[Objective] To assess the role of imbalance between regulatory T (Treg) cells and T helper 17 (Th17) cells in the pathogenesis of atopic dermatitis (AD).[Methods] Peripheral blood was obtained from 41 patients with AD and 38 age- and sex-matched healthy controls.Flow cytometry was performed to determine the percentage of Treg cells (CD4+CD25+Foxp3+ T cells) and Thl7 cells (CD4+ILl7+ T cells),real-time quantitative reverse transcription (RT)-PCR to detect the mRNA expressions of Foxp3 and RORγt,which are the specific transcription factors of Treg and Th17 cells respectively.Serum concentrations of transforming growth factor (TGF)-β,IL-17 and IL-23 were measured by enzyme linked immunosorbent assay(ELISA).Data were statistically assessed by independent-samples t test and Pearson correlation analysis.[Results] The patients with AD showed an obvious decrease in Treg cell percentage,transcription factor Foxp3 mRNA level and Treg/Th17 ratio (2.01％ ± 0.57％ vs.5.04％ ± 1.44％,t =12.47,P＜ 0.01; 0.65 ± 0.19 vs.1.71 ± 0.69,t=9.47,P＜0.01; 1.26 ± 0.61 vs.14.53 ± 5.77,t =14.11,P ＜ 0.01),but a significant increase in peripheral Th17 cell percentage and transcription factor RORγt mRNA level (1.77％ ± 0.55％ vs.0.39％ ± 0.15％,t =14.82,P ＜0.01; 5.97 ± 1.45 vs.1.49 ± 0.57,t =17.78,P ＜ 0.01 ) compared with the healthy controls.Further comparison revealed that Treg/Th17 ratio was significantly lower in patients with acute AD than in those with subacute AD (0.88 ± 0.04 vs.1.29 ± 0.11,t =4.02,P ＜ 0.01 ) and those with chronic AD (2.05 ± 0.24,t =4.83,P ＜ 0.01 ),statistically different between patients with subacute AD and chronic AD (t =2.89,P ＜ 0.05).There was no significant difference in the serum concentration of TGF-β between patients with AD and healthy controls ((15.28 ± 2.34) μg/L vs.(16.56 ± 3.27) μg/L,t =1.96,P＞ 0.05).A significant increase was observed in the serum levels of IL-17 and IL-23 in patients with AD compared with those in the healthy controls( (33.24 ± 7.06)ng/L vs.(11.68 ± 2.67) ng/L,t =17.96,P＜ 0.01; (56.35 ± 12.16) ng/L vs.(18.43 ± 3.90) ng/L,t =18.36,P＜ 0.01).In patients with moderate and severe AD,SCORing atopic dermatitis (SCORAD) index was negatively correlated with the percentage of Treg ceils (r =-0.40,P＜ 0.05 ),but positively correlated with that of Th17 cells (r =0.42,P ＜ 0.05 ).[Conclusion]s There exists a change in Treg/Th 17 ratio,mRN A expressions of RORγt and Foxp3,and serum levels of relevant cytokines in patients with AD,which may lead to immune imbalance and subsequently contribute to the development of AD.

