[Hepatitis C--progress and challenge].
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
Dong-liang Yang
Publications and source records attributed to Dong-liang Yang.
[Cloning, prokaryotic expression and polyclonal antibody preparation of HCCR: a new biomarker for hepatocellular carcinoma].
OBJECTIVES: To construct a prokaryotic plasmid expressing truncated human cervical cancer oncogene (HCCR-1(167-360)), to express and purify the recombinant protein, and to develop the polyclonal antibody against HCCR. METHODS: HCCR-1(167-360) was amplified by RT-PCR from HepG2 cells and cloned into vector pRSET-B, then expressed in E.coli BL21(DE3) pLysS, which was induced by IPTG. The recombinant protein was purified using Ni-NTA spin column and acrylamide gel electrophoresis. A polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis. RESULTS: Recombinant plasmid expressing truncated HCCR-1167-360 was constructed. A protein of 2.70 x 10(4) was successfully expressed and purified. High titer polyclonal antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein. CONCLUSIONS: The truncated recombinant HCCR-1(167-360) developed in this study is highly purified and shows strong antigenecity; the polyclonal antibody against this HCCR protein was generated by regular immunization method, showing both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of HCCR.
Animals↗
[Expression of interferon alpha family gene of Chinese marmot in eukaryotic and prokaryotic cells].
OBJECTIVE: To investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells. METHODS: Eukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity. RESULTS: The Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction. CONCLUSION: The IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.
Animals↗
Statistical analysis on 2213 inpatients with traffic injuries from January 2003 to September 2005 in Ningbo city.
OBJECTIVE: To analyze the epidemiological conditions and characteristics of inpatients with traffic trauma in order to provide objective data for basic research and clinical application of traffic trauma. METHODS: The data of 2213 inpatients with traffic trauma admitted to Lihuili Hospital, Ningbo Medical Centre, Ningbo, China, from January 2003 to September 2005 were studied retrospectively. According to the transportation ways, the patients were divided into four groups: pedestrians, bicyclists, motorcyclists, and automobilists. And the data of injured regions, combined injuries and causes of death were analyzed statistically. RESULTS: Among the 2213 patients, there were 550 pedestrians (23.5%), 521 bicyclists (24.9%), 738 motorcyclists (33.3%), and 404 automobilists (18.3%). Male patients were more than female ones, with the ratio of male to female of 2.8:1. Single region injury was found in 1663 patients (75.15%) and multiple injuries in 550 patients (24.85%). In total, 2849 regions were injured. Fracture of extremities (53.3%) occurred most often, craniocerebral trauma (19.4%) next, then followed in turn by thoraco-abdominal visceral injury (6.56%), spine fracture (5.37%), fracture of ribs (4.88%), and pelvic fracture (4.18%). The percentage of multiple injuries (33.2%) was highest and the percentage of thoraco-abdominal injury (18.0%) was higher in motorcyclists. The percentages of craniocerebral trauma in pedestrians and bicyclists were 27.8% and 28.2%, respectively. The incidence of fracture of extremities in motorcyclists reached 73.8%, but with the lowest case-fatality ratio of 1.4%. The incidence of traffic accidents caused by motorcyclists themselves (32.8%) was highest. A total of 56 patients died, with the case-fatality ratio of 2.53%. Among the deaths, 47 died from craniocerebral injury, 6 from multiple fractures combined with hemorrhagic shock, 2 from combined injury in the thoraco-abdominal region, and 1 from cervical cord injury. CONCLUSIONS: Nowadays, the patients with traffic trauma are mainly pedestrians, bicyclists and motorcyclists, and they suffer generally from fracture of extremities and craniocerebral injury. The main cause of death is craniocerebral injury. Another characteristic of traffic trauma is that the ratio of multiple injuries is higher.
Accidents, Traffic↗
[Detection of mutants of the "a" determinant region of hepatitis B surface antigen S gene among Wuhan childhood patients].
OBJECTIVE: To investigate whether the presence of HBV mutant in vaccinees simply reflects the prevalence of HBV mutant in a specific geographic area or is indeed due to the immune pressure induced by vaccination. METHODS: HBV S genes were amplified by using polymerase chain reaction (PCR) and DNA sequence analysis of the "a" determinant was performed on sera from 30 childhood patients with immunoprophylaxis and 30 patients without vaccinations. RESULTS: Mutations of the "a" determinant were detected in 8 of the 60 patients. They were all of the adw subtype. The prevalence of amino acid substitutions as detected by direct sequencing was higher in those fully-vaccinated than of those not vaccinated. In all 8 vaccinated and also with detectable mutants, the mean age was older than the other vaccinated children. CONCLUSION: The prevalence of mutants is related to HBV subtypes and genotypes. Universal vaccination has accelerated an accumulation of HBsAg "a" determinant mutants with amino acid changes critical for immune escape in vaccinated children who became carriers. This suggests that new vaccination strategies should be considered.
Child↗
[Expression of human T-cell immunoglobulin mucin 3(TIM-3) on eukaryotic cells and establishment of stable transfectant cell line].
