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<p><b>BACKGROUND AND OBJECTIVE</b>Interphase fluorescence in situ hybridization (FISH) and real-time quantitative reverse transcription PCR (RQ-PCR) are the common methods for monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients. This study was to assess the value of monitoring BCR-ABL fusion gene level in CML patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) using FISH and RQ-PCR.</p><p><b>METHODS</b>BCR-ABL fusion gene levels were detected in the bone marrow of 31 patients with CML before and 3-48 months after allo-HSCT using FISH and RQ-PCR.</p><p><b>RESULTS</b>BCR-ABL positive cells detected by FISH were decreased 3-30 months after allo-HSCT and BCR-ABL/ABL mRNA was reduced by 2 logarithmic units in RQ-PCR (P < 0.05). While no BCR-ABL positive cell was detected by FISH 30 months after allo-HSCT, BCR-ABL/ABL mRNA was detected by RQ-PCR and declined by more than 3 logarithmic units, (P < 0.05).</p><p><b>CONCLUSIONS</b>Dynamic monitoring of BCR-ABL gene on molecular level in CML patients after allo-HSCT is useful in the early prediction of susceptibility to recurrence in the patients and in designing intervention, and is thus helpful in improving the overall survival rate after transplantation.</p>
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Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Adulto Joven , Proteínas de Fusión bcr-abl , Genética , Metabolismo , Trasplante de Células Madre Hematopoyéticas , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva , Metabolismo , Patología , Terapéutica , Neoplasia Residual , ARN Mensajero , Metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
<p><b>OBJECTIVE</b>To study the clinical characteristics and outcomes of BCR/ABL-positive acute lymphoblastic leukemia (BCR/ABL360888725-ALL) and screen the prognostic factors for BCR/ABL360888725-ALL.</p><p><b>METHODS</b>From January 2001 to May 2008, 59 patients (median age of 32 years ranging from 3 to 69 years) with the diagnosis of BCR/ABL360888725-ALL by fluorescence in situ hybridization received induction chemotherapy with VDLP-/+Ara-C regimen. The patients who failed to respond to the chemotherapy received subsequent consolidation chemotherapy with imatinib (400-800 mg/day) (17 cases) or allogeneic hematopoietic stem cell transplantation (allo-HSCT) (16 cases).</p><p><b>RESULTS</b>Of the 59 patients, 32 (58.3%) achieved complete remission (CR) after the first induction cycle. In patients with peripheral white blood cell (WBC) count <30=10(9)/L, 30-99.9(9)/L and > or =100(9)/L, the CR rates were 75.0% (18/24), 56.3% (9/15) and 26.3% (5/19) (P=0.006), and the overall survival probability of 2 years ( OSs of 2-yrs) was 24.7%, 22.5% and 21.1%, respectively (P=0.180). According to the FAB classification, 56 cases were divided into L1, L2 and biphenotypic acute leukemia (BAL) subgroups, and their CR rates were 66.7% (6/9), 63.2% (24/38) and 22.2% (2/9) (P=0.029), with OSs of 2-yrs of 22.2%, 27.0% and 22.0%, respectively (P=0.623). In terms of immunophenotype grouping by EGIL, the patients with ALL, myeloid antigen-positive ALL and BAL had CR rates of 61.1% (11/18), 60.6% (20/33) and 12.5% (1/8) (P=0.039), and the OSs of 2-yrs of 22.7%, 21.0% and 18.8%, respectively (P=0.643). In 55 patients with known karyotype, the CR rates were 71.4%(5/7), 70.8% (17/24) and 37.5% (9/24) in normal, sole t(9;22) abnormality, t(9;22) with additional abnormalities groups (P=0.046), with the OSs of 2-yrs of 42.9%, 34.0% and 7.3%, respectively (P=0.000). The patients complicated by septicemia had significantly lower OSs of 2-yrs than those without septicemia (0% vs 38.8%, P=0.005). The OSs of 2-yrs were significantly higher in patients with consolidation chemotherapy with imatinib than those without (48.0% vs 11.2%, P=0.001), and allo-HSCT was associated with significantly higher OSs of 2-yrs than exclusive chemotherapy (54.2% and 8.5%, P=0.000).</p><p><b>CONCLUSION</b>BCR/ABL360888725-ALL with WBC> or =100 x 10(9)/L, presence of BAL diagnosed by FAB or FACM, t(9;22) with additional chromosome abnormalities all adversely affect the treatment results, and additional chromosome abnormalities and septicemia are associated with lower OSs of 2-yrs. Imatinib treatment and allo-HSCT can both improve the OSs of 2-yrs of the patients with BCR/ABL(+)-ALL.</p>
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Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Protocolos de Quimioterapia Combinada Antineoplásica , Usos Terapéuticos , Benzamidas , Terapia Combinada , Genes abl , Genética , Trasplante de Células Madre Hematopoyéticas , Mesilato de Imatinib , Piperazinas , Usos Terapéuticos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Genética , Terapéutica , Pirimidinas , Usos Terapéuticos , Resultado del TratamientoRESUMEN
