Objective To evaluate the effect of diammonium glycyrrhizinate extracted from the Chinese traditional medicine licorice root on the growth of human hair follicles cultured in vitro,and to detect the expression of wnt/β-catenin signaling pathway-related molecules.Methods Isolated hair follicles were cultured with diammonium glycyrrhizinate at different concentrations of 0.1,0.01,0.001 and 0.000 1 μmol/L for 10 days,and the hair follicles cultured in Williams' E medium without diammonium glycyrrhizinate served as a control group.The length of hair follicles was measured under a microscope every day,the morphologic changes of hair follicles were observed,and photos were taken.Immunofluorescence assay was performed to assess the proliferation of hair matrix cells,as well as to determine the expression of β-catenin,glycogen synthase kinase 3β (GSK3β),phosphorylated GSK3β (p-GSK3β) and lymphoid enhancer factor-1 (Lef1) in the Wnt/β-catenin signaling pathway.Statistical analysis was carried out by repeated-measures analysis of variance and one-way analysis of variance.Results As repeated-measures analysis of variance showed,only 0.01 μmol/L diammonium glycyrrhetate showed significantly promotive effect on the growth of hair follicles compared with the medium alone (P ＜ 0.05),and there were no significant differences in the length of hair follicles between the other concentration groups and the control group.Compared with the control group,the transition to the catagen phase of human hair cycle was delayed in the 0.01-μmol/L diammonium glycyrrhetate group,while it did not change in the other diammonium glycyrrhetate groups and control group.Immunofluorescence assay showed that the number of ki67-positive hair matrix cells was obviously increased in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups compared with the control group,while there was no difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group.One-way analysis of variance revealed that the expression of β-catenin,p-GSK3β and Lef1 significantly differed among all the groups (F =12.604,16.65,15.266 respectively,P ＜ 0.05),while no significant difference in the expression of GSK3β was found among these groups (F =1.472,P ＞ 0.05).Least significant difference (LSD)-t test revealed that the expre-ssion of β-catenin,p-GSK3β and Lef1 in the hair matrix cells was significantly higher in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups than in the control group (all P ＜ 0.05),but there was no significant difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group (P ＞ 0.05).Conclusion Diammonium glycyrrhetate at the concentration of 0.01 μmol/L shows markedly promotive effect on the in vitro growth of hair follicles,and can increase the proliferative activity of hair matrix cells and delay the transition to the catagen phase,which may be associated with the activation of Wnt/β-catenin signaling pathway.

Objective To determine the expression of cathepsin D and advanced glycation end products (AGEs)in skin tissues from patients of different ages or skin tissues with different degrees of sun exposure,to evaluate their correlation,and to preliminarily investigate the role of cathepsin D in the degradation and accumulation of AGEs in photoaged skin.Methods Skin tissues were collected from sunexposed and sun-protected body sites in patients aged 15-20 years,35-40 years,55-60 years or 75-80 years.These skin tissues were divided into 8 groups according to age of patients and degrees of sun exposure,and there were 6 specimens in each group.Immunohistochemical and immunofluorescent methods were used to measure the expression of cathepsin D and AGEs in the skin tissues.Statistical analysis was carried out by factorial design analysis of variance,Wilcoxon rank sum test and Kruskal-Wallis rank sum test for analyzing associations of the expression of cathepsin D and AGEs with age and sun exposure,as well as by Pearson correlation analysis for assessing the correlation between cathepsin D expression and AGEs expression.Results Immunohistochemical study showed that the expression of cathepsin D markedly decreased along with the increase of age,but the accumulation of AGEs gradually increased along with the increase of age.In the same age group,the cathepsin D expression was lower in the sun-exposed skin tissues than in the sun-protected skin tissues,while the accumulation of AGEs was more in the sun-exposed skin tissues than in the sun-protected skin tissues.Factorial design analysis of variance showed that sun exposure could decrease the expression of cathepsin D (F =58.70,P ＜ 0.001),but increase the accumulation of AGEs (F =158.18,P ＜ 0.001).Moreover,the increase of age could lead to decreased expression of cathepsin D (F =79.49,P ＜ 0.001),and increased expression of AGEs (F =106.06,P ＜0.001).Compared with the sun-protected skin tissues,the sun-exposed skin tissues in all the age groups showed significantly lower absorbance value of cathepsin D (35-40 years:0.020 ± 0.005 vs.0.032 ± 0.005;55-60 years:0.012 ± 0.004 vs.0.026 ± 0.002;75-80 years:0.002 ± 0.001 vs.0.013 ± 0.004;all P ＜0.001),but higher absorbance value of AGEs (35-40 years:0.030 ± 0.008 vs.0.010 ± 0.003;55-60years:0.066 ± 0.010 vs.0.021 ± 0.004;75-80 years:0.085 ± 0.015 vs.0.035 ± 0.009;all P ＜ 0.001)except the age group of 15-20 years.No matter whether the skin tissues were sun-exposed or sunprotected,there were significant differences in the expression of cathepsin D and AGEs among different age groups (all P ＜ 0.001).The results of double immunofluorescence staining were similar to those of immunohistochemical study.Pearson correlation analysis showed that the expression of cathepsin D in the sun-exposed skin tissues was highly negatively correlated with the accumulation of AGEs (r =-0.915,P ＜0.05),while they were moderately negatively correlated in the sun-protected skin tissues (r =-0.730,P ＜0.05).Conclusions Along with the increase of age,the expression of cathepsin D in skin tissues decreased,but the expression of AGEs increased.In the sun-protected skin tissues,the expression of cathepsin D was moderately negatively correlated with the expression of AGEs,while they were highly negatively correlated in the sun-exposed skin tissues,suggesting that cathepsin D may play an important role in the degradation and accumulation of AGEs in photoaged skin.

