Objective To explore the therapeutic effect of dual perfusion embolization and chemotherapy via hepatic artery and portal vein(combmation treatment) in the treatment of unresectable PHC.Methods Eighty-one cases of unresectable PHC were randomly divided into two gronps: (1) Combination treatment group.Forty-one cases,These cases received embolization and chemotherapy via hepatic artery and portal vein through a drug delivery system intraoperatively,and then embolization and chemotherapy via the drug pump were given periodically. (2) TACE group.Forty cases.These cases were treated with Seldinger's technique, the dosage of drugs were the same as used in the former group during laparotomy. After 3 times of treatment, AFP, the size of tumor, liver function, body weight, abdominal perimeter, survival time of the two groups were compared.Results The weight, AFP, decrease of tumour size in combination group were much better than those in TACE group( P 0.05). The median survival time in the two groups were 18.0 months and 11.1 months ( P =0.0001). The accumulating survival rate of 6, 9, 12, 24 months were 87.8%, 78.0% , 68.2%,31.7% in combination group, and 70.0%, 52.5%, 30.0%, 5.0% in TACE group, respectively . The factors affecting survival were therapeutic method, liver function, size of tumour.Conclusions Combination treatment is simple, convenient with less complications, and the effect is better than TACE. So it is an effective method for the unresectable hepatic carcinoma.

OBJECTIVETo prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein.

METHODSTIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs.

RESULTSThe prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein.

CONCLUSIONThe TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

Animals ; Antibodies, Monoclonal ; immunology ; Cloning, Molecular ; Humans ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; immunology

OBJECTIVETo demonstrate molecular characterization of a newly isolated enterovirus.

METHODSVirus were isolated from patient with unknown-causing disease and tested by reverse transcription-polymerase chain reaction (RT-PCR) and 5'3'RACE (rapid amplification of cDNA ends, RACE), in an attempt to obtain the sequence of this newly isolated enterovirus.

RESULTSSequence analysis showed that this newly isolated enterovirus shared 83%-94% nucleotide identity and 91%-100% amino acid identity with enterovirus 89. Phylogenetic analysis indicated that it was probably a new subtype of enterovirus 89.

CONCLUSIONThis newly isolated enterovirus in the stool specimen from patient has the same serotype with enterovirus 89, but it was probably a new subtype of enterovirus 89.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Enterovirus ; classification ; genetics ; isolation & purification ; Feces ; virology ; Genome, Viral ; genetics ; Humans ; RNA, Viral ; genetics ; Sequence Analysis, DNA

OBJECTIVETo investigate HBV mutations in reverse transcriptase (RT) gene and precore/basal core promoter (PC/BCP) regions in a chronic hepatitis B patient and to analyze the link between the mutations and drug resistance or HBeAg sero-conversion.

METHODSEighteen serum samples were collected from a chronic hepatitis B patient during his 14 hospitalizations from June 2002 to September 2007. HBV DNA was extracted and nested PCR was employed for amplification of target gene fragments. Direct sequencing of PCR products was performed followed by analysis with NTI software. The significance of the results was analyzed in combination with the clinical data of the patient.

RESULTSSeveral mutations were identified in succession, including LAM-resistant mutations M204I/V and L180M+M204V, ETV-resistant mutation S202G, and HBeAg nonsense mutation G1896A. The results were in accordance with the disease progression of the patient.

CONCLUSIONSequencing of HBV RT and PC/BCP regions is valuable for comprehensively checking the viral mutations and thus it is helpful in the surveillance of patients in clinics as a way for adopting reasonable antiviral therapy.

Adult ; Antiviral Agents ; pharmacology ; DNA, Viral ; genetics ; Drug Resistance, Multiple, Viral ; genetics ; Genotype ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Mutation

OBJECTIVETo construct an off-line hybrid bioartificial liver supporting system with human liver cell line, and study it's effect on the plasma from patients with liver failure.

