Expressions of desmoglein 1 and desmoglein 3 in primary keratinocytes and two keratinocyte cell lines HaCaT and A431 / 中华皮肤科杂志

Objective To detect the mRNA and protein expressions of desmoglein 1 (DSG1) and DSG3 in different types of keratinocytes (KCs).Methods Two keratinocyte cell lines HaCaT and A431,as well as primary keratinocytes from human abdomenal skin served as the object of this study.Direct immunofluorescence assay was performed to observe and quantify the expressions of DSG1 and DSG3,and quantitative PCR (qPCR) to determine the mRNA expressions of DSG1 and DSG3,in these cells.Results Both DSG1 and DSG3 were expressed in all the three types of keratinocytes,and the fluorescence intensity of DSG1 and DSG3 in HaCaT cells was higher than that in primary keratinocytes but lower than that in A431 cells.Similarly,all the keratinocytes expressed DSG1 and DSG3 mRNA,with the relative expression levels of DSG1 and DSG3 mRNA in primary keratinocytes being 291.7％ and 237.4％ of those in HaCaT cells respectively (both P ＜ 0.01),and those in A431 cells being 0.1％ and 18.8％ of those in HaCaT cells respectively (both P ＜ 0.05).Conclusions HaCaT cells,A431 cells and primary keratinocytes all can be used for the study of DSG1 and DSG3,of which,A431 cells show the strongest expressions of DSG1 and DSG3,and primary keratinocytes display the highest expressions of DSG1 and DSG3 mRNAs.

Effects of paeonol on the proliferation and apoptosis of A375 human melanoma cells / 中华皮肤科杂志

Objective To study the effect of paeonol on the proliferation and apoptosis of A375 human melanoma cells and its mechanism.Methods Cell counting kit-8 (CCK8) was used to evaluate the proliferative activity of A375 cells treated with paeonol of 0.5,1,2,4,8 mmol/L for 24,48 and 72 hours respectively.Subsequently,A375 cells were treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours followed by double staining with annexin V and propidium iodide for the detection of cell apoptosis,fluorometric assay for the estimation of caspase 3,caspase 8 and caspase 9 activity,and Western blot for the determination of the levels of p53,nuclear factor-κB proteins and some of their target proteins.The A375 cells receiving no treatment served as the blank control group.Statistical analysis was carried out by t test.Results Within the investigated concentration and time ranges,paeonol significantly inhibited the proliferative activity of A375 cells in a concentration-and timedependent manner.Compared with the blank control group,a significant increase was observed in the early apoptosis rate in A375 cells treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours (13.74％-± 1.73％,25.95％ ± 0.57％ and 46.44％ ± 0.81％ vs.3.11％ ± 0.53％,P ＜ 0.05 or 0.01),as well as in the activity of caspase 3,8 and 9 in A375 cells treated with paeonol of 2.5 and 5 mmol/L for 24 hours (P ＜ 0.05 or 0.01).After 24-hour treatment,the protein levels of p53 and Bax were elevated,but those of nuclear factor-κB,Bcl-2 and Bcl-XL were decreased in A375 cells with the increase of paeonol concentration.Conclusions Paeonol can inhibit the proliferation but induce the apoptosis of A375 cells,and the apoptosis-inducing effect may be realized through intrinsic and extrinsic pathways by modulating nuclear factor-κB and p53 genes.

Fluctuations in anti-desmoglein antibody levels and their relationship with disease activity in patients with pemphigus / 中华皮肤科杂志

Objective To investigate the relationship between disease severity and enzyme-linked immunosorbent assay (ELISA) index values of anti-desmoglein (Dsg) 1 and-Dsg3 antibodies in 56 patients with pemphigus,and to characterize fluctuations in anti-Dsg1 and-Dsg3 antibodies in different forms of pemphigus.Methods Fifty-six patients with pemphigus (including 36 cases of pemphigus vulgaris (PV) and 20 cases of pemphigus foliaceus (PF)) were enrolled in this study.Blood samples were obtained from these patients before treatment and at the following four time points:when the condition was relieved and the taper of glucocorticoids began,the dose of glucocorticoids was tapered to half of their initial dose,the maintenance treatment started,and when the duration of maintenance treatment reached two years.ELISA was performed to determine the levels of anti-Dsg1 and-Dsg3 antibodies in these serum samples.Spearman correlation analysis was carried out to assess the relationship between disease severity and ELISA index values,and independent sample's t test to compare the levels of anti-Dsg antibodies among these time points.Results The severity of pemphigus was correlated with anti-Dsg antibody ELISA index values.Both anti-Dsg1 and anti-Dsg3 ELISA index values were significantly reduced at the remission of pemphigus compared with those before treatment (all P ＜ 0.01).At the end of the two-year maintenance treatment,10 (50％) patients with PF and 7 (19.4％) patients with PV became negative for anti-Dsg1 ELISA,whereas only 1 (2.7％) patient with PV became negative for anti-Dsg3 ELISA.Conclusions Anti-Dsg antibody ELISA index value is correlated with disease severity in patients with pemphigus,which may serve as a useful marker for assessing disease severity and activity as well as evaluating therapeutic efficacy.

