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Objective@#To investigate the curative effect of local application of CpG-oligodeoxynucleotide (CpG-ODN) combined with 4-1BB monoclonal antibody in hepatoma-bearing mice, and to evaluate the effect of 4-1BB monoclonal antibody on CpG-ODN immunotherapy.@*Methods@#H22 single cell suspension was injected subcutaneously into the axilla and four limbs of the BALB/c male mice to establish a tumor-bearing mice model. After 7 days, 30 mice with corresponding tumor-bearing volume were screened and randomly divided into model control group, CpG group and CpG+4-1BB group, and the drug was injected into the tumors of left lower extremity. The same batch of normal mice was selected as normal control group. Survival of mice was recorded. Tumor-bearing volume and organ index were calculated. Serum levels of interleukin (IL) - 12 and interferon (IFN) gamma and spleen CD8+T lymphocyte ratio were measured. The measurement data were analyzed by analysis of variance. The survival rate of each group of mice was analyzed by log-rank test.@*Results@#Mice in the model control group with tumor-bearing volume had a sustained growth before the execution. CpG group and the CpG+4-1BB group [(976.08 ± 29.55) mm3, (47.25 ± 0.93) mm3)] tumor-bearing volume was decreased than model group [(1 336.52 ± 39.40) mm3] (F = 5 329.273, P < 0.05). CpG+4-1BB group distant tumor-bearing volume [(611.83 ± 113.02) mm3] was decreased than model group and CpG group [(1 406.62 ± 51.09) mm3, (1 380.01 ± 51.44) mm3] (F = 247.160, P < 0.05), but there was no significant difference between the CpG group and the model group (P > 0.05). Serum IL-12 concentration (23.90 ± 2.33 pg/ml), IFN-γ concentration (103.02 ± 6.10 pg/ml) and spleen CD8+T cell ratio (4.54 ± 0.62%) in the model group were lower than those in the normal group (P < 0.05). Serum IL-12 concentration in CpG group and CpG+4-1BB group (29.21 ± 2.23 pg/ml, 37.04 ± 1.49 pg/ml), IFN-γ concentration (116.12 ± 4.08 pg/ml, 138.65 ± 1.72 pg/ml), CD8+T cell ratio (6.65 ± 0.64%, 12.73 ± 0.88%) were higher than the model group, while CpG+4-1BB group was higher than the CpG group (P < 0.05). The survival rate of CpG+4-1BB group was higher than that of model group and CpG group (χ2 = 25.544, P < 0.05), but there was no significant difference between CpG group and model group (P > 0.05). There was no significant difference in organ index between the four groups (P > 0.05).@*Conclusion@#4-1BB monoclonal antibody combined with CpG-ODN therapy can shrink hepatoma-bearing capacity, inhibit the growth of distant tumors and significantly prolong the survival time of mice.
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Objective To investigate the effects of reverse transcription loop-mediated isothermal amplifica-tion(RT-LAMP)method and RT-PCR method for detecting Mycobacterium tuberculosis(MTB)in cerebro-spinal fluid(CSF)to provide a basis for its rapid diagnosis and clinical pharmacodynamic evaluation.Methods Eighty-five cases of CSF sample in the Bethune International Peace Hospital of PLA from December 2015 to April 2017 were selected for conducting the study and divided into the tuberculous meningitis(TBM)group(46 cases),suspected TBM group(25 cases)and control group(16 cases).The 16S rRNA region of MTB was used to design the specific primers.Then RT-LAMP and RT-PCR detection technological systems were estab-lished.Then the detection results by using the these two methods was analyzed.Results The positive detec-tion rates of the TBM group were 97.8% and 75.0% respectively,which of the suspected TBM group were 76.0% and 40.0% respectively,and which of the control group were 0.0% and 12.0% respectively,the posi-tive detection rate of each group in the RT-LAMP method was higher than that in the RT-PCR method,the difference was statistically significant(P<0.01);in the control group,adopting RT-PCR detection found non-specific amplification,while which was not found by adopting RT-LAMP method,indicating that the specifici-ty of RT-LAMP method was stronger than that of RT-PCR;the sensitivity of RT-PCR was 10.0 CFU/mL, which was higher than 1 CFU /mL of RT-LAMP.Conclusion RT-LAMP has the advantages of simpleness,sensitivity,rapidness and detecting viable bacteria,compared with PCR,which has strong specificity,easy op-erating,low cost and short time-consuming,is expected to be a routine detection tool of basic level and field medical institutions and developing countries.