<p><b>OBJECTIVE</b>To investigate the feasibility and efficacy of rituximab combined with high-dose chemotherapy supported by autologous peripheral blood stem cell transplantation (ASCT) in patients with aggressive B-cell non-Hodgkin lymphoma (NHL).</p><p><b>METHODS</b>Twenty-eight patients with aggressive B-cell NHL (22 newly diagnosed, 6 relapsed) were enrolled in this study. The high-dose chemotherapy included CHOP regimen (CTX + ADM + VCR + PDN) for the newly diagnosed patients and DICE (DEX + IFO + DDP + VP-16) or EPOCH (VP-16 + PDN + VCR + CTX + ADM) for the relapsed patients. Each patient received infusion of rituximab at a dose of 375 mg/m(2) for four times, on D1 before and on D7 of peripheral blood stem cell mobilization, and on D1 before and D8 after stem cell reinfusion.</p><p><b>RESULTS</b>Complete remission was achieved in all patients after high dose chemotherapy and ASCT. At a median follow-up of 37 months, the estimated overall 4-year survival and progression-free survival rate for all patients were 75.0% and 70.3%, respectively, while both were 72.7% for the previously untreated patients. The therapy was generally well tolerated with few side-effects attributable to rituximab.</p><p><b>CONCLUSION</b>These results suggest that adding rituximab to high-dose chemotherapy with peripheral blood stem cell transplantation is feasible and may be beneficial for patients with aggressive B-cell non-Hodgkin lymphoma.</p>
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Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Anticuerpos Monoclonales de Origen Murino , Usos Terapéuticos , Antineoplásicos , Usos Terapéuticos , Protocolos de Quimioterapia Combinada Antineoplásica , Usos Terapéuticos , Cisplatino , Usos Terapéuticos , Terapia Combinada , Ciclofosfamida , Usos Terapéuticos , Dexametasona , Usos Terapéuticos , Supervivencia sin Enfermedad , Doxorrubicina , Usos Terapéuticos , Etopósido , Usos Terapéuticos , Fiebre , Ifosfamida , Usos Terapéuticos , Linfoma de Células B Grandes Difuso , Terapéutica , Trasplante de Células Madre de Sangre Periférica , Prednisolona , Usos Terapéuticos , Prednisona , Usos Terapéuticos , Estudios Prospectivos , Inducción de Remisión , Rituximab , Tasa de Supervivencia , Vincristina , Usos Terapéuticos , VómitosRESUMEN
<p><b>OBJECTIVE</b>To investigate the expressions of cell surface differentiation antigen CD56 and CD11b antigen in acute monocytic leukemic (AML-M(5)) cells and their clinical significance.</p><p><b>METHODS</b>A total of 113 cases of de nove adult AML-M(5) were examined genetically and immunologically using G-banding technique, interphase fluorescence in situ hybridization (I-FISH) and flow cytometry immunophenotyping, and the results were analyzed in relation to their clinical data.</p><p><b>RESULTS</b>Of the 113 cases, the expression rates of CD56 and CD11b was 28.32% and 73.45%, respectively. The CD56(+) patients had high CD11b expression, and the expression levels of CD11b and CD56 were positively correlated (P<0.05). The incidence of karyotypic abnormalities was 48.57% (55 cases) in these patients, including 25 (22.12%) with 11q23 aberrations. Twenty-five cases were positive for MLL gene abnormalities as found by I-FISH analysis. Compared with the patients positive for both CD56 and CD11b, those negative for both CD56 and CD11b showed increased peripheral blood white blood cell (WBC) count and also increased blast and progenitor cells in the bone marrow (P<0.05); the former patients often had karyotypic abnormalities, commonly involving 11q23 aberrations (P<0.05), whereas the latter patients presented more likely with extramedullary infiltration and refractory leukemia (P<0.01) with lowered complete remission rate and shortened median survival time (P<0.01). CD56-positive patients were more likely to have karyotypic abnormalities and refractory leukemia than CD11b-postive patients (P<0.05), but the peripheral blood WBC counts, bone marrow blast and progenitor cells, extramedullary infiltration, complete remission rate or median survival time showed no significant differences between them (P>0.05).</p><p><b>CONCLUSION</b>AML-M(5) patients with CD56 positivity and high expression of CD11b often have aberrant karyotypes, commonly involving 11q23/MLL gene abnormality. These patients frequently develop extramedullary infiltration and refractory leukemia often with poor prognosis.</p>
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Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Antígeno CD11b , Genética , Metabolismo , Antígeno CD56 , Genética , Metabolismo , Regulación de la Expresión Génica , Cariotipificación , Leucemia Monocítica Aguda , Diagnóstico , Genética , Metabolismo , Patología , Recuento de Leucocitos , PronósticoRESUMEN
