转化生长因子α对鼠视网膜Muller细胞谷氨酸转运体调控作用的研究

Regulation of transforming growth factor alpha on glutamate transpoter of retinal Muller cells in mice

目的 观察转化生长因子α（TGFα）对鼠视网膜Muller细胞谷氨酸转运体（GLAST）mRNA、蛋白质表达及摄取活性的调控作用。方法取出生后7～10d的新生昆明小鼠视网膜组织，体外进行Muller细胞的培养。同一原代细胞的第3～4代细胞培养后随机分为2组：①TGFα干预组：分别加入浓度为50、75、125、150ng／ml的TGFα，每种浓度3孔；②对照组：仍用含20％胎牛血清的Dulbeccon改良Eagle培养基培养。采用L-3H-谷氨酸摄取分析方法观察不同浓度TGFa对Muller细胞GLAST摄取活性的影响；逆转录聚合酶链反应方法分析MOiler细胞GLAST mRNA表达的差异；免疫组织化学染色方法检测Muller细胞GLAST蛋白质表达的变化。结果随着TGFα浓度的增加，L-3H-谷氨酸的摄取量及GLAST mRNA表达量均逐渐增加，TGFα浓度为125ng／ml时，L-3H-谷氨酸的摄取量达到最大值，为对照组的266％，同时GLAST mRNA表达量也达到最大值，为对照组的近4倍。免疫组织化学染色显示当TGFα浓度为125ng／ml时，干预组Muller细胞内的GLAST蛋白表达量明显高于对照组，差异具有统计学意义（P＜0．05）。结论TGFα可通过上调GLAST mRNA和蛋白质的表达增加视网膜Muller细胞谷氨酸转运体GLAST的摄取活性。

Objective To observe the regulation effect of transforming growth factor alpha （TGFα） on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7-10 day, and then cultured Muller cells according to literature. The 3-4 generation cultured cells of the same primary cell were divided into two groups at random： ① TGFα group： maintained in different concentrations of TGFα as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;②Control group： cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFα on GLAST activity in Muller cells were observed by L 3H-glutamate uptake detection; the expression of GLAST mRNA in Muller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFα concentration, both L-3H-glutamate uptake and GLAST mRNA expression were increased. The L 3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFa on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant （P 〈 0.05）. Conclusion TGF a can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.