Effects Of 5-aminolevulinic Acid Photodynamic Therapy On Normal Human Keratinocyte

Objective:In this study the effects of different concentrations and incubation time of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on cultured normal human keratinocytes cells (KC) were investigated, which can provide theoretical basis for topical application in the treatment of psoriasis.Methods:1. Dispase isolated enzymes and Trypsin digestion two-step method was used to prepare and cultivate normal human foreskin keratinocytes.2. HE staining and cell dynamic growth curve were used to identificate cells and their vitality.3. MTT, flow cytometry and fluorescent microscopy were used to detect the best time and concentration of ALA-PDT on the apoptosis of keratinocytes. The light source was 635nm diode laser,30mW,12J/cm2.4. Hoechst33342/PI double staining, Acridine Orange staining, and Annexin V/PI double staining methods were used to observe ALA-PDT on the apoptosis of keratinocytes.5. Cell cycle was used to detect the effects of ALA-PDT on keratinocytes.6. The results were statistically analyzed by SPSS 16.0.Results:1. Cell morphology was observed. Cells were in typical epithelial-like characteristics, with high nucleocytoplasmic ratio, tightly packed, and a clear outline of refraction, which are the required keratinocytes. After inoculation, cells with a better activity adherenced rapidly. The initial cells were mainly rounded, and gradually become irregular-shaped or polygonal. Many small colonies of keratinocytes surrounded by satellite-like keratinocytes can be seen after 3 days, with good homogeneity and transparency. Up to 90% of cell conjugationed after one week, which showed cells were in good condition. After HE staining, in the optical microscope, cells were pink cytoplasm and blue nucleus.2. Keratinocytes were incubated with 0.1 mmol/L,0.6 mmol/L,1.2 mmol/L,1.8 mmol/L,3.6mmol/L of ALA for 0.5h, 1h,3h, and 5h separately at 37℃in the darkness before being exposed to 635nm laser. In MTT results, compared with the control group,0.6 mmol/L ALA-PDT group had the lower OD value, lowest at 1h. The difference was statistically significant (P<0.05). Fluorescence cells were observed by fluorescence microscopy,0.6 mmol/L of ALA incubation at 37℃in the darkness for 1h had largest number of positive cells (P<0.05). FCM detection showed that 0.6mmol/L of ALA incubation at 37℃in the darkness for lh and 3h had more absorption, with no significant difference between the two groups (P>0.05).3. Compared with the control group,0.6mmol/L ALA-PDT group can significantly promote keratinocyte apoptosis. The apoptosis rate was (27.3±1.9%), P<0.05 for Hoechst33342/PI staining and (28.2±1.0)%, P<0.05 for AO staining. And the early apoptotic rate was (16.2±0.8)%, P<0.05 for Annexin V/PI double staining.4. After the 0.6mmol/L ALA-PDT treatment, the DNA content of G1 phase was increased, the DNA content of S phase and G2 phase in keratinocyte cell cycle were decreased, and PI values was also decrease.Conclusion:1.0.6mmol/L of ALA and 1h incubation at 37℃in the darkness were the best concentration and incubation time.2. Keratinocytes were incubated with 0.6mmol/L of ALA for 1h at 37℃in the darkness before being exposed to 635nm laser can significantly promote cell apoptosis. This can also change the cell cycle, and inhibit cell proliferation. The DNA content of G1 phase was increased, the DNA content of S phase and G2 phase in keratinocyte cell cycle were decreased, and PI values was also decreased.