Objective To observe the effect of hyaluronic acid on the proliferation of and tyrosinase activity in melanocytes.Methods Normal primary human melanocytes were isolated from infant foreskin tissue and cultured.Different concentrations(0 to 10 g/L)of hyaluronic acid wero added to the culture medium immediately or 8 hours after the inoculation of melanocytes.MTT assay was performed to detect the proliferation of melanocytes,and tyrosinase activity was determined to evaluate the effect of hyaluronic acid on the melanin synthesis by melanocytes.Results The proliferation level (absorbance at 490 am,A490)of melanocytes was 0.14±0.02,0.37±0.08,0.45±0.11,0.49±0.07,0.55±0.12,0.52±0.11,0.49±0.07,0.39±0.05,0.19±0.03 and 0.01 4-0.01 when treated with hyaluronic acid of 0,0.008,0.016,0.313,0.625,1.250,2.500,5.000,7.500 and 10.000 g/L,respectively.The hyaluronic acid of 0.08 to 5 g/L markedly accelerated the proliferation of melanocytes,while that of 10 g/L inhibited their proliferation.The tyrosinase activity in melanocytes was promoted by hyaluronie acid of 0.2 to 5 g/L,but suppressed by that of 10 g/L.The proliferation of melanocytes treated with hyaluronic acid immediately after the inoculation was more rapid than that treated with hyaluronic acid 8 hours after the inoculation.Conclusion The hyaluronic acid of 0.2 to 5 g/L can enhance the proliferation of and tyrosinase activity in melanocytes.

OBJECTIVE To investigate the urogenital tract Ureaplasma urealyticum infection and drug resistance profile from 2001 to 2003.METHODS The results of U.urealyticum culture and drug sensitivity tests were explored from 2001 to 2003.RESULTS From the 1 386 cases in 2001,1 003 were infected with U.urealyticum;of the 1 015 cases in 2002,801 were infected with U.urealyticum;of the 908 cases in 2003, 691 were infected with U.urealyticum.Among the three years,the drug resistance to doxycycline and minomycine kept lower as 4%,the drug resistance to josamycin increased greatly in 2003,the drug resistance and intermediate sensitivity to roxithromycin and azithromycin were high;and the drug resistance to ofloxacin,ciprofloxacin and spectinomycin kept higher.CONCLUSIONS The drug resistance of U.urealyticum evolves with the time.Monitoring drug resistance of U.urealyticum is of great importance.Treatment for U.urealyticum infection should be based on the results of drug sensitivity tests and the pharmacokinetics of the drugs.

Objective To analyze the active principle of Melagenine and its effect on melanocytes of guinea epidermis.Methods The content of endothelins and sialic acid of Melagenine were determined by radioimmunoassay and spectrophotometer.The skin of the back of guinea pigs was treated with Melagenine and irradiated with infrared-ray.Then the biopsy specimens were taken from the treated and untreated back skin,sections and supe-rthin sections were prepared for special staining and TEM examination,respectively,the number melanocytes and melanin content index(MCI)were measured.Results The contents of en-dothelins and sialic acid in melagenine were210.5?30.1pg/mL and147.9?12.1?g/mL,respectively;the number of melanocytes,the keratinocytes with melanin granules and the melanin content index were all in-creased significantly in the treated skin.Conclusions There are endothelins,carbohydrates and glycolipids in melagenine,endothelins can promote the proliferation of melanocytes,while carbohydrates and glycolipids can enhance intercellular recognition and adhesion.The result of the study shows that melagenine promotes the proliferation of melanocytes and synthesis of melanin in the skinof guinea pigs.

