| Due to primary and secondary prevention, the mortality rate of coronary atherosclerotic heart disease (CAD) has been decreased dramatically in recent years. CAD is still one of the three major causes of deaths. Complications of CAD make heavy burden on patients, family and society. In order to increase survival rate of CAD patients, many new methods have been used in clinical practice. However, most of them are not useful in the repair of muscle and blood vessels. Mesenchymal stem cells (MSCs) are regarded as seed cells which migrate to injured tissues in vivo, then differentiate into different tissue. As a source of cells repairing the structure and function of injured lesions, MSCs rarely suffer the immunological rejection, which make it become an ideal target cells for the treatment of ischemic diseases. Angiopoietin-1 (Ang-1) is an important growth factor, which acts on endothelial cells and participates in angiogenesis. In the case of ischemia, Ang-1 can promote angiogenesis and remodel vessels by interfering with the interaction among endothelial cells, smooth muscle cells and pericytes. In this study, we transferred the Ang-1 gene to MSCs mediated by Lentivirus (LV) vector, then the gene-modified MSCs were implanted into rats suffering from LAD occlusion, observed the effect of gene modified MSCs implantation and investigated the mechanism of this effect. The study included the following three parts.The first part was to verify the feasibility and stability of Ang-1 gene modified rat mesenchymal stem cells (rMSCs) by lentivirus. Methods:Lentiviral vector plasmid recombined by Ang-1 gene was reconstructed and identified. The constructed transfer plasmid pNL-Ang-IRES2-EGFP (or pNL-IRES2-EGFP, as mock group) with the packaging plasmid pHELPER and the envelop plasmid pVSVG were co-transfected into 293T cells. The rMSCs were infected by the LV vectors and Ang-1 protein expression was identified by RT-PCR, immunohistochemistry staining, and Western Blotting. Results:Ang-1 gene modified rMSCs were successfully constructed. The Ang-1 expression could be observed by immunostaining, Western Blotting and RT-PCR. Conclusions:Ang-1 gene modified rMSCs were successfully constructed. They could survive, stable secret Ang-1. The second part was to charaterize the influence of grafted Ang-1 gene modified rMSCs on acute myocardial infarction (AMI) in angiogenesis, heart function, infarction area and cell apoptosis. Methods:Mouse model of AMI was established by ligating the left anterior descending coronary artery of the F344 rats. Saline, Angl-rMSCs or pNL-rMSCs were injected into edge region surrounding the infarction anteriorly and laterally.28 days after transplantation, cell apoptosis and differentiation in the heart lesion were observed, and echocardiogram analysis of the improvement of rat heart function, RT-PCR analysis of Ang-1 mRNA and immunostaining analysis of the angiogenesis density were used. Results:The mouse model of AMI was successfully established. Two month after implatation the GFP(+) rMSCs could still be observed and differentiate into cell with myocardial markers. The expression of Ang-1 mRNA was significantly increased compared with the controls after 1 month. Compared with the pNL-rMSCs implanation, Angl-rMSCs implantation significantly improved the heart function with higher capillary density(Vwf(+)Cell/HP,20.3±4.6/HP vs13.4±4.2/HP, p<0.05, vs Angl-rMSCs;α-SM(+)Cell/HP 14.3±3.4/HP vs 11.5±4.3/HP,p<0.05, vs Angl-rMSCs); fewer cell apoptosis(Tunel(+) Cells/103Cells; 24.3±7.1/103Cells vs49.3±9.6/103Cells,p<0.05, vs Ang1-rMSCs); and lower infarction area(Ang-1-rMSCs vs pNL-rMSCs,47.5±3.1% vs 50.6±4.2%, p<0.05). Conclusions:Ang1-rMSCs implantation significantly improved the heart function with higher capillary density, fewer cell apoptosis and lower infarction area.The third part was to investigate the function of integrin in Ang-1 related to MSCs anti-apoptosis effect. Method:24-well flat-bottom-tissue culture plates were coated with Ang-1, the adhesion effect of Ang-1 on MSCs was observed at different time points. After serum starvation, Hoechst staining and Annexin V/PI were applied to investigate the anti-apoptosis effect of Ang-1 on rMSCs. Western Blotting, and combing integrin antibody (RGD antibody) and signal transduction blocker (wortmannin) were applied to check the expression of pAkt and Bcl-2 and clarify the signal pathway associated with anti-apoptosis. Results:(1)Ang-1 can promote the adhesion of rMSC. This effect can be inhibited by EDTA,β1 antibody and RGD antibody. (2)Ang-1 promotes rMSCs survival and proliferation, which protest rMSCs from apoptosis induced by taxinol. (3)The expression of pAkt and Bcl-2 are increased by Ang-1, RGD antibody can inhibit both of them, while wortmannin can repress pAkt expression only, but not Bcl-2. Conclusions:Ang-1 can promote the adhesion and survival of MSCs which can be inhibited by EDTA, subunit of integrinβ1 antibody and RGD antibody. p-Akt and Bcl-2 may play a role in the anti-apoptosis function of Ang-1. |
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