Objective To investigate the effect of QS Alex 755 nm laser on the p16INK4a expression of melanoma cell lines. Methods Cultured melanoma cell lines, HTB 66, Sk-mel-24, G361 were irradiated with different doses of QS Alex 755 nm laser (0.85 ~ 2 J/cm2) and the irradiated cells were harvested 24 h after irradiation. The expression of p16INK4a protein and mRNA levels were detected by flowcytometer and RT-PCR respectively. Results Compared with the nonirradiated cells the level of p16INK4a protein in HTB 66 cell line increased significantly after laser irradiation, and no change in p16INK4a-negative cell line (Sk-mel-24) and less laser-sensitive cell line (G361). Loss of p16 functional activity leading to disruption of the cell cycle arrest was observed in cell line HTB 66. RT-PCR analysis indicated that p16INK4a mRNA increased in a dose-dependent manner in HTB 66. The p16INK4a mRNA expression was not changed in p16INK4a protein negative Sk-mel-24 and G361 cell lines. Conclusions QS Alex 755 nm laser increases the expression of p16INK4a protein in HTB 66 cell line, which may imply that survival cells after laser therapy may undergo DNA damage. Hence, the laser therapy should not be recommended to treat congenital nevi among those with high risk of aberrant p16.

Port wine stains affect patients on either cosmetic or functional, and even psychological aspects. Although many therapeutic modalities have been applied, the effective clearance rate of this bothersome problem is still limited to some extent. Photodynamic therapy is a state-of -the-art option for patients with port wine stains. In this article, clearance rates and adverse effects of different photo-dynamic therapy measures (including photosensitizers and light sources) for Chinese patients were re-viewed.

Objective To investigate the clinical efficacy of photodynamic therapy using low concentration of photosensitizer, short incubation time and red light source in the treatment of ache. Methods Thirty patients with facial ache were equally randomized into two groups, one group applying 5% 5-aminolevulinic acid (ALA) to the facial lesions on the right side and placebo agent on the left side as control, the other applying the same concentration of ALA to the facial lesions on the left side and placebo agent on the right side as control. The whole face of every subject was irradiated with red light once a week for 4 sessions. The lesions were counted by a dermatologist, sebum production was measured by a sebum meter (SHP88) and pigmentation index by Mexameter MX 18, at the baseline and on week 2, 4, 8 after the last treatment. Adverse effects were also recorded at each visit. Results Two weeks after the last treatment, a sixty percent or more clinical improvement was achieved in the ALA-treated side of 25.9% patients, but no patients experienced sixty or more percent clinical improvement in control side, and the efficacy of ALA was superior to that of the placebo (P<0.05). A significant reduction was noticed in the count of every kind of lesions, including comedo, papules, pustules, cysts/nodules, as well as the total count of these lesions compared with the baseline (all P<0.05); meanwhile, only the pustule count and total count of lesions were decreased in the control side (both P<0.05). Conclusions Both ALA (5%)-PDT and red light-irradiation alone can reduce the number of acne lesions, but the former is more effective than the latter with few side reactions.

Fractional radiofrequency(FRF)is an aesthetic technique that utilizes electric current emanating from electrode or microneedle arrays to heat the dermis in a fractional pattern, with only little damage to the epidermis. The recovery process following the heat damage involves multiple heat shock proteins, matrix metalloproteinases, cytokines, etc, which can stimulate the proliferation of collagen and elastic fibers in the dermis. It has been applied to treat wrinkles, skin laxity, acne scars and other aesthetic skin problems, and has proved to be a safe and effective cosmetic method for the improvement of atrophic acne scars, inflammatory acnes and postinflammatory erythema. It has few adverse effects, including tolerable pain, transient erythema, edema and mild crusting, so the downtime is short. FRF is more suitable for populations with dark complexions because of low risks of postinflammatory hyper?pigmentation. Recently, it has been used in combination with other devices such as lasers, or been used to assist transdermal drug delivery, and has shown remarkable therapeutic effects and favorable application prospects.

