OBJECTIVE: To investigate the effect of interferon-? and verapamil on the reversal of multidrug-resistance(MDR) in breast cancer cell line.METHODS: Drug-sensitive MCF-7 and ADM-induced MDR phenotypic MCF-7/ADR were taken as in vitro experimental models,which were treated with interferon-? and verapamil in combination or alone,then the cell survival rate,IC50,drug-resistant times and reversal times of different experiment groups were measured by MTT test.The quantitive expression of P-170 on the surface of cells was detected by flow cytometry.RESULTS: Treated by interferon-? and verapamil in combination,the IC50 of adriamycin-resistance was lowered to 0.32 ?mol?L-1,superior to verapamil(1.23 ?mol?L-1) or interferon-?(2.29 ?mol?L-1) alone;the reversal times increased to 51.88 in combination group,superior to interferon-? and verapamil alone(7.25 and 13.49 respectively) and the expression of P-170 were lower than in groups treated with interferon-? and verapamil alone.CONCLUSION: The efficacy of interferon-? and verapamil exhibited a more remarkable efficacy on the reversal of multidrug-resistance(MDR) in breast cancer cell line when used in combination than alone.

Objective:To establish an HPLC method for the simultaneous determination of minoxidil and tretinoin in compound minoxidil gel. Methods:A Shim-Pack VP-ODS C18(250 mm × 4. 6 mm,5 μm)column was used with the mobile phase of methanol and 2% acetic acid solution by gradient eleution . The detection wavelength was respectively set at 280 nm and 350 nm,and the flow rate was 1. 0 ml·min -1 . The column temperature was 35℃ and the injection volume was 10 μl. Results:Minoxidil and tretinoin had a good linear relationship within the range of 4. 0- 240. 0 μg·ml-1 and 0. 05- 3. 00 μg·ml-1(r =0. 999 6 and 0. 999 3),respectively. The average recovery was 99. 56%(RSD = 0. 42% ,n = 9)and 99. 36%(RSD = 0. 50% , n = 9),respectively. Conclusion:The established method is simple,and suitable for the simultaneous determination of the two constituents in compound minoxidil gel.

Objective:To establish a method to determine vitamin B 6 and fluocinonide in compound vitamin B 6 gel.Methods:The contents of vitamin B6 and fluocinonide in the gel were determined by HPLC with a Lichrospher CN C 18column (250 mm ×4.6 mm, 5μm).The mobile phase was heptane sulfonate solution (adding 6.8 g potassium phosphate monobasic and 1.0 g heptane sulfonate into 1000 ml water) -methanol –triethylamine (35:65:0.2,adjusting pH to 3.2 with phosphoric acid).The detection wavelength was 238 nm and the column temperature was 25℃.The injection volume was 20 μl.Results: Vitamin B6 had a good linear relationship within the range of 15.0-300.0 μg· ml-1(r=1.000 0), and the average recovery was 99.65% (RSD=0.3, n=9).Fluocinonide had a good linear relationship within the range of 0.4-8.0 μg· ml-1 (r=0.9990), and the average recovery was 99.35% (RSD=0.85, n=9).Conclusion:The method is simple and reproducible, and the result is accurate.The method can be used for the deter-mination of the gel.

OBJECTIVE:To investigate the effects of TanshinoneⅡA sodium sulfonate injection on levels of P-selectin,glial fi-brillary acidic protein (GFAP),vascular endothelial growth factor (VEGF) and neurological function in patients with acute cere-bral infarction. METHODS:A total of 114 patients with acute cerebral infarction selected from Lianyungang First People's Hospi-tal during Apr. 2013-Apr. 2016 were divided into control group and observation group according to random number table,with 57 cases in each group. Control group was given routine treatment. Observation group was additionally given Tanshinone ⅡA sodium sulfonate injection 40 mg 0.9％ sodium chlonride injection 250 mL,ivgtt,qd. A treatment course lasted for 7 d,and both received 2 courses of treatment. NIHSS scores,the levels of serum P-selectin,GFAP and VEGF were compared between 2 groups before treatment and after 7,14 d of treatment. The occurrence of ADR was also compared. RESULTS:Before treatment,there was no statistical significance in above indexes between 2 groups(P>0.05). Compared to before treatment,NIHSS score,the levels of se-rum P-selectin and GFAP in 2 groups were decreased significantly after 7,14 d of treatment,while the serum level of VEGF was increased significantly. These indexes of 2 groups after 14 d of treatment were significantly better than 7 d of treatment,except for NIHSS score. Above indexes of observation group was significantly better than those of control group during corresponding period, with statistical significance (P<0.05). No obvious ADR was found in 2 groups. CONCLUSIONS:For acute cerebral infarction, Tanshinone ⅡA sodium sulfonate injection can significantly reduce the levels of serum P-selectin and GFAP,improve VEGF level and promote the recovery of neurological damage with good safety.