Expression of CD200 in peripheral lymphocytes and hair follicles from patients with alopecia areata and its significance / 中华皮肤科杂志

Objective To investigate the expression of CD200 in peripheral lymphocytes and hair follicles from patients with alopecia areata (AA).Methods Flow cytometry was used to detect the expression of CD200 in peripheral lymphocytes from 43 patients with AA and 43 healthy controls.Immunohistochemistry was carried out to quantify the expression of CD200 and cytokeratin 15(CK15,a marker for basal cells of the outer root sheath) in resected scalp specimens from 8 patients with AA and 8 healthy controls.Differences in the expression of CD200 and CK15 between the patients and controls were assessed by independent-samples t test and rank sum test.Data were processed by the software SPSS17.0.Results The percentage of CD200-expressing cells in peripheral blood lymphocytes and T lymphocytes was significantly lower in the patients with AA than in the healthy controls (5.73％ ± 3.46％ vs.12.01％ ± 4.90％,8.85％ ± 4.80％ vs.12.31％ ± 3.12％,t =6.865,3.964,respectively,both P ＜ 0.05).However,no significant difference was observed in the percentage of CD200-expressing cells in peripheral blood B lymphocytes between the patients and controls (74.68％ ± 8.12％ vs.75.75％ ± 9.45％,t =0.570,P ＞ 0.05).Further more,the patients showed a lower expression of CD200 (P ＜ 0.05),but a similar expression of CK15 (P ＞ 0.05) in hair follicles compared with the controls.Conclusion The decrease in CD200 expression in peripheral lymphocytes and hair follicles may be involved in the pathogenesis of AA.

Effect of microRNA-21 and microRNA-494 on cell cycle of and apoptosis in a human melanoma cell line A375 / 中华皮肤科杂志

Objective To optimize the concentration of a microRNA-21 (miR-21) inhibitor and a miR-494 mimic for the transfection of A375 human melanoma cells,and to estimate the effect of the miR-21 inbihitor and miR-494 mimic on the proliferation of A375 cells.Methods A miR-21 inbihitor and a miR-494 mimic were designed and constructed.To optimize the concentration of the miR-21 inbihitor and miR-494 mimic for transfection,six concentrations (70-250 nmol/L) of the inbihitor and mimic were transfected into A375 cells separately by using LipofectamineTM2000.Then,quantitative fluorescence-based PCR was performed to determine the expression of miR-21 and miR-494 in A375 cells.Some A375 cells were classified into five groups:Mock blank control group remaining untransfected,miR-21 inhibitor group transfected with the miR-21 inhibitor,miR-21 control group transfected with the miR-21 inhibitor negative control,miR-494 mimic group transfected with the miR-494 mimic,and miR-494 control group transfected with the miR-494 mimic negative control.Mter another 48-hour culture,the cells were collected for the analysis of cell apoptosis and cycle by using flow cytometry.Meanwhile,Cy5-labelled miR-494 mimic negative control was transfected into A375 cells for the evaluation of the transfection efficiency by using an inverted fluorescence microscope.Results miRNAs were successfully extracted from A375 cells.As quantitative PCR revealed,the A375 cells transfected with the miR-21 inhibitor at 120 nmol/L showed the lowest expression level (2-△△Ct) of miR-21 (average:0.80; range:0.65-0.92),and those transfected with the miR494 mimic at 250 nmol/L displayed the highest expression level of miR-494 (average:126.82; range:111.52-144.22).The transfection efficiency in A375 cells was higher than 90％.Compared with the corresponding negative control groups,the miR-21 inhibitor group and miR-494 mimic group showed increased apoptosis rate ((27.74 ± 1.39)％ vs.(12.93 ± 0.65)％,(34.30 ± 2.35)％ vs.(15.54 ± 1.02)％,both P ＜ 0.01),percentage of G1-phase cells ((61.61 ± 3.25)％ vs.(50.34 ± 5.62)％,(61.05 ± 3.17)％ vs.(49.95 ± 2.58)％,both P＜ 0.05),but decreased proliferation index ((38.39 ± 3.25)％ vs.(49.66 ± 5.62) ％,(38.95 ± 3.17)％ vs.(50.05 ± 2.58)％,both P ＜ 0.05).Conclusions Both the miR-21 inhibitor and miR-494 mimic can promote the G1-phase arrest and apoptosis in A375 cells,and miR-21 may act as a protooncogene accelerating the proliferation of A375 cells,while miR-494 may founction as a tumor suppressor inhibiting the proliferation of A375 cells.

