Objective: To establish the method for detecting duck hepatitis B virus (DHBV) DNA using fluorescence quantitative PCR. Methods: Three primers derived from DHBV DNA S gene were designed . The semi - nested primer was labelled by AmpliSen-sor using coupling reagent.The standard curve of the positive standards of DHBV DNA was established after asymmetric preamplifi-cation ,semi - nested amplification and on - line detetion. 70 samples of duck serum were tested by fluorescent quantitative DHBV DNA PCR method and dot blot hybridization assay using digoxigenin - labelled DHBV DNA probe. The correlation of results between the two methods was analysed. Results : After 30 cycles , amplification products showed two bands about 180bp and 70bp by 2% a-garose gel electrophoresis. The concentration of amplification products was in direct proportion to the initial concentration of the positive standards. The magnitude of detection index was in direct proportion to the amount of amplification products accumulating at the current cycle. The initial concentration of the positive standards was inversely proportional to the number of cycles needed for the amount of amplification products.The correlation coefficient of results was 0. 97 (P

Objective:To study the therapeutic effect of the purine nucleoside analog adefovir against duck hepatitis virus (DHBV) in vivo and anti-HBV activity in vitro.Methods:The Chongqing duck hepatitis B animal model was treated with adefovir by intragastric administration once a day for 10 days.DHBV DNA in serum and liver were monitored by serum dot-blot hybridization and Southern blot.DHBsAg in serum was detected simultaneously by ELISA.The anti-HBV activity of adefovir was observed in 2.2.15 cells (human hepatocellular carcinoma cells stably transfected with HBV).Results:Adefovir could significantly reduce DHBV DNA level.After stopping the treatment for 3 days,DHBV DNA level in serum and liver returned significantly.The O.D values of DHBsAg in duck serum did not change at the period of the treatment.After 12 days of continuous exposure to adefovir at the concentration of 20?g/ml in vitro culture,the inhibitions of HBsAg,HBeAg,HBV DNA were 17.01%,26.80%,and 26.92% respectively.Conclusion:The study confirms the safety and potent dose-dependent antihepadnaviral activity of adefovir in vivo.The anti-HBV effect is not demonstrated in 2.2.15 cells.

5R-5-hydroxytriptolide (LLDT-8) is a new drug candidate which is in clinical trial treating rheumatoid arthritis. Polymorph screening of the compound was carried out in this study. Polymorph of LLDT-8 was prepared by evaporative crystallization and antisolvent crystallization methods and was characterized by powder X-ray diffraction (p-XRD), infrared spectrometry (IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). It was found that p-XRD patterns, DSC curves, TG curves and IR spectra of the LLDT-8 samples prepared by the above recrystallization methods were all consistent. The 20 of main peaks in the p-XRD patterns appeared at 7.58 degrees, 8.14 degrees, 8.66 degrees, 15.46 degrees, 16.46 degrees, 29.54 degrees, 31.16 degrees and 38.26 degrees, while the infrared absorption peaks appeared at 3 471.3, 2 962.2, 2 887.0, 1 762.6, 1 677.8, 1 432.9, 1 365.4, 1 247.7, 1 080.0, 1 031.7 and 877.5 cm(-1). LLDT-8 was decomposed at 271.2 degrees C based on the determination from DSC and TG. It was showed in single crystal X-ray diffraction study that LLDT-8 crystal was monoclinic with the space group being P2 (1). The cell parameters were found to be: a = 11.460 1 (11), b = 6.320 5 (6), c = 13.028 1 (12), alpha = 90.00, beta = 115.557 (2) and gamma = 90.00. The crystal was a hydrogen-bonded dimmer. The slurry experiments, which were further conducted in solvents with different polarities, confirmed the stability of solid state of LLDT-8 based on the p-XRD determination. The polymorph of LLDT-8 made assurance of its efficacy consistence during its clinical trials.

Objective　we studied the effect of the Purine mucleoside Valaciclovir on anti-duck hepatitis virus(DHBV) in vivo to provide an experimental basis for clinical treatment of patients with hepatitisB.Methods　The Chongqing duck hepatitis B virus model was treated with Valaciclovir once a day for a month at the doses of 50mg.kg－1、100mg.kg-1、200mg.kg－1of body weight per day. Serum DHBV DNA was detected four times in the course of the treatment,ALT and AST in serum and DHBV DNA in liver were detected simultaneously.Results　Valaciclovir could signsificantly lower the serum DHBV DNA level. Serum ALT of several ducks in serum rose slightly during the treatment,but became normal after 1 week stopping Valaciclovir. Examination of DHBV DNA in liver with Southern Blot indicated Valaciclovir could inhibit DHBV DNA replication,but could not completely eliminate DHBV SC DNA.Conclusion　The study confirms the safety and potent antihepaticviral activity of Valaciclovir in vivo.

