【作者】 王祥；

【导师】 曾秋棠；

【作者基本信息】 华中科技大学 ， 心血管病， 2006， 博士

【摘要】 冠心病是一种常见的心脏疾病,冠心病病人由于心脏冠状动脉器质性病变和或功能性改变,导致心肌血供不足,从而产生一系列临床症状和心脏结构及功能的改变。研究已证实,病变冠状动脉的病理变化主要包括冠状动脉壁出现脂质条纹、纤维斑块与复合病变三种情况。上述三种情况的基础主要包括管壁细胞的种类、数目、结构与功能发生改变,尤其是血管内皮细胞和血管平滑肌细胞的变化。目前,有关冠心病的发病机制主要有脂浸润学说、损伤反应学说和单克隆学说、血栓形成和血小板聚集学说等。根据这些发病理论,目前临床上所用的治疗方法主要在于努力增加冠脉的供血从而减少心肌缺血的问题。目前临床上所使用的治疗方法虽然在一定时间内显著改善了缺血心肌的血供并提高了患者的生存时间与生存率,但尚不能改变冠脉病变的根本原因。近年来,随着分子生物学研究的迅速发展,特别是人类基因组项目的成功实施,使我们能够从分子到蛋白水平探讨冠状动脉粥样硬化发病的原因以及相关的治疗方法。目前已知,肝细胞生长因子(Hepatocyte growth factor ,HGF)是一种多功能的细胞生长因子,分子量为103KD,其前体是由728个氨基酸残基组成的单链,经蛋白酶水解后可产生有生物活性的异二聚体,其作用受体为细胞膜上的C-MET受体。种属分析表明,人和大鼠的HGF具有高度的同源性。晚近的研究表明,HGF对缺血心肌细胞具有保护作用,然而其保护作用的机制并不十分清楚。为了探讨肝细胞生长因子治疗缺血性心脏病的作用机制,我们进行了本项实验研究工作,共分为三部分。第一部分肝细胞生长因子基因重组腺病毒载体的构建目的利用AD-EASY TM系统,构建高效而又安全的肝细胞生长因子(HGF)基因腺病毒载体,以研究HGF基因在心脏内的表达,并建立心肌缺血模型,观察该基因载体对心肌缺血的治疗作用。方法从人胎肝中抽提总RNA,并以该肝细胞总RNA为摸版,利用合成的一对引物,采用RT-PCR技术获得HGF基因的cDNA,并引入酶切位点,先转入感受态质粒,再转化大肠杆菌进行扩增,筛选阳性菌落,经酶切及测序,证实目的HGF基因已经转入,再将该基因转入pUC18质粒,pUC18质粒与AD-EASY质粒进行同源重组,用PacI酶切成线性化DNA,利用脂质胺转染293细胞,经三轮扩増,制备高效表达并用绿色荧光标志HGF基因重组腺病毒载体(Ad-HGF)。并将该载体作实验组,与HGF组作比较,观察对VEC的抗凋亡作用。结果在荧光显微镜发现293细胞发光率为100%,HGF基因重组腺病毒载体的HGFcDNA经过测序鉴定与基因库序列一致。Ad-HGF组与HGF组间无明显差异(P>0.05)。结论Ad-HGF和HGF具有其相同生物学效应,由于HGF合成昂贵,Ad-HGF合成成本低,效率高,为大量制备该载体提供依据.第二部分Ad-HGF在急性心肌缺血时对心肌细胞凋亡的影响及对新生血管的作用目的研究心肌发生急性缺血时,直接转染Ad-HGF后心肌缺血的变化情况。方法12周的雄性WKY大鼠60只,随机分成二组,每组30只,常规消毒麻醉后,制造心肌缺血模型,采用直接转染方法,分别转染Ad-HGF(实验组)及注射DMEM培养基(对照组)到缺血大鼠心肌组织。其后,在不同的梗死时间取出大鼠的心脏组织,使用免疫组化方法,研究Ad-HGF对不同梗死时间点大鼠心脏组织中各个区域心肌细胞凋亡数目及新生血管数目的影响。结果1.Ad-HGF转染组3天后HGF开始表达,第7天时表达效率最高,第14天时表达基本消失。免疫组化也证实Ad-HGF感染组有HGF表达,DMEM组无任何HGF表达信号;2.两组新生血管数于梗死后的前7天无明显差别,14天时差异显著(P<0.05)。3. Ad-HGF感染组心肌细胞凋亡数明显少于对照组(P<0.01)。结论Ad-HGF能够促进梗死区域血管的生长,减少细胞凋亡的发生,减少心肌梗死面积。第三部分Ad-HGF对急性心肌梗死后心功能及心室重构的影响目的观察Ad-HGF对心肌梗死后心功能及左室重构的影响。方法60只新西兰兔随机分为3组,结扎左冠状动脉前降支复制急性心肌梗死模型。分别在心室缺血区内注射Ad-HGF(实验组),注射DMEM(对照组),注射生理盐水(空白对照组)。结果4周后应用超声心动图测心功能、左室重构指标,取出心脏,以梗死区/缺血区重量比为梗死范围。结果,实验组与对照组及空白对照组相比,左室舒张末容积、左室相对重量、左室壁厚度均显著降低(P<0.05);左室短轴收缩率(LVFS)、左室射血分数(LVEF)均显著升高(P<0.05)。与对照组相比,Ad-HGF干预组LVESV、LVEDV显著降低,而心肌梗死范围明显缩小(P<0.05)。结论Ad-HGF能减少心肌梗死范围、并能限制AMI后的左室重构,改善心功能。其作用机制可能与其抗心肌细胞凋亡有关。更多还原

【Abstract】 Coronary heart disease is a kind of very common myocardial ischemia heart disease, which is the cardiac muscle injury due to the imbalance between blood supply for coronary artery and blood demands of cardiac muscle caused by coronary artery functional pathological changes or organic pathologic changes. Now investigation has comfirmed that pathological changes are that fatty streak, fibrous plaque and complicated lesion appear in the coronary artery wall. However, the basic pathological changes are the changes of type,number,structure and functions of wall cells, especially the changes in vascular endothelial cell and vascular smooth muscle cell. The main pathogenesis is fatty infiltration,thrombosis and platelet aggregation,injury response and monoclonal theories,etc. According to these morbility theory,presently, the common therapeutic methods are: major exertion increase blood supply for coronary artery,decrease myocardial ischemia,treat and