Objective To replace the endogenous CTL epitope LLD in HBsAg with EYILSLEEL of glypican(GPC3)in order to prepare a GPC3-HBsAg multiple peptides vaccine for HBV infection.Methods The HBsAg/GPC3 DNA was amplified by gene splicing by overlap extension(SOEing)PCR from pcHBsAg plasmid and then inserted into pBSSK+ vector to construct a pBSSK/GPC3 vector.The vector was then identified by PCR,double digesting and sequencing.The fragment encoding GPC3 CTL epitope EYILSLEEL was obtained by double digesting the plasmid pBSSK/GPC3 and then inserted into pcDNA3.1+ vector.Results Sequencing and restrict endonulease digestion indicated that eukaryotic expression vector pcDNA-HBsAg/GPC3 was constructed successfully.Conclusion The endogenous CTL epitope LLD of HBsAg is replaced by the EYILSLEEL,and an eukaryotic expression vector pcDNA-HBsAg/GPC3 is constructed.

Objective To assess the behavior of children with tic disorder (TD),and to analyze the behavioral characteristics among children with TD at different clinic conditions.Methods Sixty-three children with TD were evaluated with Child behavior checklist (CBCL).ANOVA and t-test were used to analyze the difference in the total and individual scores of CBCL in the children classified according to the different clinical types,the severity of TD,and comorbid attention-deficit hyperactivity disorder(ADHD).Results There were no significant differences among the total and individual scores of CBCL in the patients of the different clinic types( P ＜ 0.05 ) ;the scores of body complain in the patients in moderate to severe conditions (4.15 ± 2.34) were higher than that of those in mild condition ( 2.68 ± 2.22 ) ( t =- 2.540,P =0.014) ; the scores of attention problem (9.94 ± 3.57 ),disciplinary offence ( 3.94 ± 3.06 ),aggressive behavior ( 15.39 ± 5.12 ),exportoriented behavior problems ( 13.98 ± 7.34)and behavior problem (47.89 ± 17.51 )in TD comorbid ADHD were higher than in simple TD group ( 7.31 ± 3.34,2.44 ± 2.22,7.24 ± 4.93,9.78 ± 6.55,37.07 ± 17.98 ) ( t =- 2.774,- 2.166,- 1.930,- 1.956,- 2.174,P =0.007,0.034,0.048,0.04 1,0.034 ).Conclusion Children with TD at different clinical conditions have varied behavioral problems and behavioral characteristics,while comorbid ADHD is the most significant factor to affect TD patient's behaviors.

AIM To study the chemical constituents from Securidaca inappendiculata Hassk..METHODS The dichloromethane and ethyl acetate fractions of S.inappendiculata 95％ ethanol extract were isolated and purified by silica,gel column and recrystallization,then the structures of obtained compounds were identified by physicochemical properties and spectral data.RESULTS Ten compounds were isolated and identified as 8-hydroxy-1,3,4-trimethoxyxanthone (1),1,2,7-trimethoxyxanth-one (2),4-hydroxy-3,5-dimethoxybenzaldehyde (3),sesamin (4),2-methoxy-3,4-methylenedioxybenzophenone (5),1,3,7-trihydroxy-4-methoxyxanthone (6),1,3,7-t-rihydroxy-2-methoxyxanthone (7),2-hydroxy-1,7-dimethoxyxanthone (8),3,8-dih-ydroxy-1,4-dimethoxyxanthone (9),oα-spinasterol (10).CONCLUSION Compound 1 is first found in the form of natural product,compounds 2-4 are isolated from genus Securidaca for the first time.

Objective To evaluate the regulatory role of acetylcholine receptor in muramyl dipeptide (MDP)-induced activation of Nod-like receptor 2/receptor-interacting protein 2 (2NLR2/RIP2) pathway in macrophages of mice.Methods RAW264.7 cells at the logarithmic growth phase were seeded in 12-well plates (density 1 × 106 cells/ml,2 ml/well),a total of 108 wells.The cells were randomly divided into 3 groups (n =36 each) using a random number table:control group (group C),MDP group (group M),and GTS-21 (a7nAChR specific agonist) group (group G).The cells were routinely cultured in group C.MDP with the final concentration of 10 μg/ml was added to the culture medium in group M.MDP with the final concentration of 10μg/ml and GTS21 with the final concentration of 50 μg/ml were added to the culture medium in group G.The cells were incubated for 24 h.At 1,6 and 24 h of incubation with MDP,12 wells were chosen and the cell suspension was obtained for measurement of NLR2 mRNA expression (by real-time fluorescent quantitative PCR),RIP2 expression (by Western blot),and concentrations of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) in the culture media (by ELISA).Results Compared with group C,the levels of NLR2 mRNA,RIP2,TNFα and HMGB1 were significantly increased at each time point in group M (P ＜ 0.05).Compared with group M,the levels of NLR2 mRNA,RIP2,TNF-α and HMGB1 were significantly decreased at each time point in group G (P ＜ 0.05).Conclusion Acetylcholine receptor can suppress MDP-induced transduction of NLR2/RIP2 pathway in macrophages of mice.

