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1.
Mem. Inst. Oswaldo Cruz ; 115: e190405, 2020. graf
Article in English | LILACS | ID: biblio-1091247

ABSTRACT

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.


Subject(s)
Humans , Cell Transformation, Viral/genetics , Oncogene Proteins, Viral/metabolism , DNA-Binding Proteins/metabolism , Human papillomavirus 18/metabolism , Oxidation-Reduction , Gene Expression Regulation, Neoplastic , Cell Survival , Cell Line, Tumor/virology , Cell Proliferation
2.
Clinics ; 73(supl.1): e558s, 2018. tab, graf
Article in English | LILACS | ID: biblio-974945

ABSTRACT

The name of the family Polyomaviridae, derives from the early observation that cells infected with murine polyomavirus induced multiple (poly) tumors (omas) in immunocompromised mice. Subsequent studies showed that many members of this family exhibit the capacity of mediating cell transformation and tumorigenesis in different experimental models. The transformation process mediated by these viruses is driven by viral pleiotropic regulatory proteins called T (tumor) antigens. Similar to other viral oncoproteins T antigens target cellular regulatory factors to favor cell proliferation, immune evasion and downregulation of apoptosis. The first two human polyomaviruses were isolated over 45 years ago. However, recent advances in the DNA sequencing technologies led to the rapid identification of additional twelve new polyomaviruses in different human samples. Many of these viruses establish chronic infections and have been associated with conditions in immunosuppressed individuals, particularly in organ transplant recipients. This has been associated to viral reactivation due to the immunosuppressant therapy applied to these patients. Four polyomaviruses namely, Merkel cell polyomavirus (MCPyV), Trichodysplasia spinulosa polyomavirus (TSPyV), John Cunningham Polyomavirus (JCPyV) and BK polyomavirus (BKPyV) have been associated with the development of specific malignant tumors. However, present evidence only supports the role of MCPyV as a carcinogen to humans. In the present review we present a summarized discussion on the current knowledge concerning the role of MCPyV, TSPyV, JCPyV and BKPyV in human cancers.


Subject(s)
Humans , Tumor Virus Infections/virology , Polyomavirus/pathogenicity , Polyomavirus Infections/virology , Neoplasms/virology , Virus Activation , Cell Transformation, Viral , Polyomavirus/classification , Polyomavirus/physiology
3.
Arch. argent. pediatr ; 113(5): 411-418, oct. 2015. graf, tab
Article in Spanish | LILACS | ID: lil-757062

ABSTRACT

Introducción. Existen evidencias de la asociación de determinantes sociales con la salud infantil. Objetivo. Identificar características sociodemográficas asociadas a desigualdades en la salud infantil y evaluar el efecto acumulado sobre la salud de factores de riesgo basados en estas características. Población y métodos. Evaluamos niños de 4-13 años, de Bariloche, entre junio de 2008 y mayo de 2009. Características sociodemográficas consideradas: nivel socioeconómico, educación materna, embarazo adolescente, cobertura médica, inseguridad y hábitos familiares. Valoramos la percepción parental de la salud física y socioemocional, el estado nutricional y la salud bucal en relación con dichas características y con la acumulación de factores de riesgo. Utilizamos encuesta, antropometría y examen bucal. Resultados. Participaron 180 escolares. El nivel educativo materno se asoció con la salud física, socioemocional y bucal del niño. El porcentaje de niños con piezas faltantes o caries fue 77% entre aquellos cuyas madres, como máximo, habían completado el primario, comparado con 13% entre aquellos cuyas madres habían completado estudios terciarios/universitarios. La posibilidad de percepción de salud física y socioemocional no óptima aumentó con cada factor de riesgo 1,8 y 1,4 veces, respectivamente, y la posibilidad de caries o piezas faltantes se duplicó con cada factor de riesgo adicional. El 27,3% de los escolares presentó sobrepeso y el 8,7%, obesidad, y no se encontró asociación con características sociodemográficas. Conclusiones. El bajo nivel socioeconómico familiar y educativo materno se asoció con una mayor prevalencia de resultados de salud desfavorables. Múltiples factores de riesgo tienen un efecto acumulado sobre la percepción parental de la salud física y socioemocional y la salud bucal.


