缺氧条件下1-磷酸鞘氨醇对人视网膜色素上皮细胞的促增生和抗凋亡作用

Promoting proliferation and inhibiting apoptosis effects of sphingosine-1-phospate on human retinal pigment epithelium cells under the hypoxic condition

背景 1-磷酸鞘氨醇（S1P）是一种具有生物活性的脂质,是细胞内重要的信使分子,参与多种生物过程的调节,如细胞增生、迁移、分化、凋亡等.缺氧不仅是脉络膜新生血管（CNV）的关键始发因素,也是许多眼部疾病的病理基础,而视网膜色素上皮（RPE）细胞也参与缺氧后新生血管的形成,缺氧条件下S1P对人RPE细胞增生和凋亡的影响尚不清楚. 目的 观察缺氧条件下S1P对人RPE细胞增生和凋亡的影响,从而揭示S1P在CNV中的作用机制.方法 体外培养人RPE细胞株并进行传代,3～5代的RPE细胞分为6个组,空白对照组细胞进行常规培养,缺氧组细胞用200.00 μmol/L CoCl2培养细胞2h,不同浓度S1P干预组（0.01、0.10、1.00、10.00 μmol/L）用200.00 μmol/L CoCl2培养细胞2h后加入相应浓度的S1P,采用WST-1试剂盒检测各组细胞的吸光度（A）值;采用Hoechst染色检测各组细胞的凋亡情况,计算并比较各组细胞的凋亡率.结果 培养的细胞生长良好,可见细胞质内的色素颗粒.WST-1试剂盒检测显示,空白对照组细胞增生值（A值）为0.91 ±0.08,缺氧组为0.37±0.09,0.01、0.10、1.00和10.00 μmol/L S1P组分别为0.46±0.08、0.52±0.09、0.61 ±0.06和0.70±0.10,各组间的差异有统计学意义（F=21.104,P=0.000）,不同浓度S1P组A值均明显高于缺氧组,且随S1P浓度的升高而增加,差异均有统计学意义（均P＜0.05）;不同浓度的S1P组细胞存活率均明显高于缺氧组.Hoechst染色显示,空白对照组RPE形态正常,可见少数凋亡细胞;缺氧组可见大量凋亡细胞,细胞核呈淡蓝色,染色质浓缩;S1P干预后凋亡细胞数较缺氧组减少,随着S1P浓度的增加,凋亡细胞数逐渐减少.空白对照组凋亡率为（1.21±0.08）％,缺氧组为（8.99±0.09）％,0.01、0.10、1.00和10.00 μmol/L S1P组的细胞凋亡率分别为（6.60±0.08）％、（5.95±0.09）％、（4.81±0.06）％和（3.96±0.10）％,6个组间细胞凋亡率的总体差异有统计学意义（F=25.070,P=0.000）,与缺氧组比较,各S1P组细胞凋亡率均明显降低,差异均有统计学意义（均P＜0.05）,且随S1P的浓度升高其凋亡率逐渐降低.结论 缺氧条件下S1P对人RPE细胞的增生有促进作用,对细胞凋亡有抑制作用.

Background Sphingosine-l-phospate （S1P） is a bioactive lipid and important messenger molecule in cells.It participates in the regulation of many biological processes,such as cell proliferation,migration,survival,differentiation,apoptosis,etc.Hypoxia is a trigger factor of choriod neovascularization （CNV） and pathological basis of many diseases,and retinal pigment epithelial （RPE） cells are involved in formation of CNV.However,the effects of S1P on proliferation and apoptosis of RPE cells are below understood.Objective This study was to investigate the influence of S1P on proliferation and apoptosis of human RPE cells under hypoxic conditions.Methods Human RPE cells line-D407 cells were cultured and passaged and generation 3-5 cells were used and divided into 6 groups.The cells were regularly cultured in the blank control group using DMEM containing 10% fetal bovine serum.CoCl2（200.00 μmol/L） was added into the colture medium for 2 hours in the hypooxic group.S1P of different concentrations （0.01,0.10,1.00,10.00 μmol/L） were added in culture medium 2 hours after the affection of 200.00 μmol/L CoCl2.The proliferative values of the cells were detected using WST-1 method as the absorbance （A value） and the proliferative rate of different groups were calculated.The apoptosis of the cells was assayed by Hoechst staining.The results were compared among different groups.Results Cultured cells showed the round-like in shape with clear nuclei and pigment.The proliferative values （A value） was 0.91 ±0.08,0.37±0.09,0.46±0.08,0.52±0.09,0.61 ±0.06,0.70±0.10 in the blank control group,hypoxic group and 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups （F=21.104,P=0.000）,and A values in various S1P groups were higher than those in the hypoxiac group （all at P〈0.05）.The proliferative rate was gradually raised with the increase of dose of S1P.Hoechst staining exhibited a few apoptosis cells in the blank control group,but in the hypoxic group,a lots of apoptosis cells were seen with the light-blue nuclei and condensable chromatin.However,the number of apoptosis cells was significantly decreased in various concentrations of S 1 P groups.The apoptosis rates were （1.21 ±0.08） %,（8.99 ±0.09） %,（6.60 ±0.08） %,（5.95 ±0.09） %,（4.81 ± 0.06）% and （3.96±0.10）% in the blank control group,hypoxic group and the 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups （F =25.070,P =0.000）.Compared with the hypoxia group,the cellular apoptosis rates of various S1P groups were lower （all at P〈0.05）.Conclusions Under the hypoxia condition,S1P can promote the proliferation of human RPE cells and inhibit apoptosis.