Effects of palmitate-stimulated macrophages on invasion and migration of HepG2 cells / 中国病理生理杂志

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.

Laparoscopic total splenectomy vs partial splenectomy for splenic benign tumors / 中华普通外科杂志

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.

Surgical experience on Roux-en-Y pancreaticojejunostomy after local pancreatic head resection for benign tumors of the pancreatic head / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the value of Roux-en-Y pancreaticojejunostomy after local pancreatic head resection in treating benign tumors of pancreatic head (BTPH).</p><p><b>METHODS</b>The clinical data of 12 patients diagnosed as BTPH and treated by Roux-en-Y pancreaticojejunostomy after local pancreatic head resection in Department of General Surgery, Peking University Third Hospital from November 2006 to October 2013 were retrospectively analyzed.Of the 12 cases, 5 patients were male, 7 patients were female, the age of patients ranged from 21 to 64 years(average 42.3 years). Diameter of tumors was 3.0-4.8 cm.Diameter of pancreatic wound after resection was 5.1-7.9 cm, and main pancreatic duct injury happened in 1 case.</p><p><b>RESULTS</b>Two cases of mucinous cystadenoma, 2 insulinoma, 3 solid pseudopapillary tumor and 4 nonfunctional pancreatic neuroendocrine tumors were confirmed histopathologically.No mortality and pancreatic leakage occurred during the perioperative period.All the 12 patients had no sign of recurrence.Experienced good life quality without occurrence of diabetes during the follow-up period of 24-108 months(more than 60 months in 4 cases).</p><p><b>CONCLUSIONS</b>Roux-en-Y pancreaticojejunostomy after local pancreatic head resection is a reasonable choice for benign tumors of the pancreatic head as long as the patient is properly selected.</p>

Role of FAT/CD36 in high-fat diet-induced adipose tissue inflammation / 中国病理生理杂志

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.

Role of tumor necrosis factor-alpha in the anti-HBV activity of tetracycline / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>A 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.</p>

Construction of pGL3-SM22-SCAP (D443N) eukaryotic expression vector and its expression in CHO cells / 生物工程学报

The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.

Therapeutic effect of adefovir anti-duck hepatitis B virus in vivo and anti-HBV activity in vitro (2.2.15 cells) / 重庆医科大学学报

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.

Establishment and evaluation of the method for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>The 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.</p>

Experimental study on the effect of valaciclovir on antiduck hepatitis B virus / 重庆医学

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.

Establishment and application of the method for detecting DHBV DNA with fluorescence quantitative PCR / 重庆医科大学学报

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