| Objictive: To elucidate the effect of hematoporphyrin monomerthyl ether(HMME), a domestic new generation photosensitizer product, mediated photodynamic therapy on nontumorous conditional immune system and provide some theoretical guidance for HMME-PDT clinical use. Preliminary studies were performed on hematoporphyrin monomerthyl ether (HMME) absorption characterization by cutaneous resident and infiltrating cell types and the effect of HMME-PDT on these immune cells proliferation, the effect of HMME-PDT on irradiated skin antigenicity and systemic immunity, the specific biomarker of the skin reaction to PDT.METHODS: (1) Lymphocytes were separated from human peripheral blood by density gradient centrifugation with Ficoll and lymphocytes were activated with polyclonal stimulators PHA or PBD + Ion. Both of the activated and resting lymphocytes were incubated with a serial doses (0~ 40μg/ml) of HMME for lh or were incubated with HMME at 10μg/ml for various times (0~160min), then HMME absorption were measured by flow cytometry after immuno-staining with anti-CD3 antibody. The different sensitivity of activated T cells and resting T cells to HMME-PDT were analyzed by MTT method 24h after PDT. (2) Exponentially growing keratinocyte cell line Hacat were incubated with a serial doses of HMME (0~40μg/ml) for lh or incubated with 10μg/ml HMME for various times (0~120 min). The effect of incubation concentration and incubation time on absorbtion of HMME were measured by flow cytometry. To test the inhibitory effect of HMME-PDT, Hacat cell were incubated with a serial doses of HMME (2.5-40 μg/ml ) for 2h, then exposured to frequency-doubled Nd:YAG laser (532 nm, 20 mW/cm~2) for 0, 2.5, 5, 10, 20 min, respectively. Cell viability was assessed by MTT assay 24 h after PDT. (3) Female BALB/c mice were irradiated by 532 nm laser at power density 50mW/cm~2 or100mW/cm2 for 20 min after intravenous injection ,via tail vein, of lOmg/kg HMME immediately. At different time points before and after PDT treated, The murine allergic contact dermatitis was induced by topical application of dinitrofluorobenzene, ear thickness measurements were made prior to and 24h after challenge with an engineer's micrometer, then the ear was cut for H-E staining . The irradiated skin was harvest 24h after PDT, then was treated with neutral protease to get epidermal cell single-cell suspensions and enriched for LC by density gradient centrifugation. LC were analyzed with flow cytometry for the changed in surface antigen molecules of CD80 and CD86. Nylon wool-enriched C57 spleen lymphocytes were cocultured with epidermal cell single-cell suspensions for mixed epidermal cell lymphocyte reation and the proliferation of lymphocytes were dectected by MTT 3d later. (4) Female BALB/c mice were irradiated by 532 run laser at power density 50mW/cm2 for 20 min after intravenous injection of lOmg/kg HMME immediately. Skin tissue was harvested and then homogenized in lysis buffer, soluble protein was quantitated and used for SELDI-TOF-MS.RESULTS: (l)The HMME absorption-dose curve and absorption-time curve were shifted to right and up in the activated T cells as compared to resting T cells. HMME absorptions by activated T cells were statistic significantly larger than that of resting T cells in the doses between 5^g/ml to 20^g/ml. HMME absorptions of either activated T cells or resting T cells underwent a gradual increase with the incubation-time in HMME concentration of 10p.g/ml. HMME absorptions of activated T cells were statistic significantly larger than that of resting T cells in the incubation-time between 15 to 60min. At the concentrations from 0.313 to 5ug/ml, HMME-PDT had selective killing effect on activate T cells. (2) The absorption of HMME by Hacat cells was increased with incubation concentrations incubation and times. HMME-PDT could inbinition the proliferation of Hacat cells significantly. At centain power density, the inhibitory effect of PDT on Hacat cells were in proportion to the HMME concentration and light dose. (3) PDT could significantly down-regulated the expression of CD86 on LC, and significantly suppressed the intensity of MELR. A significantly reduction in the CHS response to DNFB wereobserved when PDT was given prior to DNFB sensitization. However, the magnitude of the CHS response was unaffected if PDT was given after DNFB sensitization. (4) HMME-PDT could effect the expression of irradiated local skin mass spectometry significantly, 11 distinct proteins were found with 2 proteins up-regulated and 9 proteins down-regulated in PDT group and 1 protein down-regulated in light irradiated group when compared with control group.CONCLUSION: (1) The differences of HMME absorption between activated T cells and resting T cells are time-dependent and dose-dependent. HMME absorptions by activated T cells were significantly larger than that of resting T cells in certain incubation-times and doses. At certain concentration , activated T cells are more sensivtive to HMME-PDT treatment. The results suggest that incubation-time or/and dose associated with HMME-PDT therapeutic windows will be created for selective deletion of activated T cells. (2) The absorption of HMME by Hacat cells are also dose-dependent and time-dependent. Incubation concentration and laser energy density are two important factors affect the inhibitory effect of HMME-PDT. At the same power density, the inhibitory effect of HMME-PDT on Hacat cells is positive correlation with incubation concentration and laser energy density. (3) Cutaneous HMME-PDT can produce both skin local and systemic immune suppression. (4) Proteomic analysis of skin following PDT treatment or light irradiating resulted in significant changing the expression pattern of skin proteins, suggesting involvement of these proteins in the cutaneous response mechanism to PDT stress or temperature stress.
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