Effects of triptolide on the proliferation of and apoptosis in a human melanoma cell line M14 / 中华皮肤科杂志

Objective To study the effects of triptolide on the apoptosis in and proliferation of a human melanoma cell line M14.Methods M14 cells were cultured with the presence of 5 concentrations (12.5,25,50,100,200 nmol/L) of triptolide for 24,48 and 72 hours respectively,and cell counting kit-8 (CCK-8) was used for the detection of cell proliferation.Some M14 cells were treated with triptolide at 10 nmol/L,20 nmol/L and 30 nmol/L for 48 hours followed by the analysis of cell cycle by flow cytometry and detection of cell apoptosis by flow cytometry following annexin V-fluorescein isothiocyanate (FITC)/propidium iodide double staining.The morphological changes of M14 cells treated by triptolide at 30 nmol/L for 48 hours were observed by Hoechest 33258 staining.Results Compared with untreated M14 cells,an increase of cell population in S phase was observed in triptolide-treated cells,along with a decline in cell population in G2/M phase.The apoptosis rate was (2.92 ± 0.17)％,(20.99 ± 0.40)％,(34.28 ± 2.04)％ and (63.38 ± 0.71) ％ respectively in M14 cells treated with triptolide at 0,10,20 and 30 nmol/L for 48 hours,suggesting that triptolide enhanced the proliferation of M14 cells in a dose-dependent manner.After treatment with triptolide of 30 nmol/L,M14 cells showed morphological changes characteristic of apoptosis.Conclusion Triptolide could inhibit the proliferation of and induce the apoptosis in M14 human melanoma cells.

Methylation status of CpG islands in the promoter region of IGFBP7 gene in human melanoma cell lines / 中华皮肤科杂志

Objective To assess the relationship of methylation status of CpG islands in the promoter region of insulin-like growth factor binding protein 7 (IGFBP7) gene with the expression of IGFBP7 gene in human melanoma cell lines and primary melanocytes.Methods Primary melanocytes from human forcskin tissue as well as 4 human melanoma cell lines,including A375,M14,SK-MEL-1 and MV3,were used in this study.Bisulfite sequencing PCR (BSP) was applied to detect the methylation status of 54 CpG sites in the 5'-flanking promoter region of IGFBP7 gene in all of the melanoma cell lines and primary melanocytes.Results As hierarchical cluster analysis showed,IGFBP7-positive cells (including A375,M14 and SK-MEL-1 ) differed significantly from IGFBP7-negative cells (including MV3 cells and primary melanocytes) in the methylation pattern of IGFBP7 gene promoter region.Conclusion The methylation status of CpG island in the promoter region of IGFBP7 gene may be associated with its expression in melanoma cell lines.

Detection of promoter methylafion of three human leukocyte antigen (HLA) class I genes (HLA-A, -B and -C) in patients with psoriasis vulgaris / 中华皮肤科杂志

ObjectiveTo investigate the correlation between the methylation status of HLA class Ⅰ genes(HLA-A, -B and -C) in psoriatic epidermis and disease severity in patients with psoriasis vulgaris. MethodsDNA specimens were obtained from the lesional and nonlesional epidermis of 46 patients with psoriasis vulgaris and from the normal skin of 28 human controls. Methylation specific PCR (MSP) was conducted to detect the methylation status of CpG islands in the promoter region of HLA-A, -B and -C genes. The severity of psoriasis was evaluated by psoriasis area and severity index(PASI) scores. ResultsThe percentage of promoter methylation of HLA-B and HLA-C genes was 4.35％(2/46) and 21.74％(10/46), respectively in nonlesional epidermis, 4.35％ (2/46) and 4.35％ (2/46), respectively in lesional epidermis from these patients. No methylation was observed for the promoter of HLA-A, -B or -C gene in the normal control epidermis or for that of HLA-A gene in the nonlesional or lesional epidermis from the patients. The frequency of HLA-C gene promoter methylation in the nonlesional epidermis was significantly higher than that in the lesional epidermis and control epidermis, but was uncorrelated to the disease severity. No significant difference was observed for the methylation frequency of HLA-A or -B gene promoter among the three groups of specimens. Conclusion Abnormal methylation of HLA-C gene promoter is observed in patients with psoriasis vulgaris.

