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OBJECTIVE@#To analyze the clinical efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for paroxysmal nocturnal hemoglobinuria (PNH), and preliminarily explore the role of an improved post-transplantation cyclophosphamide (PTCy) based conditioning regimen in PNH patients receiving transplantation.@*METHODS@#Clinical related data of PNH sufferers receiving allo-HSCT in Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology were collected, and hematopoietic reconstitution, chimerism, PNH cloning, graft-versus-host disease (GVHD), infection, and survival were analyzed.@*RESULTS@#Totally five PNH patients receiving allo-HSCT were enrolled, including 1 case with classic PNH, 3 cases with aplastic anemia-PNH syndrome, 1 case with myelodysplastic syndrome, three of them (case 1-3) received the improved PTCy based conditioning regimen before HSCT. All sufferers engrafted successfully within 28 days, the median time of neutrophil and platelet engraftment was 11 days and 12 days, respectively, no patient occurred acute or chronic GVHD, after a median follow-up of 16 months, all recipients survived and completely eliminated PNH cloning.@*CONCLUSION@#Allo-HSCT can completely clear PNH cloning and restore hematopoietic function with controllable complications, and the improved PTCy based conditioning regimen is proved to be effective in PNH transplantation.
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Humanos , Anemia Aplástica/terapia , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Hemoglobinúria Paroxística/terapia , Condicionamento Pré-TransplanteRESUMO
The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.
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The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.
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Humanos , Antígenos de Neoplasias , Metabolismo , Linhagem Celular Tumoral , Complexo Multienzimático de Ribonucleases do Exossomo , Metabolismo , Células K562 , Leucemia Eritroblástica Aguda , Metabolismo , Proteínas de Neoplasias , Metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA , Metabolismo , Frações Subcelulares , Metabolismo , Proteínas WT1 , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen (HBsAg) on viral antigenicity and replication.</p><p><b>METHODS</b>Hepatitis B virus (HBV) DNA was obtained from patients with HBsAg-positive chronic hepatitis B (CHB) infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B (AB073826) and genotype C (AF286594). Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site-directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants' effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay (CMIA) and Southern blot hybridization, respectively. The viral antigenicity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp-Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein's surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone.</p><p><b>RESULTS</b>Two CHB patients harbored HBV with insertion mutations in HBsAg: one with two (NT) and one with three (NTT) inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA (P = 0.02). The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites (with an average increase of 0.13). The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability (range: -0.55 to 2.97; affecting eight proximal amino acids) and the flexibility (range: -0.01 to 1.1; affecting five proximal amino acids).</p><p><b>CONCLUSION</b>The NTT three amino acids insertion in the HBsAg open reading frame, between 114 and 115 of the wild-type sequence, detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.</p>
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Adulto , Humanos , Masculino , Pessoa de Meia-Idade , DNA Viral , Genética , Genótipo , Antígenos de Superfície da Hepatite B , Genética , Vírus da Hepatite B , Genética , Alergia e Imunologia , Fisiologia , Mutagênese Insercional , Replicação ViralRESUMO
<p><b>BACKGROUND</b>Little is known about the prognosis of coronary artery disease (CAD) in Chinese patients with abdominal aortic aneurysm (AAA). The aim of this study was to evaluate the predictors of in-hospital all-cause mortality of severe CAD in Chinese patients who were hospitalized for AAAs.</p><p><b>METHODS</b>From January 2003 to August 2009, 368 patients were operated on for AAAs. The clinical characteristics were retrospectively collected. The primary outcome was the in-hospital all-cause mortality. The clinical risk factors were subjected to a multivariate analysis to determine the predictors of in-hospital all-cause mortality.</p><p><b>RESULTS</b>During their hospitalization, 23% (85/368) of the patients underwent coronary angiography, which revealed significant lesions in 93% (79/85) of the patients. In 25 cases, coronary artery bypass grafting (CABG) was performed before the AAA repair and in 16 cases of percutaneous coronary intervention (PCI) was performed. Ten patients with AAA alone died before discharge, and eight patients diagnosed with AAA combined with CAD died. There was no statistical difference in the postoperative death between the two groups. The logistic analysis showed that age > 70 years and CAD (vessels ≥ 2) were the significant factors in predicting the adverse clinical outcome.</p><p><b>CONCLUSIONS</b>The prevalence of severe CAD in Chinese patients with AAAs seemed lower than those that were reported. Myocardial evaluation and subsequent revascularization before AAA surgery could improve the clinical outcome for these patients who have severe CAD.</p>
