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 explore the effect of mosapride combined with polyethylene glycol electrolyte powder on cleaning intestinal tract before colonoscopy in patients with chronic constipation.Methods A total of 127 patients with chronic constipation for colonoscopy were randomly divided into observation group of 64 cases,oral administration of mosapride 10 mg and polyethylene glycol electrolyte powder; control group of 63 cases,oral polyethylene glycol electrolyte powder for bowel preparation for colonoscopy.The intestinal cleanness,first defecation time and adverse reaction was compared between two groups.Results The Boston bowel preparation score was (8.32 ± 0.86) scores in observation group,and (7.69 ± 0.95) scores in control group,and there was significant difference between two groups (t =3.918,P＜ 0.05).The first defecation time after taking the medicine was (45.69 ± 13.57) min in observation group and (54.63 ± 11.78) min in control group,and there was significant difference between two groups (t =3.966,P ＜ 0.05).After taking the medicine,5 cases of nausea and vomiting and 11 cases of abdominal distension in observation group,13 patients of nausea and vomiting and 23 cases of abdominal distension in control group,and there was significant difference between two groups (x 2 =4.29,6.04,P ＜0.05).Conclusion Mosapride combined with polyethylene glycol electrolyte powder can improve the intestinal tract cleaning quality,shorten the time of first defecation time,and reduce adverse reaction.

Objective To detect the levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-l (sICAM-1) in the sera of patients with systemic lupus erythe-matosus (SLE) and their clinical significance was analysed. Methods Serum level of sVCAM-1 and sICAM-1 of 30 controls and 60 SLE patients were measured by enzyme linked immunosorbent assay (ELISA). Results 1 Serum levels of sVCAM-1 were significantly increasd in patients with SLE compared with those in normal controls (P

Objective To investigate the relationship between the serum level of soluble vascular cell adhesion molecule-1(sVCAM-1) and soluble intercellular adhesion molecule-1(sICAM-1) and the disease activity in systemic lupus erythematosus(SLE). Methods The serum concentrations of sVCAM-1 and sICAM-1 were measured by ELISA in 60 SLE patients and age- and sex-matched normal controls. Results ① Serum levels of sVCAM-1 and sICAM-1 were significantly increased in SLE patients compared with those in normal controls (P

ObjectiveTo observe the efficacy of a Levofloxacin-containing 10-day sequential therapy for Helicobacter pylori eradication.Methods Totally 86 Helicobacter pylori-infected patients with chronic gastritis or peptic ulcer diagnosed by gastroscopy were randomized into two groups: the trial group and the control group (n =43 in each group).The trial group was treated with Esomeprazole 20 mg bid plus Amoxicillin 1000 mg bid at the first 5 days,and followed by Esomeprazole 20 mg bid,Levofloxacin 500 mg qd and Furazolidone 100 mg bid,at the last 5 days;The control group was treated with standard triple regimen: Esomeprazole 20 mg,Amoxicillin 1000 mg and Clarithromycin 500 mg,all bid for 7 days.The eradication rates of Helicobacter pylori in the two groups were compared after completion of treatment.ResultsAfter treatment,the eradication rates of Helicobacter pylori were significantly different between the trial and control group(93.0％ vs 74.4％,P ＜0.05)Conclusion The 10-day sequential therapy with.Levofloxacin-containing regime can achieve a higher eradication rate of Helicobacter pylori infection.

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.

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 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 evaluate the therapeutic effect of transplantation of autologous melanocytes cultured with individualized medium in vitiligo. Methods Donor skin was obtained by suction blisters from a normally pigmented area of the abdomen of 155 patients with vitiligo. The roof of the blisters was clipped and digested with trypsin, then the suspension of epidermal cells and melanocytes were cultured in Hu16 medium.The cell division time (DOT) and melanin content of cultured melanocytes were measured followed by the adjustment of concentration of fetal calf serum, cytokines and cAMP elevating agents based on the DOT,melanin content and morphology of melanocytes for the individualized culture of melanocytes. After 2 - 5 passages, melanocytes were harvested and inoculated into ultrapluse CO2 laser-denuded lesions. All patients were followed up for at least 6 months. Results One hundred and fifty-five vitiligo patients with 204 lesions were treated with transplantation of autologous melanocytes. Of the 155 patients, 119 received 1 session of transplantation, 36 received 2 to 4 session of transplantation. Cells were expanded by 50 - 80 times in vitro after individualized culture. Repigmentation was more than 50% in 84.8% of these lesions, more than 90% in 52.94% of the lesions. A homogeneous skin color was obtained in repigmented skin, and no scarring or other side effects were observed. No influence was noted on the outcome of transplantation for sex, age, course of disease or lesion size of patients. Segmental vitiligo showed better response than vitiligo vulgaris: the effective rate and cure rate were 93.62% and 65.96% respectively for segmental vitiligo, 82.16% and 49.04% respectively for vitiligo vulgaris. Lesions located on the arms and legs (not including elbows and knees) showed the best response, with a cure rate of 73.08%, whereas acral sites were the most difficult area to repigment, with a cure rate of just 25.93%. Conclusions Individualized culture can significantly increase the success rate of melanocyte culture and expanding times of melanocytes. Transplantation of cultured autologous melanocytes is an effective modality deserving clinical application in the treatment of stable vitiligo, with the advantage of treating large depigmented area with melanocytes from a small donor site.