Study On The Effect Of Angiogenesis Of Myocardial IRI Rats By The Bmscs Transplantation Induced In Vitro By Jiaweidanshenyin Medicated Serum

Objiective:Establish rat models of myocardial IRI, observe the improvement of heart function of myocardial IRI rats through BMSCs transplantation induced in vitro by JWDSY Medicated Serum, and investigate the dynamic effect and function mechanism of the angiogenesis of myocardial IRI rats.Methods:Test1:cultivate BMSCs of rats by the whole bone marrow stick wall of training, perform passage curture and amplification, observe the morphology and growth characteristics of cells, draw the growth curve, analysize phenotypes of CD34, CD45, CD90through the flow cytometry.Test2:detect toxic effects and proliferous influence of JWDSY Medicated Serum with different concentration on BMSCs by MTT method, mix BMSCs of rats cultivated by the whole bone marrow stick wall of training with JWDSY Medicated Serum and blank serum, induce in vitro to differentiate3d, establish rat models of myocardial IRI, which are randomly divided into four groups:Group SO (sham operated), Group model, Group BMSCs induced by blank serum, and Group BMSCs induced by JWDSY Medicated Serum. Use the approach of transplanting by direct injection in myocardium, inject the induced transplanting cells in the fringe area of myocardial infarction while Group model and Group SO are injected with the same amount of L-DMEM in three parts immediately after the animal model succeeding, and observe the general performance of the ligation part macroscopically. All observations are carried out by the three time points of3d,7d and14d respectively after transplantation. Assay the change of myocardial infarct size in the left ventricle of rats by TTC staining method, observe the morphologic change of cardiac muscular tissue with HE staining staining method, observe pathological changes of ischemia area, determine the level of cardiac enzyme CK and CK-MB by the automatic biochemistry analyzer, detect expressions of Ⅷ factor and CD34which are specific landmark of vascular endothelial cells in the edge of the infarction parts with the method of immunohistochemical staining, calculate and compare myocardial microessel-density (MVD), and then evaluate its heart protection and effect on angiogenesis.Test3:the transplanting method of induced BMSCs is the same with that in test2. All detections are processed by the three time points of3d,7d and14d respectively after transplantation. Detect protein expression of VEGF, Ang-1and PDGF-β with immunohistochemical method, Serum IGF-1level with radioimmunoassay, VEGFmRNA and Ang-1mRNA expression with Real-Time PCR in fringe area of myocardial infarction. Evaluate the ability of angiogenesis by intramyocardial transplantating of BMSCs that have been induced in vitro by JWDSY Medicated Serum, and investigate the relationship between the changes of various angiogenesis factors expression and angiogenesis.Results:Test1:according to the identification of FCM (flow cytometry), cells tends to have the shape of coincident spindle and grows in parallel or vortex after3times passage with BMSCs of rats cultivated by the whole bone marrow stick wall of training. The purity is high. Positive cells of CD90are above95%with CD34and CD45less than5%, which is in line with the requirements.Test2:the result of MTT method shows that20%of JWDSY Medicated Serum is the best concentration to induce BMSCs. After rats ligatured, ST segment and T wave increase abnormally. But after reperfusion, ST segment and T wave fall back quickly, and pathological Q wave appears, which indicates that IRI model of rat is successful. Observing by the three time points after BMSCs transplantation, it has found that the general situation of Group model is worse than Group SO, and the volume of heart increases obviously. Then serum myocardial enzymes CK and CK-MB also increase significantly. After the rat in Group BMSCs receives treating, its general situation has been improved with the shape and volume of heart being close to Group SO. The Group BMSCs induced by JWDSY Medicated Serum performs more obviously than the Group BMSCs induced by blank serum in narrowing fringe area of myocardial infarction (P<0.05). Comparing with Group model, Group BMSCs induced by JWDSY Medicated Serum and Group BMSCs induced by blank Serum have improved the level of CK and CK-MB, and Group BMSCs induced by JWDSY Medicated Serum performs more obviously. The Microvessel density (MVD) in fringe area of myocardial infarction of Group BMSCs that has been induced by JWDSY Medicated Serum and blank serum is higher than Group model (P<0.05) and Group SO (P<0.05). And Group BMSCs induced by JWDSY Medicated Serum increases more greatly than the Group BMSCs induced by blank serum (P<0.05) Test3:using immunohistochemical method to determine IRI rats VEGF, Ang-1and PDGF-(3protein expression of IOD in fringe area of myocardial infarction and radioimmunoassay to detect the level of IGF-1, it has found that the protein expression of the growth factor in Group model enhances more obviously than Group SO (P<0.05). Therefore, it can be considered that it arises from compensatory mechanism of organism on ischemia and anoxic of the tissue, proving that the model succeeds. The expression level of Group BMSCs induced by JWDSY Medicated Serum and blank serum is higher than Group model (P<0.05). Group BMSCs induced by JWDSY Medicated Serum increases more significantly (P<0.05), and it shows a general continuous trend (P<0.05). Applying Real-Time PCR to detect the expression of Ang-1mRNA, it has found that the heart tissue of Group BMSCs has the expression of VEGFmRNA and Ang-1mRNA in3d,7d and14d after transplantation. Group SO and Group model show weak expression. And Group BMSCs induced by blank serum and JWDSY Medicated Serum show a peak expression of VEGFmRNA and Ang-1mRNA in7d after reperfusion (P<0.05), then it decreases. The expression of VEGFmRNA and Ang-1mRNA in Group BMSCs induced by JWDSY Medicated Serum is stronger than Group BMSCs induced by blank serum at the same time point (P<0.05). The high expression of growth factor shows some relevance with MVD which reflects the strength of neovascularization.Conclusion:1. the method of the whole bone marrow stick wall of training can cultivate BMSCs with high purity and good activity.2. BMSCs transplantation induced by JWDSY Medicated Serum can improve heart function of myocardial IRI rats, reduce infarction area, increase MVD of ischemia area, and reduce serum myocardial enzymes CK and CK-MB. All the improvements are more significant than BMSCs transplantation induced by blank serum.3. BMSCs transplantation induced by JWDSY Medicated Serum can obviously promote angiogenesis in fringe area of myocardial infarction. The effects may be relevant with the protein expression of VEGF, Ang-1, PDGF-β and Serum IGF-1level and the expression of VEGFmRNA and Ang-1mRNA in local Ischemic Myocardium which is raised steadily and continuously. The effect is better than that of BMSCs transplantation induced by blank serum. BMSCs transplantation induced in vitro by JWDSY Medicated Serum has cooperative and stimulative role on angiogenesis.