OBJECTIVE:To determine plasma concentration of caffeine,dapsone and chlorzoxazone in rats,and to calculate pharmacokinetic parameters. METHODS:6 rats were given the mixture of caffeine,dapsone and chlorzoxazone intragastrically, 1.5,2 and 3 mg/kg,respectively. 0.2-0.3 ml blood were collected before medication and 0.5,1,2,3,4,6,8,12,24 h after medication.The plasma sample was treated with solid phase extraction. The plasma concentration of caffeine,dapsone and chlorzoxa-zone were determined by HPLC using N-(2-Hydroxyethyl) phthalimide as internal standard. The pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS:The linear ranges of caffeine,dapsone and chlorzoxazone were all 0.2-30 μg/ml (r were 0.996 4,0.996 1,0.998 8,respectively). The limit of quantitation were 0.2 μg/ml. The recoveries of low-concentration, medium-concentration and high concentration were(84.8±3.6)%-(111.4±10.2)%(RSD were 4.3%-9.8%,n=3),(107.0±13.3)%-(113.5±8.1)%(RSD were 7.1%-14.0%,n=3),(104.2±10.8)%-(111.1±12.2)%(RSD were 8.0%-11.0%,n=3). Pharmacoki-netic parameters were as follows as tmax(1.70±0.99),(1.50±1.00),(1.92±0.80)h;t1/2(0.73±0.22),(2.77±1.35),(2.78±2.34) h;cmax (2.60 ± 0.50),(5.78 ± 1.19),(9.76 ± 1.37) mg/L;AUC0-t (8.43 ± 0.79),(20.68 ± 1.91),(26.71 ± 2.45) mg·h/L(n=6). CONCLUSIONS:The method is simple,sensitive and accurate,and can be used for the plasma concentration determination and pharmacokinetic study of caffeine,dapson and chlorzoxazone.

ObjectiveTo detect the expression levels of macrophage migration inhibitory factor (MIF) in peripheral blood mononuclear cells (PBMCs),sera and skin tissue fluid from patients with vitiligo vulgaris,and to investigate their clinical significance.MethodsThirty-nine patients with vitiligo vulgaris and 31 age- and sex-matched normal human controls were recruited in this study.Real-time reverse transcription-PCR was employed to assess the expressions of MIF mRNA in PBMCs,enzyme-linked immunosorbent assay (ELISA) to detect the concentrations of MIF in sera and skin tissue fluid from these subjects.ResultsPatients with vitiligo vulgaris showed a significantly higher level of MIF mRNA in PBMCs (6.70 (2.64 - 8.65) vs.1.67 (1.24 - 2.45),Z=5.895,P＜ 0.05),MIF protein in sera (32.76 (10.67 - 40.98) μg/L vs.7.89 (6.13 - 9.54) μg/L,Z=5.936,P ＜ 0.05 ) and skin tissue fluid ( 167.80 ( 107.40 - 219.60) μg/L vs.42.44 (32.29 - 49.74) μg/L,Z =4.715,P ＜ 0.05) compared with the normal human controls.The expression levels of MIF mRNA in PBMCs,and MIF protein in sera and skin tissue fluid were also higher in patients with progressive vitiligo than in those with stable vitiligo (7.89 (3.89 - 9.12) vs.5.62 (2.23 - 7.29),Z=2.213,P＜ 0.05; 37.80 (29.50 - 45.70) μg/L vs.22.70 (9.36 - 37.78) μg/L,Z=2.141,P＜ 0.05; 211.50 (131.70 - 248.75) μg/L vs.144.65 (89.13 - 167.30) μg/L,Z =2.100,P ＜ 0.05).In addition,the vitiligo area severity index (VASI) score was positively correlated with the expression levels of MIF mRNA in PBMCs (r =0.486,P ＜ 0.05)and MIF protein in sera (r =0.453,P ＜ 0.05).ConclusionMIF might play a certain role in the pathogenesis of vitiligo vulgaris.

Objective To investigate the inhibitory effect of immunosuppressive agent FRY720 on hepatocellular carcinoma Hepal-6. Methods Hepal-6 cells were cultured, and divided into 4 groups, namely control group and 0.1 μg/ml, 10 μg/ml, 100 μg/ml quality concentration groups. The cells were treated by the drugs for 24 to 48 hours respectively. The inhibitory rate of the cells was measured by MTT assay, and cell cycle and cell apoptotic rate were detected by flow cytometry (FCM). Results The ability of tumor cell growth were inhibited by FTY720 after 48 h. The maximal inhibition rate was 62.10 %, The apoptosis ratio was increased when FTY720 was 0.1-100 μg/ml, and it was 4.07 %, 8.16 %, 19.84 % respectively. FTY720 significantly prolonged cell G1 phase. Conclusion FTY720 could inhibit the growth of hepatocellular carcinoma, arrest the cell in G1 phase, and increase apoptosis.

