Experimental Study Of Myocardial Reconstruction By Transferring MyoD Gene Into Cardiac Fibroblasts In Infarcted Myocardium

PART ONE Construction of recombinant adenoviral vector carrying MyoD geneObjective: To construct recombinant adenoviral vector carrying MyoD gene. Methods: To extract MyoD cDNA fragments from plasmid pEMSV-MyoD, PCR was used to clone the whole-length gene of MyoD and the agarose gel electrophoresis was performed. After adding CACC sequence at 5' end , MyoD gene was cloned into adenoviral expression vector DNA via pENTR/D-TOPO vector. The recombinant adenoviral vectors transfected into HEK293A cells were packaged and amplified. The viral titer was determined by counting plaque-forming units in HEK293A cells .The MyoD cDNA fragment inserted was certificated by PCR and sequencing . Results: The result of PCR and sequencing confirmed the right length of the inserted DNA, which consisted with the sequence reported. The viral titer was 1.3-4.8×l0¹¹pfu/ml. Conclusion: The recombinant adenoviral vector carrying MyoD gene was constructed successfully.PART TWO Experimental study on the differentiation of rat cardial fibroblasts into myoblasts induced by MyoD gene in vitroFirst section Collection and culture of rat ventricular fibroblasts...