| Objective:1. To construct recombinant adenovirus vector Ad5-Sema3A.2. To investigate the effects of Sema3A on the sympathetic sprouting at peri-infarct zone in rats after myocardial infarction.3. To investigate the effects of Sema3A on the electrical remodeling at peri-infarct zone in rats after myocardial infarction.Methods:1. Xba I and Nhe I double digestion Sema3 A plasmid by aims bands. And then by Xba I and Nhe I double digestion pDC316 plasmid vectors are linearized bands, clips, transformed and screened restructuring pDC316-Sema3A plasmid. Using PCR and restriction enzyme digestion followed the method of recombinant plasmid was identified pDC316-Sema3A. Then, use automatic DNA sequencing analyzer to confirme connected correctly. To produce large scale of plasmid DNA, the competent cells were prepared. Then the plasmids were transfected into 293 cells to produce recombinant adenovirus Ad5-Sema3A. After amplification and purification the Ad5-Sema3A, calculate the virus titer.2. Adult male Wister rats were divided randomly into 4 groups:sham-operated rats treated with sham operation (sham group, n=30), myocardial infarction (MI) rats received of saline (MI group, n=40), Ad-GFP virus (Ad-GFP group, n=40) or Ad5-Sema3A (Ad-Sema3A group, n=40). MI was induced by left coronary artery ligation.8 weeks after surgery, Western blot and RT-PCR for analying growth-associated protein 43 (GAP43) and tyrosine hydroxylase (TH) protein and mRNA in infarcted border zone, respectively. Immunohistochemistry analysis for observing changes of GAP43 and TH distribution in infarcted border zone.3. Adult male Wister rats were divided randomly into 4 groups:sham-operated rats treated with sham operation (sham group, n=30), myocardial infarction (MI) rats received of saline (MI group, n=40), Ad-GFP virus (Ad-GFP group, n=40) or Ad5-Sema3A (Ad-Sema3A group, n=40). Eight weeks after MI, heart rate variability (HRV), monophasic action potential duration (MAPD), effective refractory period (ERP) and the inducibility of ventricular arrhythmia at the peri-infarct zones were evaluated and compared with MI rats. After these studies, the expression of Kv1.4, Kv4.2, KChIP2, Kir2.1 and SERCA2a were analysis by western blot and real-time RT-PCR.Results:1. We successfully constructed the recombinant containing the Sema3A pDC316-Sema3A plasmid, and used PCR, enzyme digestion, sequencing and BLAST methods were identified, that is properly connected; obtained E. coli strains carrying Sema3A gene; carrying Sema3A gene was successfully constructed defective proliferation of new recombinant Ad5-Sema3A, and was amplified and purified. The virus titer was measured 3.4×1011 VP/ml, virus activity units (TCID50) was 5.2×109 IU/ml.2. Eight weeks after MI, GAP43 and TH protein and mRNA increased significantly in the MI group and Ad-GFP group, the distribution of nerve fibers in infarcted border zone were chaotic and the fibers was immunoreactive to GAP43 and TH after MI. Sema3A decreased GAP43 and TH protein and mRNA expression significantly and reduced the density of sympathetic nerve.3. Compared with the sham group, rats of the MI group and Ad-GFP group showed a decrease in HRV, MAPD and ERP prolonged in the MI group and Ad-GFP group. The protein levels of Kv4.2, Kv4.3, KChIP2, Kir2.1 and SERCA2a were all decreased in the MI group and Ad-GFP group. Compared with the MI group and Ad-GFP group, Sema3A increased HRV, shorted MAPD and ERP and increased Kv4.2, Kv4.3, KChIP2, Kir2.1 and SERCA2a protein and mRNA expression.Conclusions:1. Reconstructed adenovirus vector Ad5-Sema3A was successfully constructed.2. The present study show that Sema3A can ameliorate sympathetic sprouting at peri-infarct zones after MI.3. The present study show that Sema3A can ameliorate electrical remodeling at peri-infarct zones after MI. |
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