RESUMEN
Since evidence suggests that transplantation of bone marrow stem cells with the CC chemokine receptor type 5 (CCR5)Δ32/Δ32 genotype may cure patients infected with human immunodeficiency virus (HIV)1, the present study aimed to reproduce the CCR5Δ32 mutation in cluster of differentiation (CD)4+ U87 cells using genome engineering methods. A modified transcription activatorlike effector nucleases (TALENs) technique, combined with homologous recombination for sitespecific, sizecontrolled and homozygous DNA deletions, was used to reproduce the homozygous CCR5Δ32 mutation in CD4+ U87 cells. The results indicated that the frequency of the TALENstargeted mutation reached 50.4% without any selection, whereas homologous recombination from CCR5 to CCR5Δ32 occurred in 8.8% of targeted cells. Notably, a HIV1 challenge test demonstrated that CCR5Δ32/Δ32 CD4+ U87 cells were resistant to HIV infection. In conclusion, engineered CCR5Δ32/Δ32 mutations endowed CD4+ U87 cells with resistance against HIV1 infection; this sitespecific, sizecontrolled and homozygous DNA deletion technique was able to induce precise genomic editing, i.e., the deletion or insertion of a predetermined length of DNA sequence at a specific locus throughout the genome.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Resistencia a la Enfermedad/genética , Infecciones por VIH/genética , VIH-1 , Homocigoto , Receptores CCR5/genética , Eliminación de Secuencia , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Resistencia a la Enfermedad/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Modelos Biológicos , Unión ProteicaRESUMEN
Patients with human immunodeficiency virus-1 (HIV-1) infection often present with hematopoietic failure. As the important hematopoietic support cells in the bone marrow (BM), the BM mesenchymal stem cells (BMSCs) can be impacted by HIV proteins that are released by infected cells within BM. In this study, we tested whether HIV protein p55-gag could induce senescence of BMSCs and reduce their capacity to support expansion of hematopoietic stem cells in vitro. BMSCs were chronically treated with p55-gag (BMSCgag ) for up to 20 days, and their proliferative activity and senescence makers were compared to nontreated cells (BMSCcon ). Then, we analyzed the hematopoietic support function of BMSCcon and BMSCgag by determining cellular proliferation, colony-forming ability, and primitive hematopoietic populations of hematopoietic progenitors grown on the BMSCs. In addition, we compared the gene expression patterns for supporting hematopoiesis of BMSCcon and BMSCgag. The results show that when compared to BMSCcon , BMSCgag reduced their proliferative activity and underwent senescence. The ability of BMSCgag to support the expansion of committed hematopoietic progenitors from umbilical cord blood-derived CD34+ cells may be impaired, while the expression of genes associated with maintaining and enhancing hematopoiesis appeared to be decreased in treated BMSCs compared to control BMSCs. In conclusion, senescence induced by p55-gag resulted in decreased hematopoietic support function of BMSCs through reducing a series of hematopoietic cytokine expression.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , VIH-1/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Precursores de Proteínas/farmacología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Sangre Fetal , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/fisiología , Precursores de Proteínas/toxicidadRESUMEN
OBJECTIVE: To explore the viral etiology of human breast cancer to determine whether there are novel molecular targets for gene therapy of breast cancer and provide evidence for the research of gene therapy and vaccine development for breast cancer. METHODS: PCR was used to screen HPV16 and HPV18 oncogenes E6 and E7 in the SKBR3 cell line and in 76 paraffin embedded breast cancer tissue samples. RNA interference was used to knock down the expression of HPV18 E6 and E7 in SKBR3 cells, then the changes in the expression of cell-cycle related proteins, cell viability, colony formation, metastasis, and cell cycle progression were determined. RESULTS: HPV18 oncogenes E6 and E7 were amplified and sequenced from the SKBR3 cells. Of the patient samples, 6.58% and 23.68% were tested to be positive for HPV18 E6 and HPV18 E7. In the cell culture models, the knockdown of HPV18 E6 and E7 inhibited the proliferation, metastasis, and cell cycle progression of SKBR3 cell. The knockdown also clearly affected the expression levels of cell cycle related proteins. CONCLUSION: HPV was a contributor to virus caused human breast cancer, suggesting that the oncogenes in HPV were potential targets for gene therapy of breast cancer.
