ALA—PDT对正常人角质形成细胞生物效应的实验研究

目的：探讨ALA—PDT抑制角质形成细胞（KC）增殖的可行性和最佳效果。方法：新鲜包皮组织经两次酶消化法进行KC分离与培养，分设对照组、单纯ALA组、单纯照光组及0．1mmol／L、0．6mmol／L、1．2mmol／L、1．8mmol／L、3．6mmol／LALA五个浓度组，经0．5h、1h、3h、5h四个避光孵育时间后PDT。酶标仪检测PDT后KC存活率、FCM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；吖啶橙染色和Annexin V／PI双染法检测KC凋亡率和生长周期的影响。结果：0．6mmoL／LALA（用药1h）-PDT组为最佳药物浓度和最佳用药孵育时间，显示Pp Ⅸ荧光强度表达与KC凋亡率最高，能明显抑制S期与G2期的细胞增殖，使细胞增殖指数降至最低。结论：0．6mmol／LALA（用药孵育1h）-PDT能明显抑制KC增殖，更有效地促进正常KC凋亡。

An Experimental Research on ALA-PDT Inhibit Normal Human Keratinocytes Cell Proliferation

Objective.To explore the feasibility as well as the optimum clinical effect of ALA-PDT inhibiting the proliferation of kerationocytes cell （ KC）. 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 0.1 mmol/L, 0.6 mmol/L, 1.2 mmol/L, 1.8 mmol/L, 3.6 mmol/L five concentrations of ALA-PDT groups having 0.5 h,1 h,3 h,5 h four periods of incubation time. We used the enzyme mark instrument to examine KC survival and fluorescence microscope was used to examine the fluorescence intensity of protoporphyrin IX of KC. Then the effect of ALA-PDT on KC apoptosis and its growth cycle were observed by using AO staining fluorescence examination and Annexin V / PI double staining methods. Results ： 0.6 mmol/L of ALA-PDT group was the optimal concentration and 1 h 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. Conclusion：0.6 mmol/L ALA-PDT with 1 h incubation time can significantly inhibit the proliferation of KC, and effectively promote normal human KC apoptosis.