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Tuberculosis is a chronic infectious diseases caused by Mycobacterium tuberculosis(MTB).As the drug-resistance characteristics are different in patients with various genotypes,thus,the gene polymorphism study have critical clinical significance.Among the all kinds of techniques,some have been used to analyze polymorphism for a long time and new development in that respect has also been made recently.On the other hand,some techniques are e-merging but demonstrate promising application prospects.This study summarizes the gene polymorphism study of MTB which have been used or are emerging in recent years,and points out a few shortcomings briefly.Our object is to make a contribution to theoretical basis and knowledge accumulation in the drug-resistance and epidemiological survey field.
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Objective To explore the effects ofBuzhong Yiqi Pills on HBV-related decompensated liver cirrhosis patients.Methods A total of 176 patients of HBV-related decompensated liver cirrhosis were enrolled in the study from January 2007 to January 2010 and were divided into treatment group (82 cases) and control group (94 cases) according to patient's wishes. Patients in both groups were given antiviral therapy. According to the liver function and complications, patients were given glycyrrhizin to protect liver, Kuhuang Injection to treat jaundice, and spironolactone and furosemide for diuretic treatment. Patients in the treatment group receivedBuzhong Yiqi Pills, one bag for each time, twice a day, four weeks as a treatment session, three sessions each year, with five-year follow-up. Effects ofBuzhong Yiqi Pills on the hepatorenal function, blood coagulation, blood routine, complications and survival rate in patients with decompensated liver cirrhosis were observed.ResultsBuzhong Yiqi Pills could effectively improve the hepatorenal function, blood routine and coagulation disorders of HBV-related decompensated liver cirrhosis patients (P<0.05,P<0.01). The rate of complications with hydrothorax and ascites (46.34% vs. 88.30%), upper gastrointestinal hemorrhage (39.02% vs. 69.15%), infection (31.71% vs. 57.45%), hepatic encephalopathy (23.17% vs. 54.26%), hepatorenal syndrome (6.10% vs. 18.09%) and chronic hepatic failure (9.76% vs. 25.53%) in the treatment group and the control group were with statistical significance (P<0.05,P<0.01). The five-year survival rates were significantly higher in the treatment group (79.27%) compared with the control group (64.89%), with statistical significance (χ2=5.353,P=0.021).ConclusionLong term use ofBuzhong Yiqi Pills can significantly decrease the complications of HBV-related decompensated cirrhosis and improve survival rate of patients.
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We investigated the carrying status of the virulence genes of Salmonella from different sources in Shijiazhuang City,China,to provide the basic data for the further risk assessment of Salmonella.A total of 186 isolates of Salmonella from different sources were collected and identified serotypes in the area of Shijiazhuang from 2011 to 2016.PCR was performed for eight virulence genes (invA,sopE,agfA,spvR,hilA,stn,pefA,shdA).These Salmonella bacteria were detected in 13 kinds of serotypes.Enteritidis is a significant advantage of the group.The above 8 virulence genes were analyzed,and the virulence genes hilA,stn and invA were the most frequently carried,their respective carrying rate were 90.3％ (168/186),86.6％ (161/186) and 82.8％ (154/186) respectively.We found the virulence genes of Salmonella from different sources were different.It is necessary to take measures to strengthen the food hygiene supervision and prevention and control of the storage and sale of raw poultry stalls in the morning market in Shijiazhuang area.
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Objective To investigate the differences and characteristics of virulence genes carried by Salmonella enteritidis from different sources in Shijiazhuang City.Methods One hundred and twenty-four strains of Salmonella enteritidis isolated from morning markets of raw and poultry stalls,slaughterhouses and food poisoning specimens in Shijiazhuang area were collected.Eight virulence genes (invA,sopE,agfA,spvR,hilA,stn,pefA,shdA) were detected by polymerase chain reaction (PCR).Results Salmonella enteritidis might have different virulence gene profiles.The above eight virulence genes were detected in different strains.The carrying rate of virulence genes invA,sopE,stn,hilA,spvR and pefA in the food poisoning strains was higher than 94％.There was no difference in the carrying rate of 8 virulence gene between the morning raw poultry stalls isolates and the patient strains,but was different with the slaughterhouse strains.Conclusion There were more risks of food poisoning caused by Salmonella enteritidis from morning markets,and the hygiene supervision should be strengthened to prevent and control foodborne disease.
