RESUMO
The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been described as a possible prognostic marker in patients with acute myeloid leukemia (AML). However, the results in this field are not reproducible in different cohorts. In this study, we investigated WT1 mutations, expression levels and SNP rs16754 in a cohort of 122 adult patients with AML. As the major allele (65.6%) in a Chinese population, WT1(GG) was associated with younger age (≤ 60) and lower percentage of blasts than WT1(GA/AA). Meanwhile, improved overall survival (OS, p = 0.035) and disease-free survival (DFS, p = 0.021) were observed in WT1(GG) compared with WT1(GA/AA). We then found that WT1 mutation, occurring in 8% of patients with AML, did not predict clinical outcome. Finally, WT1 levels were higher in patients with WT1(GG) than in those with WT1(GA/AA). However, high levels of WT1 (> median) predicted worse OS (p = 0.015) and DFS (p = 0.034) than low levels of WT1 (≤ median). However, further studies are required to elucidate the mechanism of why WT1(GG), which was associated with higher median expression of WT1 that predicts worse OS and DFS compared to low expression of WT1, predicted better OS and DFS compared with WT1(GA/AA). In summary, WT1 rs16754 and WT1 expression have a significant impact on clinical outcome in patients with AML.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/genética , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas WT1/genética , Doença Aguda , Adolescente , Adulto , Idoso , Povo Asiático/genética , China , Feminino , Frequência do Gene , Genótipo , Humanos , Quimioterapia de Indução/métodos , Estimativa de Kaplan-Meier , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/etnologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/etnologia , Leucemia Promielocítica Aguda/genética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Modelos de Riscos Proporcionais , Indução de Remissão , Adulto JovemRESUMO
OBJECTIVE: To construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20 positive primary chronic lymphocytic leukemia (CLL) cells and provide a promising tool for tumor adoptive immunotherapy. METHODS: The recombinant vectors were transduced into PA 317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection for one week. Then transduced T lymphocytes and primary CLL cells were co-cultured. The status of primary chronic lymphocytic leukemia cells were observed by microscope. The level of IL-2 and IFN-gamma in the culture medium were measured. RESULTS: Primary T cells expressing anti-CD20scFv/IgGFc/CD80/CD28/zeta could be constructed successfully. These T cells were able to lyse CD20+ targets and secrete high levels of IL-2 (1301.00 pg/ml) and IFN-gamma (602.18 pg/ml) in vitro. CONCLUSION: (1) Recombinant gene modified T cells can be constructed successfully. (2) Recombinant gene modified T cells can specially kill CD20 positive primary CLL cells in vitro.
Assuntos
Antígenos CD20/genética , Linfócitos T/imunologia , Transfecção , Antígeno B7-1/genética , Antígenos CD28/genética , Vetores Genéticos , Humanos , Imunoterapia Adotiva , Interferon gama/metabolismo , Interleucina-2/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Retroviridae/genética , Linfócitos T/metabolismo , Células Tumorais CultivadasRESUMO
AIM: To construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/zeta gene and detect its expression in Jurkat cells. METHOD: CD28-zetacDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT- PCR. RESULTS: The recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT-PCR. CONCLUSION: Recombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/zeta gene was successfully constructed and objective protein could be expressed in Jurkat cells.
Assuntos
Antígenos CD20/metabolismo , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Região Variável de Imunoglobulina/metabolismo , Linfócitos T/metabolismo , Antígenos CD20/genética , Antígeno B7-1/genética , Antígenos CD28/genética , Vetores Genéticos , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Células Jurkat , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , TransfecçãoRESUMO
OBJECTIVE: To construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy. METHODS: CD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 gene. The recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radio-activation cytotoxicity assay and ELISA respectively. RESULTS: The recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro. CONCLUSION: Recombinant gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.
Assuntos
Antígenos CD20/imunologia , Antígeno B7-1/imunologia , Antígenos CD28/genética , Linfócitos T/imunologia , Antígenos CD20/genética , Antígeno B7-1/genética , Antígenos CD28/imunologia , Linhagem Celular , Citotoxicidade Imunológica , Vetores Genéticos , Humanos , Imunoterapia Adotiva , Plasmídeos/genética , Linfócitos T/metabolismo , TransfecçãoRESUMO
OBJECTIVE: To study the effect of hydroquinone on apoptosis of bone marrow mononuclear cells, and to evaluate the toxic effect of benzene on stem cells. METHODS: Cell morphology was observed by HT fluorescent stain method, and DNA fragments were analyzed by agarose gel electrophoresis. Anti-Annexin V FITC plus PI staining for apoptotic and necrotic rate was examined by flow cytometer. RESULTS: After adding different concentrations of hydroquinone to the cells for 6 h culture, the fluorescent intensity of nucleus increased, the color of nucleus became deep and inhomogeneous, and the chromatin was condensed and distributed around the neucleus. DNA ladder was detected in all samples. Cell apoptotic rate in different concentration of hydroquinone groups was significantly higher than that in blank control group (P < 0.05). With the increase of the concentration of hydroquinone, the apoptotic and necrotic rate also increased. The optimal concentration of hydroquinone was 50 micro mol/L. When it was >or= 75 micro mol/L, the necrotic rate increased significantly. Hydroquinone-induced apoptosis was associated with culture time at the concentration of 50 micro mol/L, and the peak apoptotic time was 10 h, then the apoptotic rate decreased and necrotic rate increased. CONCLUSION: Hydroquinone can induce apoptosis of bone marrow mononuclear cells in vitro with dose-effect and time-effect relationship.