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Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Aneurisma da Aorta Abdominal , Mortalidade , Cirurgia Geral , China , Epidemiologia , Doença da Artéria Coronariana , Epidemiologia , Mortalidade , Cirurgia Geral , Mortalidade Hospitalar , Modelos Logísticos , Análise Multivariada , Prevalência , Estudos RetrospectivosRESUMO
<p><b>OBJECTIVE</b>To analyze the correlation between early lymphocyte count (lymphocyte count on day 30, LC30) post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) and transplant prognosis in leukemia patients.</p><p><b>METHODS</b>The data from 124 consecutive patients undergoing allo-HSCT for leukemia from January 2003 to April 2011 was analyzed retrospectively. LC30 post-allo-HSCT correlated with 5-year overall survival (OS), 5-year relapse rate (RR), 5-year nonrelapse mortality (NRM), accumulative rate of acute graft versus host disease (aGVHD) and chronic graft versus host disease (cGVHD) was studied.</p><p><b>RESULTS</b>Univariate analysis indicated that patients with LC30 ≥ 0.40×10(9)/L had higher 5-year OS than those with LC30 < 0.40×10(9)/L \[(62.2 ± 5.8)% vs (37.0 ± 8.6)%, P = 0.003\], lower 5-year RR\[(13.9 ± 4.7)% vs (32.0 ± 8.4)%, P = 0.027\], lower 5-year NRM \[(31.3 ± 5.8)% vs (45.0 ± 9.3)%, P = 0.048)\], and higher cGVHD cumulative incidence \[(82.9 ± 4.6)% vs (62.7 ± 11.1)%, P = 0.042)\]. Multivariate analysis also suggested that LC30 was associated with 5-year OS, 5-year RR, 5-year NRM, and cGVHD cumulative incidence. At the same time disease risk stratification was associated with prognosis.</p><p><b>CONCLUSIONS</b>Early lymphocyte count (LC30) post-allogeneic hematopoietic stem cell transplantation in leukemia is highly associated with prognosis, which can be the independent prognosis index after allo-HSCT in leukemia and can identify a group of patients who might be suitable candidates for early interventions treatment.</p>
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Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Transplante de Células-Tronco Hematopoéticas , Leucemia , Diagnóstico , Cirurgia Geral , Contagem de Linfócitos , Prognóstico , Estudos RetrospectivosRESUMO
This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.
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Animais , Humanos , Coelhos , Anticorpos , Alergia e Imunologia , Metabolismo , Antígenos de Neoplasias , Alergia e Imunologia , Células Cultivadas , Escherichia coli , Metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , Alergia e Imunologia , Vetores Genéticos , Glutationa Transferase , Células Endoteliais da Veia Umbilical Humana , Biologia Celular , Proteínas de Ligação a RNA , Alergia e Imunologia , Proteínas Recombinantes de Fusão , Alergia e ImunologiaRESUMO
<p><b>OBJECTIVE</b>To analyze mutation patterns in the RT region of hepatitis B virus (HBV) P gene in patients treated with nucleoside or nucleotide analogs.</p><p><b>METHODS</b>Viral DNA was extracted from 227 serum samples of chronic hepatitis B patients from September, 2005 to March, 2007. The RT region of HBV P gene was PCR-amplified and sequenced.</p><p><b>RESULTS</b>Known resistant mutations were found in 111 cases (48.9%) among 227 samples, 75 cases with clear therapy history. Novel mutations, including A222T, L229V and S256C, were also found. The incidence of multi-drug resistance in patients sequentially treated with lamivudine and adefovir was 25% (4/16), and none of the patients treated with lamivudine plus adefovir in combination shown multi-drug resistance.</p><p><b>CONCLUSION</b>The patterns of mutation is complex in nucleotide analogue treated patients. Switching to adefovir in lamivudine resistant patients may lead to multi-drug resistance. Novel mutations identified in this study need further investigation.</p>
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Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Antivirais , Farmacologia , Usos Terapêuticos , Análise Mutacional de DNA , DNA Viral , Genética , Farmacorresistência Viral , Produtos do Gene pol , Genética , Genes Virais , Variação Genética , Vírus da Hepatite B , Genética , Hepatite B Crônica , Tratamento Farmacológico , Genética , Virologia , Inibidores da Transcriptase Reversa , Farmacologia , Usos Terapêuticos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , MétodosRESUMO