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Neoplasias de la Mama/terapia , Terapia Genética/métodos , Proteínas Oncogénicas Virales/genética , Papillomaviridae/fisiología , Infecciones por Papillomavirus/terapia , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Neoplasias de la Mama/genética , Femenino , Humanos , Persona de Mediana Edad , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/genética , Alineación de SecuenciaRESUMEN
Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.
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Regulación hacia Abajo , Infecciones por VIH/virología , VIH-1/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Línea Celular , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Infecciones por VIH/terapia , VIH-1/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
OBJECTIVE: To research the relationship between human herpesvirus 7 (HHV-7) viral Load and the etiopathogenisis of hemophagocytic syndrome, in order to provide evidence for the clinical diagnosis of hemophagocytic syndrome and anti-virus therapy. METHODS: Peripheral blood of patient with hemophagocytic syndrome during different treatment periods, extracted DNA, Syntheticed the primers of HHV-7, gene sequence of PCR amplified fragments detected, determined HHV-7 viral Load by Real-time fluorescent quantitative PCR and the ferritin concentration in peripheral blood detected by chemiluminescence. RESULT: The sequence result indicated that PCR amplified fragment was a part of HHV-7 gene, the ferritin concentration viried with the load of HHV-7. CONCLUSION: The occurrence of hemophagocytic syndrome is connetted with the load of HHV-7.
Asunto(s)
Herpesvirus Humano 7/aislamiento & purificación , Herpesvirus Humano 7/fisiología , Linfohistiocitosis Hemofagocítica/virología , Carga Viral , Ferritinas/metabolismo , Herpesvirus Humano 7/genética , Humanos , Linfohistiocitosis Hemofagocítica/metabolismoRESUMEN
OBJECTIVE: To Construction of P and NP genes eukaryotic expression vectors of Newcastle Disease Virus LaSota strain,study its reverse genetics and functional genome of NDV. METHODS: P, NP genes were amplified and cloned into pGEM-T easy vector and then subcloned into pcDNA3.1 (+) expression vector respectively, the recombinant plasmids were named pcDNA3.1 (+)-P and pcDNA3.1 (+)-NP, Recombinant plasmids were transfected into 293 and BHK-21 cells respectively and were detected using IE and Western blot analysis. RESULTS: Expression of P, NP genes were detected and confirmed by the IE and WB analysis. CONCLUSION: The recombinant eukaryotic plasmids pcDNA3. 1(+)-P, pcDNA3.1 (+)-NP were expressed in 293 and BHK-21 cells successfully. This research may be helpful for further study of reverse genetics and functional genome of NDV.
Asunto(s)
Expresión Génica , Virus de la Enfermedad de Newcastle/genética , Nucleoproteínas/genética , Fosfoproteínas/genética , Proteínas Virales/genética , Animales , Línea Celular , Cricetinae , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Virus de la Enfermedad de Newcastle/metabolismo , Proteínas de la Nucleocápside , Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Virales/metabolismoRESUMEN
OBJECTIVE: To explore the correlation between the polyomavirus DNA load and the dose of immunosuppressant in patients with allogene bone marrow transplantation (allo-BMT) for preventing the development of post-transplantational hemorrhagic cystitis. METHODS: Serial blood and urine samples from 122 cases of allo-BMT recipients were obtained and DNA was extracted from urine samples. Polyomavirus DNA-specific probe was synthesized and Fluorescence quantitative polymerase chain reaction was used for detecting the polyomavirus DNA loads and Fluorescence polarization immunoassay (FPIA) was performed for determining the dose of immunosuppressant Cyclosporin A (CsA) in blood. RESULTS: The altered polyomavirus DNA load in urine was followed by concentration of CsA in blood. When the concentration of CsA in blood was higher than 86-105 ng/ml, the positive rate of polyomavirus DNA load was significantly increased and both presented the linable correlation. CONCLUSION: In immunosuppression condition, polyomavirus DNA load correlated to the dose of immunosuppressant, which increased the risk of post-transplantational hemorrhagic cystitis.