Introduction. There is evidence of an association between social determinants and child health. Objective. To identify sociodemographic characteristics related to child health inequalities and to analize the cumulative effect on health of risk factors based on these characteristics. Population and Methods. We evaluated 4-13 year-old children in Bariloche between June 2008 and May 2009. The following sociodemographic characteristics were taken into account: socioeconomic level, maternal education, adolescent pregnancy, medical coverage, unsafeness, and family habits. We assessed parental perception of physical, and social and emotional health, nutritional status and oral health in relation to these characteristics and the accumulation of risk factors. We used survey, anthropometry and oral examination. Results. One hundred and eighty students participated. The level of maternal education was associated with the child's physical, social and emotional, and oral health. The percentage of children with missing teeth or cavities reached 77% among those whose mothers had, at most, completed primary school, compared to 13% among those whose mothers had completed tertiary school or university. The possibility of perceiving a non-optimal physical, and social and emotional health increased 1.8 and 1.4 times with each risk factor, respectively, and the possibility of having missing teeth or cavities was twice as much with each additional risk factor. Overweight and obesity was observed in 27.3% and 8.7% of students, respectively, and no relationship was found with sociodemographic characteristics. Conclusions. A low family socioeconomic level and a low maternal education level were associated with a higher prevalence of unfavorable health outcomes. Multiple risk factors have an cumulative effect on parental perception of physical, social and emotional, and oral health.


Subject(s)
Humans , Cell Transformation, Viral/genetics , Gene Expression Profiling , Transcriptome , Antigens, Surface/genetics , Antigens, Surface/metabolism , Cell Line, Transformed , Cell Proliferation , Cells, Cultured , Flow Cytometry , Gene Expression , Genes, Viral , Genotype , /genetics , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Transcription, Genetic , Virus Latency
4.
Article in English | WPRIM | ID: wpr-61562

ABSTRACT

Epstein-Barr virus, a ubiquitous human herpesvirus, can induce both lytic and latent infections that result in a variety of human diseases, including lymphoproliferative disorders. The oncogenic potential of Epstein-Barr virus is related to its ability to infect and transform B lymphocytes into continuously proliferating lymphoblastoid cells. However, Epstein-Barr virus has also been implicated in the development of T/natural killer cell lymphoproliferative diseases. Epstein-Barr virus encodes a series of products that mimic several growth, transcription and anti-apoptotic factors, thus usurping control of pathways that regulate diverse homeostatic cellular functions and the microenvironment. However, the exact mechanism by which Epstein-Barr virus promotes oncogenesis and inflammatory lesion development remains unclear. Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases often have overlapping clinical symptoms as well as histologic and immunophenotypic features because both lymphoid cell types derive from a common precursor. Accurate classification of Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases is a prerequisite for appropriate clinical management. Currently, the treatment of most T/natural killer cell lymphoproliferative diseases is less than satisfactory. Novel and targeted therapies are strongly required to satisfy clinical demands. This review describes our current knowledge of the genetics, oncogenesis, biology, diagnosis and treatment of Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.


Subject(s)
Cell Transformation, Viral , Epstein-Barr Virus Infections/complications , Herpesvirus 4, Human/physiology , Humans , Killer Cells, Natural/immunology , Lymphoproliferative Disorders/diagnosis , T-Lymphocytes/immunology
5.
Article in English | WPRIM | ID: wpr-358135

ABSTRACT

Xerostomia is a severe side effect of radiation therapy in head and neck cancer patients. To date, no satisfactory treatment option has been established. Because mesenchymal stem cells (MSCs) have been identified as a potential treatment modality, we aimed to evaluate stem cell distribution following intravenous and intraglandular injections using a surgical model of salivary gland damage and to analyse the effects of MSC injections on the recruitment of immune cells. The submandibular gland ducts of rats were surgically ligated. Syngeneic adult MSCs were isolated, immortalised by simian virus 40 (SV40) large T antigen and characterized by flow cytometry. MSCs were injected intravenously and intraglandularly. After 1, 3 and 7 days, the organs of interest were analysed for stem cell recruitment. Inflammation was analysed by immunohistochemical staining. We were able to demonstrate that, after intravenous injection, MSCs were recruited to normal and damaged submandibular glands on days 1, 3 and 7. Unexpectedly, stem cells were recruited to ligated and non-ligated glands in a comparable manner. After intraglandular injection of MSCs into ligated glands, the presence of MSCs, leucocytes and macrophages was enhanced, compared to intravenous injection of stem cells. Our data suggest that injected MSCs were retained within the inflamed glands, could become activated and subsequently recruited leucocytes to the sites of tissue damage.


