Hypoxia-inducible Factor 1α Gene Transfected Into Skeletal Muscle Cells And Effect On The Proliferation Of Vascular Endothelium Cells In Vitro

ObjectiveHypoxia-inducible factor-1α (HIF-1α) gene were transfected into primary cultured skeletal muscle cells of rats in vitro. Then the VEGF protem secreted in culture medium were measured and the function of secreted VEGF was evaluated by studying its effect on the proliferation of primary cultured endothelium cells.Methods1. Muscles were obtained from the limbs of 1-3d Wistar rats, then digested by trypsin, finally transported into culture medium. The cells were observed under inverted microscope every day. The cultured cells were identified by immunohistochemical staining with anti-factor desmin, immunocytochemical staining with anti-α-sarcometric actin and transmission electron microscope;Thoracic aorta were obtained from the Wistar rats which weight about 120g,then primary cultured with explant. The vascular endothelium cells were identified by immunocytochemical staining with anti-factor Ⅷ related antigen.2. Extraction and amplification the plasmid of pHOX/HIF-1α, The plasmid was comfirmed using restriction analysis, transfected plasmid into primary cultured skeletal muscle cells with cationic liposome (Lipofectamine^TM2000) , Transfection efficiency were identified, To demonstrate the secreted VEGF protein concentration in the supernatant by Enzyme-Linked Immunosorbent Assay (ELISA) , the function of secreted VEGF was evaluated by studying its effect on the proliferation of primary cultured vascular endothelium cells with the methods of MTT.Resultsl.Rat skeletal muscle cells were isolated and cultured successfully in vitro and were identified by immunocytochemical stain and immunohistochemical stain with high purity;Rat aortic vascular endothelium cells were isolated and cultured successfully in vitro and were identified by immunohistochemical staining with high purity.2. HIF-la were transfected into primary cultured skeletal muscle cells successfully, green fluorescent protein(GFP) were observed through fluorescence microscope, VEGF protein were detected in culture medium, 1308.40±l<^ 1449.2±69.86> 1802.3±96.7K 1494.95±86.24 (pg/ml )VEGF protein were detected in culture medium after skeletal muscle cells were transfected 24h% 48h> 72h and 96h respectively. Furthermore, the secreted VEGF protein has the ability to stimulate the proliferation of vascular endothelium cells. The VEGF protein which HIF-la transfected skeletal muscle cells secreted could promote the proliferation of primary cultured vascular endothelium cells dose-dependently. The proliferation of VEGF groups were significantly greater than that of control group from 24h to 72h(/K0. 05) except lOpg group at 24h.Conclusions1. Rat skeletal muscle cells were isolated and cultured successfully in vitro.2. Rat endothelium cells were isolated and cultured successfully in vitro.3. HIF-la were transfected into primary cultured skeletal muscle cells successfully and the secreted VEGF protein can promote the proliferation of primary cultured vascular endothelium cells.