| Ischemic disease is always a hotpot research object in the region of Cardiovascular. Itis threatening the health of human being seriously. The current pharmacotherapy can onlyalleviate the contradiction between supply and demand on myocardial blood and it couldnot solve the problem of myocardial ischemia fundamentally. The mechanical myocardialrevascularization treatment, such as coronary artery bypass surgery, CABG, PTCA andstent placed inside coronary artery, produces a good curative effect in most of patients. But,it still has the risks like blood vessels to narrow again in the future. It is more importantthat the revascularization treatment can’t be applied to the patients with the stenosis ofdiffuse coronary artery distal branch and vascular bypass restenosis after CABG surgery.However, a huge of this kind of patients exists there. People are looking for newtreatments.With the developing of angiogenesis factor research in recent years, it brings the hopeto solve the problem. Based on the large amount of experiments on animals, angiogenesisfactor gene therapy techniques has been used and it has obtained affirmed effect oninducing the form of myocardial new blood vessels, enhancing the compensatory capacityof collateral circulation and realizing myocardial endogenous revascularization. But, thesemethods could not resolve the problem of myocardial ischemia fundamentally. Oncemyocardial infarction, the process to adapt to myocardial ischemia changes is too slow ifonly relying on their own new blood vessels. And, usually it is only part of thecompensation and it is difficult to alleviate the heart damage and aggravated symptoms.Recently, when we did the second heart surgery for the patients, we found that extensiveadhesion existed between heart and heart-sac, pericardial artery nets hyperplasia entermyocardium and form collateral anastomosis with internal myocardial blood vessels. Fromthis, we consider whether the pericardial artery nets hyperplasia can be used to provide fullof blood to ischemic myocardium. In1955, Plachta A and others did similar study. It is thefirst time that they inject complex magnesium silicate (talcum powder) into the pericardialcavity to make the heart-sac and heart occur adhesion in the stimulation of chronicinflammation and use pericardial artery nets hyperplasia to supply blood for ischemicmyocardium when it enters the myocardium. Although the generation of new blood vessels can be observed, the heart-sac and heart extensive adhesion is obviously growing thick andit seriously limits the diastole and shrinkage of the heart. Therefore, we are trying to find away to make heart-sac to adhesion in part mostly and control its excessive thickening. Atthe same time, we provide blood vessel growth factors to promote the pericardial arterynets hyperplasia, form wide collateral circulation network with internal myocardium bloodvessels, save hibernate myocardium and sudden stop myocardium, prevent the death ofprogressive myocardium cell, non-infarction myocardial hypertrophy and fibrosis andreduce ventricle remodeling, so as to prevent the occurrence of cardiac insufficiency.The artery of pericardial artery nets comes from the thoracic artery, the thymus artery,thyroid artery, thyrohyoid stem, bronchial artery, up septal artery and down septal artery.The blood supply is very rich. In addition, the preparation technology of blood vesselgrowth hormones (ANG) to restructure adenovirus is mature. The laboratory has alreadyproved its strong effect on vascularization and new reagents application on promotion thereaction of inflammation. All of these is the foundation of our experiment. This content,method and results of this research are as below:1. High titers of angiogenin (ANG), preparation and identification ofrestructured adenovirusThe Ad.ANG this experiment used is a monoclonal ANG recombinant adenovirus. Itis from our laboratory and it is made by using adenovirus DNA end peptide complexes andrecombinant cosmid to transfect293cells.Getting monoclonal Ad.ANG liquid which was made by our laboratory, making itunfrozen and infecting it to293cells, collecting cells and nutrient liquid and multigelationcentrifugation, then we could get a new generation of recombinant adenovirus venom. Byusing the PEG/NaCl precipitation purification, we can get Ad.ANG whose titer is3.2x1010pfu/ml.2. In vitro transcript of ANG restructuring adenoviruswhen MOI is100, we can find ANGmRNA transcription after the first day, the thirdday and the seventh day when ANG recombinant adenovirus transfection ECV304in vitroand RT-PCR. We can find184bp band after its reaction product electrophoresis.Meanwhile, we don’t see ANG gene transcription in the control