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Ciclosporina/efectos adversos , Cistitis/virología , Inmunosupresores/efectos adversos , Infecciones por Polyomavirus/virología , Poliomavirus/aislamiento & purificación , Carga Viral , Trasplante de Médula Ósea/efectos adversos , Ciclosporina/sangre , Cistitis/prevención & control , ADN Viral/genética , ADN Viral/orina , Relación Dosis-Respuesta a Droga , Humanos , Inmunosupresores/sangre , Poliomavirus/genética , Infecciones por Polyomavirus/prevención & control , Trasplante Homólogo/efectos adversosRESUMEN
OBJECTIVE: To study the correlation between polyoma virus load and hemorrhagic cystitis after allogeneic stem cells transplantation for prevention of hemorrhagic cystitis. METHODS: Blood and urine specimens were collected from 40 healthy persons, 40 patient with stem cells transplantation and 20 cases complicated with hemorrhagic cystitis for determination of VP1 gene of polyomaviruses BK virus (BKV)/Jamestown Canyon virus (JCV) and simian virus 40 (SV40) by polymerase chain reaction (PCR) and EvaGreen stain fluorescence quantitative assay. RESULTS: In the peripheral blood, all genes of BKV/JCV and SV40 were negative, while BKV gene in urine and blood from healthy persons and patient with stem cells transplantation was 15% (6/40) and 100% (40/40), respectively. The gene of JCV was positive in 10% (4/40) and 12% (5/40), the gene of SV40 was negative. CONCLUSION: Genes of BKV and JCV was detectable in urine specimens of healthy persons and there was a correlation between the load of polyomavirus and incidence of hemorrhagic cystitis.
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Cistitis/diagnóstico , Hemorragia/diagnóstico , Infecciones por Polyomavirus/virología , Poliomavirus/crecimiento & desarrollo , Proteínas de la Cápside/genética , Cistitis/etiología , Cistitis/virología , ADN Viral/sangre , ADN Viral/genética , ADN Viral/orina , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Hemorragia/etiología , Hemorragia/virología , Humanos , Reacción en Cadena de la Polimerasa/métodos , Poliomavirus/genética , Infecciones por Polyomavirus/complicaciones , Carga ViralRESUMEN
BACKGROUND: Study on the promotive effects of N-nitrosopiperidine on carcinogenesis process was performed, based on the immortalization of human fetal esophageal epithelium induced by human papillomavirus (HPV) 18E6E7 genes. METHODS: The immortalized esophageal epithelium SHEE was induced by HPV18E6E7. The cells at 17th passages were cultured in 50 ml flasks. The N-nitrosopiperidine (NPIP) 0, 2, 4, 8 mmol/L added to the cultured medium of SHEE cells for 3 weeks. The morphology, proliferation and apoptosis of the cells were studied by phase contrast microscopy and flow cytometry. Modal number of chromosomes was analyzed by standard method. Tumorigenicity of the cells was assessed by soft agar colony formation and by transplantation of cells into nude mice. Expression of HPV was detected by Western blot. RESULTS: When cells were exposed to high concentration (8 mmol/L) of NPIP, cell death was increased, leaving a few live cells. In normal cultural medium instead of NPIP proliferative status of the cells restored after 4 weeks and the cells progressed to the proliferation stage with continuous replication and atypical hyperplasia. At the end of the 8th week, the cells appeared with large colonies in soft-agar and tumor formation in transplanted nude mice. When the cells were cultured in 2, 4 mmol/L NPIP the doubling passage was delayed and without tumor formation in transplanted nude mice. Modal number of chromosomes was 61-65, in 8 mmol/L NPIP group and control group, 56-61. Expression of HPV18 appeared in experimental and control groups. CONCLUSION: NPIP promotes malignant change of the immortalized esophageal epithelial cells induced by HPV18E6E7. HPV18E6E7 synergy with NPIP will accelerate malignant transformation in esophageal epithelium.
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Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Viral/efectos de los fármacos , Papillomavirus Humano 18/fisiología , Nitrosaminas/toxicidad , Animales , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Esófago/citología , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Oncogénicas Virales/metabolismoRESUMEN
OBJECTIVE: To investigate the mechanism of tetra-arsenic tetra-sulfide (As4S4) in inducing apoptosis of acute promyelocytic leukemia (APL) cells. METHODS: The gene expression patterns in NB4 cells pre- and post-treatment with As4S4 were analyzed by cDNA microarray, and differentially expressed genes related with apoptosis were identified. The mRNA expression levels of these apoptosis related genes in the peripheral blood of APL patients treated with As4S4 pre- and post-remission were examined by RT-PCR. RESULTS: Among the differentially expressed genes in NB4 cells pre- and post-treatment with As4S4, two genes were related with cellular apoptosis, which were Apaf-1 and PNAS-2. Apaf1 ratio between pre- and post- As4S4 in NB4 cells was 2.910, PNAS-2 ratio was 0.420. RT-PCR results showed that the expression ratios of Apaf-1, caspase-9 and PNAS-2 in APL patients pre- and post-remission were 2.31 and 3.21, 0.99 respectively. CONCLUSION: As4S4 induced cellular apoptosis in NB4 cells involves the expression changes of the two genes: the up-regulation of Apaf1 and down-regulation of PNAS-2. The increased expressions of Apaf1 and caspase-9 indicate that As4S4 induced apoptosis in APL cells is through mitochondrial pathway, but not the death-receptor pathway. The role played by PNAS-2 in cellular apoptosis needs to be clarified.
