三氧化二砷对表皮生长因子诱导的视网膜色素上皮细胞增生和迁移的影响

The effects of arsenic trioxide on epidermal growth factor-induced proliferation and migration of retinal pigment epithelial cell

背景细胞因子失衡所导致的视网膜色素上皮（RPE）细胞的异常增生和迁移是增生性玻璃体视网膜病变（PVR）的主要病理变化之一。三氧化二砷（As2O3）是中国传统中药中的有效成分，可有效抑制肿瘤细胞的增生和迁移。但As2O3对细胞生长因子引起的RPE细胞增生和迁移的影响尚未明确。目的探讨As2O3对表皮生长因子（EGF）诱导的ARPE-19细胞增生和迁移的影响。方法用无血清培养基对RPE细胞系ARPE-19细胞进行培养，将终浓度为0、0．5、1．0、2．0、5．0、10．0和20．0μmol／L的As2O3分别加入到无血清培养基和含10mg／LEGF的ARPE-19细胞培养液中作用24h和48h，通过噻唑蓝（MTT）比色法检测各培养组ARPE-19细胞活性的吸光度（A）值，以探讨As2O3对细胞的药物毒性作用，并筛选安全、有效的As2O3作用浓度。用10mg／LEGF加入培养基诱导ARPE-19细胞迁移，分别在培养板中加入0、0．5、1．0、2．0μmol／L As2O3作用24h和48h，并通过划痕试验和Transwell试验检测As2O3对EGF诱导的ARPE-19细胞迁移的影响。结果MTT法检测发现不同浓度As2O3组作用24h和48h后，无血清培养组细胞A值随As2O3浓度的升高而逐渐下降，总体差异有统计学意义（F浓度=38．269，P=0．000；F时间=0．874，P=0．358）。与空白对照组（0μmol／L As2O3组）比较，0．5～5．0μmol／L As2O3组ARPE-19细胞A值的差异均无统计学意义（P〉0．05）。对含10mg／LEGF组的ARPE-19细胞，药物对细胞4值的影响呈现浓度和时间依赖性（F浓度=152．155，P=0．000；F时间=51．649，P=0．000）。与对照组比较，0．5～2．0μmol／L As2O3加入24h和48h后，A值的变化差异均无统计学意义（P〉O．05），而0．5、1．0、2．0μmol／L As2O3加入10mg／LEGF诱导的ARPE-19细胞中作用24h和48h后，A值的变化差异均无统计学意义（F浓度=2．215，P=0．126；F时间=2．230，P=0．155）。5．0～20．0μmol／L As2O3作用于EGF诱导的ARPE-19细胞中作用后，细胞A值明显下降，与空白对照组比较差异均有统计学意义（P〈0．05），5．0～20．0Ixmol／LAs，O，作用24h后，对EGF诱导的ARPE-19细胞增生抑制率分别为12％、32％、37％；作用48h后细胞抑制率分别为39％、44％和53％。划痕试验结果显示，0．5—2．0μmol／L As2O3对EGF诱导的ARPE-19细胞的横向迁移具有抑制作用。Transwell试验结果表明，0．5～2．0μmol／L As2O3对10mg／LEGF诱导的ARPE-19细胞纵向迁移有明显的抑制作用，0．5、1．0、2．0μmol／L As2O3作用12h对ARPE-19细胞的抑制率分别为22％、33％和46％。结论As2O3在一定浓度范围内对ARPE-19细胞无毒性作用，2．0μmol／L以下浓度的As2O3对EGF诱导的ARPE-19细胞增生无明显影响，但可影响细胞的迁移能力，5．0μmol／L以上浓度的As2O3可明显抑制ARPE-19细胞的增生。

Background Growth factor-induced proliferation and migration of retinal pigment epithelium （RPE） cells are the major pathological changes of proliferative vitreoretinopathy （PVR）. Arsenic trioxide （ As2O3 ） is an active ingredient of Chinese traditional medicines, which has an inhibition on proliferation and migration of tumor cells. However,it is not clear whether As2O3 could inhibit growth factor-induced proliferation and migration of RPE cells. Objective This study was to explore the effects of As2O3 on epidermal growth factor （EGF）-induced proliferation and migration of ARPE-19 cells. Methods RPE cell line （ARPE-19 cells） were cultured. Different concentrations of As2O3 （ 0,0.5,1.0,2.0,5.0,10.0,20.0 μmol/L） were added in the culture plate to treat ARPE-19 cells with or without 10 mg/L EGF in serum-free group for 24 and 48 hours,respectively. The MTT colorimetric assay was used to check the cell viability and evaluate the drug toxicity. The effects of As2O3 on EGF-induced proliferation of ARPE-19 ceils were analyzed to get an effective and avirulent concentrations of As2O3. The effeets of As2O3 on EGF-induced migration of ARPE-19 cells were observed by scratch-wound assay and the Boyden chamber assay. Results MTT assay showed that the A values were gradually declined with the increase of As2O3 concentrations after As2O3 treatment without EGF for 24 hours and 48 hours （ Fgroup = 38. 269, P = 0. 000 ; Ftime = 0. 874, P = 0. 358 ）. Compared with the control group, no significant differences were seen in the A values of ARPE-19 cells in 0.5 - 5.0 μmol/L groups （ all P〉0.05 ）. Meantime, As2O3 reduced the A values of ARPE-19 cell with 10 mg/L EGF in dose- and time-dependent manner （ Fgroup = 152. 155, P = 0. 000 ; Ftime = 51. 649, P = 0. 000 ）. There were not significant differences in 10 mg/L EGF-indueed cell growth after 0.5,1.0,2.0 μmol/L As2O3 was added for 24 and 48 hours （Fgroup =2. 215,P =0. 126;Ftime =2. 230,P =0. 155）. However,when 5.0-20. 0 μmol/L As2O3 added,the A values of 10 mg/L EGF-indueed ARPE-19 cells lowed,showing a significant difference in comparison with the control groups （all P〈0.05） ,with the cellular inhibiting rate 12% ,32% ,37% in 24 hours and 39% ,44% and 53% in 48 hours. Scratch-wound assay showed that EGF-indueed horizontal migration of ARPE-19 cells was slow after 0.5-2.0μmol/L As2O3 treated,and the same results also appeared in cell lognitudinal migration by Boyden chamber assay, with the inhibitory rates 22% , 33% and 46% respectively. Conclusions As2O3 is avirulent on ARPE-19 cells within definite concentration range. At ≤2.0 μmol/L concentrations, As2O3 dose not affect EGF-indueed proliferation of ARPE-19 cells, hut it suppresses EGF-induced cell migration. At≥ 5.0μmol/L concentrations, As2O3 plays an inhibitory role to EGF-indueed proliferation of ARPE-19 cells.