Expression and significance of CXCR7 chemokine receptor in cutaneous malignant tumors and their cell lines / 中华皮肤科杂志

Objective To investigate the expression of CXCR7 in several cutaneous malignant tumors including cutaneous squamous cell carcinoma (SCC),basal cell carcinoma (BCC) and invasive cutaneous malignant melanoma and their cell lines,as well as its significance.Methods Tissue specimens were obtained from the lesions of 30 patients with cutaneous squamous cell carcinoma,25 patients with basal cell carcinoma and 30 patients with cutaneous malignant melanoma.Immunohistochemistry was performed to detect the expression of CXCR7 protein in these tissue specimens and several cell lines (A375 human melanoma cells,M14 human melanoma cells,A431 human epidermoid carcinoma cells,HaCaT human keratinocytes).The mRNA expression of CXCR7 in these cell lines was measured by reverse transcription PCR.Results CXCR7 protein was apparently expressed in invasive cutaneous malignant melanoma.The high expression rate of CXCR7 protein was significantly elevated in cutaneous malignant melanoma tissue specimens compared with SCC and BCC tissue specimens [80％ (24/30) vs.26.67％ (8/30) and 8％ (2/25),x2 =17.16,28.36,both P ＜ 0.05].CXCR7 mRNA was expressed in A375,M14 and A431 cells,but not in HaCaT cells,with the strongest expression observed in A375 cells.Immunohistochemistry revealed the expression of CXCR7 protein only in A375 cells.Conclusions CXCR7 is highly expressed in cutaneous malignant melanoma and A375 cells,which may be involved in the malignant invasion and metastasis of melanoma.

Effects of 5-aza-dc on the expression of insulin-like growth factor binding protein 7 (IGFBP7) in and proliferation of melanoma cells / 中华皮肤科杂志

Objective To study the effects of 5-aza-dc on the expression of IGFBP7 in and proliferation of melanoma cell lines A375 and M14.Methods Reverse transcription-PCR and immunocytochemistry were performed to detect the mRNA and protein expression of IGFBP7 in A375 cells and M14 cells after treatment with 5-aza-dc of 10μmol/L for 48 hours,and MTT assay to measure the proliferation of both cell lines treated with 4 different concentrations (2.5,5,10,20μmol/L) of 5-aza-dc for various durations.Results The treatment with 5-aza-dc restored IGFBP7 expression at both mRNA and protein levels.The four concentrations of 5-aza-dc inhibited the proliferation of A375 and M14 cells in a dose-dependent (F=561.12,271.43,respectively,both P<0.01) and time-dependent (F=141.35,549.33,respectively,both P<0.01) manner.Conclusions DNA methylation may be involved in the modulation of aberrant IGFBP7 gene expression in melanoma,and 5-aza-dc could inhibit the proliferation of A375 and M14 cells.

Transcription activator protein 1 (AP-1)-related study in basophils from patients with chronic idiopathic urticaria / 中华皮肤科杂志

Objective To investigate the role of AP-1 in the pathogenesis of chronic idiopathic urticaria (CIU). Methods By using immunomagnetic separation technology, peripheral blood basophils were isolated from 10 CIU patients and 10 normal human controls followed by the extraction of nuclear protein from the basophils. TransAMTM AP-1 family kit was used to detect the DNA binding activity changes of AP-1 family transcription factors in basophils, and Western blotting to detect the expression of P-c-jun protein. Results There were some differences in the DNA binding activity of AP-1 family transcription factors in basophils between CIU patients and normal controls. The DNA binding activity of Phospho-c-jun, c-fos, Fos-B, Jun-B and Jun-D factors was increased in CIU patients compared with the controls, and the increase in that of P-c-jun and Jun-D was statistically significant (both P < 0.05). There was an insignificant decrease in the DNA binding activity of Fra-1 factor in the CIU patients compared with the controls (P > 0.05). The P-c-jun (Ser73) protein expression was higher in CIU patients than that in the controls (0.527 ± 0.312 vs. 0.435 ± 0.042, P < 0.05),whereas there was no significant difference in the P-c-jun (Ser63) protein expression level. Conclusion Some changes in DNA binding activity of AP-1 and overexpression of P-c-jun (Ser73) protein in basophils may be involved in the pathogenesis of CIU.

