氧对5-氨基酮戊酸光动力疗法诱导HaCaT细胞凋亡和死亡的影响

目的 探讨基态氧和单线态氧对5-氨基酮戊酸光动力疗法（ALA-PDT）诱导HaCaT细胞凋亡和死亡的影响。方法 取对数生长期的HaCaT细胞，分别与不同浓度的ALA避光孵育4 h，随后给予相应剂量的He-Ne激光照射，照射后12 h以MTT法检测细胞存活率；PI单染法检测细胞凋亡率；二氯荧光素二乙酸酯（DCFH-DA）标记法测定细胞内活性氧基（ROS）水平。为了造成光动力反应前后细胞的化学缺氧以及淬灭产生的单线态氧基（1O2），将不同浓度的氯化钴（CoCl2）和叠氮钠（NaN3）分别加入培养基，比较ALA-PDT处理组与未处理组HaCaT细胞凋亡和死亡率以及细胞内ROS水平的变化。结果 MTT结果表明，ALA-PDT诱导HaCaT细胞的凋亡与死亡率与ALA浓度和He-Ne激光照射剂量呈正相关。经400 μmol/L CoCl2处理的细胞，胞内ROS水平显著降低，细胞死亡率未处理组为57.65% ± 2.88%，处理组降至16.68% ± 1.86%，细胞凋亡率则分别为43.80%和15.40%。10 mmol/L NaN3几乎能完全清除ALA-PDT诱导产生的ROS，增强HaCaT细胞对ALA-PDT诱导的细胞死亡或凋亡的抵抗。结论 改变光动力反应前后细胞内的基态氧与单线态氧含量，能调控ALA-PDT诱导的HaCaT细胞凋亡和死亡。

Effect of oxygen on the induction of apoptosis and necrosis of HaCaT cells by 5-aminolevulinic acid-based photodynamic therapy

Objective To investigate the effect of ground state and singlet oxygen on the induction of HaCaT cell apoptosis and necrosis by 5-aminolevulinic acid-based photodynamic therapy （ALA-PDT）. Methods HaCaT cells were cultured and treated with various concentrations （0.5, 1.0, 2.0, 4.0 mmol/L） of ALA for 4 hours in the darkness, then irradiated with different doses （1.5, 3.0, 6.0, 12.0 J/cm2） of He-Ne laser. Twelve hours after the irradiation, MTT assay was used to measure the survival rates of the HaCaT cells, flow cytometry with PI staining to detect the cell apoptosis rate, and the level of intracellular reactive oxygen species （ROS） was determined by 2′,7′-dichlorofluorescin diacetate labeling. To mimic chemical hypoxia or singlet oxygen quenching in HaCaT cells before or after photodynamic reaction, the cells were pre-treated with various concentrations of cobalt chloride （CoCl2） or sodium azide （NaN3）, respectively, in addition to ALA-PDT. The changes in apoptosis and necrosis rates as well as ROS level in pretreated cells were compared with those without pretreatment. Results The rates of apoptosis and necrosis of HaCaT cells positively correlated with the concentrations of ALA and doses of He-Ne laser within the test range. The pretreatment with CoCl2 at 400 μmol/L significantly decreased the ROS level in cells, and the cell necrosis rate was downregulated from 57.65% ± 2.88% to 16.68% ± 1.86%, apoptosis rate from 43.80% to 15.40%. NaN3 of 10 mmol/L almost eliminated all the ROS from the cells induced by ALA-PDT, and enhanced the resistance of the cells to necrosis and apoptosis induced by ALA. Conclusion The alteration to intracellular ground state and singlet oxygen content can regulate the necrosis and apoptosis of HaCaT cells induced by ALA-PDT.