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Effect of resveratrol on the proliferation of malignant melanoma cell lines in vitro

Dongsheng LI ; Yiqan DUAN ; Weizhen WANG ; Liuqing CHEN ; Chenghui ZHANG ; Yongyan CAO ; Zhongsheng TONG

Chinese Journal of Dermatology.2009;42(12):839-842. doi:10.3760/cma.j.issn.0412-4030.2009.12.014

Objective To investigate the anticancer activities of resveratrol on malignant melanoma cells in vitro and involved mechanisms. Methods A375 human malignant melanoma cells and B16-F1mouse malignant melanoma cells were cultured and treated with various concentrations of resveratrol for different durations. The cell proliferation, apoptosis and cycle of both B16-F1 and A375 cells were detected with MTT assay, Annexin V-F1TC/propidium iodide (PI) double staining flow cytometry and propidium iodide flow cytometry, respectively. Western blot analysis was performed to measure the expression of Bcl-2 and Bax protein in both cells. Results Resveratroi inhibited the proliferation of A375 and B16-F1 cells in a time- and dose- dependent manner. The apoptosis rate of A375 cells was (16.7±2.1 )%, (17.2±1.7)% and (52.3±4.1 )% after treatment with resveratrol of 25 μmol/L for 24 hours, resveratrol of 100 μmol/L for 12 and 72 hours, respectively;, and resveratrol of 100 μmol/L induced the apoptosis of B16-F1 at a rate of ( 18.4±1.6)%, (39.6±3.3 )% and (56.7±4.5 )% at 12, 24 and 72 hours, respectively. Flow cytometry showed that A375 and B16-F1 cells treated with resveratrol were arrested in the G1 phase of cell cycle, and the blocking effect increased in a dose-dependent manner. The percentage of A375 and B16-F1 cells in G1 phase was (40.51±3.97 )% and (41.34±3.12 )%, respectively, after 24-hour treatment with resveratrol of 25 μmol/L,(55.64±4.95)% and (53.93±5.12)%, respectively with resveratrol of 100μmol/L for the same duration.The expression of Bcl-2 protein was decreased in malignant melanoma cells treated with resveratrol,while that of Bax protein increased. Conclusions Resveratrol can effectively inhibit the proliferation of malignant melanoma cells by regulating the cell cycle and inducing cell apoptosis, which seems to be associated with the regulation of Bcl-2 and Bax expressions.

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Polymorphism of intefferon-γ receptor 2 gene in patients with psoriasis vulgaris in Chinese Hans

Ningyan GU ; Heng GU ; Xu YAO ; Wuqing ZHOU ; Pangen CUI ; Min CHEN

Chinese Journal of Dermatology.2009;42(12):824-827. doi:10.3760/cma.j.issn.0412-4030.2009.12.008

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.

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Immune responses induced by recombinant major outer membrane protein of Chlamydia trachomatis serovar E

Caihong SHENG ; Yuanjun LIU ; Yanfei LI ; Shuping HOU ; Weifeng YAO ; Manli QI ; Quanzhong LIU

Chinese Journal of Dermatology.2009;42(12):817-820. doi:10.3760/cma.j.issn.0412-4030.2009.12.005

Objective To investigate specific immune responses in mice induced by recombinant major outer membrane protein(rMOMP)of C.trachomatis serovaf E.Methods Thirty-six female BALB/cmice aged 3 to 4 weeks Were divided into three groups.i.e.,adjuvant group vaccinated、with purified rMOMP and Freund's adjutant,solitary group vaccinated with rMOMP only and control group vaccinated with phosphate buffered saline(PBS).All the mice were intramuscularly vaccinated on week 0,2 and 4.Blood samples and vaginal washes were obtained from these mice on week 6,then,mice were challenged with elementary body(EB)of C.trachomatis serovar E at the footpad followed by the observation of delayed hypersensitivity.On week 7.mice were genitally infected with C.trachomatis EB;one week later,blood samples and vaginal washes were obtained again;six weeks later,spleen lymphocytes were isolated from the mice and stimulated bv C.trachomatis or ConA followed by the detection of cell proliferation with MTT assay.In vitro neutralization assay was also performed.ELISA was used to determine the titers of Chlamydia-specific IgO antibody in sera and IgA antibody in vaginal washes,as well as the level of IFN-γ in culture supernatant of lymphocytes and sefa of mice.Vaginal swabs were collected after genital challenge and subjected to C.trachomatis culture.Results The absorbance at 405 ms of Chlamydia-specific IgG antibody and proliferation index of lymphocytes were 0.641±0.059 and 5.085±1.291.respectively,in mice immunized with rMOMP and Frennd's adjuvant.significantly higher than those in mice immunized with rMOMP only(0.424±0.015 and 3.123 ±0.840.both P<0.05).The thickness of right hind footpad increased by 0.324±0.054 mm and 0.272±0.064 mm,respectively,in solitary group and adjuvant group,respectively,with significant difference between the two groups(P<0.05).A significant increase was also observed in the adjuvant group compared with the control group in the above three parameters(all P<0.01).Conclusion The rMOMP of C.trachomatis could efficiently induce Chlamydia-specific humoml and cellular immune responses in mice.

