Objective:To establish a determination method for syringin, ferulic acid and salvianolic acid B in Huoluo Xiaotong tabletsbyHPLC.Methods:ThecolumnwasKromasilC18(150mm×4.6mm,5 μm),themobilephasewasmethanol-0.2% phos-phoric acid solution with gradient elution at the flow rate of 1. 0 ml·min-1 , the detection wavelengths were 265nm for Sgnigin, 321nm for Ferulic acid and 286nm for Salvianolic acid B. The column temperature was 25℃. Results: Syringin, ferulic acid and salvianolic acid B showed good linear relationship within the range of 64. 500-1. 032 × 103 , 7. 525-120. 400, 65. 250-1. 044 × 103 μg·ml-1 with the average recovery of 100. 75%, 99. 56% and 99. 75%, respectively. Conclusion:The method is simple, accurate and rapid.

OBJECTIVE: To prepare double-layer sustained release ornidazole films and investigate its release rate in vitro.METHODS: The double-layer sustained release ornidazole films film were prepared by the method of homogenate.The formula of the films were optimized by orthogonal experiment with the mass ratio of polyviol to CMC-Na(factor A),the total amount of polyviol and CMC-Na(B) and the amount of glycerol(C) as factors.The concentration of ornidazole was determined by ultraviolet spectrophotometry and its release rate was also computed.RESULTS: The double-layer sustained release film was well-shaped.The optimized formula of the film was as follows: factor A=1∶1,B=4.5 g,C=4.0 g.The standard curve of ornidazole was linear within a range of 1～20 ?g?mL-1(r=0.999 5,n=6),with an average recovery of 100.3%(RSD=1.3%,n=5).A continuous releasing of 11.0 hours was achieved for the prepared films and its cumulative release amount in vitro within 8 hours reached 81.50%.CONCLUSIONS: The preparative method of the films was simple,and the film had a slow-release property.

Objective To evaluate the efficacy of metal stent placement for the treatment of malignant gastroduodenal obstruction. Methods Medical records of 24 patients with gastroduodenal obstruction secondary to malignant tumors (gastric cancer, 19 patients; pancreatic cancer, 4 patients; postsurgical biliary duct cancer, 1 patient) from October 2002 to November 2004 were reviewed. Under radioscopy, a self-expanding metal stent was placed endoscopically into the stenosis site of the stomach or the duodenum. Results All the metal stents were placed at the expected position. After operation the patients were given fluid diet at 1 day and soft diet at 3 days. No stent-related gastrointestinal perforation or hemorrhage was observed. Follow-up for 1~24 months in 21 patients found re-obstruction in only 2 patients and no migration of metal stents. Conclusions Self-expanding metal stent placement for malignant gastroduodenal obstruction is safe and effective.

Objective The aim of the study WSfl to establish the method using CDl27 as the new biomarker to identify regulatory T cells(Treg cells)and apply the CD 127 to detect the Treg cells in patients with gastric cancer.Methods The phenotypes of Treg cells were analyzed using five-color flow cytometry method Foxp3.FITC/CD127-PE/CD4-PerCP/CD25-APC/CD3-PC7.The mRNA and protein expression of Foxp3 in isolated CD+4 CD25high CD127-/low Treg cells were detected.The relationships between Foxp3 and CD127 protein expression in CD4+ T cells from aduh human peripheral blood were investigated.PBMCs,Ascltes,turnor-infihration lymphocyte and tumor-draining lymph nodes in 35 patients with gastric cancer and PBMCs in 20 normal healthy donors were evaluated for the proportion of Treg ceils,as well as the percentage ot the total CD +4 cells.Results CD4+ CD25 high CD-127 low ceils expressed the high Foxp3 in protein level(87.1%)and mRNA level.Within the CD4+CD+25 population,there was a significant correlation between Foxp3 and the CD127low phenotype(r=0.985,P<0.01).Compared with healthy olunteers,patients with gastric malignancies had a higher proportion of CD4+4 Cdhigh 25 CD-low127 cells in peripheral blood(t=2.542,P<0.05).The Dereentages of Treg cells were more abundant in ascites(t=2.357,P<0.05),TIL(t=6.174,P<0.01) and tumor-draining lymph nodes(t=5.481,P<0.01)of individuals with gastric cancer than that in their blood.There were significant differences in the prevalence of Treg ceils between the early and advanced disease stages in gastric cancer[(6.04±2.31)%in stageⅠ+Ⅱ VB(10.16±2.29)% Ⅲ+Ⅳ,t=2.473,P<0.05].Conclusions The CD127 biomarker can be used to selectively enrich human Treg cells for in vitro functional studies.The populations of CD4+ CD high25 CD127-/low Treg cells increased ith tumor stage in individuals with gastric cancer.

