In Vitro Experiment Study On Treatment Of End-stage Coronary Heart Disease Using Human VEGF₁₆₅ Gene Transfected Skeletal Myoblasts

ObjectiveEnd-stage coronary heart disease is difficult to treat because of serious myocardial ischemia and life-threatening heart failure caused by cardiaomyocyte irreversible lose. On the basis of hypothesis that transplantation of VEGF-expressing myoblasts could effectively proceed the myocardium revascularization and cellular cardiomyoplasty, we transfected human VEGF165 gene into adult human skeletal myoblasts and tested the expression and biologic activities of VEGF165 in gene transfected cells.MethodThe first section: pcDNA3.1-VEGF165plasmid were transfected into CHO cells by electroporation and clones were screened by G418.The transcription and expression of VEGF165 in CHO cells were tested by RT-PCR, ELISA and Western-blot. The biologic activities of VEGFI65 secreted by CHO cells were investigated by Mile's assay and endothelial cells MTT assay.The second section: Skeletal myoblasts of adult human were isolated, cultured and purificated from skeletal muscle tissue. And cells were detected by optic microscope, electron microscope and immunohisto -chemistry.The third section: pcDNA3.1-VEGF165 plasmid were transfected into skeletal myoblasts by Lipofectin. RT-PCR and endothelial cells MTT assay were done to test expression and biologic activities of VEGF165 secreted by Skeletal myoblasts.ResultsThe first section: We Obtained anti-G418 CHO cells mono-clones. RT-PCR, ELISA and Western-blot prove that there was transcription and expression of VEGF165 in anti-G418 CHO cells. The expressed VEGF165 could increase the vessel permeability and promote the proliferance of endothelial cells.The second section: Adult human skeletal myoblasts looks like "fibroblast" under optic microscope. Immunohistochemistric stain showed Desmin(+). Electron microscope showed immature myogenous cell.The third section: RT-PCR proved that skeletal myoblasts transfected pcDNA3.1-VEGF165 plasmid by Lipofectin could transcript VEGF165. MTT assay proved that the surpernatant of transfected cells could promote the proliferance of endothelial cells. Conclusions1. pcDNA3.1-VEGF165 plasmid could express active VEGF165 well in mammalian cells. 2. Adult human skeletal myoblasts could be isolated, cultured and purificated well. 3. Adult human skeletal myoblasts transfected pcDNA3.1-VEGF165 by Lipofectin could produce active VEGF165 .4. Adult human skeletal myoblasts had little essential secretion of VEGF165 during culture in vitro.