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.

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.

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.

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 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.

Objective To study the level of expression of RAR?/RXR?mRNA in skin lesions of patients with psoriasis vulgaris.Methods The skin biopsies were collected fro mskin lesions in 20cases of psoriasis vulgaris and 10normal controls.Expression o f RAR?/RXR?mRNA was detected by RT -PCR.Results Expression levels of RXR?mRNA were 0.19760?0.0933in epiderm ides from psoriatic lesions,which were markedly lower than those in normal controls(0.5867?0.0132)(P0.05).Conclusion The results indicate that decreased expression of RXR?mRNA might be related to the pathogen esis of psoriasis.

Objective To study level of the expression of RAR? mRNA in the skin lesions of patients with psoriasis vulgaris. Methods The biopsies were taken from skin lesions in 20 patients with psoriasis vulgaris and the skin of 10 normal controls, and levels of RAR? mRNA were investigated with RT-PCR. Results The level of RAR?mRNA was significanlly lower in the epidermis of patients with psoriasis vulgaris than that in the control(P

Objective To construct the short hairpin RNA (shRNA)-expressing plasmid vectors specific for BRAF gene, and to test their effects in BRAF knockdown in human melanoma cell lines. Methods Two pairs of specific BRAF shRNA oligoes and a pair of randomly synthesized non-specific shRNA oligo were synthesized and inserted into plasmid pGenesil-1. Their fidelity was confirmed by double endonuclease digestion and sequencing. The constructed plasmids were transfected into human melanoma cell lines A375 and M14. The expression of BRAF mRNA and BRAF protein were detected by RT-PCR and Western blotting, respectively. Results The designed shRNA oligoes were precisely cloned into the plasmid pGenesil-1. The expression of BRAF mRNA and protein were down-regulated by specific plasmid braf 1 and braf 2, except to non-specific plasmid neg. The plasmid braf 1 was more effective, reducing BRAF gene expression by 90 per cent. Conclusions Plasmid mediated shRNA could successfully knockdown BRAF expression in human melanoma cells, and the suppression of the gene expression could maintain for 1 month at least.

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.

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.