ObjectiveTo establish an individualized culture system for melanocytes, and to estimate its efficacy for the treatment of large-area vitiligo. MethodsHu 16 medium was used for in vitro primary culture of melanocytes isolated from patients with stable segmental vitiligo.Doubling time(DOT), melanin content (M), melanin production(MP) and number of dendrites were examined to evaluate the biological activity of melanocytes. To obtain melanocytes with better biological activity, the components of Hu16 culture medium were adjusted. Ultra pulse CO2 laser was utilized to shave the vitiligous lesions and remove the epidermis followed by autologous transplantation. Follow-up was carried out. ResultsMelanocytes were obtained from 10 patients with stable segmental vitiligo and cultured. The melanocytes from 6 patients showed relatively short DOT, stable M and MP during the first and seventh passage, and were considered to be at initial or growth stage and applicable to transplantation. The remaining melanocytes from the other 4 patients had displayed long DOT, instable M, MP and dendrite quantity since the third passage; by adjusting the components of culture medium, these cells were induced into growth stage and finally applied to transplantation. A 12-month follow-up revealed that the repigmentation rate was higher than 90％ in 7 patients, ranged between 70％ and 80％ in the remaining 3 patients, with the transplantation area being 116.8 + 75.6 cm2. ConclusionsThe individualized culture system with adjusted components in culture medium yields melanocytes with satisfying biological activity, which are proved to be effective for the treatment of large-area, segmental and stable vitiligo.

Objective To evaluate the relationship between cell seeding density and clinical efficacy of autologous cultured melanocyte transplantation in the treatment of vitiligo.Methods A total of 632 patients with vitiligo were enrolled in this study,and randomly classified into 4 groups to be treated with transplantation of autologous cultured melanocytes at 4 different seeding densities respectively,i.e.,(3.0-4.9)× 104/cm2 (n =201),(5.0-7.9) × 104/cm2 (n =303),(8.0-9.9) × 104/m2 (n =82),(10.0-12.0) × 104/cm2 (n =46).Epidermal sheets were obtained by suction blister biopsy from the normal skin of the vitiligo patients,and subjected to the isolation and culture of melanocytes.After 2 to 5 passages,the cultured autologous melanocytes were transplanted at different seeding densities to vitiligous lesions,which were abraded previously by ultra-pulsed CO2 laser,of these patients.All the patients were followed for 6-12 months.Results At 6 months after the transplantation,52.85％of these patients achieved more than 90％ repigmentation,and 82.28％ more than 50％ repigmentation,with no differences in the cure rate and response rate between the 4 groups (both P ＜ 0.05).The percentage of patients obtaining excellent color matching was significantly higher in the group treated with transplantation of melanocytes at a seeding density of (5.0-7.9) × 104/cm2 than in the other 3 groups at 6,12 and 24 months after treatment (all P ＜ 0.05),and higher in all the 4 groups at 12-and 24-month points compared with the 6-month point (all P ＜ 0.05),but no statistical difference was observed between the 12-and 24-month point in any of these groups (all P ＞ 0.05).Conclusions The transplantation of autologous cultured pure melanocytes is effective for the treatment of stable vitiligo with the optimal cell seeding density of melanocytes being (5.0-7.9) × 104/cm2,and the color matching appears to improve with time.

Objective To investigate the relationship between the efficacy of autologous cultured melanocyte transplantation and serum levels of interleukin-17 (IL-17) and FoxP3 in patients with vitiligo.Methods Forty patients with stable vitiligo vulgaris were included in this study,and received autologous cultured melanocyte transplantation.Six months after the transplantation,treatment efficacy was evaluated,and patients were classified into the successfully treated group (n =25) and unsuccessfully treated group (n =15).Peripheral blood was collected from all the patients before the transplantation,and enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum levels of IL-17 and FoxP3.Statistical analysis was done using two independent samples t-test with the SPSS software (version 17.0).Results The successfully treated patients showed lower serum levels of IL-17 ((15.29 ± 7.86) vs.(43.88 ± 13.02) ng/L,P ＜ 0.05),but higher serum levels of FoxP3 ((6.08 ± 2.03) vs.(3.37 ± 1.81) ng/L,P ＜ 0.05) than the unsuccessfully treated patients.Conclusion The increased serum IL-17 and decreased serum FoxP3 may contribute to the failure of autologous cultured melanocyte transplantation in patients with vitiligo.