Influence of IgG in bullous pemphigoid blister fluid on the secretion of chemokines by human keratinocytes / 中华皮肤科杂志

Objective To investigate the effect of immunoglobulin G (IgG) in bullous pempbigoid (BP) blister fluid on the secretion of chemokines by human keratinocytes. Methods IgG was obtained from the blister fluid of patients with bullous pemphigoid and sera of normal human controls, then purified by sequential precipitation with caprylic acid and ammonium sulfate. The immunological activity of blister fluid was tested before and after the purification by BP180 ELISA kit. Keratinocytes were isolated from the foreskin tissue of yong adults, and subjected to primary culture. After 3 passages, the primary keratinocytes were harvested and subcultured in the presence of purified IgG of 0.5, 1, 2, 4 and 8 g/L, respectively, for 24 hours, or IgG of 4 g/L for 3, 6, 12, 24 hours, respectively, followed by the detection of levels of eotaxin, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 in the supemate of keratinocytes by ELISA. Results The valence of IgG remained unchanged after the purification with caprylic acid and ammonium sulfate. Compared with IgG from the sera of normal controls, that from bullous pemphigoid blister fluid sig- nificantly enhanced the secretion of IL-8 by keratinocytes in a time- and dose-dependent manner (both P < 0.01 ). Neither eotaxin nor MCP-1 was detected in the supemate of control IgG-treated, BP IgG-treated or untreated keratinocytes. Condusions The IgG in BP blister fluid has been proved to stimulate the secretion of IL-8 by cultured human keratinocytes, which may be involved in the pathogenesis of BP.

Inhibitory effects of siRNA targeting survivin on the growth of a human melanoma cell line,M14 / 中华皮肤科杂志

Objective To study the inhibitory effects of siRNA targeting survivin on the expression of survivin,as well as the apoptosis,proliferation and invasion of a human melanoma cell line,M14.Methods Two siRNAs targeting survivin were designed,chemically synthesized,and used to construct the recombinant plasmids,pRAT-H1.1/neo-survivin-siRNA1 and pRNAT-H1.1/neo-survivin-siRNA2.Then,recombinant plasmids were transfected into M14 cells mediated by Lipofectamine 2000 reagent.Those cells untransfected or transfected with empty vector served as the control.After culture over various periods of time.cells were collected for the detection of mRNA and protein expression of survivin with RT-PCR and Western blotting,respectively,and for the examination of apoptosis and proliferation of M14 cells by flow cytometry and MTT methods,respectively.Also,Transwell assay was performed to detect the invasive capability of M14 cells.Results A statistical decrease in the mRNA and protein expressions of survivin was observed along with an increase in apoptotic rate(x2=31.55,P＜0.01)in M14 cells transfectcd with siRNA-containing plasmid compared with untransfected and empty vector-transfected cells.As MTT assay indicated,on day 4 after the transfcorion,the proliferation of M14 cells was inhibited by(55.4±4.3)%,(34.5±4.3)%and(13.3±4.6)%,with pRNAT-H1.1/neo-survivin-siRNA1,pRNAT-H1.1/neo-survivin-siRNA2 and empty vector,respectively:there was a significant difference among the three groups(P＜0.05).Decreased invasive capability was noticed in M14 cells transfected with siRNA-containing plasmid compared with untransfected cells(all P＜0.05).Conclusions The plasmid containing siRNA against survivin can specifically inhibit the expression of sarvivin,proliferation and invasion of tumor cells,and induce cell apoptosis.The inhibition of survivin expression by siRNA may be a rational approach to the gene therapy for malignant melanoma.

In vitro anti-proliferation effect of a histone deacetylase inhibitor,chidamide,on a malignant melanoma cell line,A375 / 中华皮肤科杂志

