| 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.Sectionâ…¡Affection 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.Sectionâ…¢Affection 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.
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