Objective To investigate the minimal persistent pigment dose (MPPD)of normal skin to UVA in Guangzhou city,and to observe its relationship to sex,age,skin type,seasons,ITA,and the years lived in Guangzhou.Methods 316 healthy subjects were exposed to Solar 601-300,and the MPPD was measured and observed by two professional technicians after 2-3 hours.Results The average MPPD value of all subjects was (9.61±2.57) J/cm2.In male and female,the average MPPD values were (11.09 ± 2.82) J/cm2 and (9.01 ± 2.20) J/cm2 respectively,and male was significantly higher than female (P＜0.01).There was significant difference of the MPPD value in different seasons (P＜0.01).The MPPD value in winter (10.66± 2.71) J/cm2) was significantly higher than spring (9.37±2.39) J/cm2,summer (9.53±2.66) J/cm2 and autumn (8.98±2.25) J/cm2.There was significant difference of the MPPD value between different ITA groups (P＜0.01).ITA grade-3 (10.72± 2.84) J/cm2 was significantly higher than grade-1 (8.50±1.45) J/cm2 and grade-2 (9.12±2.31) J/cm2 (P＜0.01),but there was no significant difference from grade-4 (11.87±2.73) J/cm2 (P =0.93).The MPPD value in subjects lived in Guangzhou over 10 years (8.97± 1.88) J/cm2 was significantly lower than those whose residence was less than 1 year and from 1 to 5 years,respectively (P＜0.01),but there was no significant difference from those lived from 5 to 10 years (P =0.47).Conclusions The MPPD value of the subjects in Guangzhou is related to gender,seasons,ITA grade and the years lived in Guangzhou city,while there is no correlation with age and skin types.

Objective To evaluate effectiveness of peripheral Mohs micrographic surgery for the treatment of extramammary Paget′s disease (EMPD). Methods A total of 28 patients with EMPD were treated with peripheral Mohs micrographic surgery. The depth and extent of tumor infiltration were evaluated before the surgery. One day before the surgery, 20% aminolevulinic acid hydrochloride was topically applied to determine and label surgical margins under a Wood′s lamp. After fluorescence-based localization, peritumoral skin tissues were resected and underwent frozen-section examination according to the protocol for Mohs micrographic surgery. Meanwhile, the tumor was resected. After surgery, patients were followed up every 3 - 6 months to detect local recurrence and metastasis. Results Of the 28 patients, 25 were male and 3 were female. Six patients each underwent 3 sessions of frozen-section examination, and 12 patients each received 2 sessions, with an average of 1.86 sessions for each patient. During the follow-up for 5 - 72 months, local recurrence occurred in 3 cases, and 1 patient died of tumor metastasis and uremia after 2 years of follow-up. Conclusion Peripheral Mohs micrographic surgery is a time-saving and effective treatment for EMPD.

