RESUMO
We constructed the human dickkopf 1 (DKK1) eukaryotic expression plasmid and expressed, purified, and identified its expression product. We extracted cancer cells from cervical cancer tissue, followed by extraction of mRNA. Reverse transcription-polymerase chain reaction was conducted to obtain DKK1 gene fragments. Using these fragments, we prepared the recombinant plasmid pCMV-HA2/DKK1. The recombinant plasmid was restriction enzyme-digested and sequenced, and using liposome vectors, was transiently transfected into Free-Style 293-F cells (serum-free medium). DKK1 protein was detected by western blotting. The amplification product showed the expected size. Restriction enzyme digestion and sequence analysis showed that the recombinant plasmid was PCMV-HA2/DKK1. The expression product was verified properly by western blotting using an anti-DKKI antibody. The successful cloning of the DKKI gene and expression of DKKI protein will be useful for studying the biological activity of tumorigenesis.
Assuntos
Carcinogênese/genética , Proliferação de Células/genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , RNA Mensageiro/biossíntese , Linhagem Celular Tumoral , Eucariotos/genética , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Plasmídeos/genética , RNA Mensageiro/genéticaRESUMO
The study was performed to analyze the proteomic profiling of doxorubicin-treated H9c2 cardiomyocytes in order to identify novel protein biomarkers associated with doxorubicin-induced cardiomyopathy. The protein profiling of H9c2 cells in response to doxorubicin at an apoptosis-induced concentration of 0.5 µM were compared using iTRAQ analysis. Western-blot analysis was used to confirm differentially expressed proteins identified in the proteomic study. A total of 22 differently expressed proteins were identified in doxorubicin-treated H9c2 cells including 15 up-regulated and 7 down-regulated proteins. Gene Ontology (GO) analysis revealed that 10 altered proteins were enriched in the process of apoptosis. We further validated the expression of cathepsin B and its possible regulator nuclear factor kappa B (NF-κB) in H9c2 cells were increased during doxorubicin treatment using Western-blots. Differentially expressed proteins might provide clues to clarify novel mechanisms underlying doxorubicin-induced cardiomyopathy. Our results also suggest that increased cathepsin B expression might be associated with NF-κB up-regulation, and the exact mechanisms need to be clarified.
Assuntos
Catepsina B/metabolismo , Doxorrubicina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteômica/métodos , Animais , Linhagem Celular , NF-kappa B/metabolismo , RatosRESUMO
Campylobacter jejuni of different subtypes were identified in chicken, dairy cattle and diarrhoea patients in China from 2005 to 2006, using multiplex PCR and RFLP. The results indicated that, of the three types of samples, C. jejuni was most frequently detected in poultry of the three types of samples, with an average isolation rate of up to 18.61% and a flock contamination rate of 86.67%. The average incidence of C. jejuni in overall cattle and environmental samples, milk cows, heifers and diarrhoea patients was 7.77, 5.02, 8.70 and 4.84%, respectively. A higher prevalence was detected in outpatients than ward patients (P<0.01), and in patients aged <7 years than in older patients (P<0.01). The 265 isolates of C. jejuni were classified into 20 distinct types by PCR-RFLP analysis of the flaA gene, with the genotype distribution in humans overlapping that in poultry and cattle. This suggests that certain C. jejuni strains circulate between humans and domestic animals such as cattle and poultry.