氯胺酮对原代培养星形胶质细胞缝隙连接的影响

Effects of ketamine on the gap junction in primary cultured astrocytes

目的 观察不同浓度氯胺酮对原代培养星形胶质细胞缝隙连接蛋白43（Cx43）功能及表达的影响.方法 原代培养的星形胶质细胞纯化后用星型胶质细胞特异性标志物胶质纤维酸性蛋白（GFAP）鉴定纯度.根据氯胺酮药物浓度不同将培养好的细胞分为4组：对照组（Ctrl）、Ket0.1（氯胺酮0.1μmol/L）、Ket1（氯胺酮1.0 μmol/L）、Ket10组（氯胺酮10.0 μmol/L）.纯化培养的星形胶质细胞分别与0.1、1.0、10.0 μmol/L浓度的氯胺酮共同孵育24 h后,使用细胞接种荧光示踪法测定星形胶质细胞缝隙连接功能的变化,Western blot检测Cx43蛋白表达水平.结果 原代培养的星型胶质细胞纯化后细胞纯度达（90.2±5.6）％.荧光示踪法显示与Ctrl组比较,Ket10组荧光传递能力降低0.48 ±0.12（P＜ 0.05）,Western blot结果也显示Ket10组Cx43蛋白表达水平较Ctrl组降低0.50±0.15（P＜ 0.05）;而Ket0.1组、Ket1组荧光传递能力分别降低0.08 ±0.13、0.11±0.15,荧光传递能力和Cx43蛋白表达水平与Ctrl组比较差异无统计学意义（P＞0.05）.结论 高浓度氯胺酮对星形胶质细胞缝隙连接功能及Cx43蛋白表达具有抑制作用,这可能是氯胺酮神经毒性作用的机制之一.

Objective To observe effects of different concentrations of ketamine on the function of gap junction and the expression of connexin 43 （Cx43） in primary cultured astrocytes.Methods Primary cultured astrocytes were isolated and enriched by shaking off and differential adhesion.The purity of astrocytes was identified by an astrocytic marker glial fibrillary acidic protein （GFAP）.The cultured cells were divided into 4 groups： control group （Ctrl）, Ket 0.1 group （ketamine 0.1 mol/L）, Ket1 group （ketamine 1.0 mol/L）, Ket10 group （ketamine 10.0 mol/L） according to the concentrations of ketamine.After 24 h of incubation with ketamine, the function of gap junction in cultured astrocytes was determined by parachute assay, and the expression levels of Cx43 protein were detected by Western blotting.Results The purity of enrichment cultured astrocytes was about （90.2 ± 5.6） % by GFAP immunofluorescence staining.The number of received cells in Ket10 group decreased by 0.48 ± 0.12 as compared with the control group.Both the number of received cells and the expression of Cx43 protein decreased by 0.50 ± 0.15 in Ket10 group （P 〈 0.05）.The number of received cells reduced by 0.08 ±0.13 in Ket0.1 and 0.11 ±0.15 in Ket1 groups, and there were no significant differences between Ket0.1, Ket1 and control groups （P 〉 0.05）.Conclusion The gap junction function and the expression of Cx43 protein were inhibited by a high concentration of ketamine in primary cultured astrocytes, which may be one of the mechanisms of ketamine neurotoxicity.