Objective To summarize the experience in nursing patients undergoing skin biopsy on head and face . Method One hundred and twenty patients with head facial dermatosis undergoing skin tissue biopsy were nursed from August 2012 to October 2013. Result All operation for biopsy were completed smoothly, with the cure rate at the first phase 100.0%, healing time 5~7 days, averaged (7.0 ± 3.0)d and no complications. Conclusion For those patients having undergone facial skin tissue biopsy, the nursing measures like psychological nursing, health education before operation, preoperative relief of mental disorder, postoperative wound nursing and health education, prevention of complications are critical for their recovery.

Objective To investigate the minimal erythema does (MED) of normal skin to UV in Guangzhou city,and to observe its relationship to sex,age,skin type,seasons and the years lived in Guangzhou.Methods 621 healthy subjects were exposed to Solar Simulator (GS2004) and the MED was measured and observed by two professional technicians after (24±2) hours.Results The average MED value of all subjects was (1170.2±333.2) mJ/cm2.In male and female group,the average MED values were (1132.8-339.4) mJ/cm2 and (1182.1 ± 330.7) mJ/cm2,respectively,and there was no significant difference between male and female (P=0.20).The MED value in subjects aged from 30 to 50 (1014.7 ± 359.7) mJ/cm2 was significantly lower than those aged from 20 to 29 (1222.9±304.3) mJ/cm2 and over 50 years (1179.0±374.3) mJ/cm2 (P＜0.01).The MED value in skin type Ⅱ (673.53±228.3) mJ/cm2 was significantly lower than those in type Ⅲ (1224.3±254.2) mJ/cm2 and Ⅳ(1363.1±278.5) mJ/cm2(P＜0.01).There was significant difference of the MED value between different seasons (P＜0.01).The MED value in spring (969.2±355.8) mJ/cm2 was lowest,and followed by summer (969.2± 355.8) mJ/cm2.However,there was no significant difference between autumn and winter (P＞0.05).The MED value in subjects lived in Guangzhou from 5 to 10 years was significantly lower than those whose residence time was from 1 to 4 years and over 10 years,respectively.Conclusions The MED value of the subjects in Guangzhou is quite different from other cities of China,and related to age,skin types,seasons and the years lived in Guangzhou city,while there is no correlation between MED value and gender difference.

Objective To investigate the three methods for sun protection factor determination in vivo in Chinese under the same condition, and to evaluate the feasibility and reliability of the three methods. Methods There were 45 healthy volunteers in Guangzhou area were involved in the study. All of them were randomly divided into three groups. Among them 15 volunteers were in the current method group, 5 volunteers in the simplified method group, and 25 volunteers were in the single-exposure method group. Two standard sunscreens of SPF 4 and SPF 15 and one sunscreen labeled as SPF 20 were tested by all the three methods in the study. Two experienced observers read results, respectively, under the same conditions. Results The volunteers in the current method group received the largest dose of UV light.The simple method was next to it and the single-exposure reduced 4-5 folds of UV dose received. The results of the two observers in the current method group and the single-exposure group were very close. But the test results of P3 were not completely coincident between the two observers in the simplified method group. SPFs value of the three sunscreens were 4. 12, 16.09, and 22.60 obtained by the observer 1 and 4. 18, 15.92, and 21.97 obtained by the observer 2, respectively. In the simplified method group, SPFs values of ＜4, ＞15, ＞20 and ＜4, ＜15, ＞20 were obtained by the two observers. In the single-exposure method group, SPFs values of the three sunscreens were 4.04, 15.34, 21.33 by the observer 1 and 3. 96, 15.82, 21.33 by the observer 2, respectively. Conclusion The single-exposure method is convenient and easily to operate and may replace the current method some day. The simple method is not valuable in determination of SPF labeled on the packages of sunscreens, but may be useful in supervision of the market sunscreen products.

