| Acute myocardial infarction (AMI) is a clinical syndrome which, on the basis of coronary artery disease, myocardial necrosis occures as a result of coronary plaque rupture, acute occlusion of coronary artery and the blood flow, sudden decrease or interruption of blood supply, causing acute ischemia and serious and persistent hypoxia on the corresponding myocardium.Currently the treatment regimen of AMI includes medical therapy, percutaneous coronary intervention, and coronary artery bypass grafting surgery. The evolving of new treatment approaches as well as the industrial improvement have played a positive role to improve the prognosis of AMI, yet AMI remains a serious threat to human safety of life.The clinical staff must attach great importance to emergency control and best treatment methods.It has been generally known that myocardial infarction repair process is closely related to the status of blood vessels. Rapid angiogenesis and the re-opening of residual vascular bed are vital factors for establishing good collateral circulation to improve the blood supply to the infarcted area and myocardium survival after myocardial infarction. Angiogenesis is a multi-step and complex process highly regulated by a variety of factors and synergies in terms of time and space, jointly promoting the angiogenesis process. In recent years, with the deepening research on the angiogenesis and vascular growth factors in the infarcted area, regulation of expression of the various angiogenesis related factors to stimulate the growth of small blood vessels and to promote the formation of collateral circulation in ischemic areas has become a very attractive, intuitive and reasonable method of treatment for AMI.The bone marrow mesenchymal stem cells (BMSC) are multipotent adult stem cells, with the multipotent differentiation potential of developing into endothelial cells, cardiac cells, cartilage cells, etc. The BMSC have very strong paracrine abilities. They can stimulate the proliferation of local blood vessels and cardiac cells as well as inhibition of cardiomyocyte apoptosis in ischemic area through the autocrine or paracrine of multiple pro-angiogenic cytokines such as VEGFs in the myocardial microenvironment. The micro RNA, or the miRNA, has been a recently discoved endogenous, small single strand, non-coding RNA of a length of18to26nucleotides. With its highly conservative sequence, and through adjustment of the gene expression at the transcriptional level via sequence-specific interaction of the target gene, the miRNA plays negative regulatory role by complementary match to the3’non-coding region of its target mRNA molecule (3’-untrans-latedregion,3’-UTR). It participates in a variety of biological processes. In recent years, some scholars believe that miRNAs can act as an access to stem cell transplantation therapy for AMI. The miR-126, highly expressed in heart and lung tissue, is one of the reported miRNAs which has close relations to vascular endothelial cells and vascular function. Its absence would delay the sprout of the blood vessels, resulting in the damage to blood vessel integrity.We speculate that miRNA-126, by regulating bone marrow mesenchymal stem cells, may mediate and control the expression of a variety of angiogenesis related factors so as to stimulate the growth of blood vessel and to promote the formation of collateral circulation in the schemic region. For this purpose, we attempted to use miR-126-BMSC for the mine-swine’s AMI model, evaluated its results and tried to explore its mechanisms.And we hope to discover a new way for the treatment of AMI with stem cell transplantation technique.PART1Establishment of the Bone Marrow Mesenchymal Stem Cells with Lentivirus-mediated Stable Overexpression of miR-126Purpose1. To establish the isolation and cultivation methods for the bone marrow mesenchymal stem cells (BMSC), and to investigate the biological features and phenotypic characteristics of their growth and proliferation in vitro.2. To construct the pCDH-miR-126lentiviral expression vector, and to establish the miR-126-BMSC with stable overexpression of miR-126. Methods1. Mini-Swine bone marrow mesenchymal stem cells were separated through the density gradient centrifugation technique by application of the Percoll cell separation medium. It was cultured and passaged thereafter. The cultured cell’s growth curve was calculated with the MTT method. Flow cytometry was applied to identify the bone marrow mesenchymal stem cells.2. Lentiviral-expressed plasmid pCDH-miR-126was constructed. BMSC cells were infected by matured