A20 Effectively Inhibits Cell Death In Central Nervous System In Vivo And In Vitro

A20 is a TNF-induced primary response gene which encodes a novel zinc finger protein which can inhibits tumor necrosis factor (TNF)-mediated apoptosis in many cell lines. But little work has been done in central nervous system. AIM: To evaluated the antiapoptotic effect of A20 gene in both primary hippocampal neurons and the cell line SY-SH-5Y derived from CNS in vivo and in vitro. METHODS: The rat El8 hippocampal primary neurons were freshly prepared by hippocampus dissection and were identified with neuron-filament by immunocytochemical staining. Then the neurons were transfected with A20 gene by a new electroporation transfection method: Nucleofector? the transfection rate was confirmed by immunocytochemical staining with anti-A20 IgG; on the other hand, A20 was delivered into the cell line SY-SH-5Y too by liposome,then the positive clone was picked by G418. The expression of A20 protein was ensured by RT-PCR and Western Blot. Then the cells overexpressing A20 were exposed to TNF- a stimuli, the mortality was measured by fluorescence-activated cell sorting (FACS), in addition TUNEL staining, DNA fragmentation were alao used to the degree of the cell death. In vivo test, the A20-neurons and pCDN A3-neurons which had been labeled by Hoechst33258 in advance were transplanted into both the penumbra zone following middle cerebral artery occlusion (MCAO) in the adult rat and the same area in the normal rats as the control respectively. 3 and 7days after operation, cryostat sections were made, fluorescence was visualized by an inverted microscope, the number of survival cells was counted. RESULTS: The immunocytochemical staining for the neuronal marker NF showed that cells grown in Neurobasal supplemented with B27, and then transfected, were mostly neurons; the transfection efficiency reached over 50% (52.46% 5.26%). And the SY-SH-5Y cell line which had been picked by clone filtration proved to be mostly A20 positive. The test in vitro showed that the apoptosis rates of A20 -neurons and SY-SH-5Y were 28.46% A20 effectively inhibits cell death in central nervous system in vivo and in vitro Abstract3.87% and 9.59% 3.88%respectively, much lower than the ones of control groups which were 53.06% 5.36% amd 41.52% 3.29%under the stimuli of TNF- a (P<0.05); the results of TUNEL and DNA fragmentation also consistented with the FACS. In vivo, much more cells survived could be found in the A20 group than the pCDNA3 group on both 3 and 7 days after transplantation. CONCLUSION: A20 gene can exihibit an anti-apoptosis effect in cells of CNS, and may play a key role for the treatment of many diseases of nervous system which apoptosis is their main manifestation.