reduce complications. Although clinically therapeutic roles can improve ischemia, increase acromegalic live time and survival rate in a period of time,but they can not change coronary artery fundamental reasons.For the past few years, gene therapy is a fast developing biological technology,especially after the establishment of human gene pool doing well. We can investigate coronary atherosclerosis pathogenetic reason and ralative therapia from molecule to protein level. Nearly researches indicate that HGF can protect myocardial cell, but the protect mechanism is not clear. It is well known, hepatocyte growth factor (HGF) is a kind of cell growth factor with multi-effects. In recent years,research on cardiovascular decease is increasing gradually. The molecular weight of HGF is 103KD, its precursor consists of monochain with 728 amino acid residues,which will produce heterodimer with bioactivity after protease hydrolysis,and the HGF in human and rat is of high homology,it’s functional receptor is C-MET receptor on the cell membrane. In order to research the mechanism of action about therapy in ischemia heart disease, we are going to this research project, which includs three sections.Section I Construction of human HGF gene recombination adenovirus vectorObjective With AD-EASY TM system, set up high efficient and safety AD-HGF in order to research the expression of HGF gene in the heart, as well as the effect on VEC, VSMC and MC, and set up the myocardial ischemia model to observe the therapy effect of this vector to myocardial ischemia. Methods Extract total RNA from human fetal liver, take the total RNA of hepatocyte as model, use a pair of synthetic primers, adopt RT-PCR technology to obtain cDNA of HGF gene and insert enzyme cutting site, turn to competence plasmid first, and then to transfer the colibacillus to amplify, screen the positive colony, after enzyme cutting and sequencing, ensure the targeted HGF gene being transferred, then transfer the gene into pUC18 plasmid, carry out homologous recombination with pUC18 plasmid and AD-EASY plasmid, enzyme cutting into linearization DNA with PacI, use fatty amine to transfection 293 cell, amplify three times, prepare high effective expression and had been labed by fluorecent adenovirus vector (Ad-HGF) of HGF gene recombination, and take this vector as the experiment group, compare with the HGF group and observe the anti-apoptotic effect of VEC. Results It is found that 293 cell luminance is 100% under the fluorescence microscope, HGF recombination adenovirus vector as the gene pool sequence with the acquisition, there is no obvious difference between the Ad-HGF group and HGF group(p>0.05).Conclusions Ad-HGF and HGF have the same biological effect. Since the synthetic cost of HGF is expensive and that of Ad-HGF is cheaper with high efficiency, it provides a basis for mass preparation of this vector.SectionⅡAffection of Ad-HGF to Myocardial Cell Apoptosis and Angiogenesis After AMIObjective For the purpose of research on the occurrence