Treatment Of Myocardial Infarction By Transplantation Of Adiponectin Gene-Modified Stromal Vascular Fraction Cells From Adipose Tissue

Objective1. To construct recombinant lentiviral vector of human adiponectin (HapM1) gene.2. To modify Stromal vascular fraction cells (SVF cells) by HapM1 gene and test the expression of HapM1 gene in SVF cells.3. To investigate the effects of HapM1 gene-modified SVF cells on myocardial infarction and observe the survival and differentiation of transplanted cells.Methods1. Construction and identification of lentiviral vector plasmid recombined with HapM1 gene.(1) Primers were designed according to the HapM1 gene sequences reported in Genbank. The HapM1 gene was obtained with RT-PCR. DNA fragment encoding HapM1 was cloned into pMD18-T vector. The recombinant TA cloning plasmid was then identified with restriction endonuclease and sequencing.(2) The recombinant lentiviral vector plasmid pNL-HapM1-IRES2-EGFP was constructed with double digestion and gene recombination,then identified with restriction endonuclease and sequencing.2. Culture and construction of HapM1 gene-modified SVF cells.(1) The constructed transfer plasmid pNL-HapM1-IRES2-EGFP with the packaging plasmid pHelper and the envelop plasmid pVSVG were co-transfected into 293T cells. The lentivirus was collected after transfection and was concentrated by centrifugation. The insersion of HapM1 gene in the lentivirus was identified by PCR using the genome extracted from the lentivirus as template.(2) The rat Stromal vascular fraction (SVF) cells were separated and cultured with the combination of collagenaseâ… and adherent culture,and the expressions of CD31 and CD34 in SVF cells were detected with immunofluorescence assay.(3) The SVF cells were infected by the lentivirus and identified with fluorescent microscope,RT-PCR, and western blot. The biological activities of HapM1 protein were tested by the migration of vascular endothelial cells.3. Treatment of myocardial infarction by transplantation of HapM1 gene-modified SVF cells.(1) The rat myocardial infarction model was established by ligation of the left anterior descending coronary arteries,and the operation was validated by ECG Changes. The gene-modified SVF cells were injected into the heart.(2) The cardiac function was evaluated with noninvasive ultrasound and invasive hemodynamic measurement 4 weeks after transplantation.(3) The histological changes of heart slices were observed with HE stain.(4) The distribution of grafted SVF cells were observed under fluorescence microscope. The differentiation of grafted SVF cells was observed with immunohistological fluorescent staining using the antibody of anti-CTNI as first antibody. The expression of HapM1 was observed with immunohistology using the antibody of anti-HapM1 as first antibody.â…§factor related antigen was marked to show the vascular density.Results1. The target 735bp HapM1 gene fragment was completely identical to the sequence of HapM1 in GeneBank. The pNL- HapM1-IRES2-EGFP plasmid was confirmed by agarose gel eletrophoresis and sequencing.2. EGFP expression in 293T cells after co-transfected was identified with fluorescent microscope. The HapM1 gene included in the recombinant lentivirus was identified by PCR. Immunofluorescence assay showed that the cultured SVF cells were CD34 positive, but CD31 negative. EGFP expression in SVF cells after infection was identified with fluorescent microscope. HapM1 expression in SVF cells after infection was identified by PCR and western blot. HapM1 protein expressed in constructed SVF cells shown the biological activity inducing migration of vascular endothelial cells. 3. The myocardial infarction rats treated with HapM1 gene-modified SVF cells recovered better in function, heart histology andâ…§factor related antigen than the control group,and than the rats treated only with SVF cells. EGFP labeled SVF cells were found in the location of myocardial ischemia. It was also observed that the grafted SVF cells could express the CTNI markers of myocardium.Conclusion1. A recombinant lentiviral vector containing pNL- HapM1-IRES2-EGFP was successfully constructed. The target gene HapM1 could be packed in the recombinant HapM1 lentivirus, which would provide a powerful tool in the study of HapM1 gene function.2. SVF cells were successfully modified with HapM1 gene. The functional gene HapM1 and reportor gene EGFP could co-express by the constructed SVF cells, which would provide an intuitionistic method in the study of HapM1 gene-modified SVF cells.3. The HapM1 gene was integrated into SVF cells genome, transcribed and expressed correctly. HapM1 protein expressed in reconstructed SVF cells had the biological activity of chemoattractant effect of inducing migration of vascular endothelial cells.4. SVF cells carrying HapM1 gene may differentiate into Myocardial Cells.5. SVF cells carrying HapM1 gene could promote the recovery of myocardial infarction siginificantly. SVF cells carrying HapM1 gene may differentiate into myocardial cells.