This study was aimed to explore the single nucleotide polymorphism (SNPs) of breakpoint cluster region of bcr gene in Chinese people and the relationship between SNPs and chronic myelogenous leukemia (CML). A 3.12 kb region spanning from exon 13 to exon 15 in the bcr region were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were verified by sequencing. The results indicated that 6 novel SNP sites and 2 bases different from reference sequence were confirmed in the region studied, and the frequency of 6 novel SNP sites in studied population was obtained, one SNP of which was found in exon 13 and caused a nonsynonymous mutation. The gene frequencies of novel SNPs had no significant difference between CML and control people. It is concluded that sequence polymorphisms in the major breakpoint cluster region of bcr gene are found, most of which are SNPs, No relationship can be confirmed between SNPs and CML disease.
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Humanos , Secuencia de Bases , Rotura Cromosómica , Proteínas de Fusión bcr-abl , Genética , Leucemia Mielógena Crónica BCR-ABL Positiva , Genética , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-bcr , GenéticaRESUMEN
The study was aimed to explore whether there are leukemic characteristics in the bone marrow mesenchymal stem cells (BMMSC) from leukemic patients as compared with normal controls. The mesenchymal stem cells from bone marrow of normal volunteers and patients with APL and CML were isolated, then cultured and proliferated in vitro. The morphology, growth curve and cell surface markers of two different sources mesenchymal stem cells were investigated for detecting whether the bone marrow mesenchymal stem cells derived from leukemia patients have the specific abnormal fusion gene of leukemia cells through fluorescent in situ hybridization. The results indicated that there was no significant difference between the mesenchymal stem cells derived from different subjects, the bone marrow mesenchymal stem cells derived from leukemia patients did not have the clonal malignant fusion gene as seen in the leukemia cells. Taken altogether, mesenchymal stem cells derived from leukemia patients had no biological differences as compared with those from normal volunteers, and no malignant clonal abnormality was found. It is concluded that mesenchymal stem cells derived from leukemia patients as an alternative vehicle may be used for assistant of autologous hematopoietic stem cell transplantation or cell therapy and gene therapy.
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Humanos , Células de la Médula Ósea , Biología Celular , Células Cultivadas , Proteínas de Fusión bcr-abl , Genética , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva , Genética , Patología , Leucemia Promielocítica Aguda , Genética , Patología , Células Madre Mesenquimatosas , Patología , Proteínas de Fusión Oncogénica , GenéticaRESUMEN
To study the gene expression profile in K562 cells treated by IFN-alpha, so as to provide some information about the potential mechanism of IFN-alpha curing CML, the changes of gene expression were examined with the DNA array in K562 cells before and after treatment with IFN-alpha. The results showed that no gene expression difference more than 2.5 times in K562 cells was found on the first day after treatment with IFN-alpha (200 U/ml), then the genes significant expression difference increased step by step, and reached the peak on the forth day. In all examined genes, 97 genes significant expression difference were detected, 86.60% (84/97) gene of interest out of those gene were up-regulated, 13.40% (13/97) were down-regulated. In these 97 genes with significant expression difference, cell regulator protein genes accounted to 23.71% (23/97), surface receptor genes 14.43% (14/97), oncogenes and tumor suppressors 11.34% (11/97), extracellular communication proteins 9.28% (9/97), cell adhesion molecular genes 8.25% (8/97) and the other genes accounted to 32.99% (32/97). JAK1 was up-regulated to 3.78 times, JAK2 to 15.43, STAT1 and STAT2 were up-regulated to 11.98 and 8.11 times respectively, and these genes are components of JAK-STAT pathway. The number of different genes began to decrease on the fifth day. There were still 9 genes that had expression difference more than 3 times on the twenty-first day. It is concluded that when concentration of IFN-alpha was 200 U/ml, the forth day should be considered as the best time to examine change of gene expression in K562 cells treated by IFN-alpha. IFN-alpha realizes its biological functions through the JAK-STAT pathways and it may be one of the mechanisms for curing CML with IFN-alpha.