Objective To investigate the protective effect of acetylated epigallocatechin gallate (AcEGCG) against H2O2-induced oxidative damage to human epidermal melanocytes,and to explore its possible mechanism.Methods Human epidermal melanocytes were isolated and cultured in vitro.Some melanocytes were classified into a H2O2 group induced by H2O2 only,EGCG groups and AcEGCG groups induced by H2O2 after pretreatment with different concentrations of EGCG and AcEGCG,respectively.Three concentrations (10,20 and 40 μmol/L) of EGCG or AcEGCG were used to treat melanocytes for 1 hour in MTS assay and lactate dehydrogenase (LDH) leakage assay and for 2 hours in Western blot assay,while only one concentration (40 μmol/L) was used to treat melanocytes for 0.5,1,2 and 4 hours respectively in flow cytometry assay.Some melanocytes treated with only culture medium and 0.1％ dimethyl sulphoxide (DMSO) served as the control group.After additional culture,MTS assay was performed to determine cell survival rate,flow cytometry to detect the level of reactive oxygen species (ROS) in melanocytes,Western blot to measure the expressions of caspase-9 and caspase-3 proteins.Lactate dehydrogenase (LDH) kit was used to detect the leakage of LDH to culture medium.Statistical analysis was carried out by using one-way analysis of variance for comparisons of multiple group means followed by Student-Newman-Keuls-q (SNK-q) test for multiple comparisons.Results Compared with the control group,the H2O2 group showed significantly decreased cell survival rate (22.99％ ± 0.53％,P ＜ 0.01),but increased LDH leakage level (36.58％ ± 0.73％,P ＜ 0.01),intracellular ROS level (19.08 ± 0.57,P ＜ 0.01),as well as caspase-9 (2.65 ± 0.079,P ＜ 0.01) and caspase-3 (2.36 ± 0.057,P ＜ 0.01) expressions.In comparison with the H2O2 group,the cell survival rate was significantly higher in the 10-,20-and 40-μmol/L AcEGCG groups (79.50％ ± 3.62％,86.52％ ± 5.13％,97.81％ ± 5.21％,respectively,all P＜ 0.01) and EGCG groups (43.19％ ± 1.68％,63.34％ ± 3.60％,70.82％ ± 2.1％,respectively,all P ＜ 0.01).However,the 10-,20-and 40-μ mol/L AcEGCG groups and EGCG groups all showed a significant decrease in the expression levels of caspase-9 (AcEGCG groups:1.44 ± 0.067,1.26 ± 0.059 and 1.10 ± 0.072 respectively;EGCG groups:2.31 ± 0.085,2.13 ± 0.091 and 1.35 ± 0.064 respectively,all P ＜ 0.05) and caspase-3 (AcEGCG groups:1.70 ± 0.053,1.57 ± 0.057 and 1.24 t 0.068 respectively,all P＜ 0.05;EGCG groups:2.09 ± 0.076,1.98 ± 0.093 and 1.79 ± 0.056 respectively,all P ＜ 0.05) compared with the H2O2 group.Similarly,a significant reduction was observed in the leakage level of LDH in these AcEGCG and EGCG groups (all P ＜ 0.01) and in ROS levels in the 40-μmol/L AcEGCG and EGCG groups when compared with the H2O2 group.Conclusions AcEGCG has a stronger protective effect against H2O2-induced oxidative damage to human epidermal melanocytes compared with EGCG,which may be realized through clearance of free radicals,antioxidant effects,and decrease of caspase-9 and caspase-3 expressions.