A 48-year-old man presented with a 4-day history of fever and 10-year history of papulovesicles on the face, neck, trunk and limbs which had been aggravated 10 days prior to the presentation.Skin biopsy showed a dermal infiltration of numerous small- to medium-sized atypical lymphocytes, which was mainly located around blood vessels or appendages, with the involvement of subcutaneous fat tissue and destruction of blood vessels. The infiltrating atypical cells stained positive for CD45RO, CD8, CD56, T-cell intracellular antigen-1, granzyme B, Epstein-Barr virus-encoded small nuclear RNAs (EBER), but negative for CD20, CD79a, CD3, CD4 or CD30. Cytoplasmic CD3ε was also observed in these cells. Laboratory examinations on admission revealed a progressive decrease in peripheral erythrocytes, white cells and platelets, persistent increase in serum aminotransferase and bilirubin, and decline in serum fibrinogen and hypertriglyceridemia. The B-mode ultrasound of the abdomen showed hepatosplenomegaly. Based on the above findings,the diagnosis was made as extranodal nasal type NK/T-cell lymphoma of skin complicated by hemophagocytic syndrome.

Objective To investigate the expression changes of aspartic proteinase (cathepsin D) and cysteine proteinase (cathepsin K) in photoaged fibroblasts. Methods The senescence of human fibroblasts was induced via culture in the presence of 8-methoxypsralen (MOP) of 50 mg/L in darkness for 24 hours followed by irradiation with UVA of 80 kJ/m~2. Then, aged fibroblasts were confirmed by senescence-associated β-galactosidase (SA-β-gal) staining. Real-time RT-PCR and Western blot were carried out to detect the mRNA and protein expressions of cathepsin D and cathepsin K in photoaged and normal control fibroblasts, respectively. Results Western blot showed a significant difference between photoaged and control fibroblasts in the grey scale of cathepsin D and cathepsin K (3.25 ± 0.33 vs 14.18 ± 2.25, f = 30.61, P < 0.01; 2.39 ± 0.66 vs 29.38 ± 4.62, t = 12.63, P< 0.01). The △Ct values for cathepsin D and cathepsin K mRNA were 2.79 ± 0.17 and -0.92 ± 0.06, respectively, in photoaged fibroblasts, significantly lower than those in the control fibroblasts (4.54 ± 0.34, 2.57 ± 0.13, t = 20.78, 28.50, respectively, both P < 0.01). According to the value of 2~(-△△Ct), the expression of cathepsin D and cathepsin K mRNA decreased 0.24 ± 0.021 and 0.09 ± 0.005 folds, respectively, in photoaged fibroblasts compared with the control fibroblasts. Conclusion The expression of cathepsin D and cathepsin K is decreased in photoaged fibroblasts.

Objective To investigate the expression of cathepsin B in photoaging skin and its signifi-cance.Methods Skin specimens were obtained from the right forearm(sun-exposed sites)and buttock (unexposed sites)of 6 healthy volunteers with informed consent and subjected to immunohistochemistry for the detection of cathepsin B expression.Primary human fibroblasts derived from the prepuce of children aged 3 to 6 years were cultured in vitro;after 10 to 15 passages,cells were divided into four groups to be treated with methoxsalen of 50 mg/L for 24 hours followed by ultraviolet A(UVA)exposure(premature senescence group),phosphate buffered saline(PBS)only(control group),UVA exposure only(UVA group),methoxsalen only(methoxsalen group).Then,the protein and mRNA expressions of cathepsin B were detected by Western blot and RT-PCR,respectively,in these fibroblasts 1,2,3 weeks after the treatment.Results Cathepsin B was observed in both exposed and unexposed sites of all volunteers,and the average absorbence of cathepsin B was significantly lower in exposed sites than in unexposed sites(0.2130±0.7997 vs 0.4520±0.5921,t=5.37,P<0.05).Decreased protein expression of cathepsin B was also noted in the premature senescence group compared with the other three groups.Moreover,the gray ratio between cathepsin B protein and glyceraldehyde phosphate dehydrogenase(GAPDH)in premature senescence group reduced from 28.099±O.054 before treatment to 25.1 03±0.102 in week 1 and 17.693±0.099 in week 3 after UVA exposure.RT-PCR revealed that the mRNA expression level of cathepsin B in fibroblasts of premature senescence group decreased by 36 percent compared with that in the control group.Conclusions The expression of cathepsin B decreases in photoaged skin as well as in UVA-exposed fibroblasts in a time-dependent manner,which may be associated with the self-repair of photoaged skin.