lentivirus particles, which were produced by the packaged HEK293T cells, resulting in the BMSC with stable overexpression of miR-126. Detection of the expression level of miR-126was accomplished with the RT-PCR.ResultsThe primarily cultured BMSC kept adherent within the frist24, hours and stretched into polygonal or spindle shape. It was in a relative inhibition status in the first3days, following which, a gradual acceleration of cell proliferation occurred logarithmically till the7th day when it reached the platform phase with cell coverage up to about95%. In two weeks time when the third generation cells were obtained, the BMSC count reached106, a state that cell numbers has met the needs for transplantation. The characteristics of BMSC were investigated with flow, cytometry, and the results showed that they were CD44positive, and CD34negetive, which corresponded with the features of BMSC.PCR and sequencing proved successful construction of pCDH-miR-126plasmid. After HEK293T cell virus packaging and BMSC infection, compared with the control group, the miR-126expression level was significantly increased in the miR-126-BMSC group.ConclusionsWith the Percoll cell separation medium, and through density gradient centrifugation approach, a more uniform and pure population of bone marrow-derived mesenchymal stem cells with stable growth in vitro was successfully isolated. A relative fast cell growth was noticed after passage.The pCDH-miR-126lentiviral plasmid was successfully constructed, and bone marrow mesenchymal stem cell lines miR-126-BMSC with stable expression of miR-126established. A useful cell model could be offered for the study on the effects and the mechanism of the miR-126in the angiogenesis of mini-swine after acute myocardial infarction.PART2The Establishment and Evaluation of Mini-swine’s Model of Acute Myocardial InfarctionPurposeTo establish a model of acute myocardial infarction by ligating the left anterior descending coronary artery (LAD) in mini-swine.MethodsA total of12experimental mini-swine were used for the study. General anaesthesia was applied and traechal intubation was performed in a routine fashion. The pig was put lay flat on the DS A operation table with the extremitie fixed as per protocol. Real time ECG and invasive blood pressure moniter were carried out Transeasaphageal echocardiography probe was inserted and connected to the main unit (Philips iE33). Heparin was delivered and femeral artery puncture was performed. Selective coronary artery angiography was accomplished to check if the left and other coronary arteries were normal without any stenosis or occlusion. The left anterior chest wall was de-haired and thoracotomy was undertaken, followed by pericardiotomy and exposure of the left anterior descending (LAD) coronary artery. Ligation suture was placed around the LAD about1.5-2cm distal to the ostium of the first diagonal branch. An acute myocardial infarction model was constructed. It was considered successful when the following features were demonstrated. ST segment elevation was noticed on multiple ECG leads. The corresponding myocardium of the anterior left ventricular wall and apexs turned dark, purple, and white instead of the normal red. Hypokinesis or akinesis developed in the apical wall. And coronary angiogram showed total occlusion of the LAD. Hemostasis was performed prior to chest wound closure. Tracheal extubation was provided when the pig’s spontaneous breath was back to normal. Myoccardial damage markers such as serum CK, CK-MB, and cTnl were checked at different time intervals. Feeding was offered from the2nd postoperative day, and antibiotics delivered as per the protocol during the first3days after surgery. Myocardial nuclein perfusion imaging (ECT) was undertaken in the2nd week. Cardiac arrest took place at the6th week by intravenous injection of potassium chloride. The heart was removed from the pericardial cavity. The normal and ischemic myocardium corresponding to the LAD supply was dissected and sent for pathological evaluation of histological changes.ResultsNine out of the12mini-swines have survived the process, for a survival rate of75%. After complete ligation of the left anterior descending (LAD) coronary artery and total occlusion of blood flow, the following ECG changes which were characteristic to acute myocardial infarction were noticed. Those included ST-segment elevation, the upright T wave, ST segment elevation arched upward for more than0.5h. Twenty-four hours after the ligation of the left anterior descending coronary artery, the serum myocardial enzymes, including