Forty cases of type Ⅱ diabetes mellitus were treated by puncturing point Neiguan (PC 6), and the effect on their cardiac vegetative nerve functions were observed at 20 min, 40 min and 60 min after acupuncture respectively. The findings showed that all heart rate variables improved remarkably at the three time periods after acupuncture treatment, with significant differences (P＜0.01); but there was no significant difference in the curative effects among the three time periods.

AIM: To investigate the role of fatty acid translocase/CD36 (FAT/CD36) in adipose tissue in-flammation induced by a high-fat diet.METHODS:C57BL/6J mice were fed with a normal-chow diet ( NCD) or a high-fat diet ( HFD) for 14 weeks.The content of free fatty acid ( FFA) in the serum was measured by ELISA.The expression of CD36, cytokines and chemokines at mRNA and protein levels in the adipose tissues was determined by real-time poly-merase chain reaction and Western blotting.Immunohistochemical staining was used to examine the macrophages infiltration in the adipose tissues.The inflammatory responses in CD36 knockout mice and wild type mice with high-fat diet were ana-lyzed.RESULTS:The levels of FAT/CD36 were higher in HFD group than that in NCD group.HFD feeding enhanced the mRNA and protein expression of IL-1β, IL-6, TNF-α, MCP-1 and MIP-1, as well as promoted macrophage infiltration in the adipose tissues.Interestingly, as fed with HFD, the expression of cytokines/chemokines and macrophage infiltration were significantly reduced in adipose tissues of the CD36 knockout mice, compared with the wild type mice.CONCLU-SION:High-fat diet promotes adipose tissue inflammation in the mice in a FAT/CD36-dependent manner.

AIM:To investigate the impact of palmitate-stimulated macrophages on the invasion and migration of HepG2 cells and to explore the underlying mechanism .METHODS:Human acute monocytic leukemia cell line THP-1 were induced to macrophages by phorbol myristate acetate and were stimulated with palmitate (0.16 mmol/L).The culture supernatants were collected and used to incubate HepG 2 cells.The effect of palmitate on migration of the macrophages was detected by Transwell chamber assay .The mRNA expression of target genes was measured by RT-qPCR.The invasion and migration of the HepG 2 cells were assessed by invasion assay and scratch test .RESULTS:Palmitate promoted the migra-tion of the macrophages and increased the mRNA levels of interleukin -1β( IL-1β) , interleukin-6 ( IL-6 ) , tumor necrosis factor-α(TNF-α) and monocyte chemotactic protein-1 (MCP-1) in the macrophages.The invasion and migration of the HepG2 cells incubated with conditioned media from palmitate-stimulated macrophages were greater than those of the HepG 2 cells incubated with conditioned media from macrophages without palmitate .The media of palmitate-stimulated macrophages up-regulated the mRNA expression of cytokines and N-cadherin, and down-regulated the mRNA expression of E-cadherin in the HepG2 cells.CONCLUSION:Palmitate-stimulated macrophages promote the invasion and migration of HepG 2 cells through paracrine/endcrine loop.

Objective To evaluate laparoscopic partial splenectomy (LPS) for benign splenic tumors.Method Data of 55 patients undergoing laparoscopic partial splenectomy (20 cases) vs total splenectomy (LTS in 35 cases) at Peking University Third Hospital from August 2008 to July 2016 were collected and retrospectively analyzed.Results There was no difference in sex,BMI,preoperative H GB,preoperative PLT,operation time,operative blood loss and hospital stay between two groups.Age in LPS cases was younger than LTS group,while the tumor size was larger.On the 4th day postoperatively,PLT level was significnatly higher in LTP group.More patients in LTS group suffered from thrombocytosis.Conclusions Laprtoscopic partial splenectomy is a safe and effective procedure for the management of splenic benign tumors.

OBJECTIVETo establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).

METHODSThe probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed.

RESULTSThe sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01).

CONCLUSIONSThe method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase ; chemistry ; metabolism ; Animals ; DNA Probes ; chemistry ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; blood ; diagnosis ; virology ; Hepatitis B virus ; genetics ; Humans ; Molecular Diagnostic Techniques ; methods ; standards ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).

METHODSThe Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR.

RESULTSThe TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA.

CONCLUSIONA Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.

DNA, Viral ; biosynthesis ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; physiology ; Humans ; Tetracycline ; pharmacology ; Transfection ; Tumor Necrosis Factor-alpha ; genetics ; Virus Replication