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Granrlar parakeratosis: a ease report

Zhenying WANG ; Xiulian XU ; Jihe LIU ; Jianfang SUN

Chinese Journal of Dermatology.2009;42(12):807-809. doi:10.3760/cma.j.issn.0412-4030.2009.12.001

A case of granular parakeratosis is reported. A 31-year-old woman presented with a 23-year history of pruritic erythema and erosion in the left axilla. On examination, there was a ring-like annular erythematous patch sized 8 cm×10 cm in the left axilla. Bright mauve, cone-shaped, millet-like papules were observed in the center of the lesion, some confluenced into plaques. Erythema was present in the pedlesional region along with mild erosion, exudation and small numbers of grain-sized pustules. Scar formed in some perilesional areas. No lesions were noted at any other intertriginous regions. Fungal microscopy of lesion secretions was negative. Histological examination of biopsy specimens from the center of the left axilla revealed psoriasiform hyperplasia of epidermis and thickened stratum comeum with hyperkeratosis and parakeratosis. Most cells in the stratum comenm retained nuclei and contained numerous basophilic granules. Granular layer could be noted under the parakeratotic cells with cytoplasm vacuolization of some cells. There was a perivascular, mixed inflammatory infiltration predominated by lymphocytes and hemangiectasis in the dermis. A diagnosis of granular parakeratosis was made.

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Rosiglitazone inhibition of A375 human malignant melanoma cell invasion

Lili NIU ; Jin HAO

Chinese Journal of Dermatology.2009;42(12):831-834. doi:10.3760/cma.j.issn.0412-4030.2009.12.011

Objective To explore the effect ofrosiglitazone (RGZ), a ligand of peroxisome proliferatoractivated receptor γ (PPARγ), on the invasiveness of A375 cells in vitro and its mechanism of action.Methods A375 human melanoma cells were cultured in vitro and treated with different concentrations of RGZ. The proliferation of the cells, mRNA expression levels of matrix metalloproteinase (MMP) 2 and tissue inhibitor of metalloproteinase (TIMP) 2, protein expression of MMP2 in A375 cells were detected by MTT assay, semi-quantitative RT-PCR and Western-blot, respectively. The invasiveness of A375 cells was detected by Matrigel invasion assay. Results MTT assay showed that the proliferation of A375 cells was inhibited by (4.86±0.31 )% and (5.15±0.52)% under the 24-hour treatment with RGZ of 10 and 25 μmol/L, respectively, and no evident cytotoxity was observed for RGZ. Compared with untreated A375 ceils, a significant decrease was observed in the mRNA and protein expression of MMP2 in A375 ceils treated with RGZ of 10 and 25 μmol/L (all P < 0.05), along with an increase in the mRNA expression of TIMP2 (both P < 0.01 ).The count of A375 cells transmigrating through matrigel was 154.1±7.7 and 87.3±8.1 under the treatment with RGZ of 10 and 25 μmol/L, significantly lower than that of those without treatment (210.7±14.9,both P < 0.01 ). Conclusions RGZ could inhibit the transmigration of A375 ceils, likely by down-regulating the mRNA and protein expression of MMP2.

