心梗面积小于细胞组与基因组的心梗面积（P＜0.05）;Buxco检测心功能显示LVSP、+dp/dt_max明显高于其他各组,LVEDP、-dp/dt_max明显低于其他各组;Ⅷ因子染色示联合组动物心梗区毛细血管密度高于细胞组和对照组（P＜0.05）,较基因组也有一定程度增加（P=0.054）;Brdu、肌钙蛋白T双染示各治疗组心梗区心肌细胞数量不同程度多于对照组,部分为双染阳性细胞;RT-PCR显示联合组HIF-1α的体内表达高于其他各组。结论:1.成功构建了HIF-1α-pcDNA3.1真核表达载体。2.HIF-1α-pcDNA3.1转染大鼠MSCs后能有效增加HIF-1α表达。3.HIF-1α转染MSCs移植可显著改善缺血心脏功能、促进血管发生、增加心梗区心肌细胞数量,其综合疗效优于单独基因治疗或细胞治疗。更多还原

【Abstract】 Ischemic heart disease(IHD)is the disease that the myocardial blood-deficiency occurs when the coronary artery blood circulation can not match the heart’s need.IHD seriously damages mankind’s health due to its high morbidity and mortality.The basic pathology is characterized by the massive cardiaomyocytes irreversible loss,which is replaced by non-contractile fibrous cells.Ventricular remodeling process develops in the remaining surviving myocardium if the ischemia persists,and eventually the progressive deterioration of congestive heart failure is inevitable.Current therapy including drug treatment,percutaneous coronary intervention(PCI)and coronary artery bypass graft(CABG) mainly aim at saving the viable cardiomyocytes,improving the function of survival cardiomyocytes and reversing ventricular remodeling.The ability to regenerate damaged myocardium presents a major challenge in the treatment of myocardium infarction.Therapeutic angiogenesis,which have become leading fields in the research of IHD therapy worldwide,is a potentially viable therapeutic strategy designed to amplify the native angiogenic process and enhance the reperfusion for reversing the ischemic remodeling process,compensate for the loss of ventricular mass and contractility,eventually restore cardiac function.There are 3 approaches to induce angiogenesis and vasculogenesis:recombinant protein,cells and gene therapy.Hypoxia-inducible factor-1α(HIF-1α)is a protein with transcription activity.HIF-1 can not only promote the transcription and expression of many angiogenisis factor,such as VEGF and TGF-β,to increase the regeneration of vesscle and enhance the reperfusion,but also improve the adaption of ischemic myocardial cell in anoxia condition,intensify the process of zymolysis,and supply myocardial cell with more energy.So it is possible that the therapeutic effect of HIF-1 would be better than that of VEGF or FGF treatment simply.Bone marrow mesenchymal stem cells(MSCs),the early cell of mesoderm,which can keep the potent plasticity of differentiate to multi-tissue have the advantage of can be isolated easily from a variety of sources,genetic stability,no immunologic and ethical concerns.So they were considered as ideal candidate donor cells for stem cell therapy and target cells for gene transfer.Thus we put forward the hypothesis that the combination of MSCS transplantation and Hypoxia-inducible factor-1α(HIF-1α)gene transfer might improve the function of gene or cellular treatment mutually and would be superior to either strategy alone for treatment of chronic myocardial ischemia.In this study,we investigate the effects of MSCs transferred with HIF-1αgene transplantation for IHD therapy in rat myocardial ischemia model and provide experimental principles for further clinical application for human ischemic heart disease.This experimental study is composed of three parts.PartⅠGene cloning of HIF-1αand construction of the HIF-1α-pcDNA3.1 expression plasmid.Objective:To construct and identify eukaryotic expression vector of human HIF-1α(HIF-1α-pcDNA3.1).Method:HIF-1α- pcDNA3.1 is constructed with the technology of gene recombination.Then the recombined plasmid is identified by enzyme digesting and DNA sequencing.Result:HIF-1α-pcDNA3.1 eukaryotic expression vector is constructed successfully.PartⅡExpression of HIF-1αgene in Rat mesenchymal stem cells in vitroObjective:To investigate transfection and HIF-1αgene expression of HIF-1α-pcDNA3.1 eukaryotic expression vector in cultured mesenchymal stem cells.Methods:MSCs of Wistar rat was isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic. Detections of cell surface marker phenotype,including CD34,CD45, CD44 and SH3(CD73),were performed using flow cytometry.The vector HIF-1α-pcDNA3.1 was transfected into MSCs with the method of lipofectamine mediated.The expression of HIF-1αin the transfected cells was detected by RT-PCR,ELISA and Western blot analysis.Results:1.MSCs were CD34~-,CD45~-,CD44~+,SH3~+.They can differentiate into bone and adipocytes successfully.2.The expression of HIF-1αgene in the transfected rat MSCs was demonstrated by RT-PCR, ELISA and Western blot analysis. PartⅢEffects of myocardial transplantation of MSCs transfected with HIF-1αgene for heart functionObjective:To evaluate the effect of MSCs transfected with HIF-1αgene for heart function restoration and mitigation of the adverse effects of ventricular remodeling after myocardial infarction,compare the therapeutic difference between the combinated therapy and single cell or gene therapy.Methods:Wistar rat ischemic heart animal model was constructed by left anterior descending coronary artery ligation.80 ligated animals were divided into 5 groups(16 each).2 weeks later,4 groups were injected at the heart infarct zone with HIF-1α-transfected MSCs(Combo group),MSCs(Cell group),HIF-1α-pcDNA3.1(Gene group),or medium (Control group).The left one(Model-assessment group)was served as model assessing.Another 16 rats without ligated were used as a sham operation group.4 weeks after the injection,the rat cardiac function was measured by Buxco system.Brdu labeling andⅧfactor were identified by immunohistochemical stain;size of infarcted area was calculated by Evan’s blue stain.HIF-1αgene expression was evaluated by RT-PCR.Results:78.4%(80/102)of the ischemic models,constructed by coronary artery ligation,were qualified for the next study.2 weeks after the ligation,the animals had smaller infarcted area(P＜0.05)and better heart performance than those of 6 weeks’.4 weeks after the transplantation,the combo group animals’ infarcted heart size was (29.8±3.2)%,smaller than that of cell and gene group’s(P=0.049,0.016), which were smaller again when compared with control group;the heart function,measured by Buxco system,was better than that of both cell and gene groups’,the capillary density was similary to gene group’s but more than cell group.Brdu and Troponin-T double stain showed that there was a different increase of survived cardiaomyocytes,some were double stain positive,located at the infarcted area of combo,cell and gene group.The expression of HIF-1αin combo group was higher than that of other groups.Conclusions:1.HIF-1αwas constructed into the pcDNA3.1 vector successfully. There are no mutations by sequencing confirmation.2.HIF-1αhad been successfully transfected into MSCs and well expressed demonstrated by RT-PCR,ELISA and Western blot analysis..3.Transplantation of MSCs transfected with HIF-1αgene can improve myocardial perfusion and restore heart function.The effects was better than that of cellular or gene therapy simply.更多还原