METHODSWe established the bioreactor using Psu-2s (Fresenius) cultured with Hep G2 cell transfected with human augmenter of liver regeneration (hALR) gene, then constructed a hybrid bioartificial liver supporting system, at last using the bioartificial liver support system to purify the plasma treated 2 hours with serum bilirubin absorbent, separated from acute on chronic liver failure patients infected by hepatitis B virus.

RESULTSBioreactor was successful constructed. The cell viability in perigastrum of bioreactor is 85.2% and cell propagated rapidly. Before and after treating with bilirubin absorbent, serum total bilirubin was (176.19 +/- 54.14) micromol/L and (50.1 +/- 16.85) micromol/L respectively (P = 0.0002). While there were no significance difference in the level of albumin, urea and glucose. At the begin and end of treatment with bioartificial liver, serum total bilirubin was (50.10 +/- 16.85) micromol/L and (30.27 +/- 15.02) micromol/L respectively (P = 0.000), the urea and albumin increased, urea has significantly difference, but the change of albumin hasn't.

CONCLUSIONThe off-line hybrid bioartificial liver supporting system with human liver cell line were builded successfully and have synthesis and metabolism functions for acute on chronic liver failure patients.

Adult ; Artifacts ; Bilirubin ; metabolism ; Bioreactors ; standards ; Chimera ; End Stage Liver Disease ; physiopathology ; Hepatocytes ; metabolism ; physiology ; Humans ; Liver ; physiology ; Liver Failure ; Liver, Artificial ; utilization ; Male ; Middle Aged

OBJECTIVETo observe the immune effect of DNA vaccines encoding mutated HBV pre-c/c gene (VE2,VE4) in mice.

METHODSThree kinds of plasmid VEC(DNA vaccines encoding HBV pre-c/c gene), VE2 and VE4 were injected into the thigh muscles of different group of BALB/c mice.Blood and splenocytes from mice were isolated at 4 weeks after immunization. We also have mouse groups immunized with three of these plasmid combined with IFN-gamma gene plasmids. The anti-HBc and anti-HBe antibody in peripheral blood in mice were detected by enzyme linked immunosorbent assay (ELISA), antigen-specific cell immune responses were detected by CTL test and enzyme linked immunospot assay(ELISpot).

RESULTSWe found that anti-HBe titers of VE2 and VE4 immunizing groups are higher than VEC group (P < 0.05). We also observed that VE2 and VE4 could induce stronger antigen-specific immune responses than VEC and when combined with IFN-gamma plasmid,the antigen-specific immune responses are stronger than those without combination immunization in mice (P < 0.05).

CONCLUSIONSThe DNA vaccine VE2 and VE4 could induces stronger antigen-specific immune responses than VEC, and when combined with IFN-gamma plasmid,the antigen-specific immune responses are improved in mice.

Animals ; Female ; Hepatitis B ; prevention & control ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B Vaccines ; administration & dosage ; Hepatitis B virus ; genetics ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Mutation ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

OBJECTIVETo express and purify the HIV p24 gene in E. coli cells, and identify p24 antigen activity.

METHODSThe full length gene fragment of HIV p24 was amplified by PCR and inserted into the pRSET vector in order to construct the pRSET-p24 recombined vector. After transforming into E. coil, the purified p24 protein was prepared by metal-ligand affinity chromatography (IMAC). The accuracy of inserted gene and activity, specificity of HIV p24 proteins were detected by two enzymes digestion technology, SDS-PAGE, Western Blot (WB) and ELISA.

RESULTSThe length of the HIV p24 gene fragment was 690 bp after digesting the recombinant plasmid T-p24 and pRSET-p24 with BamH I and Hind III. The expressed proteins had a single expected band of about 24 x 10(3) in SDS-PAGE. The specificity and activity of p24 protein were tested by WB and ELISA.