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Rapid detection of pathogenic mycoplasmas in genitourinary tract using PCR-reverse dot blot hybridization

Xiujuan XUE ; Heping ZHENG ; Guoming LI ; Jiamei HUANG ; Weiying ZENG ; Yaohua XUE ; Xingzhong WU

Chinese Journal of Dermatology.2009;42(12):814-816. doi:10.3760/cma.j.issn.0412-4030.2009.12.004

Objective To develop a PCR-mverse dot blot hybridization(RDB)assay to rapidly detect pathogenic mycoplasmas in genitourinary tract.Methods Universal primers were designed and applied to amplify the 16S rRNA gone of ureaplasma parvum(Up),ureaplasma urealyticum(Uu),Mycoplasma genitalium(Mg),Mycoplasma hominis(Mh)by using nestcd PCR.Specific nucleotide probes of Up,Uu,Mg and Mh Were constructed and immobilized onto nylon membranes.PCR products were denatured and hybridized、with specific oligonucleofide probes on nylon membrane.The sensitivity and specificity of the PCR-RDB assay were evaluated based.on the hybddizafion results.Also,PCR-RDB Was utilized to detect pathogenic mycoplasmas from 60 clinical samples.Results The four probes selectively hybridized with the PCR product of corresponding mycoplasmas,and no cross hybridization was observed.The detection limit of PCR-RDB Was one colony forming unit(CFU)of mycoplasma.Out of the 60 clinical samples、19were positive for mycoplasm,Mixed infections were found in three samples,including two coinfected with Up and Uu and one with Uu and Mg.Conclusion PCR-RDB is a rapid,specific and sensitive approach to the identification of pathogenic mycoplasmas in urogenital tract.

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Effect of nitric oxide on substance P-induced scratching response in BALB/c mice

Yangping YANG ; Shuangjin ZHENG ; Lei CHEN ; Yiming FAN

Chinese Journal of Dermatology.2009;42(12):843-845. doi:10.3760/cma.j.issn.0412-4030.2009.12.016

Objective To investigate the effect of nitric oxide (NO) on the scratching behavior evoked by substance P (SP) in mice. Methods Forty BALB/c mice were randomly divided into 5 groups to receive intradermal injection of different doses of SP (0, 20, 40, 80, 160 nmol/site) into the rostral part of the back to establish the acute itch model. Another 40 mice were randomly allocated to model, spantide, L-arginine,L-NAME and aminognanidine groups injected intracutaneously with normal saline (NS), spantide, L-arginine,L-NAME and aminoguanidine, respectively, 10 minutes before SP (80 nmol/site) injection. Subsequently,the scratching behavior was observed, iNOS expression and NO level in the injected skin were detected by immunohistochemical staining and nitrate reductase assay, respectively. Results The scratching bouts per hour induced by intradermal NS and SP of 20, 40, 80 and 160 nmol/site were 4.38±4.07, 5.38±3.78,12.75±6.52, 23.50±7.84 and 42.38±15.84, respectively, and only SP at higher doses (40 - 160 nmol/site)elicited a dose-dependent scratching response in mice (P < 0.01 or 0.05) compared with NS. The scratches over 1 hour in SP, L-arginine, spantide, L-NAME and aminognanidine group were 67.13±32.79, 70.75±34.80, 10.75±8.14, 29.00±21.19 and 35.38±22.83, respectively; of them, pretreatment with spantide,L-NAME and aminognanidine significantly inhibited SP-induced scratching (P < 0.01 or 0.05), but L-arginine showed no inhibitory effect (P > 0.05). Compared with SP, the pretreatment with spantide, L-NAME and aminoguanidine significantly downregulated the iNOS expression and NO content (P < 0.01 or 0.05) in the injected skin other than L-arginine (P > 0.05 ). Conclusion Intradermal SP could increase NO synthesis by neurokinin 1 receptor activation, resulting in the scratching behavior in BALB/c mice.

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In vitro effect of cyclooxygenase (COX)-2 antisense oligonucleotide on the proliferation and expression of COX-2 in human skin squamous cell carcinoma cell line Colo-16

Jie MA ; Zhiping WEI ; Yanqun LIU

Chinese Journal of Dermatology.2009;42(12):835-838. doi:10.3760/cma.j.issn.0412-4030.2009.12.013

