Experimental Study Of Mesenchymal Stem Cells Transplantation Combining With Vascular Endothelial Growth Factor Gene Transfer On The Treatment Of Myocardial Infarction

Acute myocardial infarction (AMI) is a common disease that severely threatens the elder's health. Although thrombolysis and intervention in time in 4-6 hours after onset of AMI can restore the blood flow of the infarct-related vessels and save the dying myocytes, the reperfusion therapy only can limit the infarcted size and have no effect on the infarcted myocardium and the ischemic myocytes in the majority patients. Lacking the ability of differentiation, the infarcted myocardium can only be replaced by the fibroblasts to form the scar tissue and have no systolic function. Thus the patients are easy to suffer heart failure and sudden death with poor life quality. The cellular transplantation technique gives us a good perspective to repair the injured myocardium. Mesenchymal stem cells (MSCs) are multipotent ones that can be induced in vitro to differentiate into osteoblasts, chondroblasts, tendon, ligament, fibroblasts, nerve cells and muscles, et al. In addition, MSCs characterized by easy separation and cultivation and potential ability to expansion in vitro have been viewed as ideal donors to treat ischemic coronary diseases in stem cell transplantation. Vascular endothelial growth factor(VEGF) as heparin-link glucoprotein dimmer can stimulate cell division and proliferation of vascular endothelium and induce angiopoiesis. Its presence may account for increased blood flow of collateral circulation and capillary density, which may contribute to better blood supply and improve cardiac performance. In this research, MSCs transfected with VEGF gene in vitro were injected in infarcted area by intervention. And the effects of stem cell transplantation and gene therapy were investigated. MSCs amplified the capillary density in infarcted area combined with effective collateral circulation trigged by VEGF which may contribute to better blood and oxygen supply. Combination therapy may be ideal method for treatment of myocardial infarction.ObjectivesPart I Research in vitro(1) To construct hVEGF165 with adenovirus carrier.(2)To establish a method for isolation, purification and cultivation of MSCs and induce them into myocardium with 5-aza.Part II Research in vivo(1)To build pig model system with AMI(2) To investigate the effects of myoblast transplantation and hVEGF to pig with AMI. And our focuses were the effects of myocardial infarction cell regeneration, angiogenesis infarction regional density to myocardial apoptosis and remodeling.Methods and Results : 1. recombinant VEGF adenovirus construction : We cut the cDNA fragment of VEGF from pd-VEGF by restriction enzymes Xba I and Xho I , and inserted it into pShuttle-CMV by T4 DNA ligase enzyme, then cloned positive ones, amplifed them, extracted the plasmids, cut them with PmeⅠenzyme and then with pAdeasy-1 plasmid to transform the BJ5183 Ecoli, finally preparated for homologous recombination. Recombinant plasmid was amplificated, and plasmid DNA was separated. We taked 10ug DNA and cut it with Pac-Ⅰ. With lipid-mediated transfection to transfect HEK293 cells, we got the replication - defective recombinant adenovirus AdV-VEGF, and through DNA OD value ,we could got the titer of recombinant adenovirus virus. Recombinant adenovirus identification: HEK293 cells transfected by adenovirus vector became rounded, swelling and some floating 7-10 days ago. We placed the cell culture plate into -70 degrees refrigerator and repeated freeze-thaw 3 times. Then we got lot of recombinant adenovirus particles in the freeze-thaw supernatant. We extracted the COS-7 cells which were infected by adenovirus, isolated total RNA by RT-PCR. We found that mRNA levels of COS-7 cells transfected were significantly higher than those of non-transfected group. So our recombinant adenovirus could express foreign genes in host cells.2. experimental research of MSCs in vitro expansion and differentiation into cadiocyte : Bone marrow mononuclear cells were isolated with 1.073g/ml Percoll. MSCs were obtained by removing the non-adherent cells. Then the MSCs were purified and expanded through passaging in time. To prevent the MSCs frome differnetiating or slowing their rate of division, each primary culture was replaced to 2-3 new plates when the density within colonies became 70%~80% confluent approximately 2-3 days after replating. The cells still maintained the same shuttle-like shape. Then we detected the viability by Trypan blue staining, Drew cell growth curve; identificated the MSCs phenotype using flow cytometry, used 5-aza to induce them to myocardial cells, when the cells were amplificaed in vitro in the 2nd, 6th and 11th generations; finally tested them with immunohistochemical staining and cardiac - specific factor geneβ-MHC. The results showed that MSCs expressed