group. Therefore, it showsthat the ANG recombinant adenovirus has successfully enter ECV304cells, and can betranscribed to the corresponding mRNA. 3. Establishment of animal model and groups75New Zealand big white rabbit only health male only randomly divided into5groups of75:Group1: MI+pericardial adhesion+Ad. ANG groupGroup2: MI+pericardial adhesion+Ad. Null groupGroup3: MI+pericardial adhesion groupGroup4: MI+Ad. Null group5groups: MI+Ad. ANG groupInjecting0.025mg/kg citric acid fentanyl to the ear vein of the rabbit, we ligatured1,2diagonal branch of the main branch of the left ventricular and then we found in ECGexamination that the part between II and ST segment elevated around2mm like an arch. Itshows the model of myocardial infarction established successfully.5-0polypropylene lineclosed the pericardial cavity. Closed to cavity diaphragm, Using1ml injector of5EM-BTto inject200u l Freund incomplete adjuvant to pericardial cavity to create a model ofpericardiosymphysis. The result of pathological examination shows that the models arestable, reliable and can meet the requirements of experiment.4. The effect on schemic myocardial revascularization under the action ofcardiopericardiopexy by the angiogenin (ANG) recombinant adenovirus.Through RT-PCR detection, we still could detect the production of184bp ANGtranscripts in the cardiac muscular tissue on the28th days of transfection ANGrecombinant adenovirus. Pathological observation showed the density of each capillarywas increased along with time after myocardial infarction. The group of pericardialadhesions and the pericardial cavity to ANG recombinant adenovirus (group1) increasedsignificantly higher than the other four control groups. Especially2weeks later, the changeis more outstanding. However, the control groups have no big changes after2weeks.4weeks later, the pericardial thickness of group1was similar to the one in the2nd week.But the capillary density was significantly higher. To compare with the other4groups, theStatistical differences is very obvious. Through heart color super, we found each functionof heart were significantly weak after myocardial infarction. But, it became better andbetter with the time going on.2weeks later, each function of the heart of the group1andthe group which the pericardial cavity gave ANG recombinant adenovirus (Group5)improved significantly higher than the other three groups. After4weeks, it got further improvement. But the EF values of these two groups have no significant difference. Theresults suggest that pericardial thickening and adhesion restricts the diastole to somedegree and then cause mild limitation on heart diastole and systole. But blood supply toischemic myocardium in group1was much more abundant than the other4groups. Itprevented the death of progressive myocardial cell and reduced the remodeling of left heart.It had reached the target of improving heart function on the whole. Based above results, theconclusion is:1. cDNA gene segment of ANG, the coder created through RT-PCR technology is big.The area being used in design primers is less and its specificity is poor. Therefore, we didsome exploration on optimization in the primer design, reverse transcription and PCR. Itwill be helpful to provide some experience for the future similar work.2. The method in this experiment, using Cosmid-TPC calcium phosphateco-precipitation to transfect293cells, made us obtain high rate of gene recombinantadenovirus and low rate of false positive. At the same time, we established a feasible wayon how to judge the generation of recombinant adenovirus, how to obtain the purifiedhigh-titer recombinant adenovirus. It also provided a convenient way to get recombinantadenovirus fast and efficiently.3. This topic is aimed to research the application of Ad.ANG intrapericardial injectionway, it made recombinant adenovirus transfect, express and play its role of biologicaleffects within myocardial tissue. It proved that the ANG gene in vivo can promoteangiogenesis in ischemic myocardium.4. Based on predecessors’experience and our exploration, we found a feasible methodin the process of establishment the model of pericardial adhesion for ischemic cardiacmuscle. We established the animal model successfully and proved its advantage onimproving myocardial blood supply and maintenance of cardiac function comparing withthe control groups. It also proved that this experiment was effective on improvingmyocardial blood supply, restraining myocardial cell death, relieving ventricularremodeling, preventing cardiac insufficiency. It provided new experimental data and newideas for the treatment of ischemic heart disease. |
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