Objective:To investigate the induction effect of NPPB,a chloride channel blocker,on the apoptosis of human glioma SHG-44 cells,and to explore its mechanism. Methods:The SHG-44 cells were cultured in vitro and divided into control group and NPPB groups (50,100,200 μmol· L-1 ).The cell viability was detected by MTT assay.The apoptotic rates were detected by flow cytometry.The expression levels of Bax, Bcl-2 and caspase-3 were detected by immunohistochemical analysis and Western blotting method.Results:Compared with control group,the cell viabilities of SHG-44 cells in 100 and 200 μmol·L-1 NPPB groups after treated for 24 and 48 h were decreased significantly (P < 0.01).The results of flow cytometry showed that the apoptotic rates of SHG-44 cells in 100 and 200 μmol·L-1 NPPB groups were 24.64% and 41.85%,and they were higher than that in control group (4.17%) (P <0. 01).The immunohistochemical analysis and Western blotting results showed that the expression levels of caspase-3 and Bax proteins in SHG-44 cells in 100 μmol · L-1 NPPB group were increased (P < 0.05 or P < 0. 01 ), and the expression level of Bcl-2 protein was decreased (P < 0.05 ). Conclusion:NPPB could induce the apoptosis of human glioma SHG-44 cells by the down-regulation of the expression of Bcl-2 and the up-regulation of the expression of Bax,and the activation of caspase-3.

Objective To investigate the role and mechanism of B7-H4, a negative costimulatory molecule, in mediating the immunomodulatory effects of mesenchymal stem cells C3H10T1/2 (C3H10) on T cell polarization. Methods The lentiviral vectors that carried the shRNA targeting mouse B7-H4 were transfected into mouse mesenchymal stem cells (C3H10-B7-H4). The cells were co-cultured with PHA-acti-vated mice spleen lymphocytes before and after the transfection. ELISA was performed to detect the concen-trations of cytokines in supernatants of cell culture in order to elucidate the effects of B7-H4 expressed by C3H10 on T cell polarization. A mouse model of experimental allergic encephalitis (EAE) was established. Fifty C57BL/6 mice were divided into five groups including control group, EAE group, C3H10 group (injec-ting EAE mice with C3H10 cells), C3H10-NC group ( injecting EAE mice with C3H10-NC cells) and C3H10-B7-H4 group (injecting EAE mice with C3H10-B7-H4 cells). ELISA was performed to detect the soluble form of IL-2, IL-17, IFN-γ and IL-4 in plasma samples. Results Knocking down the B7-H4 gene with shRNA significantly decreased the expression of B7-H4 on C3H10 cells, which weakened the inhibitory effects of C3H10 cells on the secretion of IL-2, IL-17 and IFN-γ by spleen lymphocytes. The therapeutic effects of C3H10-B7-H4 cells on mice with EAE were weakened after silencing the B7-H4 gene expression, which was manifested as higher nerve function score and earlier onset and bring forwarded peak time of EAE than those of the C3H10 group. Treating EAE mice with C3H10-B7-H4 cells was less efficient in inhibiting the expression of IL-2, IL-17 and IFN-γin plasma. However, knocking down the B7-H4 gene had no signif-icant effect on the expression of IL-4 in terms of treating EAE with C3H10 cells. Conclusion The co-inhib-itor molecule B7-H4 expressed on C3H10 cells mediated the treatment of EAE with C3H10 cells by regula-ting Th1 and Th17 effector T cells.