Subject(s)
Animals , Antigens, Polyomavirus Transforming , Allergy and Immunology , Cell Culture Techniques , Cell Movement , Physiology , Cell Transformation, Viral , Clone Cells , Physiology , Flow Cytometry , Immunohistochemistry , Injections, Intralesional , Injections, Intravenous , Leukocytes , Pathology , Macrophages , Pathology , Mesenchymal Stem Cell Transplantation , Methods , Mesenchymal Stem Cells , Pathology , Physiology , Necrosis , Rats, Wistar , Salivary Ducts , Pathology , Sialadenitis , Pathology , Therapeutics , Simian virus 40 , Allergy and Immunology , Submandibular Gland , Pathology , Submandibular Gland Diseases , Pathology , Therapeutics , Time Factors
6.
Chinese Journal of Virology ; (6): 268-277, 2014.
Article in Chinese | WPRIM | ID: wpr-356604

ABSTRACT

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.


Subject(s)
Amino Acid Sequence , Animals , Betaretrovirus , Chemistry , Classification , Genetics , Physiology , Cell Transformation, Viral , Green Fluorescent Proteins , Genetics , Metabolism , Mice , Molecular Sequence Data , NIH 3T3 Cells , Phylogeny , Retroviridae Infections , Virology , Sequence Alignment , Sheep , Sheep Diseases , Virology , Transformation, Genetic , Tumor Virus Infections , Virology , Viral Envelope Proteins , Chemistry , Genetics , Metabolism
7.
Invest. clín ; 54(3): 311-324, sep. 2013. tab
Article in Spanish | LILACS | ID: lil-740328

ABSTRACT

El linfoma de Hodgkin (LH) es una neoplasia del sistema linfático. La incidencia mundial anual del LH es de 3-10/100,000 habitantes. El mecanismo mediante el cual se lleva a cabo la transformación celular no es completamente claro; sin embargo, algunas evidencias parecen indicar que ciertos virus oncogénicos como el virus Epstein Barr (VEB), pueden tener alto impacto en la patogénesis de la linfoproliferación. También algunos factores genéticos y ambientales pueden estar involucrados, pues se ha encontrado una alta incidencia de casos de LH entre individuos de una misma familia que comparten características genéticas y conviven en un mismo ambiente. En México se han realizado estudios encaminados a conocer la prevalencia del VEB en pacientes con LH y se ha encontrado la presencia de este virus hasta en el 64,2%. El VEB ha sido detectado en las Células Reed Sternberg (CRS) y en Células de Hodgkin (CH) en el 50% de los casos de LH clásico. No se ha dado hasta ahora una explicación satisfactoria, pero se ha propuesto que la variabilidad geográfica y la variabilidad inmunológica desempeñan un papel determinante en la positividad del VEB en LH. A pesar de los avances que hasta ahora se tienen, no existen suficientes evidencias que permitan establecer una clara asociación entre los factores del huésped, el medio ambiente y el agente patógeno en el riesgo de la linfoproliferación que conduce al desarrollo de LH. La presente revisión tiene como objetivo analizar algunos de los factores de riesgo que influyen durante la interacción huésped, agente patógeno y medio ambiente en la etiología del LH.