Objective To investigate the populations of CD+4CD25high Foxp3+ regulatory T cells(Treg cells)and mRNA expression of Foxp3 in peripheral and the marginal region of tumor from patients with gastric cancer,and to evaluate the prevalence of Treg cells from patients with gastric cancer in relation to the TNM stages.Methods PBMC,Ascites,tumor-infiltration lymphocyte and tumor-draining lymph nodes in 47 patients with gastric cancer(TNM I11,Ⅱ18,Ⅲ10,Ⅳ8)and PBMC in 20 normal healthy donors were evaluated for the proportion of Treg cells,as a percentage of the total CD4+ cells,by flow cytometric analysis.Levels of mRNA for Foxp3 were measured witll a real-time quantitative PCR Results The percentage of CD4+CD25high Foxp3+ Treg cells in PBMC for cases of gastric cancer were significantly higher than those for healthv donors(P<0.05).The percentage of Treg cells were more abundant in ascites(P< O.05),1rIL(P<0.01)and tumor-draining lymph nodes(P<0.01)of individuals with gastric cancer than in their blood.And the Foxp3 mean fluorescent intensity (MFI) of Treg cells in TIL and tumor draining lymph nodes with gastric caners were significantly higher than PBMC and ascites(P<0.05).The proportion 0f Treg cell in patients of gastric cancers with stage I,II,Ⅲ,Ⅳ were(5.72±1.95)%,(6.45± 2.23)%,(9.58±3.13)%,(11.70±2.30)%,respectively.There were significant correlation between Drevalenee of Treg cells and disease stages in gastric cancer(r=0.784,P<0.01).Foxp3 mRNA levels were higher in PBMC from the group with gastric cancers than in the group with healthy donors.Foxp3 mRNA levels were correlated with TNM stages(r=0.623,P<0.05).Conclusions Our results suggest that the populations of CD4+CDhigh25 Foxp3+Treg cells and Foxp3 mRNA levels in patients with gastric cancer are signifieantlv higher in comparison to those in control donors.In addition,the expression of Fox：p3 mRNA increased with tumor stage.The increased prevalence of Treg cells may be one of the explanations for impaired cell-mediated immunity in cancer bearing hosts.

Objective To investigate the effects of IL-12 coexpression level on antigen expression and immune responses induced by HBsAg DNA vaccination. Methods DNA vaccine plasmid pHBV carrying codon-optimized preS2-S gene of reference sequence CHN-HBV07-C in China was constructed. Three DNA vaccine plasmids pHBV-12i, pHBV-12l and pHBV-12h were also constructed by subcloning three different IL-12 expression cassettes with various expression strengths to plasmid pHBV, respectively. Expression levels of IL-12 and HBsAg in vaccine plasmid-tranfected 293T cells were measured by quantitative ELISA. DNA vaccines were administered intramuscularly to BALB/c mice and HBsAg-specific cellular immune responses were determined by IFN-γ ELISPOT. HBsAg-specific antibodies were tested by Chemiluminescence Quantitative Immunoassay. Results The HBsAg expression level in 293T cells was 70 ng/ml when transfected by plasmid pHBV without IL-12 expression cassette, and the HBsAg level was 18 ng/ml when transfected by plasmid pHBV-12l carrying low-level IL-12 expression cassette, whereas the HBsAg level was only 6 ng/ml when transfected by plasmid pHBV-12h carrying high-level IL-12 expression cassette.Results of DNA vaccination revealed that HBsAg-specific humoral and cellular immune responses were significantly decreased in mice administering vaccine pHBV-12h carrying high-level IL-12 expression cassette. Although HBsAg-specific antibody responses in mice inoculated with pHBV-12l were also decreased when compared with those in pHBV-vaccinated mice without IL-12 expression, the HBsAg-specific cellular immune responses were significantly increased. Conclusion High-level coexpression of IL-12 may suppress the expression of HBsAg, Whereas modest coexpression of IL-12 significantly enhanced the HBsAg-specific T cell responses induced by DNA vaccination. Therefore, it is so important to balance the expression between adjuvant and antigen to enhance the immune response.

OBJECTIVEBy studying the possibility of obtaining expression of human hepatitis B virus (HBV) genes and production in normal liver cells from heterologous species like normal primary duck hepatocytes (PDH), to investigate the species-specificity of HBV infection and replication.

METHODSTwo days after transfecting the complete HBV genome into PDH by electroporation (transfected group), HBsAg and HBeAg in the supernatants and lysates of PDH were measured by the IMX system. Meanwhile, replication of HBV in PDH was analyzed by Southern blotting and dot blotting procedures. PDH was electroporated as control.

RESULTSHBsAg in the lysate of transfected group was 9.10 (P/N values, positive?2.1), HBeAg was 1.0 (negative?2.1), both were negative in the supernatants of transfected group. dot blotting revealed that transfected group was strongly positive, whereas the control group was negative. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (RC), covalently closed circular (CCC) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, and there was no integrated HBV DNA in the cellular genome. Control groups were negative.