Objective:To evaluate the effect of emodin on liver injury in a mouse model of intestinal ischemia-reperfusion (I/R) and the role of heme oxygenase-1-mediated autophagy.Methods:Twenty-four SPF-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (Sham group), I/R group, emodin group (E group) and emodin plus HO-1 inhibitor Zinc Protoporphyrin Ⅸ (ZnPP) group (ES group). The intestinal I/R injury model was established by clamping the superior mesenteric artery for 45 min followed by 120 min of reperfusion. Emodin 40 mg/kg dissolved in 5% methylcellulose sodium was given by gastric gavage once a day for 5 days before ischemia in E group. Emodin 40 mg/kg dissolved in 5% methylcellulose sodium was given by gastric gavage once a day for 5 days before intestinal I/R, and ZnPP 7.5 mg/kg was injected via the tail vein at 12 h before ischemia in ES group. Orbital venous blood samples were collected at the end of reperfusion for determination of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations. Then the mice were sacrificed, and liver tissues were obtained for microscopic examination of the pathological changes (after HE staining) and for determination of the activity of superoxide dismutase (SOD), content of malondialdehyde (MDA), expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) mRNA (by fluorescent quantitative polymerase chain reaction), the expression of HO-1, autophagy-related protein Beclin1 and microtubule-associated protein 1 light chain 3 (LC3) (by Western blot). The LC3-Ⅱ/Ⅰ ratio was calculated. Results:Compared with Sham group, the activity of SOD was significantly decreased, the content of MDA and serum ALT and AST concentrations were increased, the expression of IL-6 and TNF-α mRNA and HO-1 was up-regulated, the expression of Beclin1 was down-regulated, the LC3-Ⅱ/Ⅰ ratio was decreased ( P<0.05), and the pathological changes of liver tissues were found in I/R group. Compared with I/R group, the activity of SOD was significantly increased, the content of MDA and serum ALT and AST concentrations were decreased, the expression of IL-6 and TNF-α mRNA was down-regulated, the expression of HO-1 and Beclin1 was up-regulated, the LC3-Ⅱ/Ⅰ ratio was increased ( P<0.05), and the pathological changes of liver tissues were significantly attenuated in E group ( P<0.05). Compared with E group, the activity of SOD was significantly decreased, the content of MDA and serum ALT and AST concentrations were increased, the expression of IL-6 and TNF-α mRNA was up-regulated, the expression of HO-1 and Beclin1 was down-regulated, the LC3-Ⅱ/Ⅰ ratio was decreased ( P<0.05), and the pathological changes of liver tissues were aggravated in ES group. Conclusions:Emodin can alleviate liver injury induced by intestinal I/R in mice, and the mechanism may be related to the activation of HO-1-mediated autophagy.

Objective To analyze the chemical compounds of Shenqi Dihuang decoction by the ultraperformance liquid chromatography coupled with linear quadrupole ion trap-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap-MS). Methods Warters ACQUITY UPLC HSS T3 (2.1 mm ×100 mm, 1.8 μm) was used as chromatographic column with mobile phase: 0.1% formic acid water (A)-0.1% formic acid acetonitrile (B) with gradient elution, and flow rate was 0.3 ml/min. Electrospray ion source (ESI) and an electrostatic field orbital ion trap mass analyzer were adopted, which was used to collect mass spectrometry fragment information with positive and negative ion modes, by comparing with the relative retention time of the reference substance. In addition, the fragment information of the mass spectrum was used to identify the compounds. The accurate identification of the chemical components in Shenqi Dihuang decoction was confirmed with literature. Results The study found that UPLC-LTQ-Orbitrap-MS technology could be used to identify 62 chemical components, including 13 aromatic acids, 9 flavonoids, 8 saponins, and 5 aromatic amines, 3 keto acids, 2 phenols, 1 aromatic quinone and other ingredients in Shenqi Dihuang decoction. Conclusion The identification analysis method in this study was efficient and accurate, which could be applied to the identification and analysis of chemical components in Shenqi Dihuang decoction and provided the important experimental data for the research on the material basis and mechanism.

