丙戊酸钠慢性作用及停药后对C6神经胶质瘤细胞GAT-3及GABA-T mRNA表达的影响

Changes in GAT-3 and GABA-T mRNA expression of C6glioma cells in response to a 2-week treatment with sodium valproate and withdrawal

目的通过研究丙戊酸钠(VPA)慢性作用及停药后对C6神经胶质瘤细胞γ-氨基丁酸转运体-3(GAT-3)和γ-氨基丁酸转氨酶(GABA-T) mRNA表达水平的影响,从星形胶质细胞的角度来探讨VPA的停药反跳机制。方法用含50 mg/L VPA的DMEM培养基将C6细胞培养2周后制成VPA慢性作用模型,采用半定量RT-PCR方法检测VPA慢性作用及停药后对C6神经胶质瘤细胞GAT-3和GABA-T mRNA表达水平的变化。结果(1)在VPA慢性作用下,相对灰度值(RV值,即GAT-3、GABA-T电泳条带与相应的β-actin灰度积分的百分比)为(39.1±0.5)%,低于正常对照组的(46±1.3)%;各停药组的RV值均低于VPA慢性作用组,其中停药24 h组的最低,为(11.7±1.6)%,停药30 min组的最高,为(38.9±0.6)%;而停药48 h组又升高到(33.5±1.1)%。(2)VPA慢性作用组[RV值为(71.31±6.91)%]与对照组[RV值为(34.77±3.26)%]相比,GABA-T mRNA表达明显上调;各停药组与VPA慢性作用组相比GABA-T mRNA的表达明显下调,其中停药12 h组[RV值为(25.36±7.68)%]降至最低。结论VPA慢性作用可使GAT-3 mRNA的表达下调,使GABA-T mRNA表达上调。VPA慢性作用后停药造成的GAT-3和GABA-T mRNA表达水平的波动可能与VPA停药反跳有关。

Objective To examine the effects of sodium valproate(VPA) treatment and withdrawal on the expression of GA-BA transporter-3 (GAT-3) and GABA transaminase (GABA-T) mRNA in C6 glioma cells, and to explore the role of GAT-3 and GABA-T in the rebound mechanism of VPA withdrawal. Methods C6 glioma cells were maintained for 2 weeks in DMEM medium containing VPA (50 mg/L) to establish the cell model of chronic exposure toVPA. Semi-quantitative RT-PCR was used to examine the changes of GAT-3 and GABA-T mRNA expression in response to VPA treatment and with-drawal. Results Chronic exposure toVPA down-regulated GAT-3 mRNA expression to 39.1%±0.5% from 46%±1.3% in the control group; After VPA withdrawal, GAT-3 mRNA expression level kept decreasing, reaching the minimum (11.7%±1.6%) 24 h after the withdrawal and with an increase to the level of 33.5%±1.1% after another 24 hr. GABA-T mRNA expression was up-regulated to 71.31%±8.39% from 34.77%±2.36% of the control level after VPA treatment, the withdrawal of which re-sulted in decreased GABA-T mRNA expression. Till 12 h after the withdrawal, the GABA-T mRNA expression level de-creased to the minimum, 25.36%±7.68%. Conclusions Chronic treatment with VPA can down-regulate GAT-3 mRNA ex-pression and up-regulate GABA-T mRNA expression in C6 glioma cells, and this undulation may involve VPA withdrawal re-bound.