Effects Of The Bone Marrow Mesenchymal Stromal Cells Combined With Gene Implantation On Cardiac Myocyte Apoptosis

Acute myocardial infarction (AMI) is one of the common clinic cardiovascular diseases.Drug,interventional therapy and surgery can reduce the fatal rate,improve the prognosis of the acute myocardial infarction patients.while if myocardial infarction were happened,local heart muscle suffered ischemic and hypoxia,much heart muscle cells occurred cellular mecrosis or apoptosis,the abnormal heart muscle were taken place by fibrosis tissue,then the cardiac ventricle were remodelled. When the heart muscle were remodeled,the heart muscle would be thinner than before,the relaxation pressure of the left ventricle woulde be increased,the volume of the left ventrile would be enlarged,then the heart failure was happened.Recently,more and more scholars dedicated their energy into looking for the new therapy to acute mycardiac infartion by gene and bone marrow stem cell transplanting.Through all those studies we concluded that if we use both of these methods we mentioned above,we could promote the neovascularization and improve the blood supply to the infarctus tissues,encourage the heart muscle's funcation and the function of the heart,then reduce the remodeling of the left ventricle.There are few studies were on the gene affiliated bone marrow transplanting to treat AMI.We observe the effets of the stem cell transplanting therapy to AMI by intercurrenting MSCs through the coronary to the infarctionings.ObjectPartⅠResearch in vitro(1) Abstract MSCs by density gradient centrifugation,amplicate the product in vitro.Then induce the MSCs into cardioblast by 5-aza.(2)Transfection MSCs cardiac myoid cells by Ad.VEGF165.Part II Research in vivo(1)To build pig model system with AMI(2) To investigate the effects of myoblast transplantation and VEGF to pig with AMI. And our focuses were the effects of Bcl-2,Bax and Caspase-3. Methods and Results :1,Experimental research of MSCs in vitro expansion and differentiation into cadiocyte : MSCs were isolated by Percoll,then the MSCs were purified, expanded and cultivated.Add 5-aza into the cultures,then the MSCs differentiate into cadiocyte.appreciate the differentiated cadiocyte by immunocytochemistry.Results:We found that the induced MSCs express positive TnI and desmin protein through the immunocytohistochemical methods,and that indicates the induced MSCs could differentiate into myocardial cells through induction.2,Adenovirus with Ad.VEGF infection from the myocardium like cells by kinds of M.O.I,and ascertain the infection rate.To determine the expression of infected VEGF by ELISA. After 24 hours when MSCs were induced by 5-aza , we add different Ad.VEGF contained MOI,then observe the expression of GFP through influorescence microscope. After infected MSCs 48 hours,we tested expression of Ad.VEGF in sarcoblast cells with ELISA. The results showed that immunohistochemistry showed that the MSCs which were transfected by recombinant adenovirus expressed VEGF and without attenuation after 2 weeks being transfected. It showed that Bone marrow stromal cells had good susceptibility to the adenovirus, had higher transfection efficiency and expression activity, can be sustained for some time to express VEGF.3,Make the minitype pig with acute myocardial infarction successfully: give a foundation anesthesia to the minitype pigs, separate its right femoral artery, puncturing, use balloon to block vessel 120min in 1/3 distal LAD. Model success criteria : ECG monitor: at least two related leads ST-segment elevating more than 0.2mV, formating of AMI ECG typical performance; after 6 hours when operation finished, cTnI and CK-MB of blood increasing twice more than normal; distal LAD occlusion from blocking area with angiography.4,The effect of stem cell transplantation and VEGF gene transfection on cardiomyocyte apoptosis and proteins'expressions that related to apoptosis ,such as: of Bcl-2,Bax and Caspase-3.One week after the establishment of acute myocardial infarction pig model, a cell transplantation and / or VEGF gene transfection was performed. 16 pigs were divided into four groups randomly: Group 1. Myoblasts +VEGF gene therapy group (n = 4) : OTW coronary balloon occluded descending branch of COSCO vessels, while VEGF gene transfection of myoblast (1.0×108/5ml) were infused by balloon catheter-reperfusion; Group 2.VEGF gene therapy group (n = 4) : the VEGF gene adenovirus was perfused by the above-mentioned manner(1010Pfu/5ml); Group 3. myoblast treatment group (n = 4) : the same number of transfected genes were perfused into muscle cells by the above-mentioned manner(1.0×108/5ml) ; Group 4. Myocardial infarction in the control group (n =4): infarction small pig coronary perfusion through the serum - free DMEM medium(5ml). 4 weeks after repeated echocardiography, catheter hemodynamic inspection and coronary angiography, the animals were killed. The heart was retrieved to observe the general changes in the structure of the heart, but leave specimens for immunohistochemical and cardiomyocyte apoptosis detection. The results showed that for the cardiomyocyte apoptosis index, Group 4 (myocardial infarction control group) was24.51±2.24%; Group 1(myoblast +VEGF) was 9.01±0.99%; Group 2. (VEGF) was14.71±1.20%; Group 3.(myoblasts) was 15.86±1.24%. Group 1 and the other three groups, had significantly differences (P <0.01); Group 2,Group 3 compared with Group 4 showed statistically different (P <0.05) Group 2 compared with Group 3 had no statistical difference( P>0.05). There are Bcl-2,Bax and Caspase-3 positive protein in the region of infarction areas.Compare to Group 4,the other group which were intervented by treatments,the expression of Bax and Caspase-3 came down (P<0.05),while the expression of Bcl-2 went up. The differences showed significantly (P <0.01) which group 1 compared with the other three groups. Group2 and Group 3 compared with Group 4 showed statistically different (P <0.05). There was no statistical difference between Group 2 and Group 3( P>0.05).Conclusions:1,MSCs induced by 5-aza could differentiated into cardiocyte in virto.2,Ad.VEGF165 which transfected into muscle cells could efficiently express VEGF.3,It was a repeatability and practical method to establish small pig model of acute myocardial infarction by coronary Intervention which had a higher rate of success.4,Transplantation of the cells transfected with VEGF gene can significantly reduce cardiomyocytes apoptosis and reverse the left ventricular remodeling.