448 nm蓝光照射致ARPE-19细胞坏死性凋亡研究

A Study on the Necroptosis of ARPE-19 Cells Induced by 448 nm Blue Light

长期超风险限值蓝光照射可能导致视网膜变性疾病,而视网膜色素上皮损伤是视网膜光损伤的关键来源。本研究探讨了448 nm蓝光对人视网膜色素上皮细胞的损伤作用及凋亡方式。25 mW/cm2蓝光照射ARPE-19细胞3、6、9、12 h,照后即刻采用钙黄绿素乙酰甲酯染色检测细胞活性,并在照后24 h采用逆转录-聚合酶链反应(RT-PCR)检测坏死性凋亡相关基因表达,同时采用蛋白质免疫印迹法(Western blot)检测坏死性凋亡和DNA损伤标志蛋白表达。9 h和12 h照射组细胞形态发生显著变化,表现为细胞肿胀、间隙增大、漂浮细胞增多、细胞碎片增加;蓝光照射3~9 h后RIPK1、RIPK3 mRNA表达量较正常组出现明显升高,且在照射9 h时达到最大值;蓝光照射6~12 h后RIPK1、RIPK3、P-MLKL坏死性凋亡蛋白标志物的表达量随蓝光照射时间增加而明显升高,并在照射12 h时达到最高值;蓝光照射3~12 h后DNA损伤标志物γ-H2AX蛋白表达量明显升高,并在照射12 h时达到最高值。蓝光照射9 h坏死性凋亡相关基因表达量达到最大,照射12 h基因表达明显下降但坏死性凋亡相关蛋白表达量累积到最大,这提示25 mW/cm2蓝光照射9~12 h可诱导ARPE-19细胞发生坏死性凋亡,并表明DNA损伤程度与细胞坏死性凋亡发生可能存在关联。

Long-term blue light exposure beyond the risk limit is an important factor that causes retinal degenerative diseases,and damage to the retinal pigment epithelium is the key source of retinal light damage. In this study, we investigated the damaging effects of 448 nm blue light on human retinal pigment epithelial(ARPE-19) cells and the mode of apoptosis. ARPE-19cells were irradiated with 25 mW/cm2 blue light for 3, 6, 9 and 12 hours, and cell activity was detected immediately by Calcein AM staining 24 h after irradiation. RT-PCR was used to examine the expression of necrotic apoptosis related genes 24 hours after irradiation, and the Western blotting was used to examine the expression of necroptosis-related and DNA-damage related proteins. The cell morphology changed significantly in the 9-hour and 12-hour groups, the cells swelled, the intercellular space was enlarged, and a large number of cell fragments and non-adhesive cells were observed. Compared with the normal group, the expression levels of the necroptosis-related genes RIPK1 and RIPK3 were significantly higher 3~9 hours after blue light irradiation, and reached the maximum value at 9 h. The expression levels of necroptosis-related proteins RIPK1, RIPK3, P-MLKL markers significantly increased with the increase of irradiation time after 6~12 h blue light irradiation, and reached the highest value at 12 h. The expression level of DNA damage-related γ-H2AX protein increased significantly after 3~12 h blue light irradiation, and reached the highest value at 12 h. The expression of necroptosis-related genes reached the maximum after 9 hours of blue light irradiation, while the expression of necroptosis-related proteins reached the maximum after 12 hours irradiation. It was speculated that 25 mW/cm2 blue light irradiation for 12 h may cause the necroptosis in ARPE-19 cells, and the degree of DNA damage may be related to the occurrence of necroptosis.