<p><b>OBJECTIVE</b>To observe the efficacy and safety of amphotericin B for treatment of invasive fungal infections (IFI) in patients with hematologic diseases.</p><p><b>METHODS</b>121 patients were given amphotericin B 5 -50 mg/d for 5 - 101 d with a median of 19 d.</p><p><b>RESULTS</b>The clinical efficacy rate was 67.3%, and fungal elimination rate 66.7%. The adverse events included rigor and fever, hypokalaemia, hepatic damage, nephrotoxicity, nausea and vomiting, phlebitis and teeter.</p><p><b>CONCLUSION</b>Amphotericin B is still a high-efficiency drug in the treatment of IFI, although it has many side effects. With monitoring of hepatic and renal function, it is still a relatively safe and effective drug.</p>
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Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Anfotericina B , Usos Terapêuticos , Antifúngicos , Usos Terapêuticos , Micoses , Tratamento Farmacológico , Resultado do TratamentoRESUMO
Suppressor of cytokine signaling (SOCS) is a new family of proteins produced in cells. It may play an important role in classic negative feedback loop to regulate cytokine signal transduction. SOCS-1 was observed and confirmed firstly. Expression of SOCS-1 can inhibit cytokine signal transduction of some cytokines, such as IL-6, LIF, OSM, INF-gamma, GH, and so on, many immune responses are regulated by them in vivo. Abnormal expression of SOCS-1 is closely related to some human diseases. It plays an important role in the development of leukemia, rheumatoid arthritis, liver cirrhosis and liver cancer. In this review, the advances of research on the relationship between SOCS-1 and cytokine, and its correlation with some diseases were summarized.
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Humanos , Metilação de DNA , Interleucina-6 , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia , Genética , Fator Inibidor de Leucemia , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , Genética , FisiologiaRESUMO
<p><b>OBJECTIVE</b>This report presented an overview on the epidemiology of enterovirus in Yunnan province, the People's Republic of China.</p><p><b>METHODS</b>A total of 210 strains of non-polioviruses isolated under acute flaccid paralysis surveillance during a 5-year study period from 1997 to 2000 and 2004 were examined. Of the 210 non-polioviruses strains, a total of 12 strains of adenoviruses were serologically identified. The remaining 198 isolates were used for molecular typing, and the viral genomes of 195 nonpolio enteroviruses (NPEVs) were translated to corresponding amino acid sequences and compared with those of the prototype strains.</p><p><b>RESULTS</b>Based on molecular typing, 5 isolates were classified into 5 serotypes of human enterovirus A species while 158 isolates into 34 serotypes of B and 32 isolates into 6 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under acute flaccid paralysis surveillance, human enterovirus B species accounted for 75.2% of the 210 isolates and was considered as the predominant one, followed by human enterovirus C (12.2%), adenovirus (5.7%), and human enterovirus A (2.4%).</p><p><b>CONCLUSION</b>Although the epidemiological characteristics of NPEVs from Yunnan province remained "unknown", the molecular typing method had provided us a breakthrough to understand the epidemiology of these viruses.</p>
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Humanos , China , Epidemiologia , Enterovirus , Classificação , Genética , Infecções por Enterovirus , Epidemiologia , Genes Virais , Epidemiologia Molecular , SorotipagemRESUMO
The purpose of this study was to construct a HA-1-DC nucleic acid vaccine and to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). The dendritic cells (DCs) were generated from HSCT donors in vitro, and its immunologic activity was studied by using flow cytometry and mix lymphocyte reaction. HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic acid vaccine. After transfecting for 48 hours, the expression of HA-1 protein was detected by Western blot. The DCs were cultured with isogenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs). The cytotoxicity of the CTLs was detected by LDH assay. The results showed that the DCs derived from peripheral blood monocytes (PBMCs) expressed the DC phenotype, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48 hours, HA-1 protein was detected by Western blot. The cytotoxity of inducing CTLs was higher than that in the control group. It is concluded that the minor histocompatibility antigen HA-1 can be considered as a target of immunotherapy against leukemia after HSCT.