Objective To investigate the in vitro anti-proliferation effect of a histone deacetylase inhibitor,chidamide,on a cutaneous malignant melanoma cell line,A375.Methods Cultured A375 cells were treated with different concentrations of chidamide(5,10,50,100,500 μmol/L)and aichostatin A (TSA)(0.1,0.25,0.5,1.0 μmol/L),respectively,for various durations(24,48,72,96,120 hours).Subsequently,cell proliferation,apoptosis and cell cycle were detected by MTT assay,annexin Vfluorescein isothiocyanate and propidium iodide double staining,and DNA ploid analysis,respectively.Results The proliferation of A375 cells was inhibited in a dose-dependent manner by chidamide of 5-500μmol/L and TSA of 0.1-1 μmol/L,and in a time-dependent manner from 0 to 120 hours after the beginning of trealment with ehidamide of 5-500μmol/L and TSA of 0.25-1μmol/L.The 48-hour 50% growth inhibition concentration(IC50)of ehidamide and TSA on A375 cells was about 250 μmol/L and 0.7μmol/L,respectively.After 48-hour treatment,the apoptosis mte was 80.27%±3.06%,79.53%±5.70%,83.13%±6.90%in A375 cells treated with chidamide of 62.5,125,250 μmol/L,respectively,16.27%±2.46%,28.83%±2.55%,83.40%±8.65%in those treated with TSA of 0.175,0.35,0.7 μmol/L,respectively,10.43%±0.96%in ontreated cells;a statistical increase was noticed in chidamide-treated cells and TSA-treated cells vs.untreated cells(all P＜0.001).A positive correlation was observed between the apoptosis rate and concentrations of TSA(r=0.955,P=0.000).Cell cycle analysis indicated that treatment with chidamide induced cell cycle arrest in G0/G1 phase,with the cell proportion in G0/G1 phase being 76.30%±6.06%,82.79%±0.74%,88.91%±5.29%in A375 cells treated with chidamide of 62.5,125,250μmol/L,respectively,versus 38.73%±3.36%in untreated cells.While after 48-hour treatment with TSA of 0.35 and 0.7 μmol/L,the proportion of cells in G2/M phases was 25.15%±2.71%and 58.71%±3.45%,respectively,compared to 15.73%±0.23%in untreated cells(P＜0.01).Conclusion Chidamide and TSA could induce cell cycle arrest and apoptosis,as well as inhibit the growth of A375 ceils in vitro.

Peptidoglycan modulation of chemokine production in normal human epidermal kerafinocytes and the role of Toll-like receptor 2 in this process / 中华皮肤科杂志

Objective To investigate the effect ofpeptidoglycan from Staphylococcus aureus on the release of several chemokines including intedeukin 8 (IL-8), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage-derived chemokine (MDC) by normal human epidermal keratinocytes (KCs) and the role of Toll-like receptor 2 (TLR2) in this process. Methods KCs were derived from the foreskin of a healthy boy and propagated. After 2 - 4 passages, KCs were collected and treated with various concentrations (3, 10, 30 and 100 mg/L) of peptidoglycan for 24 hours or with peptidoglycan of 100 mg/L for varying durations (3, 6, 12, 36 hours). A fi'action of KCs were pretreated with functional grade purified anti-TLR2 monoclonal antibody before the treatment with peptidoglycan of 100 mg/L. After additional 12-hour culture following the treatment, enzyme linked immunosorbent assay was used to detect the level of IL-8, RANTES and MDC in culture supernatants of KCs. Results KCs spontaneously released IL-8 and RANTES. Peptidoglycan increased the production of IL-8 but decreased that of RANTES by KCs. The levels of IL-8 were 209.96 ± 10.31 ng/L, 250.28 ± 9.52 ng/L, 285.11 ± 10.28 ng/L, 359.40 ± 6.93 ng/L in KCs treated with peptidoglycan of 3, 10, 30, 100 mg/L, respectively, compared to 135.41 ± 14.37 ng/L in untreated KCs (all P < 0.05). On the contrary, a significant decrement was seen in the secretion of RANTES by KCs treated with peptidoglycan of 10, 30, 100 mg/L compared with untreated KCs (110.72 ± 8.51 ng/L, 90.50 ±2.45 ng/L, 49.89 ± 13.74 ng/L vs 149.94 ± 18.71 ng/L, all P < 0.05). The monoclonal antibody to TLR-2 could markedly suppress the promotion of IL-8 production by peptidoglycan, but had no obvious influence on the inhibition of RANTES production by peptidoglycan. MDC could not be detected in the culture super-natants of KCs with or without peptidoglycan stimulation. Conclusion Peptidoglycan could inhibit RANTES secretion but induce IL-8 production by KCs likely via TLR2.

mRNA expressions of IFN-gamma receptor and TNF-alpha receptor in peripheral blood mononuclear cells of patients with psoriasis vulgaris / 中华皮肤科杂志

