优化ALA-PDT对正常人角质形成细胞凋亡的影响  

Optimizing the Effect of ALA-PDT on Normal Human Keratinocytes Cell Apoptosis

作　　者：李康英[1] 李嘉佩[1] 刘宏伟[1] 

机构地区：[1]暨南大学附属第一医院整形美容激光科,广东广州510630

出　　处：《应用激光》2010年第5期428-432,共5页Applied Laser

摘　　要：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： In this paper, the PDT biological effects of different concentrations of ALA and incubation time on in vitro normal human keratinocytes cells （KC） were investigated. We observed the fluorescence intensity of protoporphyrin Ⅸ, apoptosis and growth cycle of KC, optimizing the best concentration and incubation time of ALA, thus to explore the feasibility as well as the optimum clinical effect of ALA-PDT inhibiting the dysplasia of KC with psoriasis. Methods： Dispase isolated enzymes and Trypsin digestion two-step method were used to prepare and cultivate KC from normal human foreskin after circumcision. The experiment was divided into control group, ALA only group, light only group, and 0. lmmol/L, 0.6mmol/, 1. 2mmol/L, 1.8mmol/L, 3.6mmol/L five concentrations of ALA-PDT groups with having 0.5h,lh,3h,Sh four periods of incubation time. We first examined the fluorescence intensity of protoporphyrin IX of KC by using KMC, enzyme mark instrument and fluorescence microscope to determine the best concentration and incubation time of ALA. We further tested the effect of ALA-PDT on KC apoptosis and its growth cycle, using Hoechst33342 / PI and Annexin V / PI double staining methods. Results： 0.6mmol/L of ALA-PDT group was the optimal concentration and lh was the optimum incubation time, showing the highest fluorescence intensity and highest rate of apoptosis. It also had the greatest impact on the Phase S and Phase G2 of KC＇s growth cycle, which led to the lowest cell proliferation index. Compared with other groups, the difference was statistically significant （P〈0.05）. Conclusion： 0.6mmol/L ALA-PDT with lh incubation time can significantly inhibit the growth cycle of KC, and the optimized ALA-PDT could effectively promote normal human KC apoptosis.

关 键 词：ALA—PDT 角质形成细胞 生物效应 

分 类 号：Q255[生物学—细胞生物学]