脑源性神经营养因子对鼠视网膜Müller细胞GLAST蛋白表达及功能的调控

Regulation of brain-derived neurotrophic factor on the expression of GLAST protein and its function in retinal Müller cells of mice

目的 研究脑源性神经营养因子（BDNF）对鼠视网膜Müller细胞L-谷氨酸/L-天门冬氨酸转运体（GLAST）蛋白表达及功能的调控作用.方法 实验研究.取出生3～7 d的新生昆明小鼠视网膜组织进行Müller细胞培养,取传代后第3代Müller细胞进行后续试验.实验分为BDNF干预组和空白对照组：BDNF干预组小鼠视网膜Müller细胞分别加入50、75、100、125和150 ng/ml的BDNF培养24 h：空白对照组培养的Müller细胞不加BDNF.采用Western blot方法检测Müller细胞GLAST蛋白的表达.采用L-[3,4-H3]-谷氨酸检测100 ng/ml的BDNF干预组与空白对照组Müller细胞对谷氨酸的摄取功能的差异.对各组视网膜Müller细胞GLAST蛋白表达水平的比较采用单因素方差分析,100ng/ml的BDNF干预组与空白对照组对谷氨酸摄取量的比较采用独立样本t检验.结果 Western blot检测结果显示,空白对照组GLAST蛋白的相对表达量为0.151±0.025,50、75、100、125和150 ng/ml的BDNF干预组蛋白相对表达量分别为0.331±0.076、0.413±0.110、0.497±0.080、0.411±0.072、0.319±0.084,不同浓度的BDNF均能上调GLAST蛋白的表达水平（F=6.793,P=0.003）.当BDNF浓度为100 ng/ml时,CLAST蛋白表达量最大.100 ng/ml的BDNF组与空白对照组对谷氨酸的摄取量分别为（81 213±5982）和（68 743±2688）cpm/（mg·ml）,100 ng/ml的BDNF组能够增加Müller细胞对谷氨酸的摄取,两组差异有统计学意义（t=6.462,P=0.023）.结论 BDNF能够上调GLAST蛋白的表达,并增加细胞对谷氨酸的摄取.

Objective To study the effect of brain-derived neurotrophic factor （BDNF）on the expression of L-glutamate/L-aspartate transporter （GLAST） protein and its function in the retinal mice at 3 to 7 days postnatal were cultured by an enzymatic digestion method, and the third passage different concentrations of recombinant human BDNF （50, 75, 100, 125 and 150 ng/ml） for 24 h in group and the control group. The expression of GLAST protein was analyzed with one-way analysis of variance （ANOVA） and L-[3,4-3H]-glutamic acid uptake was analyzed with independent samples t test. Results The expression of GLAST protein in the control group was 0.151±0.025 and the expression in the BDNF group （50, 75, 100, 125 and 150 ng/ml） was 0.331±0.076, 0.413±0.110, 0.497±0.080, 0.411±0.072, and 0.319±0.084, respectively. Different concentrations of BDNF could up-regulate the expression of GLAST protein compared to the control group （F=6.793, P=0.003）.The expression of GLAST protein reached a maximum when the concentration of BDNF was 100 ng/ml.L-[3,4-3H]-glutamic acid uptake for the 100 ng/ml BDNF group and control group was 81 213±significantly higher than for the control group （t=6.462, P=0.023）. Conclusion BDNF can up-regulate the expression of GLAST protein and increase extracellular glutamate uptake.