ABSTRACT
Subject(s)
Animals , Rats , Adenoviridae , Axons , Brain , Cell Line , Clone Cells , Discrimination, Psychological , DNA Restriction Enzymes , Enzyme-Linked Immunosorbent Assay , Fluorescence , Gene Library , Half-Life , HEK293 Cells , Immunohistochemistry , Lingual Nerve , Microscopy , Models, Animal , Molar, Third , Myelin Sheath , Nerve Growth Factor , Nerve Regeneration , Neural Conduction , Neurons , Peripheral Nerves , Regeneration , RNA, Messenger , Schwann Cells , Sequence Analysis, DNA , Sucrose , Tongue , Transfection , TransplantsABSTRACT
Subject(s)
Adenoviridae , Cell Line , Cells, Cultured , Clone Cells , Cloning, Organism , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Mass Screening , Nerve Growth Factor , Neurons , Peripheral Nerves , Polymerase Chain Reaction , Regeneration , RNA, Messenger , Schwann Cells , TransfectionABSTRACT
PURPOSE: The essential triad for nerve regeneration is nerve conduit, supporting cell and neurotrophic factor. In order to improve the peripheral nerve regeneration, we used polyglycolic acid(PGA) tube and brain-derived neurotrophic factor(BDNF) gene transfected Schwann cells in sciatic nerve defects of SD rat. MATERIALS AND METHODS: Nerve conduits were made with PGA sheet and outer surface was coated with poly(lactic-co-glycolic acid) for mechanical strength and control the resorption rate. The diameter of conduit was 1.8mm and the length was 17mm. Schwann cells were harvested from dorsal root ganglion(DRG) of SD rat aged 1 day. Schwann cells were cultured on the PGA sheet to test the biocompatibility adhesion of Schwann cell. Human BDNF gene was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into E1 deleted region of adenovirus shuttle vector, pAACCMVpARS. BDNF-adenovirus was multiplied in 293 cells and purified. The BDNF-Adenovirus was then infected to the cultured Schwann cells. Left sciatic nerve of SD rat (250g weighing) was exposed and 14mm defects were made. After bridging the defect with PGA conduit, culture medium(MEM), Schwann cells or BDNFAdenovirus infected Schwann cells were injected into the lumen of conduit, respectively. 12 weeks after operation, gait analysis for sciatic function index, electrophysiology and histomorphometry was performed. RESULTS: Cultured Schwann cells were well adhered to PGA sheet. Sciatic index of BDNF transfected group was -53.66+/-13.43 which was the best among three groups. The threshold of compound action potential was between 800 to 1000microA in experimental groups which is about 10 times higher than normal sciatic nerve. Conduction velocity and peak voltage of action potential of BDNF group was the highest among experimental groups. The myelin thickness and axonal density of BDNF group was significantly greater than the other groups. CONCLUSION: BDNF gene transfected Schwann cells could regenerate the sciatic nerve gap(14mm) of rat successfully.