Objective To investigate the melanocyte lymphocyte reacion when the allogeneic lymphocytes are cultured with normal melanocytes. Methods 3H-thymidine was incorporated into the mixed culture and the transformation and proliferation rates of lymphocytes were detected by liquid scintillation counting and expressed as cpm. Electron microscopy was used to observe the ultrastructure of melanocytes after mixed culture. Results The results of lymphocyte proliferation were expressed by the stimulation indexes. The stimulation indexes in active vitiligo group was significantly different from that in stable vitiligo group and normal controls. The stimulation indexes of the melanocyte stimulated group was significantly different from that of ConA stimulated group. In the mixed melanocyte lymphocyte reaction, the allogeneic lymphocytes had little effect on the melanocytes. The ultrastructure of the melanocytes in the mixed culture showed normal morphology and normal function of synthesis of melanin. Conclusion As a specific antigen in mixed melanocyte lymphocyte reaction, melanocyte has a weak effect on the lymphocytes. The melanocytes from stable stage vitiligo patients seem more suitble to be allografted.

Objective To report a consanguineous family with lamellar ichthyosis and to detect the mutations in transglutaminase 1 (TGM1) gene in this family. Methods Genomic DNA was extracted from the blood samples of a 19-year-old male patient with lamellar ichthyosis, his family members and 100 normal human controls. PCR was carried out to amplify all the encoding sequences (15 exons) and adjacent flanking sequences of TGM1 gene followed by bidirectional sequencing. Results A C1666T mutation in the 11th exon in TGM1 gene, which resulted in the substitution of ACA (threonine) by ATA (isoleucine) at codon 529, was detected in the proband, while both his parents carried the C1666T mutation in heterozygous form, and his sister was a C/C homozygote. None of the 100 normal control individuals carried the mutation in TGMlgene. Conclusions The de novo mutation from ACA (threonine) to ATA (isoleucine) at codon 529, may contribute to the development of lamellar ichthyosis. Consanguineous marriage can increase the risk for lamellar ichthyosis by raising the probability of homozygosis of C 1666T mutation in TGM 1 gene.

Objective To detect the mutations of pigment epithelium derived factor (PEDF) gene in a human malignant melanoma cell line A375. Methods A375 cells and control melanocytes obtained from circumcised prepuce were cultured, genomic DNA was extracted from these cells. All eight exons of PEDF gene were scanned by single strand conformation polymorphism analysis ofpolymerase chain reaction products (PCR-SSCP) in both A375 cells and control melanocytes. DNA sequencing was performed for the PCR products separated into electrophoretic bands with altered mobility. Results Altered mobility was observed with SSCP analysis in amplicons of exon 3, 4, 5, 6 and 7, with the most obvious alteration occurred in exon 5 and 6. DNA sequencing revealed mutations in both exon 5 and 6. The common type of mutations was single base-deletion in exon 5 and single base-substitution in exon 6. Conclusion Mutations of PEDF gene may contribute to the development of human malignant melanoma.

Objective To study the methylation of CpG motifs and expression of lymphocyte function-associated antigen-1 (LFA-1) mRNA in patients with systemic lupus erythematosus (SLE), and evaluate their associations. Methods The peripheral blood lymphocytes were collected from 26 patients with SLE and 17 normal individuals. The methylation of CpG motifs was detected by the 5-methylcytosine antibody and flow eytometry, and the expression of LFA-1 mRNA was analyzed by RT-PCR. Results Methylation of CpG motifs in patients with SLE was lower than the control subjects (P<0.05), and a negative correlation existed between methylation of CpG motifs and SLEDAI (P<0.05). While expression of LFA-1 mRNA in patients with SLE was higher than that in the control group (P<0.05), and a positive correlation could be detected between the expression of LFA-1 mRNA and SLEDAI (P<0.05). There was a negative correlation between methylation of CpG motifs and expression of LFA-1 mRNA (P<0.05) in patients with SLE. Conclusion Hypomethylation of CpG motifs does exist in patients with SI,E, and is correlated with high expression of LFA-1, therefore, epige-netics plays an important role in the pathogenesis of SLE.