Objective To explore the immunopathological mechanism for the imbalance between the positive signal mediated by inducible costimulator (ICOS) and the negative signal mediated by programmed death-1 (PD-1) in patients with myasthenia gravis (MG).Methods Eighty-two patients with MG,56 healthy controls (HC) and 20 non-MG (NMG) patients,collected in the First Affiliated Hospital of Suzhou University from February 2014 to December 2016,were chosen to participate in the study.The expression of ICOS and PD-1 on peripheral blood mononuclear cells was detected by immuno-fluorescence staining and flow cytometry.The levels of soluble programmed death-1 (sPD-1),soluble programmed death ligand 1 (sPD-L1),IL-4 and other cytokines were detected by enzyme-linked immunosorbent assay.Results (1) Flow cytometry analysis:The co-expression of PD-1,ICOS on CD4 + T cells from MG group (9.64％ (8.82％)) was higher than in HC (1.81％ (2.10％),Z =-7.389,P ＜0.05) and NMG group (2.86％ (1.49％),Z =-4.636,P ＜ 0.05).The expression of ICOS on CD4 + T cells,ICOS ligand (ICOSL) on CD14+ monocytes and CD19+ B cells were increased in MG group comparing with that of the control groups.The proportion of PD-1 + CD4 + T cells (MG group 16.82％ (10.66％),HC 9.34％ (9.18％),Z =-4.345,P＜0.05;NMG group 7.07％ (3.40％),Z=-4.594,P＜0.05) and PD-1 Ligand (PD-L1) + CD14+ monocytes was higher in MG patients.All of these were detected by flow cytometry.(2) ELISA analysis:Serum sPD-1 expression significantly increased in MG group compared with that in the control groups (MG group (1.87 ± 0.64) ng/ml,NMG group (1.49 ± 0.70) ng/ml,t =2.04,P ＜ 0.05;HC (1.05 ± 0.50)ng/ml,t =2.08,P ＜ 0.05),while for serum sPD-L1,there was no significant difference between MG and control groups.(3) Serum cytokines detection:The expression of IL-4 was increased in MG patients (MG group (61.88 ±5.15) pg/ml,HC (32.03 ±1.84) pg/ml,t=2.50,P＜0.05;NMG group (42.62± 3.31) pg/ml,t =2.34,P ＜0.05),and there was a negative correlation between the expression of sPD-1 and the concentration of IL-4.Conclusions The increased expression of PD-1 + ICOS + CD4 + T cells suggested the subset involved in the pathological progress of MG.sPD-1 might disturb the ligation of PD-1 on T cells and PD-L1 on antigen presenting cells,while the ligation of ICOS and ICOSL passed positive signal,leading to over activity of the subsets and the progression of disease.

In the earliest days,the idea that surviving a single infection often resulted in lifelong immunity to the infecting pathogen was recorded and then led to the discovery of vaccination.We have now confirmed that such protection is primarily based on the generation of immunological memory in antibody response.With the wide implementation of more and more vaccines around the world,it is well documented that different vaccines have different potential regarding to the duration of antibody response.In clinical observations,live-attenuated vaccines often elicit long-term immunity but are also accompanied with risks in safety that are hard to avoid.In order to develop novel vaccines with both excellent potential in eliciting antibody memory and low safety risk,it is critical to further investigate the mechanism of antibody memory in the perspective of immunology.Antibody memory is mediated by certain long-lived B cells:long-lived plasma cell can secret antibody to maintain serum antibody titer while memory B cell contributes to the rapid immune response during the secondary encounter of pathogens.Cellular and molecular processes that drive the production of long-lived plasma cells and memory B cells are subjects of intensive research and have important implications for global health.Several factors in the vaccine would indeed affect and regulate these processes,including the antigen valency,vaccine kinetics and the signal integration of both antigen and danger molecules.Many studies have focused on strategies to manipulate these factors to improve or develop new vaccines.Here,we will summarize our current knowledge on how the component in vaccines will affect their potential in generating and sustaining antibody memory,and also point out the challenges we face in the route of developing a"perfect"vaccine.