Objective To investigate the role of activation of Akt/mTOR pathway in denfense against UVB-induced apoptosis in cultured human skin keratinocyte cell line HaCaT. Methods HaCaT cells were irradiated with UVB at different doses for various durations. Western blotting was performed to detect dynamic changes of Akt/mTOR pathway-related signaling molecule, such as phosphorylated-epidermal growth factor receptor (EGFR), -Akt, -4EBP1, etc; apoptosis was estimated by staining with DNA dye Hoechst 33342. To evaluate the role of signaling molecules in defense against UVB-induced apoptosis, HaCaT cells were pretreated before irradiation with EGFR inhibitor (PD153035), PI3K inhibitor (LY294002), mTOR inhibitor (rapamycin) followed by the detection of expressions of signaling molecule and apoptosis. Results UVB could activate Akt/mTOR pathway in a dose- (5 ～ 30 mJ/cm2) and time- (5 ～ 30 min) dependent manner. PD153035,LY 294002 and rapamycin could inhibit UVB-induced activation of the Akt/mTOR pathway. The apoptosis rate in HaCaT cells was upregulated by pretreatment with rapamycin and LY294002. Conclusion The activation of Akt/mTOR signaling pathway could inhibit the UVB-induced apoptosis in cultured HaCaT cells.

Objective To investigate quercetin's protective effect against oxidative stress in and impact on the biological activity of mouse B10BR melanocytes. Methods B10BR cells were cultured and treated with different concentrations of quercetin followed by additional culture. Then, cell viability was measured by using MTT assay, hydrogen peroxide-induced cell apoptosis by flow cytometry, and cell morphological changes by microscopy. The tyrosinase activity in and melanin synthesis by B10BR cells were measured by dopa oxidation assay and sodium hydroxide (NaOH)-lysis method, respectively. Results After treatment with quercetin of 33.33 μmol/L for 24 hours, the survival rate of B10BR cells reached (94.22 ± 3.36)%, tyrosinase activity (107.15 ± 10.96)%, and melanin content (111.85 ± 9.49)%. A significant difference was observed in tyrosinase activity and melanin content between hydrogen peroxide-induced and 33.33 μmol/L quercetin-treated B10BR cells and those only induced by hydrogen peroxide (both P < 0.01). Flow cytometry revealed that quercetin inhibited hydrogen peroxide-induced apoptosis in melanocytes. Conclusion The protective effect of quercetin against hydrogen peroxide-induced apoptosis in melanocytes may provide a new idea for the treatment of vitiligo.

Objective To evaluate the therapeutic efficacy of autologous melanocyte transplantation for the treatment of vitiligo in patients with abnormal thyroid function.Methods A total of 60 patients with vitiligo were enrolled in this study,including 30 with abnormal thyroid function and 30 without.Epidermal sheets were obtained by suction blister biopsy from the normal skin of all the patients followed by melanocyte isolation and culture.After 2-5 passages of subculture,the melanocytes were transplanted onto vitiliginous lesions,which were abraded previously by ultra-pulsed CO2 laser,in the corresponding patients.All the patients were followed for 6-12 months.Results Of the 30 patients with abnormal thyroid function,7 patients achieved more than 90％ repigmentation,9 patients 50％-89％ repigmentation,53.3％ more than 50％ repigmentation,with the average repigmentation rate being 47％ within 6 months after the transplantation.Meanwhile,13 out of the 30 patients without abnormal thyroid function showed more than 90％ repigmentation,11 showed 50％-89％ repigmentation,with the average repigmentation rate being 75％.Both the cure rate and response rate were significantly higher in the patients without abnormal thyroid function than in those with (cure rate,43.3％ vs.23.3％,P＜ 0.05; response rate,80％ vs.53.3％,P＜ 0.05).Significant differences were also found in the response rate for lesions on the face or neck and for those sized more than 20 cm2 between the two groups of patients (both P ＜ 0.05).The lesions transplanted with epidermal melanocytes from the waist exhibited the lowest cure rate and response rate.Conclusion Clinical or subclinical thyroid dysfunction may have a negative impact on the efficacy of autologous melanocyte transplantation in vitiligo.