AIM: To construct eukaryotic expression vector of human T-cell immunoglobulin mucin 3(TIM-3) and transfect mammalian cells to establish stable cell line. METHODS: The whole coding region of TIM-3 was amplified by PCR and inserted into eukaryotic expression vector pIRES2EGFP. The recombinant plasmid was transfected into mammalian cells by Lipofectamine. The expression product was analyzed by flow cytometry and Western blot. The stable transfectant was screened and established by flow cytometry and selective medium. RESULTS: COS-7 and CHO cells were transfected with recombinant plasmid by Lipofectamine. The expression speciality was identified by Flow cytometry and Western blot. The stable transfectant of CHO cell line was established. CONCLUSION: The whole coding region of TIM-3 was successfully subcloned into eukaryotic expression vector and expressed on the surface of mammalian cells. The stable transfectant of CHO cell line was established.
Animals↗
[Establishment of a new low-density cDNA macroarray and the application in the activity of IFN against HBV].
OBJECTIVE: To investigate the expression profile of genes which are involved in IFN antiviral activity and IFN signal transduction pathway in Hep G2 and HepG2.2.15 cells. METHODS: Genes of interest were selected from the UniGene database (http://www.ncbi.nlm.gov/UniGene/Hs.Home.html). The 5'IMAGE clones with 0.5-0.8 kb length were chosen and ordered from RZPD company. The cDNA inserts were amplified by PCR and then were spotted onto the Hybond-N+ membranes. The membranes were denatured and neutralized for Macroarray analysis. HepG2.2.15 and Hep G2 cells were treated without or with IFN-alpha for 6 h, and the total cellular RNA was isolated using Trizol Reagent. Radio-labelled cDNA was generated from 20 microgram of RNA by reverse transcription using 360 units of reverse transcriptase in the presence of 30 microCi of alpha-32P dCTP. Hybridization was performed between 32P-labelled cDNA and membrane arrays. The membranes were then scanned, and the intensity of autoradiographic spots was quantitated by Cyclone Storage Phosphor System. The images were subsequently analysed by the OptiQuant Imager Analysis Software and converted into digital data. RESULTS: The authors found that just partially IFN-inducible genes were expressed in Hep G2 and HepG2.2.15 cells, and the majority of IFN-inducible genes was lowly responsive or non-responsive to IFN-a treatment. Some interferon-stimulated genes (ISGs) were inhibited or blocked, especially in HepG2.2.15 cells. Interestingly, the authors found that the IFN signal transduction pathway (Jak-STAT) was intact and unimpaired in HepG2.2.15 cells. CONCLUSION: Differential gene expression profiles in response to IFN were found between Hep G2 and HepG2.2.15 cells.
Antiviral Agents↗
[Infection of tupaia hepatocytes with hepatitis C virus in vitro].
OBJECTIVE: Tupaia belangeri (tree shrew) has a close phylogenetic relationship with primates and has been shown to be susceptible to a variety of human viruses. This study was conducted to investigate whether or not hepatitis C virus (HCV) could infect primary tupaia hepatocytes (PTHs) in vitro. METHODS: Serum-derived HCV was cultivated with PTHs, and then positive and negative strand HCV RNA in PTHs, as well as the encapsidated HCV RNA in the culture medium were detected to evaluate the infection. Virus from the culture medium of the infected PTHs was passed to naïve PTHs, and the quasispecies of HCV were compared among the inoculum and PTHs after infection and passage. RESULTS: Both positive and negative strand HCV RNA were detected in PTHs after infection. The negative strand RNA was detectable from day 5 to day 10 after infection, while the positive strand RNA was positive up to day 14. HCV RNA, which was RNase resistant, could be detected from the culture medium of the infected PTHs from day 3 to day 14. Production of infectious virons of PTH were demonstrated by passage HCV to naïve PTHs. Compared analysis of HCV quasispecies after infection and passage showed that PTHs were selectively infected with defined HCV quasispecies, and new quasispecies emerged in PTHs after passage. CONCLUSION: The present study strongly indicates that PTHs could be infected by HCV and support HCV replication in vitro. Our results would be helpful for the establishment of a tupaia model of HCV infection.
Animals↗
[Diagnostic tests for hepatitis C].
Explore the source record for details and available documents.
Hepacivirus↗
[Correlation between sensitivity to TRAIL and expression level of DR5 on surface of tumor cells].
OBJECTIVE: To investigate the correlation between sensitivity to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and expression level of death receptor 5 (DR5) on tumor cell surface. METHODS: Anti-DR5 mAb was used to detect expression level of DR5 on surface of tumor cells by flow cytometry. Sensitivity to apoptosis induced by TRAIL was determined by TRAIL apoptosis kit. The correlation between expression level of DR5 and sensitivity to TRAIL was analyzed. RESULTS: The expression of DR5 on surface of tumor cells was approximately 97.9% in U937 cells, 95.1% in Jurkat cells, 93.8% in SW480 cells, 86.2% in HCT116 cells, 64.2% in HL-60 cells, 46.6% in HeLa cells and 13.1% in K562 cells, respectively. The apoptosis rate induced by TRAIL was 72.6% in U937 cells, 85.2% in Jurkat cells, 78.6% in SW480 cells, 70.2% in HCT116 cells, 60.1% in HL-60 cells, 45.4% in HeLa cells and 12.3% in K562 cells, respectively. There was a significant positive correlation between the expression level of DR5 with TRAIL-inducing apoptosis (r = 0.997, P < 0.001). CONCLUSION: TRAIL-inducing apoptosis is related to the expression level of DR5 on surface of tumor cells. The results confirm the importance of DR5 expression for induction of apoptosis by TRAIL.
Antibodies, Monoclonal↗