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.

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.

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.

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.

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.

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.

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.

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.

Objective To study the effects of paeonol on the tyrosinase activity and melanogenesis in co-culture model of human melanocytes and keratinocytes. Methods Melanocytes and the co-culture model of human melanocytes and keratinocytes were cultivated and the proliferation of melanocytes and the co-cultures was measured by MTT colorimetric assay. The tyrosinase activity and melanin level were measured by enzymic method. Results The melanin synthesis and tyrosinase activity were markedly suppressed by paeonol in a dose-dependent manner at the concentrations of 50?mol/L, 100?mol/L, and 200?mol/L in both melanocytes and co-cultures. The significant stronger suppression was observed with 100?mol/L and 200?mol/L of paeonol than that with controls (P

Objective To investigate the effect of Th1/Th2 cytokines and immune state on the occurrence and familial aggregation of hepatocellular carcinoma(HCC).Methods Ninety-five members whose families have had two or even more HCC patients(high-occurrence families) were selected as the case group,by matching with the same nationality,gender,residential area,age±5 years old,95 members whose families had no any cancer were selected as the control.The level of peripheral blood Th1 type cytokines such as interferon-γ(IFN-γ),interleukin-2(IL-2) and Th2 type cytokines such as interleukin-4(IL-4),interleukin-10 (IL-10) were detected by enzyme linked immunosorbent assay(ELISA).Results There was a Th1/Th2 serum cytokine imbalance profile in members of HCC high-occurrence family.The levels of IFN-γ and IL-2 were significantly lower in members of HCC high-occurrence family than that of the controls.The levels of IL-4 and IL-10 were higher in members of HCC high-occurrence family than that of the controls.Conclusion There was a poor cellular immune state in members of HCC in the high-occurrence families.Th1 type cytokines was inhibited,and Th2 type cytokines was enhanced,so more susceptible to HBV chronic infection.It might be the mechanism of HCC occurrence and familial aggregation.