EFFECT OF INTEGRIN β_8 ON NEURONAL APOPTOSIS AFTER HYPOXIA ISCHEMIA IN ASTROCYTE/NEURON CO-CULTURE SYSTEM

Objective To observe the effect of integrin β_8 on the neuronal apoptosis after hypoxia ischemia(HI) in astrocyte/neuron co-culture system.Methods Astrocytes and neurons were cultured in vitro from cerebral cortex of the P1-3days Sprague Dawley rats and El 6 days fetal rats,respectively.Immunocytochemistry staining was used to identify the purity of cells.Integrin β_8 mRNA expression was qualified in the astrocytes at 12 hours,1 day,and 2 days after HI and reoxygenation(experimental group) and in normal astrocytes(control group) by RT-PCR.Integrin β_8 small interering RNA(siRNA) system was established to specifically block astrocyte β_8 expression,the efficiency of integrin β_8 inhibition was detected by real-time fluorescent PCR.The astrocytes and neurons were co-cultured to established the astrocyte/neuron co-culture system.The neuronal apoptosis was detected with TUNEL in the normal neurons /astrocytes group(co-cultured HI group),the astrocytes infected by integrin β_8 siRNA for 2 days / normal neurons group(β_8 RNA interference group),and normal neurons in vitro with HI treatment group(HI group) at 1 day after HI and reoxygenation.The normal neurons without treatment as control(control group).Results Glial fibrillary acidic protein and neuronal nuclei staining suggested a purity of more than 90%in cultured cells.HI resulted in an increase of integrin β_8 mRNA expression at 12 hours after reoxygenation in astrocytes,which peaked at 1 day after reoxygenation,then slowly decreased and remained higher at 2 days,showing significant differences between control group and experimental group and among different time points in experimental group(P 0.05).RNA interference efficiency was most significant at 2 days after astrocytes infected with integrin β_8 siRNA(P 0.05).The neuronal apoptosis was significantly increased in HI group,co-cultured HI group,and β_8 RNA interference group when compared with control group(P 0.05).But neuronal apoptosis index(Al) was significantly decreased in co-cultured HI group and β_8 RNA interference group when compared with HI group(P 0.05).The significant difference of Al was found between co-cultured HI group and β_8RNA interference group(P 0.05).Conclusion Integrin β_8 expression can be induced with hypoxic-ischemic brain damage,leading to decreased Al of neurons and obvious protective effect.