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In recent years, new changes have occurred in the treatment of chronic hepatitis C. Direct-acting antiviral agents have shown powerful efficacy in the treatment of active hepatitis C, and even refractory hepatitis C shows a high continuous virologic response. Among these direct-acting antiviral agents, sofosbuvir (SOF) has the advantages of short course of antiviral treatment and high response rate, and some patients with certain hepatitis C genotypes have no need to receive interferon therapy. This article introduces the mechanism of action, current status of clinical application, and major adverse effects of SOF, and points out its promising future in patients with refractory hepatitis C.
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Objective To detect influenza A virus by reverse transcription‐loop mediated isothermal amplification (RT‐LAMP) assay to established a rapid ,simple and visualization nucleic acid detection method .Methods The RT‐LAMP primers were designed in accordance with the hemagglutinin gene of influenza A virus .Then ,the specificity of the primers was evaluated by detection of different influenza viruses ,and the sensitivity was confirmed by testing multiple diluted RNA samples . Hydroxynaphthol blue (HNB) was used for visually evaluation and gel electrophoresis was used for validation .Clinical samples were detected by RT‐LAMP assay .Its consistency with fluorescent quantitative polymerase chain reaction (PCR) methods was compared .Results The primers of RT‐LAMP assay had high specificity . This technique could amplify influenza A virus accurately .The detection limit of RT‐LAMP assay was 2 .5 × 103 copies/mL by detection of multiple diluted RNA samples .In addition ,the results of RT‐LAM P assay could be visually inspected using HNB by color change ,and the results was in accordance with that of gel electrophoresis . RT‐LAMP assay was in consistence with fluorescent quantitative PCR when clinically applied .Conclusions RT‐LAMP assay is a rapid ,specific ,sensitive and simple method to detect influenza A virus .
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ObjectiveTo investigate the effects and molecular mechanism of simvastatin in liver fibrosis model of non-alcoholic fatty liver disease (NAFLD) in vivo and hepatic stellate cell in vitro.MethodsFirstly,the rat liver fibrosis model of NAFLD was established by high-fat diet administration and intervened with simvastatin.The expression of endothelial nitric oxide synthase (eNOS),inducible nitric oxide synthase (iNOS) and Collagen Ⅰ at mRNA and protein level were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Secondly,quiescent phenotype of LX-2 cell line was induced by promoting adipocyte differentiation medium in vitro,and then the quiescent phenotype of LX-2 cell were treated with transforming growth factor β1(TGF-β1),Nitroso-L-arginine methyl ester (L-NAME) which was NOS inhibitor,simvastatin,TGFβ1 with simvastatin,and L-NAME with simvastatin separately.The changes of eNOS,iNOS,αsmooth muscle actin (α-SMA) and Collagen Ⅰexpressions at mRNA and protein level were determined by RT-PCR and Western blot.ResultsAs modeling time extended,the expressions of eNOS in rat's liver tissue of model group at mRNA and protein level decreased gradually,however the expression of iNOS and Collagen Ⅰ at mRNA and protein level increased gradually,compared with normal control group and the differences were statistically significant (P ＜0.05 and 0.01).By 24weeks,the expressions of eNOS in rat's liver tissue of simvastatin group at mRNA and protein level were increased,the expression of iNOS at mRNA and protein level were decreased and the expression of Collagen Ⅰ at mRNA and protein level were decreased (all P ＜0.05).The expression of eNOS in rat's liver tissue of model group negatively correlated with the expression of Collagen Ⅰ at mRNA and protein level (all P ＜0.01).The expression of iNOS positively correlated with that of Collagen Ⅰ at mRNA and protein level (all P ＜0.01).In LX-2 cell culture,L-NAME inhibited the activation of LX-2,reduced eNOS and iNOS expression and increased α-SMA and CollagenⅠexpression,consistent with the role of TGF-β1.Simvastatin could directly increase the eNOS expression both in quiescent and activated LX-2 cells,decrease iNOS expression,maintain quiescent phenotype and inhibit its activation.ConclusionsSimvastatin ameliorated the genesis and progression of liver fibrosis by increasing eNOS expression in LX-2 cells and reducing iNOS,α-SMA and Collagen Ⅰ expression.