CK, CK-MB, cTnl, etc. increased over2-folds, a statistically significant difference compared with the preoperative value (p<0.05). The postoperative Doppler echocardiography showed a significant decrease of the heart function compared with the peroperative values, including a major decrease of LVEF and FS whereas an increase of LVESD, LVEDD,LVEDV, etc. This change was statistically significant (p<0.05). Coronary angiography after modeling demonstrated total occlusion of the LAD blood flow with TIMI0, proving a successful construction of the myocardial infarction model. Postoperative pathology showed structural disorder in the myocardial infarct zone, including malalignment, reduced number of myocardial cells, irregular nucleus size, and unclear cell boundaries.Conclusions Mini-swine myocardial infarction model was constructed by means of coronary artery ligation.Dynamic ECG monitoring demonstrated ST segment elevation arched upward for over0.5h. Serum myocardial enzymes such as CK, CK-MB and cTnI increased more than2times of the preoperative value. Doppler echocardiography showed significantly reduced heart function.Coronary arteriography revealed total occlusion of the LAD distal to the ligation site. Mini-swine acute myocardial infarction model was successfully established.PART3Influence of the miR-126-BMSC on infarcted zoneangiogenesis for mini-swine with acute myocardial infarctionPurposeThe miR-126-BMSC was injected into the myocardial infarction area of the experimental miniature swine. Four weeks following the above procedure, the echocardiography, selective coronary angiography, infarct area microvessel density and vascular endothelial growth factor were checked and analysed. The effects and mechanisms of the miR-126-BMSC treatment on the angiogenesis and heart function for small pigs with acute myocardial infarction were explored.MethodsTwo weeks after molding, the experimental pigs were randomLy divided into three groups. Group I:myocardial infarction control group (n=3):PBS injection was undertaken.Group II:the control group (n=3). Injection of BMSC was carried out. Group Ⅲ:the miR-126-BMSC treatment group (n=3). Injection of miR-126-BMSC was given. Four weeks after the operation, the echocardiography, selective coronary angiography and ECT examinations were performed. Thoracotomy was performed. The heart was removed out of the pericardial cavity and sent for pathology. Heart tissue specimens were obtained to investigate the expressing of GFP protein under fluorescent microscope. Immunohistochemical detection of Ⅷ factor specific endothelial markers was perpormed, while the RT-PCR and immunoblot technique were applied on the assessment of VEGF mRNA and protein levels in each group. ResultsThe postoperative fluorescence microscopy has found green fluorescence in the tissue slices from the infarcted myocardium of both the BMSC transplantation group and miR-126group, indicating the survival of transplanted cells in myocardial infarcts. Four weeks after cell transplantation, angiogenesis was significantly enhanced in the ischemic muscle tissue of the BMSC group as well as the miR-126group, with a higher level of capillary density in the miR-126-BMSC group (p<0.05). The RT-PCR and Western blot analysis showed VEGF mRNA and protein level were significantly higher in both the BMSC group and the miR-126-BMSC group’s infarcted tissue than that in the control group, with the miR-126-BMSC group ranked the highest (p<0.05). Compared with pre-transplantion state, the post-transplantion cardiac function improved significantly in both BMSC group and miR-126-BMSC group (p<0.05), with the EF and FS significantly better in the miR-126-BMSC group than than of the isolated cell transplantation group (p<0.05). Selective coronary angiography showed obvious collateral formation along the left anterior descending artery and the first diagonal branch in the miR-126-BMSC group. ECT examination results showed a significant increase of blood flow in the miR-126-BMSC group.ConclusionsThe miR-126-BMSC injected into the myocardial infarction area of the mini-swine can significantly increase the number of angiogenesis in the myocardial infarction area, promote angiogenesis process, be conducive to the establishment of coronary collateral circulation, increase myocardial blood flow, and improve heart function. The mechanism of promoting the formation of new blood vessels by the miR-126may be closely related with the increased expression of VEGF mRNA and protein by the overexpression of the miR-126. |
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