CONCLUSIONThe HIV p24 sequence from HIV-1 gene plasmid hasbeen expressed in E. coil. This protein possessed good specificity and activity.

Blotting, Western ; Chromatography, Affinity ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Gene Expression ; HIV Core Protein p24 ; genetics ; isolation & purification ; metabolism ; Humans ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; isolation & purification ; metabolism

OBJECTIVETo construct and express anti-human RBC and HIVgp160 fusion protein for rapid detection of antibody to HIV.

METHODSThe gene of the anti human RBC ScFv and HIV antigen were constructed together into expression vector. The fusion protein was expressed in E. coli.

RESULTSThe fusion protein was proved to be able to bind both anti-RBC and HIVgp160. It could cause agglutination of human RBC when HIVgp160 was present.

CONCLUSIONThe fusion protein has the potential in rapid detection of HIV.

Antibodies, Monoclonal ; immunology ; isolation & purification ; Autoantibodies ; immunology ; isolation & purification ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; immunology ; Gene Expression ; Genetic Vectors ; genetics ; HIV Antibodies ; blood ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; metabolism ; HIV Seropositivity ; blood ; Hemagglutination Tests ; methods ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

BACKGROUNDTo establish a molecular detection and typing assay for identification and typing of human enteroviruses (HEV) which is suitable for clinical detection and epidemiologic research.

METHODSUsing both primers specific for HEV genus and HEV typing primers and reverse transcription polymerase chain reaction (RT-PCR) the authors detected preliminarily HEV by agarose gel electrophoresis and then identified serotype through nucleotide sequence analysis of RT-PCR amplicons. The monospecific antisera neutralization was applied to validate the typing results.

RESULTSThe serotype of 18 suspicious HEV samples was identified: 4 Coxsackievirus type A24 (CVA24), 3 CVB3, 1 CVB2, 1 CVA9, 1 CVA15, 1 Echovirus type 3 (E3), 1 E6, 1 E9, 1 E11, 1 E14, 1 E33 and 1 Rhinovirus type 9. The result was validated by monospecitive antisera neutralization.

CONCLUSIONThis RT-PCR assay for HEV detection and typing may be suitable for clinical detection and epidemic research since this method is sensitive and specific for direct identification and typing of HEV.

DNA Primers ; Enterovirus ; classification ; genetics ; Enterovirus Infections ; diagnosis ; Humans ; RNA, Viral ; genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Serotyping ; methods

BACKGROUNDTo evaluate an enzyme linked immunospot (ELISPOT) method for testing the specific cellular immunity of patients with hepatitis B and preliminarily investigate into the difference of cellular immunity in patients with various types of hepatitis B.

METHODSThe patients with acute hepatitis B, chronic hepatitis B liver cirrhosis, healthy persons with HBV vaccine immunization, healthy persons with past HBV infection and HBV naive persons were enrolled in this study. Their peripheral blood mononuclear cells were tested by ELISPOT to determine the number of gamma-interferon secreting cells.

RESULTSThe number of gamma-interferon secreting cells was significantly different between the patients with acute hepatitis B and those with chronic hepatitis B, and between the patients with acute hepatitis B and those with liver cirrhosis (P=0.0209 and P=0.0211).

CONCLUSIONThe specific cellular immunity in the patients with hepatitis B could be evaluated by determining the number of gamma-interferon secreting cells with the method of testing their peripheral blood mononuclear cells by ELISPOT. The specific cellular immunity was stronger in the patients with acute hepatitis B than in those with chronic hepatitis B and liver cirrhosis.

Enzyme-Linked Immunosorbent Assay ; methods ; Hepatitis B ; blood ; immunology ; Hepatitis B, Chronic ; blood ; immunology ; Humans ; Immunity, Cellular ; immunology ; Interferon-gamma ; blood ; Leukocyte Count ; Leukocytes, Mononuclear ; cytology ; metabolism ; Liver Cirrhosis ; blood ; immunology