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Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/efectos de los fármacos , Factor Apoptótico 1 Activador de Proteasas/genética , Arsenicales/farmacología , Sulfuros/farmacología , Apoptosis/genética , Arsénico/farmacología , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfuros/químicaRESUMEN
The platelet surface glycoprotein (GP) I balpha, an important part of the GP I b-IX-V complex, participates in the formation of thrombosis by initially mediating platelet adhesion under high shear stress. The purpose of present study was to investigate the association between gene polymorphism of GP I balpha (human platelet antigen 2, HPA2) and ischemic stroke in a matched case-control study. One hundred patients and 100 matched controls were enrolled in the study. The cases were divided into large- and small-vessel subtypes of ischemic stroke according to Trial of Org10172 in Acute Stroke Treatment criteria. Genotyping for GP I balpha polymorphism was documented by polymerase chain reaction amplification and restriction enzyme analysis. There were no statistically significant differences in the GP I balpha HPA2 genotype distribution between ischemic stroke group, large-vessel subtype group, small-vessel subtype group and corresponding control groups. The heterozygote genotype of GP I balpha HPA2 was more frequent in the large-vessel subtype group (16.1%) than in the small-vessel subtype group (10.1%), but the difference was not statistically significant. Ourresults suggest that the polymorphism of the GP I balpha HPA2 genotype might not be a genetic risk factor of ischemic stroke.
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Isquemia Encefálica/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Polimorfismo Genético , Adulto , Anciano , Anciano de 80 o más Años , Pueblo Asiatico/genética , Estudios de Casos y Controles , China , Femenino , Frecuencia de los Genes , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.
Asunto(s)
Astrocitos/patología , Cuerpo Estriado/patología , Hemo Oxigenasa (Desciclizante)/farmacología , Hemina/farmacología , Estrés Oxidativo , Adenoviridae/genética , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , Resistencia a Medicamentos , Ferritinas/metabolismo , Técnicas de Transferencia de Gen , Glutatión/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Humanos , Proteínas de la Membrana , Ratones , Ratones Endogámicos , Oxidación-Reducción/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Peroxisome proliferator-activated receptor (PPAR) signaling pathways have been reported to exert anti-inflammatory effects and attenuate atherosclerosis formation. However, the mechanisms responsible for their anti-inflammatory and antiatherosclerotic effects remain largely unknown. The present study tested the hypothesis that a PPARgamma agonist may exert significant endothelial protection by antioxidative and antinitrative effects. METHODS AND RESULTS: Male New Zealand White rabbits were randomized to receive a normal (control) or a high-cholesterol diet and treated with vehicle or rosiglitazone (a PPARgamma agonist) 3 mg x kg(-1) x d(-1) for 5 weeks beginning 3 weeks after the high-cholesterol diet. At the end of 8 weeks of a high-cholesterol diet, the rabbits were killed, and the carotid arteries were isolated. Bioactive nitric oxide was determined functionally (endothelium-dependent vasodilatation) and biochemically (the phosphorylation of vasodilator-stimulated phosphoprotein, or P-VASP). Vascular superoxide production, PPARgamma, gp91phox, and inducible nitric oxide synthase (iNOS) expression, and vascular ONOO- formation were determined. Hypercholesterolemia caused severe endothelial dysfunction and reduced P-VASP, despite a marked increase in iNOS expression and total NOx production. Treatment with rosiglitazone enhanced PPARgamma expression, improved endothelium-dependent vasodilatation, preserved P-VASP, suppressed gp91phox and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation. CONCLUSIONS: The PPARgamma agonist rosiglitazone exerted a significant vascular protective effect in hypercholesterolemic rabbits, most likely by attenuation of oxidative and nitrative stresses. The endothelial protective effects of PPARgamma agonists may reduce leukocyte accumulation in vascular walls and contribute to their antiatherosclerotic effect.
