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目的:本文通过不同浓度的ALA与不同用药时间对体外培养的人角质形成细胞(KC)PDT的生物效应研究,观察PpⅨ荧光强度及KC凋亡和生长周期的影响,优化ALA最佳药物浓度和用药时间,探讨ALA PDT抑制银屑病KC异常增生的可行性和最佳效果。方法:将新鲜包皮组织经两次酶消化法进行KC分离与培养,分设KC对照组、单纯ALA组、单纯照光组及0.1、0.6、1.2、1.8、3.6mmol/L ALA五个浓度组,经0.5、1、3、5h四个避光孵育时间后PDT。FCM、酶标仪、荧光显微镜检测KC中PpⅨ荧光强度,确定ALA最佳药物浓度和最佳用药时间;Hoechst33342/PI双染法和Annexin V/PI双染法检测KC凋亡率和对生长周期的影响。结果:0.6mmol/L ALA(用药1h)-PDT组为最佳药物浓度和最佳用药时间,显示PpⅨ荧光强度表达与KC凋亡率最高,能明显抑制S期与G2期的细胞增值,使细胞增殖指数降为最低,与其他各组相比差异显著(P<0.05)。结论:0.6mmol/L ALA(用药1h)-PDT能明显抑制KC增殖,优化后的ALA-PDT更迅速有效地促进人KC凋亡。
OBJECTIVE: To study the biological effect of PDT on human keratinocytes (KC) cultured in vitro with different concentrations of ALA and different administration time, observe the effect of fluorescence intensity of PpⅨ and the apoptosis and growth cycle of KC, and optimize the optimal concentration of ALA and Medication time, to explore the ALA PDT inhibition of psoriasis KC abnormal proliferation of the feasibility and the best results. Methods: The fresh foreskin tissue was separated and cultured by KC method with two enzymatic digestion methods. The KC control group, the ALA group, the pure light group and the 0.1, 0.6, 1.2, 1.8 and 3.6 mmol / L ALA groups were divided into five groups 0.5,1,3,5 h four dark PDT after incubation time. FCM, microplate reader and fluorescence microscope were used to detect the fluorescence intensity of PpⅨ in KC to determine the optimal concentration of ALP and the optimal drug time. Hoechst33342 / PI double staining and Annexin V / PI double staining were used to detect the apoptosis rate of KC and its growth cycle Impact. Results: The optimal drug concentration and the optimal drug time were 0.6mmol / L ALA (1h) -PDT group, which showed that the fluorescence intensity of PpⅨ and the apoptosis rate of KC were the highest, which could significantly inhibit the cell proliferation in S phase and G2 phase The cell proliferation index was the lowest, which was significantly different from other groups (P <0.05). Conclusion: 0.6mmol / L ALA (1h) PDT can significantly inhibit the proliferation of KC, the optimized ALA-PDT more quickly and effectively promote human KC apoptosis.