Objective To study the clinical, histopathologic and ultrastructural characteristics of achromic naevus (AN). Methods Clinical data, including sex, age, age of onset, pattern of lesions, involved sites, shape and number of lesions and associated systemic diseases, were collected from 85 patients with AN. Skin melanin index was detected in 34 lesions of 19 patients with AN, 30 lesions of 12 patients with vitiligo and 64 contralateral normal skin islands of the 31 patients. Reflectance confocal microscopy (RCM) was performed to analyze the lesion, normal skin and junctional area between lesional and normal skin of 62 patients with AN. Tissue samples were obtained from lesions and perilesional normal skin of 17 patients with AN and subjected to pathological examination as well as ultrastructural study with transmission electron microscopy; also, skin biopsy specimens were immunostained for tyrosinase, HMB45, tyrosinase-related protein-1 (TRP-1), TRP-2 and CD117. Results Of the 85 patients with AN, 23 (27.1%) developed lesions at birth, and 21 (24.7%) after 3 years of age; 72 (84.7%) had irregularly shaped lesions, 54 (63.5%) had only a single lesion. The mean melanin index and relative melanin index of AN lesions were 186.56 ± 52.86 and 80 ± 11, respectively, significantly lower than those in normal skin islands (223.88 ± 63.19 and 100, both P < 0.01), but higher than those in depigmented lesions from 12 patients with vitiligo (128.57 ± 64.31 and 60 ± 20, both P < 0.01). RCM revealed a decline in the number of melanocytes and brightness of melanin caps, even distribution of melanin in lesions, as well as obscure demarcation between lesions and normal skin from patients with AN. Fontana-Masson stain showed that the melanin content was lower in lesions than in perilesional skin (1810.12 ± 327.96 vs 2064.24 ± 260.41) from patients with AN. Microscopic examination demonstrated a decrease in melanocyte and melanosome number, presence of immature melanocytes at stage Ⅱ and Ⅲ in cytoplasm and dendrites of melanocytes and keratinocytes, aggregated melanosomes in affected keratinocytes in lesions of AN. In 17 patients with AN, the relative expression levels of tyrosinase and TRP-1 were 1827.35 ± 307.09 and 6102.54 ± 1642.64, respectively, in normal skin specimens, significantly higher than those in lesional skin (1477.35 ± 224.05, 5322.33 ± 1565.26, both P< 0.01); no statistical difference was observed in the expression levels of HMB45, TRP-2 or CD117 between lesional and normal skin. Conclusions AN is an early-onset, nonfamilial aggregated, stable leukoderma with irregular margins, and in lesions of AN, the number of both melanocytes and melanosomes is decreased with the presence of immature melanosomes. The measurement of relative melanin index and reflectance confocal microscopy may offer a non-invasive approach to the diagnosis of AN.

Objective To investigate the effects of 5-azacytidine and estradiol on the methylation of CpG motifs and expression of DNA methyltransferasel (DNMT1) mRNA in patients with systemic lupus erythematosus (SLE) and normal controls.Methods Peripheral blood lymphocytes isolated from 12 patients with SLE and 11 normal human controls were stimulated with phytohaemagglutinin for one day followed by additional 3-day culture with or without the presence of 5-azacytidine of 1 μmol/L or estradiol of 30μg/L respectively.Then.the methylation of CpG motifs was detected by flow cytometry using anti-5-methylcytosine antibody,and DNMT1 mRNA expression by real time reverse transcription-PCR Results After treatment with 5-azacytidine,a decrease wag observed in the methylation of CpG motifs, but not in the expression of DNMT1 mRNA in peripheral lymphocytes from patients with SLE (1=18.60,P<0.01;t=1.56.P>0.05) and in those from the normal controls (t=5.63,P<0.01;t=2.17,P>0.05) compared with untreated lymphocytes.Nevertheless,there were no significant changes in the methylation of CpG motifs or expression of DNMT1 mRNA in lymphocytes from patients with SLE (t=1.53,0.93,respectively,both P>0.05) and normal controls (t=1.93,0.11,respectively,both P>0.05) after the treatment with estradiol.Conclusions The methylation of CpG motifs is suppressed efficiently by 5-azacytidine,and the suppression is unlikely to be associated with the decrease of DNMT1 mRNA.Estradiol has no significant impact on the methylation of CpG motifs and expression of DNMT1 mRNA in lymphocytes.