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Antioxidantes/farmacología , Vasos Sanguíneos/efectos de los fármacos , Hipercolesterolemia/tratamiento farmacológico , Nitratos/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazolidinedionas/farmacología , Factores de Transcripción/agonistas , Tirosina/análogos & derivados , Animales , Antioxidantes/uso terapéutico , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Hipercolesterolemia/patología , Hipercolesterolemia/fisiopatología , Técnicas In Vitro , Lípidos/sangre , Masculino , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Conejos , Rosiglitazona , Transducción de Señal/efectos de los fármacos , Tiazolidinedionas/uso terapéutico , Tirosina/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
When cortical neurons are exposed to hemoglobin, they undergo oxidative stress that ultimately results in iron-dependent cell death. Heme oxygenase (HO)-2 is constitutively expressed in neurons and catalyzes heme breakdown. Its role in the cellular response to hemoglobin is unclear. We tested the hypothesis that HO-2 attenuates hemoglobin neurotoxicity by comparing reactive oxygen species (ROS) formation and cell death in wild-type and HO-2 knockout cortical cultures. Consistent with prior observations, hemoglobin increased ROS generation, detected by fluorescence intensity after dihydrorhodamine 123 or dichlorofluorescin-diacetate loading, in wild-type neurons. This fluorescence was significantly attenuated in cultures prepared from HO-2 knockout mice, and cell death as determined by propidium iodide staining was decreased. In other experiments, hemoglobin exposure was continued for 19 h; cell death as quantified by LDH release was decreased in knockout cultures, and was further diminished by treatment with the HO inhibitor tin protoporphyrin IX. In contrast, HO-2 knockout neurons were more vulnerable than wild-type neurons to inorganic iron. HO-1, ferritin, and superoxide dismutase expression in HO-2 -/- cultures did not differ significantly from that observed in HO-2 +/+ cultures; cellular glutathione levels were slightly higher in knockout cultures. These results suggest that heme breakdown by heme oxygenase accelerates the oxidative neurotoxicity of hemoglobin, and may contribute to neuronal injury after CNS hemorrhage.
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Apoptosis/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemoglobinas/toxicidad , Neuronas/efectos de los fármacos , Neuronas/enzimología , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Femenino , Ferritinas/metabolismo , Fluoresceínas , Glutatión/metabolismo , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo-Oxigenasa 1 , Hierro/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Masculino , Proteínas de la Membrana , Metaloporfirinas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo , Protoporfirinas/farmacología , Rodaminas , Superóxido Dismutasa/metabolismoRESUMEN
BACKGROUND: To construct human papillomavirus type 18 (HPV18 E6E7) adeno-associated virus (AAV) for studying the role of HPV E6E7 in the development of human cancer. METHODS: HPV18 E6E7 genes were inserted into adeno-associated virus expression vector and then infected 293 cell line. The expression of HPV18 E6E7 genes were confirmed by using RT-PCR/Southern blot assay. RESULTS: There was HPV18 E6E7 genes in the malignantly transformed cell line. The 293TL cells compared with the parent cells transformed cells grew more rapidly, lost their contact inhibition and formed more and large colonies in soft agar. CONCLUSIONS: HPV18 E6E7 AAV was successfully constructed and could induce malignant transformation. HPV18 E6E7 AAV can be use for studying the immortalization and malignant transformation of human normal epithelial cells.
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Transformación Celular Neoplásica , Proteínas de Unión al ADN , Dependovirus/genética , Células Epiteliales/citología , Proteínas Oncogénicas Virales/genética , Línea Celular , Transformación Celular Viral , ADN Viral/análisis , Células Epiteliales/virología , Feto , Humanos , Riñón/citología , Papillomaviridae/genética , Reacción en Cadena de la PolimerasaRESUMEN
In order to study the role of PML-RARalpha fusion gene and its expression product during apoptotic process in NB4 cells induced by arsenic trisulfide (As(2)S(3)), the apoptotic effects of NB4 cells were observed by cell morphology, flow cytometry and DNA electrophoresis. The change of PML-RARalpha fusion gene and its expression product were also assayed by chromosomal G banding, RT-PCR and Western blot. The results showed that arsenic trisulfide induced apoptosis of NB4 cells, during this process, PML-RARalpha fusion gene had no significant changes, but the expression of PML-RARalpha fusion protein and wild-type RARalpha were all reduced. It is concluded that arsenic trisulfide can induce apoptosis of NB4 cells, the degradation of PML-RARalpha fusion protein and wild-type RARalpha may play an important role during apoptotic process.