Objective To investigate the mRNA expressions of IFN-gamma receptor and TNF-alpha receptor in peripheral blood mononuclear cells (PBMCs) of patients with psoriasis vulgaris and their role in the pathogenesis of psoriasis. Methods Fifty patients with psoriasis vulgaris (37 cases of active psoriasis and 13 cases of stable psoriasis) and 24 healthy human controls were included in this study. PBMCs were isolated from blood samples obtained from all patients and controls. The mRNA expressions of IFN-gamma receptor and TNF-aipha receptor in PBMCs were detected by RT-PCR. The disease severity in patients was evaluated by psoriasis area and severity index (PASI). Results The mRNA expressions of IFN-gamma receptor and TNF-alpha receptor were observed in the PBMCs of all subjects. The mRNA expression levels of IFN-gamma receptor were 0.72 ± 0.17 in healthy controls, 1.11 ± 0.55 in all patients with psoriasis, 1.13 ±0.57 in patients with active psoriasis and 1.03 ± 0.52 in patients with stable psoriasis, respectively. A signifi-cant increase was observed in the expression levels of IFN-gamma receptor mRNA in all psoriatic patients and in patients with active psoriasis compared with those in healthy controls (both P < 0.05), but there was no significant difference between the healthy controls and patients with stable psoriasis (P ＞ 0.05). The expres-sion levels of TNF-alpha receptor mRNA were 2.05 ± 1.34 in healthy controls, 2.70 ± 3.80 in all psoriatic patients, 2.90 ± 4.40 in patients with active psoriasis, 2.14 ± 1.05 in patients with stable psoriasis, respectively;there was no significant difference between psoriatic patients and healthy controls (P ＞ 0.05). However, no correlation was found between the mRNA expression of IFN-gamma receptor, that of TNF-alpha receptor,and disease severity in psoriatic patients. Conclusions The mRNA expression of IFN-gamma receptor in PBMCs is up-regulated in patients with psoriasis vulgaris, which is unrelated to the activity of psoriasis.

Effects of genistein on biological activities of and collagen synthesis by human skin fibroblasts in vitro / 中华皮肤科杂志

Objective To study the effects of genistein on the biological characteristics of and collagen synthesis by human skin fibroblasts in vitro.Methods Human skin fibroblasts(HSFBs)were obtained from the prepuce of healthy adolescents,and suhjected to primary culture.Atier 5 to 15 passages of culture,HSFBs were treated with various concentmtions(0.03125,0.0625,0.125,0.25,0.5,1mg/L)of genistein for different durations.The potential of cell proliferation was detected by MTT assay and growth curves were drawn for HSFBs.Flow cytometry(FCM)and RT-PCR were used to estimate cell cycle phases and mRNA expression of type Ⅰ procollagen.respectively.Results The proliferation rate of HSFBs was 97.7%,113.8%,132.5%,116.4%,94.5%and 83.3%after treatment with genistein of 0.03125,0.0625,0.125,0.25,0.5 and 1 mg/L,respectively.The genistein of more than 0.5 mg/L displayed an inhibitive effect on the proliferation of HSFBs.while that between 0.0625 and 0.25 mg/L showed a promotive effect.Atier treatment with genistein at 0.0625,0.1 25 and 0.25 mg/L,the percentage of HSFBs in S phase and G2 phase significantly increased compared with untreated HSFBs(S phase:41.15%±2.88%,61.89%±3.16%,48.18%±1.68%vs30.12%±0.60%,P＜0.05;G2 phase:9.76%±3.99%,10.40%±0.54%,7.46%±2.47%vs 0.61%±0.16%,P＜0.05).Compared with the untreated HSFBs.the relative mRNA expression level of type Ⅰ procollagen was increased with genistein of 0.0625,0.125 and 0.25 mg/L(0.4814±0.0138,0.5767±0.0291,0.5675±0.0272 vs 0.4101±0.0236,P＜0.01),but decreased with genistein of Ⅰ and 0.5 mg/L(0.1662±0.0165 and 0.2017±0.0203 vs 0.4101±0.0236,P＜0.01).ConclusionCertain concentrations of genistein could enhance the proliferation and growth of as well as mRNA expression of type Ⅰ procollagen in HSFBs.