Objective To investigate the influence of β-catenin gene deletion on Stat-5α phosphorylation in bcr-abl induced leukemia cells. Methods The established conditonal hematopoitic β-catenin knockout mice were used to isolate bone marrow cells. Exogenous bcr-abl fusion gene was transduced to these bone marrow cells by retroviral infection with intent to transfom them to leukemia cells.Immunofluorescence was performed to detect the phosphorylation status of Stat-5α in both β-catenin deletion cells and control cells. bcr-abl transcription and protein levels were evaluated with real-time PCR and western blotting. Results Phosphorylation of Stat-5α was reduced significantly in β-catenin deletion leukemia cells on comparison with control cells despite that total Stat-5α protein showed no obvious changes. Total tyrosine phosphorylation and bcr-abl protein expression were reduced in bcr-abl induced β-catenin deletion CML cells,on the contrary, both of the reduction were not seen in bcr-abl induced β-catenin deletion ALL cells.Conclusion Loss of β-catenin inhibits both Stat-5α phosphorylationin and bcr-abl expression in bcr-abl induced leukemia cells.

Objective To investigate the therapeutic effect of esomeprazole,a new type 0f proton pump inhibitor(PPI),on patients with reflux esophagitis(RE).Methods 80 cases with RE diagnosed by endoscope were randomly divided into treatmeat group(n=40)and control group(n=40).The treatmeat group was treated with esomeprazole 20 mg bid,compared with the control group famotidine 20mg bid.The two courses of treatment both persisted for four weeks and included mosapride 5mg tid.At the end of therapy,the rate of relief of heartburn,acid reflux and substernal burning symptom,and the total effective rate confirmed by endoscope were recorded.ResuIts The clinical symptoms relief rates were 92.5%in the treatment and 62.5%in the control group raspectively and had obviously statistical difference(P＜0.05).The total effective rate was confirmed by endoscope were 82.5%and 55.0%respectively in two groups(P＜0.05).Conclusion Esomeprazole is a type of effective drug for patients with RE.

Objective To investigate the role of mutations in C region that may contribute to occult hepatitis B virus infection.Methods C genes were amplified from two OBI blood donor samples respectively.Plasmids with mutations in C region of hepatitis B virus were constructed by overlapping PCR.HBsAg and HBeAg in Huh7 cells and in the serum of Balb/c mice were detected by CMLA.HBcAg in liver tissue was detected by immunohistochemistry,while HBV-RNA was tested by RT-PCR.Results Mutations in C region significantly reduced the expression level of HBeAg and HBcAg,but had no significant effect on HBsAg and HBV-RNA.Conclusion The mutations in C region affect the expression level of HBeAg and HBcAg,which may play an important role in the occurrence of OBI.

Objective To evaluate the oxacillin disk screening test for screening Streptococcus pneumoniae by the penicillin Etest method.Methods 96 clinically isolated non-meningitis strains of Streptococcus pneumoniae were collected.The sensitivity and spe-cificity of the oxacillin disc screening test was evaluated by the penicillin Etest method as a standard method for detecting the peni-cillin susceptibility.Results Among 96 non-meningitis strains of Streptococcus pneumoniae,the penicillin-susceptible Streptococcus pneumoniae(PSSP)strains detected by the penicillin Etest method accounted for 96.9%(93/96),the penicillin intermediate Strep-tococcus pneumoniae(PISP)strains accounted for 3.1%(3/96)and no penicillin resistant Streptococcus pneumoniae(PRSP)strain was found.But 16 PSSP strains were detected by the oxacillin disc screening test with the sensitivity of 17.2% and the specificity of 100.0%,respectively.The difference between the oxacillin disc screening test and the penicillin Etest method was statistically sig-nificant(χ2 =77,P <0.01 ).Conclusion The oxacillin disc screening test has the low sensitivity for preliminarily screening non-meningitis strains of Streptococcus pneumoniae.Most of Streptococcus pneumoniae must be detected by the minimum inhibitory concentration(MIC)methods such as the penicillin Etest method.