of acute myocardial ischemia, after injected Ad-HGF,observe the changes of myocardial ischemia. Methods 60 12-week male WKY rats are randomly divided into two groups with 30 for each group, routine sterilization, urethane narcosis, animal respirator to help breathing, cut left chest horizontally through the fourth rib, expose the heart, tie descending anterior branch of left coronary artery completely, inject different medicines in myocardial ischemia, one of the group inject Ad-HGF, the other DMEM culture medium, kill the rats after 1, 3, 7, 14, 21 and 28 days respectively, take out their hearts, make frozen section and TTC tinctorial paraffin section, observe the fluorescent distribution and immune-histochemistry of cardiac muscle cell under the green fluorescence microscope,and finally the vessel count and related statistics analysis.Results Fluorescence is found in Ad-HGF group three days later , fluorescence at the 7th day is the strongest and disappear after 14 days;no fluorescence is found in DMEM group, brown sediment is found in immune-histochemistry of Ad-HGF group, no sediment is found in DMEM group;there is no obvious difference of vessel count in the first two groups at the first 7 days, there is obvious difference after 14 days (P<0.05), which has obvious meaning, decrease of ischemia area of Ad-HGF group is much smaller than DMEM group at the 28th day.Conclusions Ad-HGF has surely very good therapy effect on myocardial ischemia.SectionⅢAffection of Ad-HGF to Cardiac Function and Left Ventricular Remodeling After Myocardial ischemia Objective To observe the effect of hepatocyte growth factor(HGF) on cardiac function and left ventricular remodeling after AMI in the rabbits. Methods The rabbit model ligated proxi mal left anterior descending coronary artery (LAD) was used. Fifty-six rabbits were randoimly divided into four groups:sham-opera-transvenously infused with normal saline, and in experimental group HGF(2mg/kg.12h) were given. After LAD ligated 4 weeks, hemodynamic studies were performed in each group, and ischemia area and infarction size, the indexes of ventricular remodeling were also measured. Cell apoptosis was detected by TUNEL straining.Results Compared with sham-operation group, control group had a higher left ventricular end-diastolic volume, left ventricular weight and left ventricular wall thickness(P<0.05~0.01), and had a lower LVFS and LVEF. LVESV, LVEDV were significantly decreased, and LVFS and EF were significantly increased in experimental group as compared with those in control group. Cell apoptotic rate and infarction size in experimental group were significantly lower than those in control group. Left ventricular diameter was positively correlated with cell apoptotic rate.Conclusions 1. Exogenous HGF could decrease infarction area and the extent of cardiomyocyte apoptosis. 2. Exogenous HGF could limit ventricular remodeling after AMI and improve left ventricular function. The mechanism is probably associated with its anti-apoptotic role.更多还原