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目的应用血清药理学方法探讨补肾活血中药对体外培养的视网膜Müller细胞在缺氧及转化生长因子-β2(transforming growth factorβ2,TGF-β2)干预下谷氨酸(glutamate,Glu)代谢的影响。方法取新生7 d SD大鼠5只,颈椎脱位处死后进行视网膜Müller细胞培养。根据[3H]标记的D,L-Glu摄入量判断体外培养第2代视网膜Müller细胞的Glu的摄入功能;以酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定体外培养细胞外液中谷氨酰胺合成酶(glutamine synthetase,GS)活性。结果与正常对照组比较,12 h、24 h、48 h时缺氧组、TGF-β2+缺氧组的Glu摄入量增加(均为P<0.05),而24 h、48h时GS活性明显下降(均为P<0.05);与缺氧组比较,TGF-β2+缺氧组12 h、48 h时Glu摄入量减少,48 h时GS活性下降(P<0.05)。24 h、48 h时TGF-β2干预组较正常对照组Glu摄入量增加(均为P<0.05),12 h时GS活性增加(P<0.05)。与TGF-β2+缺氧组比较,中药血清+TGF-β2+缺氧组12 h、24 h时Glu摄入量减少(均为P<0.05),但GS的活性增加(均为P<0.05)。结论缺氧使视网膜Müller细胞的Glu摄入功能明显增强,GS活性明显降低,而TGF-β2加重缺氧条件下Müller细胞GS活性的降低;在缺氧和TGF-β2同时存在条件下,补肾活血中药血清下调视网膜Müller细胞的Glu摄入能力,增加GS的活性。
Objective To investigate the effect of Chinese herbs for replenishing kidney and activating blood circulation on the metabolism of glutamate (Glu) in vitro cultured retinal Müller cells under hypoxia and transforming growth factor-β2 (TGF-β2) intervention. Methods 5 neonatal 7-day SD rats were sacrificed and retinal Müller cells were cultured after cervical dislocation. According to [3H] labeled D, L-Glu intake to determine the second generation of retinal Müller cells in vitro glucose uptake function of Glu; enzyme linked immunosorbent assay (ELISA) determination of cultured extracellular fluid Glutamine synthetase (GS) activity. Results Compared with the normal control group, the amount of Glu intake in hypoxia group and TGF-β2 + hypoxia group increased (all P <0.05) at 12 h, 24 h and 48 h, but decreased significantly at 24 h and 48 h (All P <0.05). Compared with hypoxia group, Glu intake decreased at 12 h and 48 h in TGF-β2 + hypoxia group and decreased at 48 h (P <0.05). At 24 h and 48 h, the amount of Glu intake in TGF-β2 intervention group was significantly higher than that in control group (all P <0.05). GS activity increased at 12 h (P <0.05). Compared with TGF-β2 + hypoxia group, Glu intake decreased at 12 h and 24 h in serum + TGF-β2 + hypoxia group (all P <0.05), but GS activity increased (both P <0.05). CONCLUSION: Hypoxia enhances the uptake of Glu into the retinal Müller cells and decreases the activity of GS. However, TGF-β2 aggravates the decrease of GS activity in Müller cells under the condition of hypoxia and TGF-β2. Chinese medicine serum down regulate retinal Müller cells Glu intake, increase GS activity.