Objective To investigate the effects of COX-2 antisense oligonucleotide (AsODN) on the proliferation and expression of COX-2 in human skin squamous cell carcinoma cell line Coio-16. Methods The COX-2 AsODN was synthesized artificially, and various concentrations (50, 100, 200, 400 nmol/L) of the AsODN were transfected into Colo-16 cells with lipofectin followed by additional culture for different durations. The transfection results were observed with fluorescence microscopy. Subsequently, MTT assay,Western blotting and reverse transcription PCR were used to detect the cell proliferation, protein and mRNA expression of COX-2 in Coio-16 cells, respectively. Restults Compared with untreated cells, the proliferation of Colo-16 cells was inhibited significantly at 24, 48, 72 and 96 hours after transfection with different concentrations of COX-2 AsODN (all P < 0.05), and the COX-2 AsODN of 400 nmol/L exerted the highest inhibition rate of 60.3% at 48 hour. The average gray scale was 0.763±0.070, 0.600±0.065, 0.430±0.074 and 0.251±0.045 for COX-2 protein, 0.778±0.025, 0.602±0.041, 0.417±0.031 and 0.297±0.051 for COX-2 mRNA in Colo-16 cells transfected with COX-2 AsODN of 50, 100, 200, and 400 nmol/L respectively,significantly lower than that in untreated Colo-16 cells (all P < 0.05); there was a significant difference in the expression of COX-2 protein and mRNA among the cells transfected with the four concentrations of COX-2 AsODN and untreated cells (F = 83.54, 132.48, respectively, both P < 0.05). Conehtsions COX-2 AsODN can inhibit the proliferation, as well as the expression of COX-2 protein and mRNA in Colo-16 cells, which suggests that COX-2 AsODN has a potential therapeutic effect on skin squamous cell carcinoma.

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Detection of T cell receptor gamma gene rearrangements in mycosis fumgoides

Yiping MA ; Xianmei LU ; Guizhi ZHOU ; Xuechao CHEN

Chinese Journal of Dermatology.2009;42(12):828-830. doi:10.3760/cma.j.issn.0412-4030.2009.12.010

Objective To assess the diagnostic significance of T cell receptor gamma gene rearrangemerits in mycosis fungoides (MF), so as to develop a sensitive diagnosis tool. Methods A total of 50 specimens were collected, including 33 skin lesion specimens and 2 lymph specimens from 30 patients with MF,15 skin lesion specimens from 15 patients with inflammatory dermatoses. PCR was performed with specific primers targeting TCR V gamma 8, 9, 10, 11 to detect T cell receptor gamma gene rearrangement. Results Monoclonai rearrangements of TCR gene was observed in 88% (29/33) of specimens from patients with MF and 33% (5/15) of samples from patients with inflammatory dermatoses. Conclusions The detection of TCR gene rearrangements, as an ancillary test, is useful in the diagnosis and differential diagnosis of MF.

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Expression of angiopoietin-1,-2 and their receptor,tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2(Tie-2)in patients with psoriasis vulgaris

Quan LUO ; Xibao ZHANG ; Xin ZHOU ; Ling LIN ; Xuemei LI ; Huilan ZHU ; Yumei LIU ; Yuqing HE ; Kang ZENG

Chinese Journal of Dermatology.2009;42(12):821-823. doi:10.3760/cma.j.issn.0412-4030.2009.12.007

Objective To investigate the expressions of angiopoietin(Ang)-1,-2 and their receptor,Tie-2 in Patients with psoriasis vulgaris and their significance.Methods Tissue samples were collected from 30 patients with psoriasis vulgaris and 15 normal controls.Streptavidin-biotin complex(SABC)method was used to detect the expression of Ang-1,-2 and Tie-2 in these samples.Microvessel density (MVD)was assessed by CD34 staining.Results The expressions of Ang-2 and Tie-2 were significantly higher in patients with psoriasis vulgaris than in the normal controls(7.65±2.14 vs 2.54±0.91,6.09±1.97vs 1.95±0.62,both P<0.01),and higher in patients with active psoriasis than in those with inactive psoriasis (9.85±2.42 vs 4.86±1.63,8.43±1.98 vs 4.57±1.92,both P<0.05),whereas no significant difference was observed in the expression level of Ang-1 between Patients and controls or between patients with active psoriasis and those with inactive psoriasis(all P>0.05).Mcreased MVD was observed in patients、with psoriasis vulgaris compared with the controls(15.46±5.28 vs 2.68±0.79,P<0.01).Also,the expression levels of Ang-2 and Tie-2 were significantly difierent between patients with high MVD and those with low MVD(both P<0.05),while no difference was nomd in the expression of Ang-1 (P>0.05).In psoriatic lesions,a negative correlation was observed between the expression of Ang-1 and Ang-2(r=-0.521,P<0.05),and MVD was negatively correlated with the expression of Ang-2(r=0.656,P<0.01)but unrelated to the expression of Ang-l(r=0.237,P>0.05).Conclusions It is demonstrated that Ang-2 and Tie-2 closely correlate with the angiogenesis in psoriasis,and Ang-2 plays a dominant role in the regulation of angiogenesis in psoriasis.

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