Hodgkin lymphoma (HL) is a neoplasm characterized by malignant cells called Reed Sternberg and Hodgkin’s cells in the lymphatic system. Such cells comprise 1% of the tumor while the remainder is made up of lymphocytes, histiocytes, eosinophils and plasma non-neoplastic cells. The annual global incidence of HL is 3-10/100,000 inhabitants and is most commonly found in young adults. The mechanism by which cell transformation is accomplished is not entirely clear; however, some evidences suggest that oncogenic viruses like the Epstein Barr virus (EBV) may have a high impact on the pathogenesis of lymphoproliferation. Genetic and environmental factors could be involved, since it has been found a high incidence of HL among members of the same family. In Mexico, there have been studies to determine the prevalence of EBV in patients with HL and found the presence of this virus in up to 64.2% of the cases. EBV has been detected in the Reed Sternberg cells and Hodgkin cells in 50% of cases of classical HL. There is not a satisfactory explanation for this, but it has been proposed that geographic and immunological variabilities play a role in the positivity of EBV in HL. However, despite recent advances in the field, there is insufficient evidence to show a clear association between host factors, environment and pathogens, and the risk of lymphoproliferation leading to the development of HL. This review aims to give an overview about the risk factors that influence the interaction of host, pathogens and environment in the etiology of HL.


Subject(s)
Female , Humans , Male , Epstein-Barr Virus Infections/virology , Host-Pathogen Interactions , /physiology , Hodgkin Disease/virology , Biomarkers, Tumor , Cell Transformation, Viral , DNA, Viral/genetics , Epstein-Barr Virus Infections/immunology , Gene Expression Regulation, Viral , /genetics , /immunology , Hodgkin Disease/diagnosis , Hodgkin Disease/epidemiology , Immune Evasion , Immunocompromised Host , Risk , Risk Factors , Reed-Sternberg Cells/virology , Virus Latency , Viral Proteins/physiology
8.
Chinese Journal of Stomatology ; (12): 631-636, 2012.
Article in Chinese | WPRIM | ID: wpr-260220

ABSTRACT

<p><b>OBJECTIVE</b>To construct SD rat immortalized dental follicle cells (rDFC) induced by simian virus 40 large tumor antigen (SV40Tag) gene to provide a reliable cell source for periodontal tissue engineering research.</p><p><b>METHODS</b>The rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test.</p><p><b>RESULTS</b>Morphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in flat cloning were observed between the immortalized rDFC [34% (33/96)] and normal rDFC at passage four [22% (21/96)] (χ(2) = 3.71, P > 0.05). No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice.</p><p><b>CONCLUSIONS</b>The rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering research.</p>


Subject(s)
Animals , Antigens, Viral, Tumor , Genetics , Metabolism , Cell Differentiation , Cell Proliferation , Cell Transformation, Viral , Cells, Cultured , Dental Sac , Cell Biology , Allergy and Immunology , Metabolism , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Plasmids , Rats , Rats, Sprague-Dawley , Simian virus 40 , Genetics , Allergy and Immunology , Telomerase , Metabolism , Transfection
9.
Braz. j. med. biol. res ; 44(12): 1209-1214, Dec. 2011. ilus, tab
Article in English | LILACS, SES-SP | ID: lil-606544

ABSTRACT

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.


Subject(s)
Alphapapillomavirus/immunology , Capsid Proteins/biosynthesis , Oncogene Proteins, Viral/biosynthesis , Pichia/metabolism , Alphapapillomavirus/genetics , Antibodies, Viral/immunology , Capsid Proteins/genetics , Cell Transformation, Viral/physiology , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Viral , Oncogene Proteins, Viral/genetics , Papillomavirus Vaccines/immunology , Pichia/genetics , Pichia/virology , Reverse Transcriptase Polymerase Chain Reaction
10.
Article in Chinese | WPRIM | ID: wpr-234323

ABSTRACT

<p><b>OBJECTIVE</b>To establish immortalized lymphoblastoid cell lines of a Miao core pedigree with Bardet-Biedl syndrome (BBS), in order to provide a long-term source of material for research.</p><p><b>METHODS</b>With Epstein-Barr virus transformation of B cells and addition of cyclosporine A to inhibit the activity of T cells, fresh anticoagulated blood samples with heparin were collected from 12 members of the core pedigree, and were used to establish the immortalized lymphoblastoid cell lines of B lymphocytes.</p><p><b>RESULTS</b>Twelve immortalized lymphoblastoid cell lines of the core BBS pedigree were obtained successfully.</p><p><b>CONCLUSION</b>The immortalized B lymphoblastoid cell lines of the Miao pedigree with BBS can preserve the whole genome information and provide long-term research materials for BBS study.</p>


