氯胺酮对谷氨酸诱导大鼠大脑皮层星形胶质细胞自噬的影响

Effects of Ketamine on Glutamate-induced Autophagy of Primary Cultured Cerebral Cortex Astrocytes in Rats

目的观察氯胺酮对谷氨酸诱导的大鼠大脑皮层星形胶质细胞自噬的影响。方法取新生大鼠大脑皮层,原代混合培养、分离、纯化获得星形胶质细胞。实验分3个组:对照组(C组,加入D-Hank's液);谷氨酸组(G组,加入谷氨酸至终浓度125μmol/L);谷氨酸和氯胺酮组(GK组,先加入谷氨酸至终浓度125μmol/L,30 min后加入氯胺酮至终浓度1 mmol/L)。用相差显微镜观察细胞形态变化,免疫荧光染色鉴定细胞种类及LC3蛋白在细胞内的表达,通过Western blot法检测beclin-1、bcl-2、LC3蛋白的表达变化。结果 G组细胞内beclin-1、beclin-1/bcl-2、LC3Ⅱ/LC3Ⅰ蛋白表达较C组上升,bcl-2蛋白表达较C组下降(均P<0.05)。GK组细胞内beclin-1、beclin-l/bcl-2、LC3Ⅱ/LC3Ⅰ蛋白表达较G组下降,bcl-2蛋白表达较G组上升(均P<0.05)。结论 125μmol/L谷氨酸能促进星形胶质细胞发生自噬和凋亡,1 mmol/L氯胺酮可抑制谷氨酸诱导星形胶质细胞发生的自噬和凋亡。

Objective To investigate the efects of ketamine on the autophagy of cultured cerebral cortex astrocytes induced by glutamate at various time.Methods Primary cultures of mixed astrocytes were obtained from cerebral cortical tissue in newborn rats.Purified astrocytes were achieved by mechanical shaking and differential adhesions method.The astrocytes were then randomly divided into 3 groups： the control group（C） in which D-Hank＇s solution 366μi was added;glutamate group（G） in which glutamate was added（the final concentration was 125μmol/L）;glutamate-ketamine group（GK） in which glutamate was added first（the final concentration was 125μmol/L ）and 30 minutes later ketamine was added （the final concentration was 1 mmol/L）.Immunofluence staining was used to characterize the astrocytes and analyze the expression of LC3.The expression of beclin-1,bcl-2,LC3 were analyzed by Western blot. Results The expression of LC3E/LC3I,beclin-1 and beclin-l/bcl-2 were higher in group G than group C. The expression of bcl-2 was lower in group G than group C（all P〈0.05）.The expression of LC3D/LC3I, beclin-1 and beclin-l/bcl-2 were lower in group GK than group G.The expression of bcl-2 was higher in group GK than group G（all P〈0.05）.Conclusion One hundred and twenty-fiveμmol/L glutamine can induce autophagy and apoptosis,1 mmol/L ketamine can inhibit autophagy and apoptosis.