Objective To investigate the effect of electro-acupuncture at zusanli on Severe thermal injury-induced acute lung injury in rats.Methods Forty male SD rats weighing 200-250 g were used in this study.Thirty percent of the total body surface (TBS) was shaved chemically with 20% sodium sulfate and then exposed to 99-100℃ water for 12 s.The animals with third degree thermal injury involving 30% TBS were randomly divided into 5 groups(n=8 each):group Ⅰ control(group C);groupⅡ thermal injury;group Ⅲ electro-acupuncture at zusanli;group Ⅳ electric stimulation of non-acupoint and group Ⅴ electro-acupuncture at zusanli+α-bungarotoxin α-BGT).In group Ⅲ,Ⅳ,and Ⅴ electro-stimulation(3 v,2 ms,3 Hz) of zusanli or non-acupoint was performed for 12 min immediately after thermal injury model was established and every 8 h.hung specimens were obtained at 48 h after thermal injury for microscopic examination.The pulmonary HMGB-l protein level was measured by ELISA.The expression of HMGB-1 mRNA and protein in the lung was determined by RT-PCR and immuno-histochemistry respectively.Results Thermal injury induced leucocytosis in the interstitial capillaries,interstitial edema,intra-alveolar fibrin deposit,blebbing of type Ⅱ alveolar lining cells and decrease in lamellar body.Both expression of HMGB-1 mRNA and protein in the lung was significantly enhanced at 48 h after thermal injury.Electrical stimulation of zusanli significantly down-regalated the expression of HMGB-1 mRNA and protein in the lung.However,α-BGT pretreatment reversed the effects of electrical stimulation of zusanli.Conclusion Electrical stimulation of zusanli could significantly ameliorate severe thermal injury-induced acute lung injury through inhibition of HMGB-1 mRNA and protein expression and activation of cholinergic anti-inflammatory pathway mediated by nicotinic acetylcholine receptor α7 subunit.

Objective To investigate the humoral and cellular immune responses in patients with acute brucellosis, and evaluate dynamic changes of regulatory T-lymphocytes (Foxp3+ Treg) in the peripheral blood of patients during treatment, in order to clarify the relationship between immunosuppression and the therapeutic effect in human brucellosis.Methods Sixty-five patients with brucellosis hospitalized at the Third Department of Infectious Diseases, Heilongjiang Agriculture and Reclamation Bureau General Hospital between July 2015 and November 2015 were included.Twenty-eight patients were treated with conventional therapy (group A: patients received 3 courses of treatment.Each lasted for 20 days with one-week interval), and 37 patients were treated with conventional therapy in combination with immunopotentiator (group B).Thirty healthy volunteers were enrolled as the controlled group.The ratio of CD3+CD4+ Foxp3+ Treg cells in the peripheral blood of brucellosis patients were measured by flow cytometry (FCM) at the end of each course of treatment.Data in accordance with normal distribution were described as mean±standard deviation.Comparison between two groups was done by two sample t test.Comparison among multiple groups was performed by analysis of variance and SNK test.Data that did not fit the normal distribution were analyzed by multiple-sample nonparametric test.Results After the first (20 d), second (50 d) and third course of treatment (80 d), the ratios of Foxp3+Treg in the peripheral blood of 65 acute brucellosis patients were 2.83%, 3.77% and 4.03%, respectively, which were all significantly higher than control group (1.69%;t=5.97, 9.05 and 5.66, respectively, all P<0.01).At the end of the first course of treatment, the ratios of Foxp3+Treg in group A and B showed no statistically difference (t=0.33, P>0.05), while those were both higher than control group (t=7.09 and 4.94, respectively;both P<0.01).At the end of the second course, the ratio of Foxp3+ Treg in group B was higher than group A (t=2.22, P<0.01), and both of them were higher than control group (t=10.79 and 7.25, respectively;both P<0.01).At the end of treatment, Foxp3+ Treg in group A was also significantly higher than the other two groups (t=6.02 and 6.45, respectively;both P<0.01).Conclusions In patients with acute brucellosis treated with the standard antibiosis treatment in combination with immunopotentiator, the ratio of Foxp3+Tregs significantly increases and maintains at a high level, which suggests that extra immunopotentiator may be not helpful for the treatment of brucellosis at the very early stage.