RESUMO
BACKGROUND: Use of conventional dialysate (CD) (powdered sodium bicarbonate dissolved manually with reverse osmosis water before dialysis) is common in Chinese haemodialysis (HD) centres. However, this preparation carries the risk of degradation and contamination, potentially negatively impacting host defense. Commercially available high-purity dialysate (HPD) may decrease inflammation and improve nutritional status in HD patients. However, whether HPD affects immune cells is unclear. The purpose of this study was to investigate the in vitro effect of these dialysates on apoptosis in U937 monocytes and its possible mechanism. METHODS: Following incubation with two different types of dialysate, U937 cell apoptosis was measured by flow cytometry. Cell morphological changes were observed by Hoechst 33258 fluorescence staining. The expression of protein kinase C-δ (PKC-δ) was assayed by RT-polymerase chain reaction and western blot. Cytokine levels in U937 cells after exposure to CD or HPD for an indicated time were assayed by commercial enzyme-linked immunosorbent assay. RESULTS: CD contained more bacteria (66 ± 6 CFU/mL) than HPD (7 ± 3 CFU/mL) while there was no difference in endotoxin levels. Compared with cells exposed to HPD and phosphate-buffered saline (PBS), U937 monocytes experienced more apoptosis when exposed to CD for 24 and 48 h, while there was no significant difference between HPD and PBS. Expressions of PKC-δ mRNA and protein in U937 cells were enhanced following exposure to CD for 24 and 48 h, with increased proteolytic cleavage of PKC-δ which could be inhibited by rottlerin, a specific inhibitor of PKC-δ. Moreover, the cultured supernatant in CD-exposed cells contained significantly higher levels of interleukin-6 (4.09 ± 0.36 vs 2.73 ± 0.38 pg/mL, P < 0.01, 24 h; 4.28 ± 0.32 vs 2.83 ± 0.32 pg/mL, P < 0.01, 48 h) and tumour necrosis factor α (3.45 ± 0.79 vs 2.44 ± 0.39 pg/mL, P < 0.05, 24 h; 4.60 ± 0.57 vs 2.50 ± 0.37 pg/mL, P < 0.01, 48 h) than those of HPD. CONCLUSION: CD, but not HPD, contained more bacterial contamination, increased monocyte apoptosis in a PKC-δ-dependent manner and induced more cell inflammation. These findings suggest that impurity of dialysis fluid may be an important determinant of the elevated inflammation seen in CD-treated patients.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Soluções para Diálise/farmacologia , Inflamação/metabolismo , Monócitos/patologia , Proteína Quinase C-delta/metabolismo , Western Blotting , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , RNA Mensageiro/genética , Diálise Renal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937RESUMO
OBJECTIVE: To study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Anticorpos Monoclonais/análise , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Proteínas de Membrana/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , CoelhosRESUMO
OBJECTIVE: To study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein. METHODS: 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. RESULTS: 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. CONCLUSION: There is a correlation between La protein and HBV mRNA and the expression of HBV protein.
Assuntos
Autoantígenos/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , DNA Viral , Antígenos de Superfície da Hepatite B , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno , RNA Viral , Antígeno SS-BRESUMO
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.