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Objective To observe the therapeutic effects of recombinant human granulocyte colony stimulating factor(rhG-CSF)on CCl4 induced chronic liver injury.Methods Male BALB/C mice were randomly allocated into treatment and control groups.The mice model were established by injection with daily for 7 days,while the control mice were received the same volumes of saline.The mice were sacrificed to get weight,liver mass and spleen mass.The count of CD34+ cells and Thy-1+ cells were analyzed by flow cytometry and immunohistochemical staining,respectively.Results The ratio of liver/spleen was 15.94±1.20 and 10.52±0.66 on day 8 and 15 in treatment group,respectively,while those were 7.14±1.68 and 8.31±1.71 in control group,respectively(all P value＜0.05).But there was no significant difference in body weight and liver mass between two groups(P＞0.05)The concentration of album in treatment group was raised rapidly on day 15.The concentrations of alanine aminotransferase (ALT),aspartate aminotransferase(AST),hyaluronic acid(HA)and laminin(LN)on day 30 were significantly lower in treatment group compared to control group(P＜0.05).There was significant difference in score of liver fibrosis on day 30 between two groups(treatment group:5.49±2.16,control:8.74±1.86,P＜0.05).The number of CD34+ cell and Thy-1+ in treatment group(on day 8:9.54±2.24 and 5.10±1.25 and on day 15:8.18±1.93 and 7.53±1.39,respectively)were higher than those in control group(on day 8:5.40±0.99 and 3.25±0.75;on 15 days:4.46±0.77 and 3.35±0.86,all P value＜0.05).Conclusion The rhG-CSF may improve the reparation of chronic liver injury,and may provide a novel method in treatment of liver fibrosis.
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<p><b>BACKGROUND</b>To explore the possibility of using retrovirus vector to carry HBV vector, and to prove that replication defective HBV could be normally packaged.</p><p><b>METHODS</b>Two kinds of full length of mutant HBV gene, which express dominant negative mutants, were inserted into retrovirus vector. After recombinant retroviruses were harvested, they were used to infect Hep G2 and 2.2.15 cell line. Then the expression of HBV core antigen in the Hep G2 cell was examined by immune fluorescence, and the existence of recombinant HB virion in the culture medium was examined by PCR.</p><p><b>RESULTS</b>High titer of recombinant retroviruses were obtained in the culture medium of transfected PA317 cell line. Core antigen was detectable in the recombinant retrovirus infected Hep G2 cell. Recombinant HB virion was detectable in the culture medium of recombinant retrovirus infected 2.2.15 cell.</p><p><b>CONCLUSIONS</b>The results suggested that recombinant retrovirus could carry HBV vector and express HBV products. When structural protein is offered by wt-HBV, the recombinant retrovirus may function as HBV vector, therefore it could be used in anti?HBV gene therapy.</p>
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Humanos , Terapia Genética , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B , Vírus da Hepatite B , Genética , Recombinação Genética , Retroviridae , Genética , Células Tumorais Cultivadas , Replicação ViralRESUMO
Objective To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of antisense RNA. Methods Two parts of HBV genome were reversedly recombined back into overlength HBV genome, which can produce HBV particle, to express antisense RNA complementary to S or S promoter region respectively. HepG 2.2.15 cell lines were transfected with these constructs and the empty vector pMEP4, then positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Results The mean inhibitory rates of HBsAg were (2.74?3.83)%、(66.54?4.45)%(P
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Objective To evaluate the replication, encapsulation and expression of the recombinant HBV vector with the truncated C gene. Methods C gene truncated HBV vectors were constructed by technologies of molecular clone and PCR based site-directed mutagenesis in vitro. The expression of GFP was observed with the fluorescence microscope after transfection of the recombinant HBV vector plasmids into HepG2 cells by using the liposome method. The capability of HBV vector replication, encapsulation and progeny virus production were examined with semi nested-PCR, native agarose gel electrophoresis blot, quantitative southern blot analysis and the routine PCR assays. Results The HBV vectors with truncated C gene were constructed successfully. The gene was expressed effectively after transfection. The C gene truncated HBV vectors could be replicated and encapsulated in hepatocytes with the helper virus. The encapsulated efficiency were as 4～8 folders as the non C gene truncated HBV vectors by quantitative analysis. In addition, the mature HBV particles carrying the interesting gene of GFP could secrete out from hepatocytes by the help of adjunctive vector. Conclusions The truncation of C gene could improve the encapsulation efficiency of HBV vectors with no effect on the replication and expression of the intact HBV vectors.