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Humanos , Vacinas Anticâncer , Genética , Alergia e Imunologia , Células Cultivadas , Células Dendríticas , Biologia Celular , Alergia e Imunologia , Eletroporação , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia , Alergia e Imunologia , Terapêutica , Antígenos de Histocompatibilidade Menor , Genética , Alergia e Imunologia , Oligopeptídeos , Genética , Alergia e Imunologia , Linfócitos T Citotóxicos , Alergia e Imunologia , Transfecção , Vacinas de DNA , Genética , Alergia e ImunologiaRESUMO
In order to investigate the suppressor of cytokine signaling-1 (SOCS-1) expression in peripheral blood mononuclear cells (PBMNC) of patients with acute and chronic myeloid leukemia and analyze its clinical significance, RT-PCR method was used for detecting SOCS-1 mRNA expression in PBMNC of 50 newly diagnosed patients. The result showed that positive expressions of SOCS-1 were observed in 4 of 25 patients with AML (16.00%), in 11 of 25 patients with CML (44.00%) and none in 10 normal controls. The differences between patients with AML and normal controls, and between patients with CML and normal controls were statistically significant. In CML group, 2 out of 12 cases with non-progression (chronic phase), 9 of 13 cases with progression showed the positive expression, the difference between two subgroups was statistically significant. Those CML patients with SOCS-1 mRNA expression had poor response to IFN-alpha. When they transformed into accelerated phase, SOCS-1 mRNA expression was more persistently and frequently observed, and no response to IFN-alpha was observed. Most of them had very poor prognosis. It is concluded that the SOCS-1 mRNA can be detected in the PBMNC of the patients with acute and chronic myeloid leukemia. The SOCS-1 mRNA expression in the patients with CML is higher than that in patients with AML, and it is higher in accelerated phase and blast crisis significantly. This phenomenon is highly related to the reaction of IFN-alpha and prognosis.
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Humanos , Interferon-alfa , Metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva , Genética , Metabolismo , Leucemia Mieloide Aguda , Genética , Metabolismo , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , GenéticaRESUMO
This study was aimed to construct nucleic acid vaccine containing the coding region of the CML28 gene and to express it in human dendritic cells. The full length of CML28 cDNA was amplified from K562 by RT-PCR and subcloned into pGEM-T vector. The CML28 fragment was digested and subsequently inserted into the EcoRI-Xba I sites of pcDNA3.1HisA to construct the recombinant expression vector pcDNA3.1HisA-CML28, which was identified by restrition analysis and sequencing. Human dendritic cells (DC) were separated from peripheral blood mononuclear cells (PBMC) by culture with rhGM-CSF, rhIL-4 and assessed by flow cytometry. The constructed plasmid pcDNA3.1 HisA-CML28 was transfected into DC by electroporation. Western blot was used to detect the expression of fusion protein His-CML28. The results showed that recombinant plasmid pcDNA3.1HisA-CML28 contained the correct full CML28 cDNA identified by restriction analysis and sequencing, and can express the fusion protein His-CML28 in DCs. It is concluded that nucleic acid vaccine containing CML28 gene was constructed and expressed in DC successfully.