Polymorphism of intefferon-γ receptor 2 gene in patients with psoriasis vulgaris in Chinese Hans / 中华皮肤科杂志

Objective To assess the association between the amino acid polymorphism (Arg64Gln)within the interferon-γ receptor 2 gene (IFN-γR2) and psoriasis vulgaris in Chinese Hans. Methods Blood samples were collected from 182 patients with psoriasis vulgaris and 114 healthy human controls in Jiangsu and Anhui provinces. The amino acid polymorphism (Arg64Gin) within the IFN- γR2 was examined by PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing. Results No significant difference was observed in the amino acid polymorphism (Arg64GIn) within the IFN-γR2 between the psoriatic patients and healthy controls (P > 0.05 ). There was a significant difference between patients with nail involvement and those without in the frequency of Gln64/Gln64 genotype (57.5% vs 38.1%, X~2= 5.33, P < 0.05),andArg64 (Gln64)allele [19.3% (80.7%)vs30% (70%), X~2=5.03, P < 0.05]. The frequencies of Gln64/Arg64 genotype and Gln64/Gln64 genotype in psoriatic patients with nail involvement significantly differed from those in the controls (29.8% vs 49.1%,X~2 = 5.48, P < 0.05; 57.5% vs 35.1%, X~2= 6.23, P <0.05 ), while no significant difference was found between the psoriatic patients without nail involvement and controls. Moreover, significant difference was noted between patients with prior upper respiratory tract infection (as inducements) and those without in the frequency of Arg64/Arg64 genotype (33.3% vs 15.5%, X~2 =4.94, P < 0.05) and Gln64 (Arg64) allele [51.9% (48.1%) vs 35.2% (64.8%), X~2= 5.46, P < 0.05]. Condusion The amino acid polymorphism (Arg64Gln) within the IFN-γR2 may be associated with the nail involvement and upper respiratory tract infection in patients with psoriasis vulgaris.

Expression of neutrophil adhesion molecule CD11b and plasma levels of elastase in Patients with anaphylactoid purpura / 中华皮肤科杂志

Objective To explore the expression of neutrophil adhesion molecule CD11b,and plasma level of elastase in patients with anaphylactoid purpura during different clinical phases and their correlation with disease activity.Methods A total of 20 patients with anaphylactoid purpura were recruited into this study,along with 20 normal human controls.Two blood samples were collected from each patients at the first visit(active phase)and after 3～5 weeks of treatment(remission phase).The expression of CD11b was measured by flow cytometry in 12 patients and normal controls,and plasma levels of elastase by ELISA in 20 patients and normal controls.Results Increased CD11b expression and elastase level were noted in patients in active phase compared with those in patients in remission phase(3367.25±434.57 vs 2569.33±411.06.13.98±2.05 vs 4.29±0.80.both P＜0.01).No significant difference was found in CD11b expression between patients in remission phase and normal controls(P＞0.05).while the elastase level was higher in patients in remission phase than in normal controls(4.29±0.80 vs 3.67±0.54.P＜0.05).In active phase of anaphylactoid purpura,the expression of CD11b was positively correlated with the plasma level of elastase(r=0.73,P＜0.01),while no correlation was noticed between them in remission phase(r=0.20,P=0.54).Conclusion Peripheral neutrophils are activated in anaphylactoid purpura,which seems to be more obvious in active phase than in remission phase.

mRNA expression of Fc gamma RⅡA on neutrophils from patients with Beh(c)et's disease / 中华皮肤科杂志

Objective To investigate the mRNA expression of Fc gamma RⅡA(FcγR ⅡA)on polymorphonuclear neutrophils(PMN)from patients with Beh(c)et's disease(BD).Methods Twenty-five patients with active BD and 20 healthy human controls were included in this study.Blood samples were obtained from all patients with active BD before treatment,from 15 patients with inactive BD after treatment and from healthy controls.PMN were isolated.The FcγR Ⅱ A mRNA expression on PMNs was detected by RT-PCR,and plasma myeloperoxidase (MPO)activity which represented neutrophil activation,was measured spectrophotometricaily.Results The relative expression level of FcγR Ⅱ A on PMN and plasma MPO aetivity were 1.80 1±0.829 and 32±5 U/L.respectively,in patients with active BD,0.820±0.625 and 27±4 U/L,respectively,in those with inactive BD,and 0.745 ±0.931 and 29±5 U/L,respectively,in normal controls;the differences were significant in the two parameters between the patients with active and inactive BD (both P＜0.01),while no statistical difference was observed between inactive patients and normal human controls(P＞0.05).There was a positive eorrelation between the expression level of FcγR Ⅱ A on PMN and plasma MPO activity in patients with BD(r=0.39,P＜0.0 1).Conclusions The mRNA expression of FcγR ⅡA on neutrophils is up-regulated in patients with active BD.It is likely that FcγR Ⅱ A is involved in the activation of neutrophils in BD.