OBJECTIVE:To establish a method for the content determination of total flavonoids in Morus alba. METHODS:UV-visible spectrophometry was performed with Al(NO2)3-NaNO2-NaOH color-test at the wavelength of 510 nm with the reference of rutin. RESULTS:The linear range of rutin was 0.031 2-0.156 mg/ml(r=0.999 9);RSDs of precision,stability and reproduc-ibility tests were lower than 2%;recovery was 95.7%-101.0%(RSD=2.1%,n=6). CONCLUSIONS:The method is simple,sta-ble and reproducible,and can be used for the content determination of total flavonoids in M. alba.

OBJECTIVE:To establish a method for the determination of residuals of petroleum ether,ethanol,xylene and ace-tic acid in ecabet sodium crude drug. METHODS:Capillary GC was performed on the column of PGE-20M capillary column at the flow rate of 1.7 ml/min,detector was hydrogen flame ionization detector,carrier gas was nitrogen with high purity,column temper-ature was 45 ℃,maintaining 4 min,it increased to 80 ℃ with rate of 10 ℃/min,then increased to 135 ℃ with rate of 30 ℃/min,maintaining 3 min,the injection mode was direct injection,inlet temperature was 250 ℃,and the volume injection was 1.0 μl. RESULTS:The mass concentration was 0.050-1.952 g/L for petroleum ether,0.050-1.941 g/L for ethanol,0.024-0.948 g/L for xy-lene and 0.050-1.947g/L for acetic acid(r=0.999 1-0.999 7);RSDs of precision,stability and reproducibility tests were no more than 1.7%;recoveries were 99.3%-101.0%(RSD=0.7%,n=9),102.3%-103.7%(RSD=0.4%,n=9),101.2%-102.1%(RSD=0.3%,n=9) and 100.3%-102.2%(RSD=0.6%,n=9),respectively. CONCLUSIONS:The method is simple and accurate,and can be used for the control of residual of organic solvents in ecabet sodium crude drug.

Objective To screen and identify effective small interfering RNA (siRNAs) targeting the conserved 5’ untrans-lated region (UTR) of the enterovirus 71 (EV71) genome. Methods Double-stranded siRNAs were designed to target the 5’UTR of the EV71 genome. The cytotoxicity effect of siRNAs on rhabdomyosarcoma (RD) cells was evaluated. The cytopathic effect on EV71-infected RD cells was observed under phase-contrast microscopy and the effective siRNAs were screened out by cell viability assay and real-time TaqMan RT-PCR assay. Results All the siRNAs did not exhibit any cytotoxic effects on the viability and growth of RD cells. Transfection of si-1 and si-2 targeting the 5’ UTR in EV71 genome into RD cells signiifcantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability and reduced the EV71 RNA transcripts. And the inhibitory effect of si-1/si-2 on EV71 replication was sequence-speciifc. Conclusions The 2 siRNAs (si-1 and si-2) targeting the conserved 5’UTR of the EV71 genome may have broad spectrum antiviral effects on many epidemic EV71 strains.

