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目的:研究食管癌细胞的分化状态对ALA产生的内源性光敏剂PpIX累积水平的影响,及对PDT的应答敏感程度.方法:以高分化食管鳞癌细胞KYSE-450和低分化食管鳞癌细胞KYSE-70为研究对象,利用荧光分光光度法测定不同浓度ALA处理后细胞内PpIX的浓度;分别使用不同剂量的红光和蓝光对细胞进行照射处理,MTS法测定PDT对细胞的光毒毒性;TdT-流式细胞法测定PDT诱导的凋亡水平,并观察凋亡细胞的胞核形态.结果:高分化KYSE-450细胞比低分化KYSE-70细胞具有更强的PpIX合成或累积水平,两者PpIX绝对浓度值在高于0.5mmol/LALA时差异显著(315.2±88.3vs156.9±79.2,P<0.05);虽然高分化细胞PpIX产量高于低分化细胞,但无论是红光PDT还是蓝光PDT,其敏感性均明显差于低分化细胞,光剂量为3.6J/cm2(红光)和0.16J/cm2(蓝光)时,两者细胞存活率有极显著差异(88.0±7.4vs25.2±4.9,红光;97.4±7.3vs40.8±4.2,蓝光;P<0.01);KYSE-450凋亡率(12.5%)亦低于KYSE-70(23.2%),细胞主要以坏死方式为主.结论:食管鳞癌细胞的分化影响了内源性光敏剂PpIX的细胞内水平;PDT效应并不单纯依赖于细胞内的光敏剂含量,细胞的生物学特征如分化状态也影响了PDT的效果;高分化细胞部分通过抑制凋亡抵制了PDT作用.
OBJECTIVE: To study the effect of esophageal cancer cell differentiation status on the accumulation level of endogenous photosensitizer PpIX produced by ALA and the sensitivity to PDT.Methods: The expression of esophageal cancer cells KYSE-450 and poorly differentiated esophageal squamous cell carcinoma Cell KYSE-70 was used as the research object. Fluorescence spectrophotometry was used to determine the concentration of PpIX in cells treated with different concentrations of ALA. The cells were irradiated with different doses of red and blue light respectively. The phototoxicity of PDT was measured by MTS TdT-flow cytometry was used to detect PDT-induced apoptosis and the morphological changes of apoptotic cells were observed.Results: KYSE-450 cells had stronger PpIX synthesis or accumulation than that of poorly differentiated KYSE-70 cells, The absolute value of PpIX between the two groups was significantly higher than that of 0.5mmol / LALA (315.2 ± 88.3vs156.9 ± 79.2, P <0.05). Although the PpIX production of well-differentiated cells was higher than that of poorly differentiated cells, The PDT of blue light was significantly lower than that of poorly differentiated cells. There was a significant difference in the cell viability between the PDT and the PDT at the dose of 3.6J / cm2 (red light) and 0.16J / cm2 (blue light) (88.0 ± 7.4vs25). 2 ± 4.9, red light; 97.4 ± 7.3 vs 40.8 ± 4.2, blue light; P <0.01); KYSE-450 wither (12.5%) was also lower than that of KYSE-70 (23.2%), and the cells were mainly necrotic.Conclusion: The differentiation of esophageal squamous carcinoma cells affects the intracellular level of endogenous photosensitizer PpIX.The PDT effect is not pure Dependent on intracellular photosensitizer content, cellular biological characteristics such as differentiation status also affect PDT effect; well-differentiated cells partially resist PDT by inhibiting apoptosis.