To construct a repetitive, objective and consecutive system for evaluating the angiogenesis of vascular endothelial growth factor(VEGF) in vivo, a sterile polyvinyl alcohol sponge disc was implanted into the subcutaneous part of rat and five days later pCMV 4 VEGF 165 expressed plasmid and empty vector pCMV 4 were injected into the sponge, respectively. 7 and 11 days after injection, the tissue ingrowth in the implants and expression of VEGF gene in the serum, the tissues in the sponge and the liver were measured by RT polymerase chain reaction (RT PCR) .The pathological section of sponge was stained with hematoxylin and eosin (HE) and immunohistochemistry of Ⅷ factor specific for vascular endothelial cells. The tissue ingrowth in the implants was analysed by image analysis system and statistical analysis was done with Stata Software. The results indicated that the expression of VEGF gene was located at the injection site and promoted the tissue ingrowth in the implants. In brief, the angiogenesis of VEGF was successfully observed using the sponge implant model, which provides lays the theoretical foundation for local gene therapy of ischemic diseases.

HCV NS5B, acting as a RNA dependent replication enzyme,has emerged as an attractive protein used as a target for screenig of drugs against HCV NS5B, and plays an important role in HCV replication. In the report the gene expression of NS5B in E.coli was investigated. PCR was performed to gain the gene of HCV NS5B from plasmid pBRTM/HCV 1 which contains whole sequence of HCV, and the truncated NS5B gene containing no hydrophobic domain was cloned into pGEM Teasy vector. The gene of the truncated NS5B was cut from pGEM Teasy vector and cloned into E.coli expression plasmid pET 21b, then pET 21b NS5B was transfected into E.coli cells. The protein E.coli lysates were purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting assay. The RdRp activity of NS5B was examined by scintillation proximity assay (SPA). The truncated NS5B gene was successfully cloned into pET 21b. The results of SDS PAGE and Western blotting assay showed: ①the molecular weight of the expressed product was about 68 000 D, ②The truncated NS5B protein was existed in media of E.coli cells, ③The activity of NS5BDCT21 His from HCV 1b amounted to 6 900 cpm (total incorporation of approximately). These findings suggest that soluble NS5B can be successfully expressed in E.coli and could secret into media.

With more and more non-affiliated hospitals of universities joining the clinical teaching of infectious diseases,there have been some dilemmas on clinical teaching,such as the shortage of staff,teaching experience,poor lecture art and teaching enthusiasm,textbook lag,the shortage of case teaching abd students'fear of infectious disease etc. Countermeasures such as perfecting the organizational structure,formulating preferential policy,clearing historical mission,increasing students' interest in learning,adding new academic progress,enriching teaching methods,setting up experts'supervision will ensure the effective teaching quality.

The significance for specialized hospital's resident physicians participating in the standardized clinical resident training in general hospital is expounded and its existing prob-lems are pointed out.It is suggested that the administration division should give full play to subjective initiative,coordinate problem solving and improve training quality,and meanwhile speciality hospitals should make an active effort in the application for national subspeciality physician training base.

ObjectiveTo investigate the effects of isoflurane anesthesia on hippocampus synaptosomes proteome in aged rats.MethodsTwenty-seven 22- month-old SD rats weighing 480-550 g were randomly divided into 2 groups: control group (group C,n =6) and isoflurane group (group Ⅰ,n =21 ).In group C inhaled mixed gas containing 80％ oxygen for 2 h.In group Ⅰ the animals were endotracheal intubated after induction by 3％ isoflurane and inhaled 2％ isoflurane and 80％ oxygen for 2 h.Cognition function was evaluated by Y-maze at 24 h after anesthesia and the total training times were recorded.The total training times ＞ 75 was defined as cognitive dysfuction.In group Ⅰ the animals were divided into cognitive dysfuction group (group ⅠA) and non-cognitive dysfuction group (group IB) according to the results of Y-maze test.The animals were sacrificed and their hippocampi were removed and synaptosomes were extracted for two-dimensional gel electrophoresis.The different protein spots were analyzed by mass chromatographic analysis.ResultsSix rats had cognitive dysfuction (group IA) and another thirteen rats had no cognitive dysfuction (group IB).The total training times were significantly higher in group IA than in groups C and IB( P ＜ 0.05).There was no significant difference in the total training times between groups C and IB (P ＞ 0.05).There were 21 (11/10) different protein spots between groups IB and IA,and 19 (12/7) different protein spots between groups C and IA.Thirty-one protein spots were identified by means of MALDI-TOF-MS.ConclusionThe cognitive dysfuction after isoflurane anesthesia in aged rats may be related to the changes of energy metabolism protein,cytoskeletal structure and regulatory protein in synapse of hippocampus.

Mother-to-child vertical transmission is an important route of transmission for hepatitis B virus (HBV) infection in patients with chronic hepatitis B, and the rate of HBV infection increases with the increase in the mother′s HBV viral load. Although active and passive immunoprophylaxis has been implemented for neonates at present, the rate of mother-to-child vertical transmission of HBV remains around 10%. Therefore, pregnant women with a high viral load should receive antiviral therapy to reduce the viral load, in order to prevent and block the mother-to-child transmission of HBV and reduce the incidence rate of chronic hepatitis B. By retrieving related literature in China and foreign countries, this article describes the safety and efficacy of antiviral agents during pregnancy and provides a reliable basis for guiding medication during pregnancy.