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Humanos , Antígenos de Neoplasias , Genética , Alergia e Imunologia , Metabolismo , Antígenos de Superfície , Genética , Alergia e Imunologia , Metabolismo , Western Blotting , Células Cultivadas , Clonagem Molecular , DNA Complementar , Genética , Células Dendríticas , Biologia Celular , Alergia e Imunologia , Metabolismo , Eletroforese em Gel de Poliacrilamida , Exorribonucleases , Genética , Alergia e Imunologia , Metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , Citometria de Fluxo , Vetores Genéticos , Genética , Células K562 , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão , Genética , Alergia e Imunologia , Transfecção , Métodos , Vacinas de DNA , Genética , Alergia e ImunologiaRESUMO
The purpose of this study was to establish a SYBR Green I real-time quantitative RT-PCR method for investigating the correlation between CML28 mRNA expression levels and relapse of leukemia after allo-hematopoietic stem cell transplantation (HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitoring of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph(+) ALL. The results showed that the sensitivity of the established method was at 10(-4) (0.05 ng) level, with interassay variation and intraassay variation of standard samples both below 10%. The CML28 was highly expressed in AML and CML-BP or AP. CML28 level in newly diagnosed group was (6.58 +/- 2.34) x 10(-2), in pre-conditioning regimen group was (2.19 +/- 0.32) x 10(-2), in group that 1 month after HSCT was (1.35 +/- 1.28) x 10(-2), in group that 3 months after HSCT was (4.57 +/- 6.39) x 10(-3). CML28 can be detected 3 months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2 x 10(-2)) survived without relapse, the other 2 case with high level (>2 x 10(-2)) relapsed within one year, 1 case died and 1 case received the second time HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2 x 10(-2) and relapse has taken place. The conclusions was made that CML28 mRNA level is obviously correlated with the development of leukemia. Serial quantification of CML28 mRNA levels are necessary for HSCT recipients, and more informative than a single detection. Using of this assay to evaluate MRD in the patients received HSCT is helpful for prediction of relapse.
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Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Antígenos de Neoplasias , Genética , Antígenos de Superfície , Genética , Exorribonucleases , Genética , Complexo Multienzimático de Ribonucleases do Exossomo , Transplante de Células-Tronco Hematopoéticas , Leucemia , Genética , Patologia , Cirurgia Geral , Recidiva Local de Neoplasia , Compostos Orgânicos , Química , RNA Mensageiro , Genética , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Métodos , Fatores de TempoRESUMO
To explore the diagnoses and treatment of hemophilic pseudotumor, one case with hemophilic pseudotumor misdiagnosed and treated by operation, was observed and analyzed. The result showed that the final diagnosis of this case was following: hemophilia A (mild type) and hemophilic pseudotumor with injury of femoral nerve. The final diagnosis was given from inquiring case history and family history additionally, and drawing assistance from laboratory examination and computed tomography. After operation, the patient's wound healed very well through supplying coagulation factors positively. In conclusion, it was important for inquiring case history and family history particularly and thinking highly of laboratory examination to reduce the misdiagnosis and error of therapy for this case. If paying attention to preoperative preparation, the danger of hemorrhage during operation can be reduced and wound after operation can heal more rapidly.
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Adulto , Humanos , Masculino , Erros de Diagnóstico , Nervo Femoral , Patologia , Hematoma , Diagnóstico , Hemofilia ARESUMO
In order to study the role of telomerase and extracellular regulated protein kinase (ERK) in drug-resistance of leukemia and ovarian cancer cells, telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis were used for qualitative analysis or quantitative detection of telomerase activity respectively, and Western blot was used to detect the expression level of phosphorylatedly activated ERK(1) and ERK(2) protein in the parental and drug resistant cells of leukemia and ovarian cancer. In addition, chemotherapy sensitivity to HRT or DDP was evaluated by MTT assay. The difference of cell cycle distribution between parental cell and drug-resistant cell was analyzed by flow cytometry. The results showed that the drug resistant cells were of higher percentage in G(0)/G(1) phase compared with the parental cell lines. Telomerase activity and phosphorylatedly activated ERK(1) and ERK(2) protein expression level were higher in drug-resistant cells than in parental cell. It is suggested that the increasing number of the drug resistant cells in G(0)/G(1) phase may be considered as a sign of drug resistance. The up-regulation of telomerase activity and phosphorylatedly activated ERK(1) and ERK(2) protein expression level may play an important role in drug resistance of leukemia and ovarian cancer cell lines.
