血管抑素对视网膜微血管内皮细胞的细胞外信号调节激酶水平的影响

Effects of angiostatin on the activities of extra-cellular signal-regulated protein kinase in microvascular endothelial cells of rat′s retina

目的 观察血管抑素对体外培养的鼠视网膜微血管内皮细胞的细胞外信号调节激酶(ERK 1)活性的影响。 方法 利用l lysine耦联的sepharose 4B亲和层析柱从血浆中分离和纯化血管抑素。原代培养鼠视网膜微血管内皮细胞分为4组:对照组,血管内皮因子(VEGF) 10ng/ml组,血管抑素13 0 μg/ml组,VEGF(10ng/ml) +血管抑素(13 0 μg/ml)组,Western blotting检测血管抑素对血管内皮因子刺激后1、2、5、10、15、3 0min的视网膜微血管内皮细胞ERK 1水平的影响。 结果 与对照组相比,经血管抑素处理后1min ,ERK 1水平开始下降,10min时下降最为显著,3 0min后对照组与血管抑素组ERK水平没有显著区别。VEGF刺激后,鼠视网膜微血管内皮细胞中ERK 1被迅速激活,5min后达到高峰,VEGF组ERK水平较对照组提高了2 10 % (P <0 .0 5 ) ;3 0min后,VEGF组ERK水平与对照组比较,差异无统计学意义(P >0 .0 5 )。而血管抑素能使ERK 1的表达量降低11.9% (1min)、17.9% (2min)、3 8 .7%(5min)、49.3 % (10min) (P <0 .0 5 )、2 7.9% (15min)、1.12 % (3 0min)。 结论 血管抑素在体外能有效的阻断VEGF由细胞外向细胞核传导的信号通路。

Objective To observe the effects of angiostatin on the activity of extra-cellular signal-regulated protein kinase (ERK) of retinal microvascular endothelial cells of mice. Methods Angiostatin was separated and purified by l-lysine sepharose 4B from human plasma. The primary retinal microvascular endothelial cells were divided into 4 groups: the control group, vascular endothelial growth factor (VEGF) 10 ng/ml group, angiostatin 130 μg/ml group, and VEGF (10 ng/ml) + angiostatin (130 μg/ml) group. The expression of ERK-1 was assayed by Western-blotting method 1, 2, 5, 10, 15, and 30 minutes after the treatment of angiostatin. Results Compared with the control group, the expression of ERK-1 reduced 1 minute after treatment, reduced markedly after 10 minutes. After 30 minutes, no differences of the expression of ERK were seen between the control group and angiostatin group. The activation of ERK-1 of retinal microvascular endothelial cells occurred after stimulated by VEGF, and at the pitch at the peak after 5 minutes. The level of ERK in VEGF group increased 210% than that in the control ( P< 0.05). After 30 minutes, no significant difference of the level of ERK between VEGF and the control group. And because of angiostatin, the expression of ERK-1 decreased 11.9%(1 minute)?17.9%(2 minutes)?38.7%(5 minutes)?49.3%(10 minutes) ( P< 0.05)?27.9%(15 minutes)?1.12%(30 minutes) respectively. Conclusions Angiostatin can effectively block the signal path through which VEGF transmits from outside of the cell to cellular nuclei.