Objective To construct the eukaryotic expression vector coding HCV gene E2 fused with His-Tag, and to express fused protein in CHO cells for investigating the function of HCV envelope protein E2. Methods The gene encoding HCV envelope protein E2 was amplified from pBRTM/HCV1-3011, a plasmid containing the cDNA of HCV's ORF, by polymerase chain reaction (PCR) method and cloned into the vector pET28(a) containing His-Tag to obtain the fused HCV envelope protein E2 gene fused with His-Tag. The fused gene was cloned into pcDNA3.1 to construct the recombinant plasmid pcDNA3.1-His-E2, which will express the E2 protein, fused with His tag. This recombinant plasmid was transfected into CHO cells by Lipofactamine 2000 reagent. The fused protein was identified by indirect immunofluorescence (IIF) and Western-blot (WB) methods. Result The positive results were obtained when the fused protein of HCV E2 with His-Tag were identified by IIF and WB methods. Conclusion The eukaryotic expression vector pcDNA3.1-His-E2 was constructed successfully and the fused proteins were expressed in cells.

Objective To observe the specific immune responses and the protection against P815 mastocytoma cells stably expressing HBV surface antigen in H-2 d mice after DNA immunization of HBV surface antigen gene (pCR3.1-S). Methods The immunization was performed by intramuscular injection of DNA vaccine (pCR3.1-S). P815-HBV-S was inoculated subcutaneously into mice three weeks after DNA immunization. The tumor growth was measured every five days. HBsAg specific cytotoxic T lymphocyte (CTL) activity was measured by 51 Chromiunm release assay. Results HBV DNA vaccine can evidently inhibit the tumor growth, prolong the survival period and improve the survival rate in mice. Meanwhile, HBsAg specific CTL activity was obviously increased after DNA immunization. Conclusions The results show that the DNA vaccine, pCR3.1-S, has strong antigenecity in cellular immunity and has marked killing effect on HBV infected cells in vivo. DNA vaccine against HBV may be useful for both prophylactic and therapeutic purposes.

Perilla frutescens callus were induced from leave explants on MS medium suplemented with NAA and 2,4-D. The Rosmarinic acid (RosA) content of dried callus was 0.85%. The RosA was extracted from the callus with 80% alcohol and purified through extraction with ethyl acetate and a Sephadex LH-20 column chromatography. The parity of the final product was 95% as analyzed by HPLC RosA could inhibit the growths of Escherichia colt, Staphylococcus aueris and Rhizotonia with MICs of 300, 400, 800?g/mL, respectively.

Retmperitoneal ganglioneuroma is a rare neurogenic benign tumor.The prognosis of patients was good when the tumor was completely resected,while the surgical procedure is complicated.In March of 2013,a male patient with complex retroperitoneal ganglioneuroma was treated at the First Affiliated Hospital of Chongqing Medical University.A hypoechoic solid lesion (size,6.5 cm ×4.5 cm) adjacent to the head of the pancreas was detected by color Doppler ultra-sonography 9 months ago,and no any other clinical symptoms were detected.Perioperative abdominal computed tomography and the surgery confirmed that the tumor (size,8.5 cm × 7.5 cm × 4.5 cm) was located beneath the pancreas,encompassing thc ccliac artery,hepatic artery,splenic artery and superior mesenteric artery,surrounding the head and uncinate process of the pancreas,making it impossible to be separated.The tumor was closely connected with the portal vein,superior mesenteric vein,splenic vein and left renal vein.The tumor was separated from the major blood vessels,the body and tail of the pancreas,while the tumor could not be resected from the pancreatic head,and thus tumor resection and pancreaticoduodenectomy were performed.The surgery was extremely diffcult,but the complete removal of tumor was successfully achieved without excision of the major blood vessels and the patient recovered well.

