缺氧下外源性血管内皮生长因子对体外大鼠Müller细胞的影响

Effects of exogenous vascular endothelial growth factor on rat Müller cells under hypoxia in vitro

目的:观察缺氧条件下外源性血管内皮细胞生长因子(VEGF)对体外大鼠Müller细胞的影响。方法:体外酶消化法纯化培养并鉴定大鼠Müller细胞。台盼兰染色测定细胞活性。应用光镜、细胞计数、免疫细胞化学、MTT法、原位细胞凋亡等方法分析Müller细胞在不同的缺氧时间(0～24h)、添加不同浓度VEGF(10～100μg/L)及VEGF受体Flk-1阻断剂SU1498处理后,细胞中VEGF和Flk-1,Flt-1的表达、细胞活性及凋亡等变化。结果:纯化培养至第3代的Müller细胞,经鉴定其纯度为90%,活力是87.3%。缺氧8～12h时Müller细胞胶质源纤维酸性蛋白(GFAP)染色增强,VEGF和Flk-1,Flt-1表达增加,可见细胞肿胀、脱落。缺氧20h时,与对照组相比,细胞增生降低到79.3%,细胞凋亡指数增加了6.5倍。缺氧24h前加入75μg/LVEGF使细胞活性增强了1.3倍,凋亡指数下降到2/3。VEGF在10～75μg/L范围内此作用呈剂量依赖性。正常情况下,Flk-1,Flt-1表达很少,但是缺氧后,表达量明显上升,Flt-1更显著。加入SU1498后能部分抑制VEGF对Müller细胞的作用。结论:短时间缺氧使Müller细胞反应性增生,VEGF和Flk-1,Flt-1表达增加,外源性VEGF可能部分地借助于受体Flk-1和Flt-1对缺氧的Müller细胞起到保护作用。

AIM： To observe the effects of exogenous vascular endothelial growth factor （VEGF） on primary cultured rat Müller cells under hypoxia in vitro, METHODS： The rat Müller cells were purified by digestion of the trypsin solution, cultured and identified by cellular morphology and glial fibrillary acidic protein （GFAP）. The cell viability was detected by trypan blue staining. The cells were treated with VEGF at different concentrations （10-100μ/L） as well as with VEGF receptor （Flk-1） inhibitor SU 1498. The cells were then exposed to hypoxia for up to 24 hours, Light microscopy, MTT assay, TUNEL labeling and SABC immuno-histochemistry were used to measure the changes of cell morphology, growth, apoptosis and the expressions of VEGF, Flk-1 and Flt-l. RESULTS： The Moiler cells were purified from mixed cells （purity〉90%, viability〉87.3% in Passage 3） and passaged serially. After hypoxia for 8-12 hours, the proliferation and viability of the cells increased progressively, and the expression of GFAP, VEGF, Flk-1 and Flt-1 also increased. But the increased cells showed swelling and suspended in the culture medium. After hypoxia for 20 hours, the proliferation rate decreased to 79.3% and apoptosis index increased 6.5 fold as that of controls. When the concentration of VEGF incell culture medium reached 75μg/L, the cell viability increased 1.3 fold and the apoptosis index reduces to two third as that of controls. This effect of VEGF on the cells appeared dose dependent （10-75μg/L VEGF）. In normal conditions, Flk-1 and Flt-1 expressed at a low level, while after hypoxia, their expressions were significantly increased. The protective effect of VEGF was inhibited in part by SU1498. CONCLUSION： Hypoxia could induce reactive activity of the Moller cells and the expressions of GFAP, VEGF, Flk-1 and Flt-l. The exogenous VEGF may exert neuroprotective effects partially on the Moller cells via VEGF receptor-Flk-1 and Flt-1 in vitro.