CONCLUSIONSReplication of HBV can occur in hepatocytes from nonmammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

Animals ; Cells, Cultured ; DNA Replication ; physiology ; DNA, Viral ; biosynthesis ; Disease Models, Animal ; Ducks ; Electroporation ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; metabolism ; virology ; Humans ; Species Specificity ; Transfection ; Virus Replication

Objective To discuss influence and curative effect observation of sodium hyaluronate on serum matrix metalloproteinase-9,12 and 13 (MMP-9, 12 and 13) levels of patients with knee osteoarthritis (OA). Methods Selected 70 cases of patients with knee OA, and divided into observation group (n=35 cases) and control group (n=35 cases) in accordance at random. The patients in two groups were given oral 75 mg diclofenac sodium enteric capsules , once a day for 5 weeks. The patients in observation group were additionally given 2 mL sodium hyaluronate , injected into in-tra-articular, once a week, for 5 weeks. Excepted for sodium hyaluronate injection fluid, the patients in control group were given the same medical treatment as that in observation group. The changes of serum MMP-9 , 12 and 13 levels between the two groups before and after medical treatment were observed , and the clinical curative effect and untoward effect was compared as well. Results After 5 weeks’ treatment, the serum MMP-9, 12 and 13 levels of patients in two groups were declined more significantly than before with different degrees (P<0.05 or P<0.01), and the declining rate of patients in observation group was much higher than that in control group (P<0.05). The clinical good and excellent rate with in observation group(85.71%) was much higher than that in control group (62.86%)(χ2=4.79,P<0.05). 9 and 11 cases of untoward effect were appeared between control group and observation group respectively with light symp-tom, and after comparing the occurrence rates of untoward effect of patients in two groups, with no statistical differ-ences (χ2=0.28,P>0.05). Conclusion Sodium hyaluronate has favorable curative effect on patients with knee OA, whose mechanism of action has close effect on reducing serum MMP-9 ,12 and 13 levels.

To prepare recombinant human retinol binding protein 4 (RBP4) by using the baculovirus expression system and to detect its immunogenicity, the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual(pFBd), and the corresponding recombinant transfer plasmid was transformed into E. coli strain DH10bac, after transposition recombinant shuttle bacmid was screened out. The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated. After amplification of recombinant baculovirus, the recombinant baculovirus seeds were obtained. To express human RBP4, logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression. Finally, to prepare a batch of RBP4 protein, logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification. Finally for preparation of polyclonal antibody and evaluation of immunogenicity, the recombinant hRBP4 from insect cells and from E. coli were immunized rabbits. Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly, and subsequently recombinant RBP4-bacmid was generated successfully. SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa. The recombinant RBP4 protein could be secreted into the medium efficiently, and the expression level was calculated amount of 100 mg/L. Finally the rabbit antiserum was harvested after recombinant RBP4 immunization, therein the titer of antiserum against baculovirus recombinant RBP4 is 1:100 000 whereas the titer of antiserum against E. coli recombinant RBP4 is only 1:10 000. Overall, human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system, and high affinity antiserum was obtained. A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit.
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Humans ; Immune Sera ; Insecta ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Retinol-Binding Proteins, Plasma ; biosynthesis ; immunology ; Sf9 Cells ; metabolism ; Transfection

OBJECTIVEBy clarifying the natural history of chronic hepatitis B, to evaluate its long-term therapeutic outcome, antiviral drugs efficacy and economic significance.

METHODSA cohort of 183 (mean age of 31.75?.03 years, male/female ratio: 152:31) chronic hepatitis B patients with biopsy-proven and 247 cases of general population as control were followed up by retrospective cohort study. The follow-up time was 11.81?.08 years. This study was focused on long-term clinical outcome including the rate of liver cirrhosis, hepatocellular carcinoma and death, the long-term effect of antiviral drugs and prognostic factors.

RESULTSIn chronic hepatitis B patients, 22 (12.02%) developed liver cirrhosis, 12 (6.56%) hepatocellular carcinoma, and 20 (10.93%) died. The cumulative survival probabilities were 97.27%, 91.62%, and 84.47% in 5, 10, and 15 years, respectively. The cumulative probabilities of HCC were 0.00%, 3.19%, and 11.56% in 5, 10, and 15 years, respectively. In 247 control subjects, 6 (2.43%) died, none of them developed cirrhosis or HCC. The rates of death, liver cirrhosis, and HCC in hepatitis B patients were markedly different (P<0.005) compared with controls. The overall mortality of hepatitis B patients was 4.50 folds of the general population. Cox multiple regression analysis showed that old age, severe histological injury, and the positive HBeAg were closely related to liver cirrhosis, while old age, severe histological injury, and male were major factors leading to death. The independent variable of predicted HCC was not found.

CONCLUSIONSThe long-term outcome of hepatitis B is poor.

Adolescent ; Adult ; Aging ; physiology ; Carcinoma, Hepatocellular ; epidemiology ; etiology ; Cohort Studies ; Female ; Follow-Up Studies ; Hepatitis B e Antigens ; physiology ; Hepatitis B, Chronic ; complications ; epidemiology ; mortality ; Humans ; Liver Cirrhosis ; epidemiology ; etiology ; Liver Failure ; physiopathology ; Liver Neoplasms ; epidemiology ; etiology ; Male ; Middle Aged ; Regression Analysis ; Retrospective Studies ; Risk Factors ; Sex ; Survival Rate