| Background:C. albicans is a pathogenic fungi in common condition of human body, which can cause infections of skin or mucous membrane, as well as organ damage. Drug treatment is still an important therapy for candidiasis in clinics. Due to the wide application of antibiotics, corticosteroids and immunosuppressant, the drug resistance has increased seriously. Hence, the challenge for the resistance to drug treatment in C. albicans is growing. In recent years, some of PDT curative effect in clinic has been confirmed. PDT is a minimally invasive treatment, in which PpIX generated by photosensitizer ALA has the killing effect from singlet oxygen and play a therapeutic effect. In the practical application for a selection of photosensitizer, the concentration of photosensitizer, photosensitizer incubation time, light source, and light dose were unconfirmed. And there is no unified standard for body test. In this study, the PDT therapy dialogue the antifungal effect of candida and the concentration-response relationship was explored, which can provide experimental basis for further clinical choice on the best concentration of photosensitizer and optimal incubation time..Objectives:1. To compare two kinds of detection methods about PpIX, to explore efficient, accurate, reliable, rapid detection method.2. To explore the relation between the PpIX fluorescence intensity and the incubation time or concentration of ALA.3.To study the antifungal effect of 5-ALA-PDT on C. albicans.Methods:1. C. albicans suspension and ALA were incubated in the dark. Fluorescence spectrophotometer and confocal microscope were applied to measure the fluorescence indensity of PpⅨ.2. C. albicans suspension was prepared and incubated with ALA in the dark. Experiment is divided into two parts, one is that the different concentrations of 5-ALA were mixed with C. albicans suspension in dark for 60 min incubation; The other one is that the suspension in C. albicans and concentration of 200 mg/ml 5-ALA were mixed at 35 ℃ avoiding light irritation for incubation 15,30,45,60,90,120,150,120 mins, separately. The generation of PpIX was detected under a laser confocal microscope. The inhibitory rate of 5-ALA PDT on C. albicans was determined by MTT method.Results:1. Comparison of p<0.05 between fluorescence spectrophotometer and confocal microscope measurement PpIX showed that there is no statistical differences between those two methods. Microscopical operation is simple, convenient and quick,which can calculate the measured values directly precisely with analysis software. However, the measuring price is expensive. Compared with microscopical, Fluorescence spectrophotometer has a relatively more complicated operation and cheaper price.2. After incubation in dark, C. albicans and ALA produced fluorescent PpIX. ALA concentration was positively correlated with the intensity of PpIX. When drug concentration reached 300 mmol/L, the intensity of PpIX strength stopped increasing. No fluorescent material could be detected in control group.3. After dark incubation, both C. albicans and ALA produced fluorescent PpIX which reached the peak amount between 30 min and 90 min and began to decrease significantly after 120 min. There was no fluorescent material in control group.Conclusions:1. In measurement of fluorescence indensity, confocal microscope is more accurate and simpler than fluorescence spectrophotometer.2.The inhibitory effect of ALA PDT on C. albicans was closely related to the concentration of the photosensitizer and the different durations of incubation of a photosensitizer can provide data for treatment of C. albicans disease. |
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