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Feminino , Humanos , Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Células HL-60 , Leucemia , Tratamento Farmacológico , Metabolismo , Patologia , Proteína Quinase 1 Ativada por Mitógeno , Fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Fisiologia , Neoplasias Ovarianas , Tratamento Farmacológico , Metabolismo , Patologia , Fosforilação , Telomerase , MetabolismoRESUMO
<p><b>BACKGROUND</b>The aim of this study was to explore whether the inhibition of nuclear factor-kappaB (NF-kappaB) activation by mutant IkappaBalpha (S32, 36-->A) can enhance TNF-alpha-induced apoptosis of leukemia cells and to investigate the possible mechanism.</p><p><b>METHODS</b>The mutant IkappaBalpha gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IkappaBalpha were obtained by the limiting dilution method. TNF-alpha-induced NF-kappaB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-alpha. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM).</p><p><b>RESULTS</b>Mutant IkappaBalpha protein was confirmed to exist by Western blot. The results of EMSA showed that NF-kappaB activation by TNF-alpha in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IkappaBalpha repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-alpha, but changed very little in transfected HL-60 cells. The inhibition of NF-kappaB activation by mutant IkappaBalpha enhanced TNF-alpha-induced apoptosis. The cytotoxic effects of TNF-alpha were amplified in a time- and dose-dependent manner.</p><p><b>CONCLUSIONS</b>NF-kappaB activation plays an important role in the resistance to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB by mutant IkappaBalpha could provide a new approach that may enhance the anti-leukemia effects of TNF-alpha or even of other cytotoxic agents.</p>
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Humanos , Apoptose , Regulação Leucêmica da Expressão Gênica , Células HL-60 , Proteínas I-kappa B , Fisiologia , Inibidor de NF-kappaB alfa , NF-kappa B , Proteínas Proto-Oncogênicas c-bcl-2 , Genética , Fator de Necrose Tumoral alfa , Farmacologia , Proteína bcl-XRESUMO
In order to investigate the change of telomerase activity and phosphorylated (activated) extracellular regulated protein kinases (ERK) 1 and 2 in hepatocarcinomatous cell line SMMC7721 and leukemic cell line K562 proliferation inhibition and apoptosis, three kinds of chemotherapeutic drugs harringtonine (HRT), vincristine (VCR) and etoposide (VP-16) were selected as inducers; and MTT assay, flow cytometry analysis, telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis were used. The results showed that after treatment of HRT, VCR and VP-16 for 24 hours, the cell proliferation was inhibited, apoptosis was induced, and telomerase activity and the protein expression of phosphorylated ERK1/2 were down-regulated. In HRT treated groups, the descendent grade was the most obvious. It was concluded that the common molecular mechanism of these chemotherapeutic drugs killing SMMC7721 and K562 cell lines might be through inhibiting ERK signal transduction pathways, cutting down ERK activity, reducing the transcription of target genes of ERKs, then indirectly down-regulate telomerase activity, and cell apoptosis is the final result of durative loss of telomere.
Assuntos
Humanos , Antineoplásicos , Farmacologia , Apoptose , Carcinoma Hepatocelular , Patologia , Células K562 , Leucemia , Patologia , Neoplasias Hepáticas , Patologia , Proteína Quinase 1 Ativada por Mitógeno , Metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Metabolismo , Fosforilação , Telomerase , Metabolismo , Células Tumorais CultivadasRESUMO
In order to explore the effect of nuclear factor-kappa B (NF-kappaB) on bcl-x gene transcrtiption in drug-resistant leukemia cell line HL-60/E6, first of all, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin, and then bcl-x(L) mRNA levels were detected by RT-PCR after exposing HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA at different times. Morever, indirect immuno-fluorescence and FCM were used to demonstrate the location of NF-kappaB-RelA in HL-60/E6 cells and the efficiency of liposome-mediated ODN transfection. The results showed that RelA kept active and located at the nuclei of HL-60/E6 cells, and the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P < 0.01 in 4, 6 and 12 h). Exposure of HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA led to a maximal 40% decline of bcl-x(L) mRNA levels. No significant change of bcl-x(L) mRNA level occurred in control group. It was concluded that NF-kappaB was involved in regulating bcl-x transcription. Suppressing NF-kappaB-RelA by antisense technology may down-regulate level of bcl-x(L) mRNA.