ABSTRACT
<p><b>OBJECTIVE</b>To study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein.</p><p><b>METHODS</b>Golph2 gene was cloned by RT-PCR using RNA from WBF44 cell line as template, the point mutations of the cloned sequence were corrected by PCR, then the gene (1206 bp) was cloned into pET-21 plasmid, and the resulted plasmid was transformed into E.coli DH5a. The expression of 6xHis and Golph2 fusion protein was induced by isopropylthio-beta-D-galactoside (IPTG). The expression of fusion protein was detected by SDS-PAGE and Western blot, and was purified by Ni NTA chelating agarose. The rabbit antibody against Golph2 protein was obtained by immunizing 2 rabbits with the purified Golph2 protein. The specificity and titer of the antibody was determined by Western-blot and ELISA respectively. Sandwich ELISA was used to detect the level of serum Golph2 protein.</p><p><b>RESULTS</b>There were two replacement mutation and 1 deletion mutation in the cloned sequence contrasted to NM177937 in Genbank, including 644(T-->C, L-->P) , 970 (G-->A, V-->I) and 802 G deletion. The sequence was completely reversed by PCR. The sequence of Golph2 gene cloned into expression vector was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that Golph2 protein was expressed in E.coli DH5a. The antiserum could bind to the 52 kD recombinant protein and serum 73 kD protein specifically. The mean A450 value of ELISA for serum Golph2 protein were significantly higher in HCC and other liver diseases than that in control groups. The sensitivity and specificity for HCC were 44.5% and 82.0%, respectively, at the cut off value is more than or equal to 0.40.</p><p><b>CONCLUSION</b>The polyclonal antibody against Golph2 protein is specific. The level of serum Golph2 is significantly higher in patients with HCC and other liver diseases than that in healthy controls.</p>
Subject(s)
Animals , Humans , Male , Rabbits , Antibodies, Monoclonal , Allergy and Immunology , Base Sequence , Biomarkers, Tumor , Blood , Carcinoma, Hepatocellular , Diagnosis , Metabolism , Pathology , Cell Line, Tumor , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Methods , Escherichia coli , Genetics , Genetic Vectors , Genetics , Liver Neoplasms , Diagnosis , Metabolism , Pathology , Membrane Proteins , Blood , Genetics , Allergy and Immunology , Metabolism , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
Objective To investigate the effects of different dialysates on expression of protein kinase C-? (PKC?) and apoptosis of U937 cell line. Methods Different dialysates were added into culture fluid with U937 cell line at exponential phase of growth, and groups were divided: fluid A+fluid B group (dialysate A+dialysate B), fluid A+fluid B+rottlerin (PKC? specific inhibitor)group, fluid A+powder B group (dialysate A+powder B) and fluid A+powder B + rottlerin group. Besides, blank control group and normal control group were established. Cells were harvested 24 h and 48 h after treatment, morphological changes were observed by Hoechst33258 fluorescence staining, cell apoptosis was measured by Annexin-V-FITC/PI double staining, and expression of PKC? mRNA and protein was detected by RT-PCR and Western blotting, respectively. Results Cell apoptosis significantly increased in fluid A+powder B group, with typical morphology of apoptosis. After treatment for 24 h and 48 h, cell apoptosis rates in fluid A+powder B group were significantly higher than those at corresponding time points in blank control group, normal control group and fluid A+powder B+rottlerin group (P0.05). Conclusion Fluid A+powder B can significantly increase apoptosis of U937 cell line, the mechanism of which may be associated with the up-regulation of expression of PKC?. Compared with fluid A+powder B, fluid A+fluid B is superior in reducing apoptosis of peripheral blood monouclear cells.
ABSTRACT
<p><b>OBJECTIVE</b>To study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein.</p><p><b>METHODS</b>Four specific siRNAs were obtained by transcription in vitro. After transfection with the siRNAs into HepG2.2.15 cells for 3 days, the inhibitive effects of La protein were analyzed by Western blot; the content changes of HBsAg, HBeAg and HBV-DNA were detected by ECL and RT-PCR.</p><p><b>RESULTS</b>In comparison to normal cells, La protein was less in the cells. There was less La protein in the cells trans-infected with siRNAs. HBsAg, the HBeAg and HBV-DNA secreted by the cells transfected with siRNA were also less than that in the normal cells.</p><p><b>CONCLUSION</b>There is a correlation between La protein and HBV mRNA and the expression of HBV protein.</p>
Subject(s)
Humans , Autoantigens , Metabolism , Cell Line, Tumor , DNA, Viral , Hepatitis B Surface Antigens , Hepatitis B virus , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , RNA, Viral , Ribonucleoproteins , MetabolismABSTRACT
In order to explore the role of real-time PCR in detecting minimal residual disease in multiple myeloma and Waldenstrom's macroglobulinemia after autologous peripheral blood stem cell transplantation (APBSCT), real-time PCR was used to quantitate the IgH rearrangement in 8 patients with multiple myeloma (MM) and 1 case of Waldenstrom's macroglobulinemia before and after APBSCT. The results showed that the copies of IgH rearrangement pre- or post-APBSCT were 3108 +/- 1043 and 549 +/- 660 (P < 0.05) respectively. The number of IgH copies was positively correlated with the amount of plasmocytes in patient 's bone marrow and the M-protein in peripheral blood (r = 0.86, P < 0.05). Similar result was obtained in a case of relapsed Waldenstrom's macroglobulinemia. In conclusion, the quantitative analysis of IgH rearrangement by real-time PCR is a novel way to evaluate the therapeutic efficaciousness and predict the prognoses in MM patients.