We reported a case of hypophysitis caused by a programmed death-1 ( PD-1) inhibitor. The patient was a 59-year-old female with metastatic malignant melanoma who participated in the phaseⅡclinical trial of a PD-1 inhibitor toripalimab. More than five months after the administration of toripalimab,she experienced fatigue, depression, nausea, and anorexia. Laboratory examinations showed mild hyponatremia, secondary adrenal insufficiency, and secondary hypothyroidism. MRI revealed the enlargement of her pituitary with obvious enhancement. The patient was diagnosed as hypophysitis caused by the PD-1 inhibitor and was given replacement therapy with physiological doses of corticosteroid and levothyroxine sodium. Her symptoms were then improved. MRI revealed that her pituitary size returned to normal after 8 weeks of treatment and remained stable during every 3-month follow-up. This case reminds us of the possibility of hypophysitis when patients suffere from fatigue and anorexia during the process of PD-1 inhibitor treatment. Correct diagnosis, proper therapy, and regular follow-up are important to ensure the patients' safety, and to improve their prognosis.

Objective The aim of this study was to investigate the mechanism underlying pruritus by comparing the epidermal growth factor receptor inhibitor(EGFRI)-erlotinib mouse model with the substance P(SP)-induced pruritus mouse model. Methods Two randomized groups of mice were treated with erlotinib or SP to induce pruritus. Behavioral and skin manifestations were observed. Pathological images and neurokinin 1 receptor(NK-1R)expression of the skin were determined. Concentration of interleukin(IL)-31, IL-33, histamine, leukotriene B4, and SP was analyzed by enzyme-linked immunosorbent assay. Nitric oxide was analyzed by colorimetry. Results Transient pruritus induced by erlotinib appeared 2 to 5 days after treatment. In contrast, continuous pruritus was observed during the first hour, but was then gradually relieved. These two shared similar scratching behavior. Concentration of neurotransmitters showed similar trends in changes among the erlotinib group and SP group. Immunohistochemical expression was also consistent between the erlotinib group and SP group. Conclusions Erlotinib-associated pruritus is related to release of signaling factors through the SP/NK-1R signaling pathway.

Objective To investigate the endoplasmic reticulum stress(ERS)role in the course of liver failure induced by severe hepatitis B virus(HBV)infection and its related mechanism.Methods Liver tissue samples and clinical data [chronic hepatitis B patients(12 cases,chronic hepatitis B group),hepatic failure induced by severe hepatitis B virus(12 cases,severe hepatitis B virus liver failure group),and normal subjects(8 cases,control group)] were collected from the Beijing You'an Hospital affiliated to Capital Medical University between 2009 to 2011.Statistical analysis was performed on the clinical indicators of each group.The structure of endoplasmic reticulum in liver tissue was observed by transmission electron microscopy.Western blot and qRT-PCR were used to detect the expression of endoplasmic reticulum stress and apoptosis-related factors,including glucose-regulated protein(Grp),and C/EBP homologous protein(CHOP).Frozen sections of liver tissues were prepared for immunofluorescence test.All data were expressed as mean±standard deviation.LSD-t test was used to compare the results between groups.A p value<0.05 was considered as statistically significant.Results Transmission electron microscopy showed that the morphological structure of the endoplasmic reticulum was damaged in both groups(chronic hepatitis B and liver failure induced by severe hepatitis B virus),and liver failure induced by severe hepatitis B virus group was more critical.Western blot and qRT-PCR showed that Grp78,Grp94 and Caspase-4 were highly expressed in normal group and chronic hepatitis B group,and the relative protein expressions were 1.20±0.13 and 0.78±0.11,0.90±0.06 and 0.11±0.01,0.15±0.02 and 0.22±0.04,respectively.The expression of protein was weakened in liver failure induced by severe hepatitis B virus group(relative protein expression was 0.01±0,0.01±0,and 0.11±0.02,respectively).There was a statistically significant difference between the two groups(P<0.05).The expression of CHOP was consistent with the results of immunofluorescence,and increased with the stressing of injury.Conclusion During the course of severe hepatitis B infection,dysregulated endoplasmic reticulum stress activated mild stress in chronic hepatitis B group,while severe stress in hepatic failure induced by severe hepatitis B virus group.Therefore,endoplasmic reticulum stress plays an important and complex role in the pathogenesis of hepatic failure induced by severe hepatitis B virus.

OBJECTIVETo investigate the synergetic transactivating functions of HCV core and truncated HBV middle surface proteins.

METHODSTwo recombinant expression plasmids harboring HCV core and C-terminally truncated HBV middle surface protein gene were constructed, respectively. The plasmids were transfected into HepG2 cells and cotransfected HepG2 cells with reporter plasmid pSV-lacZ by lipofectamine plus reagents. The transient expressed viral proteins were identified at the transcription and translation levels. The activity of beta-galactosidase was detected, which reflected the transactivating function of the proteins.

RESULTSThe protein expression of plasmids was detected in soluble cell extracts of transiently transfected HepG2 cells. HCV core protein activated the beta-galactosidase expression at a value of 4.6 times higher than the control, while C-terminally truncated HBV middle surface protein activated at a value of 3.2 times. It reached 8.4 times transfected with the plasmids simultaneously. The transactivating effect was dose dependent.

CONCLUSIONSIt is suggested that the two kinds of virus proteins have transactivating effect on SV40 early promoter/enhancer, and they act synergistically. These contribute to explain the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection.

Hep G2 Cells ; Hepatitis B ; genetics ; metabolism ; Hepatitis C ; genetics ; metabolism ; Humans ; Membrane Proteins ; genetics ; metabolism ; Plasmids ; Promoter Regions, Genetic ; Transcriptional Activation ; Transfection ; Viral Core Proteins ; genetics ; metabolism ; beta-Galactosidase