Activation And Expression Of Inwardly Rectifying Potassium Channel-4.1 Of Astrocyte Of Cerebral Cortex Stimulated By Lipopolysaccharide

Objective:Cells of newborn rat Ast for differential adherent culture, the high purity was obtained after wedding Ast and carries on the appraisal;to investigatethe activation effect of lipopolysaccharide(LPS) on astrocyte(Ast) in vitro,explore the best activation state of Ast and discuss the effects of IL-1βwith inwardly rectifying potassium channel-4.1 on activation of astrocytes, and provide a new way for prevention and treatment of epilepsy.Methods: 1). Decapitate newborn SD rats within 24 h of age and remove the forebrains for getting the cerebral cortex open the skull brain hemisphere cortex,stripping meninges and blood vessels,useing scissors to cut it into 0.8 mm3 small pieces,with 0.25% trypsin digestion beat several times,with a serum containing10% cultivate beat upon termination of digestion,to the centrifugal tube abandoned after centrifugal clear liquid and planting preparation into single cell suspension culture.In 37 ℃ CO2 incubator digestion 20 min, which once every 5 min out forced oscillation, to promote the digestion, centrifugal training again after 25 min to remove fibroblasts.Cells can grow to cover the whole bottle, purification after 18 h, within the super net station after digestion cell subculture.After three batches can obtain high purity of Ast cell, by using the method of immunohistochemical test purity.2)Application of determined by MTT method to detect LPS activation of Ast:Divide cultivated cells into experimental group and control group, experimental group with different concentration of LPS stimulation Ast,determined by MTT method is used respectively to the concentration of 1.25 μg/ml, 2.5 μg/ml, 5μg/ml, 10μg/ml and role respectively 12 h, 24 h, 36 h,48 h after the cells for active observation;The control for the same conditions did not make any disposal of the Ast cells.3)By the method of ELISA detection of LPS induced Ast in the experimental group and control group, IL- 1 beta secretion and statistical analysis.4)Using RT-PCR technique, detection of Kir-4.1、IL-1beta m RNA expression in the experimental group and control group;using the relevant statistical software analyze the result of the experiment.Results: 1) GFAP immunohistochemical method to the original generation of cultivation and three batches of the cell line detecting, Ast cell of GFAP positive rate is higher, via detecting purity can reach 95.50%,To illustrate that the cells can be used in the experiment of cultivation.2) Ast activatived by LPS:Through determined by MTT test results found that LPS can induce Ast activation in vitro, and the concentration and time dependence.3) ELISA method to detect the result shows that:Compared with control group, LPS(including 5μg/ml) to deal with the Ast at different times(12 h, 24 h and 36 h), the level of IL- 1 beta in the cell culture supernatant increased significantly.(p < 0.05)4) The semi-quantitative rt-pcr test results shows that:As the extension of LPS processing time, Kir- 4.1 m RNA expression gradually decline, while IL- 1 beta m RNA expression gradually rise.Conclusions: 1) Use the original generation represented the cultivation, purification method can obtain high purity of Ast.2) LPS can Ast activation induced in vitro culture, and when the concentration of LPS to 5μg/ml, the processing time for 36 h Ast active best.3)Ast of LPS induced activation, Kir4.1 m RNA expression changes may be affected by the regulation of IL- 1beta.