Objective To investigate the adenoviral vector encoding the whole humanized antibody gene of pres_2 antigen for preventing the liver graft from infection by the hepatitis B virus. Methods The whole humanized antibody gene to pres_2 antigen was cloned into type 5 adenoviral shuttle plasmid pDC315. The corresponding recombinant virus was obtained by homologous recombination in 293 packaging cells. The virus containing the whole humanized antibody gene of preS_2 antigen was transfected into the rat liver graft during cold preservation. The effect of the recipient serum containing preS_2 antibody protecting human hepatocytes from hepatitis B virus infection was observed. Results The viral titer determined by TCID50 analysis was 5.1 ?10 10 PFU/ml. The concentration of preS_2 antibody was ( 16.7 ? 10.5 ) mg/L on the day 3 and ( 30.9 ? 13.6 ) mg/L on the day 7. The serum containing preS_2 antibody could protect human hepatocytes from hepatitis B virus infection in vitro when the concentration of the preS_2 antibody was more than 0.5 ?g/ml. Conclusions The adenoviral vector encoding the whole humanized antibody gene of preS_2 protein were constructed successfully. The whole humanized antibody was expressed. The preS_2 antibody in recipient serum protecting human hepatocytes from hepatitis B virus infection was observed, which might be a new method to prevent hepatitis B re-infection after liver transplantation.

Objective To analyze drug resistance data of Enterococcus faecalis and Enterococcus faecium,submitted by member units of Chongqing bacterial drug resistance monitoring network,and to provide the basis for our city effective application of antimicrobial agents and the reference.Methods Target bacteria identification and drug susceptibility test were performed by member units,according to the national technology scheme of bacterial drug resistance monitoring network and the results were determined according to standards published by Clinic and Laboratory Standard Institute(CLSI) in 2015.WHONET5.6 software was used to analyze drug susceptibility,and drug resistance difference was analyzed by using SPSS21.0 software.Results A total of un-repeated 1 811 strains of Enterococcus faecalis and 1 601 strains of Enterococcus faecium,accounting for 13.1% of all positive strains.The resistant rates of the two kinds of bacteria to vancomycin were 0.5% and 1.8%,to rinathiazole amine were 2.5% and 0.5% respectively.Tigecycline resistant strains were not founded.The resistant rate of Enterococcus feaclis to ouinupristin/dalfopristin was 90.1%,to tetracycline was 78.8%,to high concentration of gentamicin was 43.0%,to penicillin,ampicillin and nitrofurantoin was less than 7%.Except ouinupristin/dalfopristin and tetracycline,the resistant rate of Enterococcus faecium to the other drugs were significantly higher than Enterococcus faecalis(P<0.05).Strains isolated from children and adult patients,Intensive Care Unit(ICU) and un-ICU patients were with differences of drug resistance(P<0.05).Conclusion Most of Enterococcus infection could be caused by Enterococcus faecalis and Enterococcus faecium.Monitoring of drug resistance might be helpful for rational and effective usage of antimicrobial agents.

Objective To establish human pancreatic cancer xenografts in nude mice, and to investigate the antitumor efficacy of human endostatin expressed by replication-competent adenovirus AdTPHre-hE in vivo. Methods Pancreatic cancer cells AsPC-1 were injected subcutaneously in BALB/c nude mice to establish the xenografts. Tumor growth was observed and measured after AdTPHre-hE treatment. Expression of endostatin was detected by ELISA assay. The tumors were harvested for pathologic examination and immunohistochemical staining. Results Tumors grew more slowly in the AdTPHre-hE group and their sizes were markedly smaller than those of the Ad-hE group (P＜0.01)and control group(P＜0. 01). Endostatin levels were detected in the sera of nude mice in all treated groups, and endostatin expression in AdTPHre-hE group increased with time. The endostatin level in the AdTPHre-hE treated group was much higher(P＜0. 01)and increased faster than that in the Ad-hE treated group. Immunohistochemical staining for Hexon of adenovirus capsid showed more positive tumor cells in the tumor tissues treated with AdTPHre-hE. Immunohistochemical staining for FⅧ revealed a decreased microvessel density in the tumor tissues treated with AdTPHre-hE. Conclusion The replication-competent adenovirus efficiently expressed high-level endostatin and significantly inhibited tumor growth in vivo.

Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene(hEndo). Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which El A gene and E1B gene were driven by human telomerase reverse transcriptase (hTERT) promoter and hypoxia response element (HRE) promoter,respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3. 25 X 1010 pfu/ml.Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased (P < 0. 01).Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.

Objective To investigate the effect of a dual regulation of replicating adenovirus vector carrying human endostatin gene (AdTPHre-hEndo) on pancreatic cancer. Methods Human endostatin (hEndo) gene was cloned into the genome of replicating adenovirus specific for the tumor cells by virus recombination technology. The virus titer was 3.25 × 1010pfu/ml. A Balb/c nude mouse model carring sw1990 cells pancreatic cancer was established, the expression of human endostain and inhibition of tumor cells in vivo were detected. Results We successfully constructed AdTPHre-hEndo. The inhibition on pancreatic cancer cell line SW-1990 of AdTPHre-hEndo is better than Ad-hEndo (P ＜0. 01 ), and ONYX-015 ( P ＜ 0. 05 ). The endostatin expression of AdTPHre-hEndo group was significantly higher than Ad-hEndo group and the control group (P ＜ 0. 01 ). The intratumoral MVD also decreased significantly in the treated tumors(6. 8 ±2. 5 vs. 16. 0 ±4. 6、47. 2 ± 10. 0, P ＜0. 01 ). Conclusions The recombination adenovirus can express biologically active hEndo effectively, which results in inhibiting the growth of micro-blood vessels and proliferation slowly.

To retrospectively analyze the clinical data of 42 patients at our department with bulbar myasthenia gravis.They underwent video-assisted thoracoscopic extended thymectomy (VATET) from May 2003 to August 2010.Another 42 patients treated with medications alone were selected as controls.Compared with the control group (4.32 ± 0.23),postoperative dysphagia scores improved significantly in patients undergoing VATET(9.21 ±0.41).And so did the dysphagia assessment classification (P ＜0.05).Thus VATET could effectively and definitely relieve dysphagia in patients with bulbar myasthenia gravis.

Objective To investigate the role of PI_3 K/Akt signal pathway in Ephrin-Al gene mediated invasion,metastasis of Huh-7 cells.Methods Western blot was used to test the protein expression of phosphatidylinositol 3-kinase(PI_3 K)and mitogen-activated protein kinase(MAPK)after Huh-7 cells were treated with Ephrin-A1/Fc fusion protein.According to the protein expression,LY294002 was used to block PI_3 K/Akt pathway specifically,then p-Akt protein expression,mobility and invasive ability of Huh-7 cells were examined.Results In Huh-7 cells actived by Ephrin-Al/Fc fusion protein,p-Akt expression was higher than that in control group(t=4.564,P＜0.05),but there was no difference of p-p38MAPK expression between Ephrin-Al/Fc fusion protein group and IgG/Fc fusion protein group(P＞0.05).PI_3 K/Akt pathway was specifically blocked by LY294002,the p-Akt protein expression decreased in Huh-7 cells,and the mobility and invasive ability mediated by Ephrin-Al in Huh-7 cells decreased(P＜0.05).Conclusions PI_3 K/Akt pathway effects an important role in mobility and invasive ability of Huh-7 cells mediated by Ephrin-A1.