Objectives：To observe the effect of eukaryotic expression vectors coding IL-2 and IL-12 on immune responses induced by DNA immunization of HBV surface antigen（pCR3.1-S）in BABL/c(H-2d) and the protection against P815 mastocytoma cells stable expressing HBV surface antigen in mice after immunized with HBV gene vaccine.Methods：The immunization was performed by intramuscular injection,three weeks later,we directly inoculated P815-HBV-S into mice by subcutaneous injection .Tumor growth was measured every five days.Anti-HBs in serum was detected by ELISA and HBsAg specific cytotoxic T lymphocytes (CTLs) activity was measured by 51 Chromium release assay.Results：Eight weeks after immunization,the A value of mice serum in 450 nm and CTLs activity of mice codiog IL-2 and IL-12 eukaryotic expression vectors were significant higher(P<0.05) than that of mice intramuscular injected HBV-S DNA vaccine,these values are significant higher than that of mice injected pCR3.1(P<0.05).The spleen cells CTLs activity have decreased obviously after treated with anti-CD8+ monoclonal antibody and have no significant change after treated with anti-CD4+ monoclonal antibody.The HBV-S gene vaccine could evidently inhibit the tumor growth,prolong the survival period (>38.2 days) and improve the survival rate in mice.Conclusions：The DNA vaccine of HBV ( pCR3.1-S) had strong antigenicity in cellular and humoral immunity and had marked killing effect on HBV infected cells in vivo，which could be promoted by vector coding murine IL-2 or IL-12.CTLs activity was performed by CD8+ cells.

Objective: To investigate the correlation between FIB-4 and the clinicopathological characteristics and prognosis of patients with hepatocellular carcinoma (HCC) after curative resection. Methods: From January 2009 to December 2012, the clinicopathological and follow-up data of 245 patients with HCC after curative resection were retrospectively studied. Their survival was calculated using the Kaplan-Meier method. The Cox proportional hazard regression model was used for the multivariate analysis. Results: According to FIB-4 index, patients were divided into two subgroups: FIB-4Ⅰ(≤3.25) and FIB-4Ⅱ(>3.25). FIB-4 could predict liver cirrhosis severity (Ishak grade, Grade 1-5 vs. Grad 6, r=0.681, P<0.001). It was associated with liver function such as:aspartate transaminase (P<0.001)、total bilirubin (P=0.009)、albumin (P=0.001) and platelet count (P<0.001) other than tumor clinicopathologic features. Both univariate and multivariate analysis showed FIB-4 could predict the prognosis of HCC patients (Overall survival: P=0.037 and 0.011; Recurrencefree survival: P=0.027 and P=0.043, respectively). Conclusion: The preoperative FIB-4 index could be used as a prognostic marker for the prognosis of HCC after curative hepatectomy.

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.
Anemia/blood/*therapy ; Animals ; Base Sequence ; Blood Urea Nitrogen ; Cell Hypoxia ; Creatinine/blood ; Erythropoietin/biosynthesis/*genetics/secretion ; Gene Expression Regulation ; Genes, Reporter ; Genetic Therapy ; HeLa Cells ; Humans ; Injections, Intramuscular ; Kidney/pathology ; Luciferases, Firefly/biosynthesis/genetics ; Molecular Sequence Data ; Plasmids/*genetics ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/biosynthesis/genetics/secretion ; Response Elements ; Transcriptional Activation ; Uremia/blood/*therapy

Model informed precision dosing for warfarin is to provide individualized dosing by integrating information related to patient characteristics, disease status and pharmacokinetics /pharmacodynamics of warfarin, through mathematical modeling and simulation techniques based on the quantitative pharmacology. Compared with empirical dosing, it can improve the safety, effectiveness, economy, and adherence of pharmacotherapy of warfarin. This consensus report describes the commonly used modeling and simulation techniques for warfarin, their application in developing and adjusting dosing regimens, medication adherence and economy. Moreover, this consensus also elaborates the detailed procedures for the implementation in the warfarin pharmacy service pathway to facilitate the development and application of model informed precision dosing for warfarin.