The Experimemtal Studies Of Cell Transplantation Into Ischemic Myocardium Using Mesenchymal Stem Cells Modified By Recombinant Adenovirus Carrying VEGF₁₆₅ Gene

Objective:â…  .To construct recombinant adenovirus vector carrying the vascular endothelialgrowth factor 165 (VEGF₁₆₅) gene and amplify the adenovirus vectors in 293T cells.â…¡ .To establish the separation method of mesenchymal stem cells (MSCs) ofrabbit. Study the features of culture in vitro and biological characteristics ofMSCs.â…¢.Recombinant Adenovirus mediated vascular endothelial growth factor165 gene (vAd-VEGF₁₆₅) transfected into rabbit mesenchymal stemcells (MSCs), then the change of growth of MSCs after transfectionwas observed and the expression of VEGF₁₆₅ was estimated.â…£.To evaluate the method of establishing an economic and efficient model ofmyocardial infarction in rabbits and to determine the cardiac function aftermyocardial infarction.â…¤ .To observe the therapeutic effect of VEGF-expressing MSCs in myocardialinfarction by providing enhanced cardioprotection, followed by angiogeniceffects in infarcted myocardium.Methods:â…  .The VEGF₁₆₅ obtained by PCR was digested and inserted into adenovirus shuttle plasmid pAdtrack-CMV to generate a recombinant plasmid pAdtrack-CMV-VEGF₁₆₅, and then the plasmid pAdtrack-CMV-VEGF₁₆₅ linearized by PmeI was transferred into E.coli BJ5183 cells encoding adenovirus backbone plasmid pAdEasy-1. The identified recombinantplasmid vAd-VEGFi65 DNA was digested with Pad and transfected into 293T cells to package the adenovirus. The recombinant adenovirus were identificated by means of observation of the green fluorescence protein (GPP) expression under fluorescent microscope. After amplified in 293T cells, the obtained vAdenovirus were transfected into 293T cells again, and GFP expression was detected.II .Bone marrow of femurs was drawn by pucture from New Zealand white rabbits and MSCs were harvested through there different methods, then were cultivated, multiplied and expanded. Through detection the expression of CD44 and HLA-DR, the purity of MSCs were estimated. Primary cells and sub-cultured cells were estimated though phase-contrast microscope and eletronic microscope. The second generation MSCs were labeled with 4, 6 diamidino-2-phenylindole (DAPI).III.vAd-VEGFi65 were transfected into MSCs in vitro. The expression of VEGF165 genes in MSCs after transfection was determined by observing the expression of GFP. ELISA method was applied to assay the expression and secretion of VEGF165. MSCs were digested and passaged by trypsin, and the cells growth curve were drawn.IV .After anesthetized with 3% pentobarbital sodium, the left anterior descending branch were ligated in a sterile condition without using tracheal intubation and breathing machine. The change of ECG was recorded by biological workstation. The level of creatine kinase isoenzymes and Tropnin-T in blood-serum was estimated. Randomly chosen rabbits' myocadium was staining with TTC and Evens blue after ligation for 4 hours and calculating the left ventricle myocardial size, infarction size and area at risk respectivly. Survial animals' cardiac function was determined and compared with preoperative cardiac function after 4 weeks.V.The VEGF165 gene was transfected to cultured MSCs of New Zealand rabbits using an adenoviral vector. 2x106 VEGF-transfected and DAPI labeled MSCs(vAd-VEGF^s/MSCs ) group, MSCs (MSCs group) or serum-free medium only (control group) were injected into syngeneic rabbits hearts 4 week after left coronary artery occlusion. At 4 week after cell transplantation, MSCs were detected by DAPI staining in infarcted region. The cardiac functions were estimated by UCG The microvascular density was estimated though CD34 immunohistochemical analysis.Results:I .Recombinant adenoviral vector VEGF165 gene was constructed successfully,which was confirmed by restriction enzyme digestion, the examination of gene sequences and GFP expression.II .The purity of MSCs separated with Percoll was 92%. MSCs growthed inwhirlpool. The sub-cultured cells morphological character had little change and had good homogeneity and strong ability of proliferationIII.GFP expression could be observed under fluorescent microscope 24 h later after infection, and the expression of VEGF165 gene was confirmed by immunohistochemical staining. ELISA analysis showed the quantity of expression of VEGF165 in transfection group was higher than control group (P<0.01). Moreover, the MSCs growth curve of transfection group and control group had not significant difference.IV.The total mortality of establishing model of myocardial infarction is 14%. After ligation of LAD, the T wave was towery and the ST segment was raised in most animals.The level of creatine kinase isoenzymes and Tropnin-T in blood-serum was raised markedly compared with the preoperative level. Normal myocardium is dark blue with Even's blue staining while normal and risky myocardium was red with TTC staining. The area of infarcted myocardium is (11.6 + 3.5) % of left ventricle. The left ventricular end-systolic diameter, left ventricular end-diastolic diameter, left ventricular eject fraction and fractional shortening were descendent markedly.V .At 4 week after cell transplantation, LVEDP, ejection fraction, E /A wave ratioand capillary density of the infarcted region were most improved in the vAd-VEGFi6s/MSCs group, compared with the MSCs group and control group. DAPI positive cells were increased in the vAd-VEGFi6s/MSCs group.Conclusion:I .The recombinant adenoviral vector carrying VEGF165 was successfullyconstructed.II .The purity of MSCs could be elevated with Percoll. Rabbit MSCs have astrong activity of proliferation capability.III .Exogenous VEGF165 gene can be expressed in MSCs. And the transfection has no influence to the growth of MSCs.IV .We successfully established an efficient and economic method of establishing model of myocardial infarction and the cardiac function can be estimated by UCGV .This combined strategy of cell transplantation with gene therapy could be auseful therapy for the treatment of myocardial infarction.