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Humanos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Interferón-alfa , Farmacología , Janus Quinasa 1 , Genética , Células K562 , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción STAT1 , Genética , Factor de Transcripción STAT2 , GenéticaRESUMEN
To investigate the relationship between the single nucleotide polymorphism (SNPs) of the bcr and abl gene and chronic myelogeous leukemia (CML), the 9 sequence-tagged sites (STS) in bcr and abl gene were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were varified by sequencing. The results showed that the polymorphism sites were detected in 4 out of the 9 STS fragments and there were 3 bases different from the reference sequence found in 3 fragments. In conclusion, the novel SNP in U07000 fragment shows significantly different frequencies between CML and controled people.
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Humanos , Cromatografía Líquida de Alta Presión , Métodos , Proteínas de Fusión bcr-abl , Genética , Genes abl , Genética , Leucemia Mielógena Crónica BCR-ABL Positiva , Genética , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-bcr , Genética , Análisis de Secuencia de ADN , Lugares Marcados de SecuenciaRESUMEN
<p><b>BACKGROUND</b>We distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) effects and to investigate the distribution of T-cell receptor (TCR) V beta gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8 donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR V beta genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells.</p><p><b>RESULTS</b>The 24 TCR V beta gene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Another 4 donors expressed part of the 24 TCR V beta subfamily and 1 donor had oligoclonality. The expressions of the 24 TCR V beta subfamilies were skewed and restricted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR V beta subfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6 - 30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in V beta 2-3, 16-17, 18-19, 21 and V beta 23 in patients with GVHD and in V beta 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had V beta 15 and 16 T cell expansion after transplantation. One patient displayed V beta 18 T cell expansion after donor lymphocyte infusion (DLI).</p><p><b>CONCLUSIONS</b>Normal individuals express the entire 24 TCR V beta gene repertoire and have polyclonal distribution. However, the TCR V beta gene repertoire is only partially expressed in some donors. The TCR V beta gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR V beta gene subfamilies increases over time post-transplantation. GVHD and GVL effects may induce the proliferation of T cell clones. Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen.</p>
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Humanos , Enfermedad Injerto contra Huésped , Genética , Efecto Injerto vs Leucemia , Genética , Trasplante de Células Madre Hematopoyéticas , Leucemia , Genética , Terapéutica , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To screen and identify apoptosis related proteins and explore the mechanism of harringtonine (HT)-induced K562 cells apoptosis.</p><p><b>METHODS</b>Flow cytometry was used to distinguish K562 cells in the earlier stage of apoptosis from those in the later stage of apoptosis by annexin V and PI staining. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with computer image analysis was used to detect the changes in protein expression in the two stages of apoptosis. Proteins were identified by peptide mass fingerprint in combination with database searching.</p><p><b>RESULTS</b>K562 cells treated with HT for 5 and 24 hours were in the early and later stages of apoptosis respectively. Statistical analysis showed 3 spots disappeared, 7 spots with decreased intensity and 10 spots with increased intensity in the 24 h HT induced apoptotic cells as compared with that in 5 h HT induced ones. Ten spots were selected on the basis of the intensity and the significant changes in abundance. Among them, 5 apoptosis related proteins were successfully identified by MALDI-TOF: keratin 9, BTF3, TrpRS, RS and prohibitin.</p><p><b>CONCLUSIONS</b>Up-regulation of TrpRS, RS, prohibitin and down-regulation of BTF3 were involved in inhibition of transcription and protein synthesis in the apoptotic K562 cells induced by HT, whereas up-regulation of keratin 9 was related to apoptosis resistance.</p>
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Humanos , Apoptosis , Electroforesis en Gel Bidimensional , Harringtoninas , Farmacología , Células K562 , Metabolismo , Patología , Proteoma , MetabolismoRESUMEN
