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Objective To compare sensitive difference of docetaxel between the triple negative breast cancer(TNBC)cell line CAL-51 and non TNBC line T47D and analyze mechanisms underlying docetaxel resistance in former cells. Methods Cell activi?ty was determined by MTT method and IC50 value was calculated;Wright-Giemsa stain was used to analyze the effect of docetaxel in the morphology of CAL-51 and T47D cell lines. Flow cytometry(FCM)was performed to determine cell cycle distribution and apoptosis. Realtime fluorescence quantitative PCR was used to compare the relative gene expression levels.The anti-apoptosis protein Bcl-2 and caspase family protein expression levels were determined by Western blot. Results Wright-Giemsa stain showed significant morpholo?gy change in T47D cells by docetaxel treatment. Further flow cytometry results confirmed that docetaxel could significantly induce apoptosis in T47D cells compared to CAL-51 cells(P<0.01). The result of realtime fluorescence quantitative PCR revealed that anti-apoptosis protein Bcl-2 was significantly higher expressed in CAL-51 cells(P<0.05). Immunoblot analysis revealed docetaxel treat?ment induced the instrinsic pathways in both CAL-51and T47D cells,but the activated pathway of executioner caspase was different. Conclusion Our present study shows that docetaxel induces different intrinsic apoptosis pathway in CAL-51 and T47D cell lines. An?ti-apoprosis protein Bcl-2 is highly expressed,which might be the underlying mechanism of docetaxel resistance in TNBC cell line-CAL-51.
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Objective To select the immunodominant epitope of human serum albumin(HSA)and provide the basis for set?ting up a special and rapid detecting method of HSA. Methods Bioinformatic method was used to compare protein sequences of hu?man,pig,horse,ox,and ovine,and the immunodominant epitopes of HSA were predicted. The E.coli preferred codons were used to design the DNA sequences of the selected epitopes. The genes of the epitopes were expressed after they were inserted into the PGEX-4T-2 vector. The recombinant antigens were identified and valued by HSA antibody with indirect ELISA. Results The length of the selected epitopes was H1〔126-162 amino acid(aa)〕,H2(314-355aa),and H3(373-424aa)and the relative molecular weight was 3.01×104,3.06×104 and 3.17×104,respectively. The epitope of 373-424 aa were more active and its cross-reactivity IC50 of enzyme-labeled antibody was 1.635 mg/L,which was higher than HSA(P<0.05). Conlusion The immunodominant epitope of HSA is obtained, which is significant for developing a rapid and special reagent of HSA.
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Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.
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Objective To provide the candidate antigens for immunological diagnosis by analyzing the expression of nu -cleoprotein ( NP) of Ebola virus. Methods BioSun software was used to predict the NP epitopes. The bridging-PCR was used to synthesize the NP gene. The pBVIL1 vector was used to clone and express the NP gene. Results The 360-739 aa of NP was confirmed to be the dominant antigen by BioSun software. The recombinant NP dominant antigen was expressed in E.coli with molecular weight of 58 ×103.The specificity of ELISA based on recombinant NP was 99.24% (130/131) in negative samples. Conclusions The dominant NP antigen can be potentially used for developing Ebola virus diagnostic reagent.
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Objective To develop an antigen retrieval method for detection of human mammaglobin ( hMAM) immuno-histochemcal staining in old paraffin-embedded specimens .Methods The tissue sections in test group were put into dis-tilled water after deparaffinization and then moved into citric acid buffer ( pH 3.5) for 10-15 min.The other two meth-ods,microwave method and high pressure cooker method ,were compared as control groups at the same time .Finally, immu-nohistochemistry SP method was used to check the antibody in the sections .Results The color appearance in the test group (pH 3.5 citric solution) was better than that of microwave oven and high pressure cooker groups .In the test group, tissue sections were not easily cast off from the slices .Conclusion In this study,we have established a new and simple antigen retrieval method which will contribute to immunohistochemistry technology .
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ObjectiveTo discuss the relationship between the cross-reactivity of antibody against the hypervariable region 1 ( HVR1 ) of hepatitis C virus and early viral response ( EVR ) in patients undergoing antiviral therapy.MethodsBy ELISA and HCV HVR1 antibody cross-reactivity matrix reagent,the differences of anti-HCV hypervariable region antibody were tested in baseline serum from 46 patients with chronic hepatitis C before antiviral therapy.HCV genotyping and HCV RNA were analyzed by RT-PCR method.The HCV RNA assay was done at three time points:before treatment,pegylated interferon in combination with ribavirin therapy for 12 and 48 weeks.ResultsIn 46 cases of chronic hepatitis C patients,there were 16 cases with HCV 2a type,30 cases with l b and 33 patients obtained EVR.The EVR incidence of type 2a[ 93.8％ ( 15/16 ) ] was higher than that of type 1 b[ 60.0％ (18/30),x2 =4.316,P ＜ 0.05 ].In the EVR group of type 1b chronic hepatitis C patients,the positive number of average multi-target HVR1 antigen was ( 12 ± 4),which was significantly higher than that in the Non-EVR patients [ (7 ± 5 ),t =2.797,P ＜0.01 ].Bv the Fisher exact test,it was showed that patients'serum response to HVR1 antigens numbered 001,003,009,013,016 were higher in EVR group than those in non-EVR group,with statistically significant (P ＜ 0.05 ).ConclusionThe cross-reactivity of HVR1 antibody may play an important role in predicting the effectiveness of antiviral therapy.
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Objective To research CpG and Al(OH)3 adjuvants enhancing immunogenicity of hepatitis C virus(HCV) recombinant ptotein combined vaccine(TIE).Methods BALB/c mice were immunized with candidate vaccine TFE using CpG,Al(OH)3,Al(OH) 3 + CpG,or freund's adjuvant(FA) as the adjuvant.Five mice were sacrificed after 10 d of the last immunization.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Splenic cells were isolated and levels of IFN-γ,IL-4 and cytotoxic T lymphocyte(CTL) cytotoxicity assay were messuredin vitro.The remaining mice were subcutaneouly injected with 1 × 106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results The specific CTL activity of TFE + A1(OH) 3 + CpG group was higher than TFE + FA group and TFE + CpG group(P ＜ 0.05).The level of IFN-γsecreting cells in TFE + Al(OH)3 + CpG group was higher than that in TFE + M(OH)3 group or TFE + CpG group(P ＜ 0.05).Conclusion Combining Al(OH) 3 and CpG could enhance specific cellular immunogenicity of candidate HCV vaccine TFE.TFE + M(OH) 3 + CpG could effetively prevent the attack of tumor cell SP2/0-NS3 expressing nonstructural protein NS3 of HCV.
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Objective To detect antibodies against the hypervariable region 1(HVR1) of hepatitis C virus in blood screening.Methods The HVR1 antibodies were detected by F4HVR1 antigen,and then were compared with other antibodies of HCV.Results Among HCV-RNA positive samples,HVR1 antibody was 96.8% positive.The positive rate of HVR1 antibodies in 90 suspected HCV-infected samples was 61.1%,which was close to those against C and NS3,and higher than NS4 and NS5(P