Objective To detect the expressions of miR-155,T helper type 17 (Thl7) cells,and Th17 cellspecific transcription factor RORγt and effector cytokine interleukin (IL)-17 in peripheral blood and skin lesions from,and to evaluate their relationship in,patients with atopic dermatitis (AD).Methods Peripheral blood was obtained from 37 patients with AD and 33 age-and sex-matched healthy controls,and biopsy specimens from the lesional and perilesional skin of five patients with severe AD as well as from the normal skin of five healthy human controls.Real-time fluorescence-based reverse transcription (RT)-PCR was employed to measure the mRNA expression levels of miR-155,RORγt and IL-17 in peripheral blood mononuclear cells (PBMCs) and skin specimens,flow cytometry to detect the percentage of Th17 cells in PBMCs,enzyme-linked immunosorbent assay (ELISA) to determine the plasma concentration of IL-17.Statistical analysis was done using independent sample's t test,one-way analysis of variance followed by the least significant difference test,and linear correlation analysis.Results Compared with the healthy controls,the patients with AD showed a significant increase in Th17 cell percentage (1.78％ ± 0.52％ vs.0.47％ ± 0.15％,P＜ 0.01),mRNA expression levels of miR-155 (5.78 ± 1.78 vs.1.82 ± 0.46,P＜ 0.01),RORγt (6.08 ± 1.04 vs.1.64 ± 0.52,P＜ 0.01) and IL-17 (7.09 ± 1.75 vs.1.71 ± 0.46,P＜ 0.01),as well as in the plasma concentration of IL-17 ((2.51 ± 6.15) pg/ml vs.(11.80 ± 2.24) pg/ml,P＜ 0.01).There was a sequential decrease in the expression levels of miR-155,RORγt and IL-17 mRNA from lesional skin,perilesional skin to normal skin (F =41.803,17.040 and 37.064 respectively,all P ＜ 0.01).The miR-155 mRNA expression level in PBMCs was positively correlated with the SCORing Atopic Dermatitis (SCORAD) index,Th17 cell percentage,RORγt and IL-17 mRNA expression levels as well as IL-17 plasma concentration (r =0.405,0.426,0.402,0.410 and 0.408 respectively,all P ＜ 0.05).Similarly,the miR-155 expression level was positively correlated with RORγt and IL-17 mRNA expression levels in lesional and paralesional specimens (r =0.428 and 0.435 respectively,both P ＜ 0.05).Conclusion The up-regulated expression of miR-155,Th17 cells and their effector cytokine IL-17 may be associated with the development of AD.

The levels of macrophage migration inhibitory factor (MIF) in peripheral blood mononuclear cell (PBMC) and serum in patients with Hashimoto's thyroiditis (HT) were detected by real-time PCR and ELISA,respectively.The results revealed that the expression of MIF mRNA in PBMC ( Z =-4.276,P＜0.01 ) and protein level in serum ( Z=-5.358,P＜0.01 ) were increased in HT patients,and positively correlated with thyroid specific autoantibodies and TSH levels.

Objective:To determine the effect of Notch1 signaling pathway on the differentiation and function of Th17 cells in murine psoriasis model.Methods: BALB/c mice were randomly divided into psoriasis model group and control group.Murine psoriasis model was established by topical 5% imiquimod application in combination with intraperitoneal injection of α-2b interferon.The CD4+ T lymphocytes were isolated by magnetic activated cell sorter (MACS).Flow cytometric analysis (FCM) was performed to detect the percentage of Th17 cells.Real-time RT-PCR was employed to measure the mRNA levels of RORγt,IL-17A,Notch1 and Hes-1.The CD4+ T lymphocytes were then divided into γ-secretase inhibitor DAPT groups and control group,and the expression differences of Notch1 signaling molecule and its target gene Hes-1 mRNA levels,Th17 cell percentage,RORγt and IL-17A mRNA levels,and IL-17A concentrations in cell-free supernatant were detected.Results: The expression levels of Th17 cell percentage and RORγt,IL-17A,Notch1 and Hes-1 mRNA in CD4+ T lymphocytes of murine psoriasis model were significantly higher than control mouse[(2.97±0.86)% vs.(0.65±0.11)%,t=15.083;(5.75±0.61) vs.(1.57±0.43),t=21.630;(7.83±0.97) vs.(1.63±0.31),t=25.348;(7.10±1.37) vs.(1.47±0.34),t=17.386;(7.30±1.15) vs.(1.67±0.48),t=18.840,respectively,all P<0.01].Compared with control group,Th17 cell percentage,mRNA expression levels of Notch1,Hes-1,RORγt and IL-17A,and IL-17A concentrations in cell-free supernatant from cultured CD4+ T lymphocytes of murine psoriasis model were dramatically decreased in DAPT treated groups in a dose-dependent way (F=74.368,89.719,126.572,94.558,124.323 and 123.231 respectively,all P<0.01).Conclusion: Notch1 signaling pathway can regulate the differentiation and function of Th17 cells in murine psoriasis model,and may have potential value for the target immunotherapy of psoriasis.