<p><b>OBJECTIVE</b>To study the gene expression profile of chronic myeloid leukemia (CML) in chronic and blast phases for providing information about the potential mechanisms of blastic crisis.</p><p><b>METHODS</b>The gene expression difference of bone marrow mononuclear cells between CML in chronic and in blastic phases was examined with DNA array.</p><p><b>RESULTS</b>In the blastic phase, 68 genes were obviously up-regulated in the 1176 tested genes. Among the 68 genes, the transcription factors accounted for 23.5%, cell surface antigens 22.1%, regulation proteins 19.1% and others 35.3%. There were 17 genes expressed only in the blastic phase and 11 (16.2%) genes related to G protein.</p><p><b>CONCLUSIONS</b>Obvious difference of gene expression profile existed between chronic and blastic phases of CML. Up-regulation of gene expression is one of the characteristics of CML in blastic phase. The genes related to G protein may play an important role in the blastic transformation of CML.</p>
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Femenino , Humanos , Masculino , Crisis Blástica , Genética , Metabolismo , Células de la Médula Ósea , Metabolismo , Células Cultivadas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión GénicaRESUMEN
<p><b>OBJECTIVE</b>To detect the bcr/abl fusion gene in chronic myeloid leukemia (CML) for assisting in clinical diagnosis.</p><p><b>METHODS</b>Fluorescence in situ hybridization (FISH) and nested reverse transcription polymerase chain reaction (RT-PCR) were used.</p><p><b>RESULTS</b>Out of 16 CML patients, the results of FISH was not consistent with that of nested RT-PCR in 3 cases. When compared with the results of Northern blot, it was found that nested RT-PCR was more sensitive than FISH, but might give false positive results. Moreover, FISH was the easier method for detecting rarer types of bcr/abl transcripts. For detecting the latter occasions nested RT-PCR have to design several specific primers.</p><p><b>CONCLUSION</b>It could take their advantages of applying FISH and nested RT-PCR at the same time to make the results more accurate and reliable for the diagnosis, prognosis and monitor of minimal residual disease.</p>
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Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Masculino , Proteínas de Fusión bcr-abl , Genética , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva , Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Métodos , Sensibilidad y EspecificidadRESUMEN
<p><b>OBJECTIVE</b>To investigate the material foundation of the fusion of bcr and abl genes, and to explore the pathogenesis of chronic myeloid leukemia.</p><p><b>METHODS</b>By FISH combined with laser confocal scanning microscopy, the three-dimension (3D) distribution of bcr and abl genes in the interphase nuclei of normal and irradiated IM-9 cells was studied in each cell cycle phases.</p><p><b>RESULTS</b>abl and bcr genes distributed non-randomly in the interphase nuclei of IM-9 cells. abl gene preferably located at the outer layer and bcr near the core of the nucleus. The two genes were drawn near each other most in G(0) phase. The relative distance between the homologous genes was greater at proliferation phase than at quiescence phase. After irradiation, the relative distances from the two genes to the core and between the two genes were shortened, with the shortest distance between the two genes in S phase.</p><p><b>CONCLUSION</b>Irradiation could change the 3D-distribution of abl and bcr genes in the interphase nuclei of IM-9 cell and accelerate them to draw near each other.</p>
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Femenino , Humanos , Núcleo Celular , Genética , Efectos de la Radiación , Células Cultivadas , Proteínas de Fusión bcr-abl , Genética , Efectos de la Radiación , Fusión Génica , Efectos de la Radiación , Genes abl , Genética , Efectos de la Radiación , Hibridación Fluorescente in Situ , Interfase , Genética , Efectos de la Radiación , Linfocitos , Microscopía Confocal , Proteínas Proto-Oncogénicas c-bcr , Genética , Efectos de la RadiaciónRESUMEN
To clarify the association between HLA-DPB1 alleles and chronic myelogenous leukemia (CML) in South Chinese, the allelic types of HLA-DPB1 were detected by sequence based typing (SBT) in 86 patients with CML and 82 healthy individuals from Southern China. The results showed that the frequencies of HLA-DPB1 * 1301 and DPB1 * 20011 were higher in patients with CML in comparison with those of healthy individuals. It is concluded that positive association may exist between certain HLA-DPB1 alleles and CML.