Objective To explore the effect of wild type or mutant parkin gene expression on the growth of human hepatocellular carcinoma cell line Huh-7. Methods The parkin (wild type or mutant) expression vector and empty vector were transferred into Huh-7 cell lines with LipofectAMINE 2000 reagents. The positive clones that expressed parkin gene stably were chosen by G418 and checked by reverse transcription-polymerase chain reaction (RT-PCR) to check the DNA sequences. The cytobiological behaviors of those positive clones were analyzed by cell proliferation assay and tumorigenesis in nude mice. Results Huh-7 cell lines that expressed wild type or mutant parkin gene stably were successfully established. The growth of wild type parkin-expressed cells was obviously inhibited compared with the control cells transfected with empty vectors(t= 3. 875, P= 0. 031).The volume of tumor formed by wild type parkin-expressing cells in nude mice was also significantly reduced (t=8. 228,P=-0. 003). Mutant parkin gene expression had a slight effect on the growth of Huh-7 cells in vitro and in vivo (P＞0.05). Conclusion The re-expression of wild type parkin gene can favor the malignant phenotype revision of Huh-7 cells. Therefore, it might be a good candidate for tumor suppressor gene associated with HCC.

Objective To review video-assisted thoracoscopic thymectomy as a treatment for myasthenia gravis (MG),compare outcomes of thoracoscopic thymectomy for thymoma and non-thymoma MG,and assess the efficacy of Video-assisted Thoracoscopic Extended Thymectomy (VATET) combined with mediastinoscopy.Methods A retrospective review of 500 patientswith MG who underwent VATS thymectomy between 2001 and 2011 has been done.They were divided into three groups:118 cases of thymoma MG group,thoracoscopy for non-thymoma MG group 301cases,and VATET for non-thymoma MG group 81cases.Results There was no mortality.Thoracoscopic thymectomy was successfully performed for 495 cases.In the thoracoscopy group for non-thymoma MG,the operating time is (111.3 ± 31.6) min,11.0％ having post-operative myasthenic crises ; in the VATET group,the operating time is (145.0 ± 71.6) min,9.9％ having post-operative myasthenic crises ;in the thymoma MG group,the operating time is (128.5 ± 77.8)min,24.6％ having post-operative myasthenic crises.During the follow-up,CSR was 37.3％,36.5％ and 28.7％ in the groups of thoracoscopy for non-thymoma MG,VATET and thymoma MG respectively.However,the disease-free survival curve shows that CSR of the thymoma MG group became lower than other two groups 3 years after surgery,and CSR of the VATET group becoming higher than that of thoracoscopy for nou-thymoma MG group 5 years after surgery.CSRs of groups of thoracoscopy for non-thymoma MG,VATET and thymoma MG might reach 50％,60％ and 36％.Conclusion The VATET combined with mediastinoscopy has a better long-term outcome because the more thymus might be removed comparing with non-thymoma MG,thoracoscopic thymectomy for thymoma MG had a worse long-term outcome.

Objective To research the relationship between free/total prostate specific antigen (f/t PSA), PSA density(PSAD) and prostate cancer(PCa), to explore healthy middle and old-aged,the patients with BPH, the scope of reference value of PSA,frce PSA(fPSA), f/t PSA and PSAD in patients serum with PCa. Methods To de-tect 307 cases of healthy gerontism male,236 of BHP and 41 of PSA and fPSA in patients with PCa,to calcuLate f/t PSA and PSAD, to investigate the dependability between PSA ,I"PSA ,f/t PSA ,P SAD and healthy male, the patients ofBHP,the PCa ,to determine the scope of reference value PSA,fPSA,f/t PSA and PSAD fitting for Chinese. Results PSA and fPSA in patients with PCa are obviously higher than normal control(P <0.01). f/t PSA is obviously lower than the BPH and normal control(P < 0.01). When PSA is 9.25 and f/t PSA is 20%, there are better value of clini-cal diagnosis,when PSAD is 0.18 (AUC = 0.635), we can get best aceuration of diagnosis. Conclusions f/t PSA and PSAD are better than PSA in clinical diagnosis. When PSA is more than 9.25,f/t PSA is less than 20% or PSAD is more than 0.18 is significance for diagnosis. The normal reference value of PSA less than 9.25,f/t PSA more than 20%, PSAD less than 0.18 is fit for Chinese.