the high level of CD44 and CD105 antigen but lower of CD34. The cells formed the spindle-shaped, polynuclear and cell muscle-like structure after induction. We found that they expressed TnI, desmin andβ-MHC with Immunohistochemical metheds. The results showed MSCs could differentiate into myocardial cells through induction.3. Adenovirus with Ad-VEGF infection from the myocardium like cells : After 24 hours when MSCs were induced by 5-aza , we revaccinated the induced cell into orifice or flask, and put 0, 5,20,100,200 pfu/cells MOI of Ad-VEGF (diluted with DMEM) into them respectively and made the pbs to control. Oscillating in 37°C for 2 h, then aspirated the virus solution, put 2.5% fresh medium 1 ml per hole. After every 12 hours, we observed GFP expression under a fluorescence microscope and calculate the ratio of immunofluorescence transfection . After that we tested expression of the muscle 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. So that was useful to treatment-induced angiogenesis.4. All pigs were feeded 1 week before experiment. Before operation aspirin was taken orally and 1 mg of atropine by intramuscular injection. Ketamine, diazepam and Sumianxin as foundation anesthesia were given. Then the skin incision, separating right femoral artery, puncturing, measuring ventricular pressure and taking the coronary angiography were operated step by step. We used balloon to block vessel 120min in 1/3 distal LAD and move the ballon in a small area to make endothelial lesion. That could save the operation time. 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. The experiments showed that balloon occlusion through PTCA successfully established animal model of AMI. High survival rate, more survival time and repeated coronary angiography were the advantages of this methed. That provided good animal models to further study.5. The effect of stem cell transplantation and VEGF gene transfection on ischemic myocardial regeneration, angiogenesis, cardiomyocyte apoptosis and left ventricular remodeling: one week after the establishment of acute myocardial infarction pig model, a cell transplantation and / or VEGF gene transfection was performed. 15 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 (10×107/10ml) 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; Group 3. myoblast treatment group (n = 4) : the same number of transfected genes were perfused into muscle cells by the above-mentioned manner ; Group 4. Myocardial infarction in the control group (n = 3): infarction small pig coronary perfusion through the serum - free DMEM medium. 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 BrdU-positive cells of the donor disseminated to the entire movement infarction region in Group 1 and Group 3, and incorporated with vascular wall worked as part of organizational structure, whereas there was no dissemination to the normal region; TnI cardiac - specific protein immunohistochemical staining was positive. There were no new myocytes in Group 2 (VEGF) and Group 4 (MI control group). Meanwhile there was no significant neovascularization (3.9±1.2 acre/HPF) in neovascularization number Group 4 (MI control group); Group 1 (myoblast +VEGF) 38.8±2.8 acre/HPF, Group 2 (VEGF), 22.4±2.3 acre /HPF and Group 3 (myoblasts), 14.4±3.6 acre /HPF. To the improvements in heart function (EF%) : Myoblasts +VEGF group >"myoblast cells" VEGF group >"MI control group, P <0.01. After intervention by a group, it was showed that Myoblasts +VEGF group infarct size (3.83%±1.20%), Group 2.VEGF myocardial infarct size (8.82%±3.52%), Group 3. myoblast group infarct size (7.78%±3.14%), Group 4.MI control group myocardial infarct size (17.46%±5.39%). The differences showed significantly (P <0.01) which group I 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. For the cardiomyocyte apoptosis index, Group 4 (myocardial infarction control group) was 32.7±6.8%; Group 1(myoblast +VEGF) 10±1.20%; Group 2. (VEGF) was15±3.52%; Group 3.(myoblasts) was 14%±3.14%. Group I 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 II compared with Group III had no statistical difference. The results indicated: stem cell transplantation/VEGF gene therapy could further improve the heart function of infarction pig.Conclusions:1. 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.2. In the support of BFGF, MSCs induced by BrdU can be divide into myocardial cells directly in vitro.3. Ad-hVEGF165 which transfected into muscle cells could efficiently express VEGF.4. Myocardial infarction size was reduced by the transplantation of muscle cells differentiation from MSCs and/or VEGF gene transfection. Moreover, the combination of these two could play a notable role to improve cardiac function and reduce the myocardial infarction size. 5. Transplantation of the cells transfected with VEGF gene can significantly reduce cardiomyocytes apoptosis and reverse the left ventricular remodeling.