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Humanos , Alelos , Distribución de Chi-Cuadrado , China , Frecuencia de los Genes , Genotipo , Antígenos HLA-DP , Genética , Cadenas beta de HLA-DP , Leucemia Mielógena Crónica BCR-ABL Positiva , GenéticaRESUMEN
Objective To investigate BAALC(brain and acute leukemia cytoplasmic)gene expression in patients with de novo acute myeloid leukemia(AML)and its clinical significance. Methods BAALC expression was determined by real-time quantitative polymerase chain reaction(RQ-PCR) in 63 de novo AML patients.The association between BAALC expression and therapeutic effect was analyzed.Results The correlation coefficiencies were over 0.99 for standard curves of RQ-PCR method. BAALC expression was detected in 49(78%)AML patients.The peripheral WBC counts,hemoglobin, platelet counts and the bone mahow blast cell percentage at onset in 31 AML patients with high BAALC expression were(26.3?18.1)?10~9/L,(78.3?21.8)g/L,(76.9?64.5)?10~9/L and(61.2?22.3)% and those of 32 AML patients with low BAALC expression were(30.2?21.7)?10~9/L,(81.6?30.9)g/L, (73.9?57.2)?10~9/L,(54.3?16.3)%,respectively.No statistic differences were found between these two groups.The AML patients with normal chromosome karyotypes are more likely to have a high BAALC expression(68%)compared with those with abnormal chromosome karyotypes(23%,?~2=12.093,P= 0.001).AML patients with normal cytogenetics and high BAALC expression shows significant lower CR rate (65%)compared with those with low BAALC expression(84%,?~2=6.573,P=0.013). Conclusion High BAALC expression may define an important risk factor in AML with normal cytogenetics and predicts an adverse prognosis.
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The aim of this study was to investigate the cryobiological characteristics of placental cord blood (PCB) cryopereserved by using BioArchive auto-preserved liquid nitrogen system (BioArchive system). After Hespan depletion of red blood cells, 5 ml mixture of DMSO and 10% Dextran 40 were added into 20 ml of enriched leukocyte. 53 PCB units were cryopreserved as following protocol: pre-freeze rate 10 degrees C/min, start freeze temperature -3 degrees C, end freeze temperature -10 degrees C to -15 degrees C, post freeze rate 2 degrees C/min, and end temperature -50 degrees C. After rapid thawing at 38 degrees C, the PCB were washed with 5% human serum albumin -10% Dextran 40 and centrifuged at 400 x g, 10 degrees C for 20 minutes. The results showed that the viability of nucleated cells post-thaw was (73.3 +/- 12.5)%, the CD34(+) cell content was (0.3 +/- 0.21)% for pre-freeze PCB and (0.45 +/- 0.36)% for post-t haw PCB. No significant difference for CFU-GM/-G/-GEMM counts was found between pre-freeze and post-thaw PCB. Thawed PCB contained in two compartments (20 ml and 5 ml) of a freezing bag showed similar viability and clonogenic capacity. Differential count of white blood cell was significantly changed. For post-thaw PCB, it was dramatically decreased for the percentage of granulocytes, and highly increased for the percentage of lymphocytes and monocytes. It was concluded that the condition for cryopreservation and thawing of PCB may be harmful to mature cells, and cells with large size, such as granulocyte, but suitable to lymphocyte and monocyte, especially for the cells with small size, such as CD34(+) cells.
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Femenino , Humanos , Antígenos CD34 , Alergia e Inmunología , Conservación de la Sangre , Métodos , Supervivencia Celular , Criopreservación , Métodos , Sangre Fetal , Biología Celular , Alergia e Inmunología , Granulocitos , Biología Celular , Células Madre Hematopoyéticas , Biología Celular , Linfocitos , Biología Celular , Monocitos , Biología Celular , Placenta , Biología Celular , Factores de TiempoRESUMEN
The aim of this study was to construct recombinant mDHFR-GFP/AAV vector containing mutated dihydrofolate reductase (mDHFR) and green fluorescent protein (GFP) fusion genes and its expression in NIH3T3 cells, to investigate the resistance of the cells to methotrexate. Amplified cDNA of mDHFR and GFP segmented from their plasmid separately were linked by PCR with the aminoacetic acid linker. The fusion gene was inserted into T vector, and after enzyme cutting the fusion gene fragment was inserted into AAV vector, then packaging the vector into recombined AAV and infected NIH3T3 cells. Expression of gene fusion was observed by PCR, fluorescent microscopy and flow cytometry. mDHFR and GFP cDNA were found in NIH3T3 genomic DNA, the GFP expression rate was about 25%, and resistance of the transferred cells to MTX was increased markedly. The results showed that AAV vector can transfer mDHFR and GFP fusion gene into NIH3T3 cells and increase resistance to MTX in gene modified cells. This data provided a basis for application of mDHFR and AAV vector in gene therapy.