Subject(s)
B-Lymphocytes , Cell Biology , Bardet-Biedl Syndrome , Blood , Genetics , Cell Line , Cell Line, Transformed , Cell Transformation, Viral , China , Ethnology , Ethnic Groups , Genetics , Herpesvirus 4, Human , Humans , Pedigree
11.
Article in Chinese | WPRIM | ID: wpr-290023

ABSTRACT

<p><b>OBJECTIVE</b>To establish immortalized B lymphoblast cell lines (B-LCLs) from healthy anti-HBs antibody (anti-HBs)-positive volunteers and screen for human anti-HBs and the antibody-secreting cells.</p><p><b>METHODS</b>The peripheral blood mononuclear cells (PBMC) isolated from 3 healthy volunteers positive for anti-HBs with hepatitis B vaccine boost vaccination were infected with Epstein-Barr virus (EBV) and incubated in the presence of CpG DNA motifs and cyclosporin A (CyA). The anti-HBs in the culture supernatant of the immortalized B-cells was quantified by Architect anti-HBs assay with chemiluminescent microparticle technique. Immunocytochemistry was performed to identify the differentiation of the cell clones expressing anti-HBs.</p><p><b>RESULTS</b>Immortalized B-cell culture was successfully established from the cell clones secreting anti-HBs with EBV infection and CpG DNA stimulation. The titer of anti-HBs in the culture supernatant was at its peak at 3 weeks of cell culture and then decreased gradually. At 3 months of cell culture, the cells still retained the capacity of anti-HBs production as verified by the results of immunocytochemistry for CD20 and CD138.</p><p><b>CONCLUSION</b>Immortalized B-cell culture secreting anti-HBs from volunteers receiving boost hepatitis B vaccination has been successfully established by modified EBV immortalization technique.</p>


Subject(s)
B-Lymphocytes , Allergy and Immunology , Cell Line , Cell Transformation, Viral , Hepatitis B , Hepatitis B Antibodies , Allergy and Immunology , Hepatitis B Surface Antigens , Allergy and Immunology , Hepatitis B Vaccines , Allergy and Immunology , Herpesvirus 4, Human , Allergy and Immunology , Humans , Immunization, Secondary , Vaccination
12.
Article in English | WPRIM | ID: wpr-635138

ABSTRACT

Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells (IPSCs), which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.


Subject(s)
Cartilage/cytology , Cell Proliferation , Cell Transformation, Viral , Cells, Cultured , Fetus , Simian virus 40/genetics , Stem Cells/cytology , Transfection
13.
Arab Journal of Gastroenterology. 2009; 10 (4): 117-124
in English | IMEMR | ID: emr-99946

ABSTRACT

At each stage of the life cycle of the virus, hepatitis C virus [HCV] interferes with the cellular antiviral mechanisms of the host. Therefore, HCV infection represents a fencing match between the virus and the host cell. The host's defense depends primarily on activation of the immune response, including activation of interferon [IFN] signalling, expression of cytokines [TNF-alpha, IL-12, IL-10, IFN-alpha] and stimulation of cellular immune response [CIR] and humoral immune response [HIR]. HCV offense relies on envelope mutation, evasion of the host immune response and interference with the endogenous cellular antiviral factors


Subject(s)
Antiviral Agents/immunology , Interferons , Cell Communication , Cytokines , Immunity, Cellular , Immune System , Immunity, Humoral , Virus Replication , Interleukin-10 , Interleukin-12 , Tumor Necrosis Factor-alpha , Virus Internalization , Virus Assembly , Cell Transformation, Viral
14.
Alexandria Journal of Veterinary Sciences [AJVS]. 2009; 28 (1): 71-80
in English | IMEMR | ID: emr-99709

ABSTRACT

In this study we use 20 sheep divided into four groups [one group was injected with the vaccine, the second was taken the vaccine and ampicillin, the third group was taken the vaccine and cephalosporin and the fourth act as a control]. The immunity was measured humoral immunity to sheep pox virus vaccine by neutralization test and cell mediated immunity by lymphocyte transformation assay as well as phagocytosis. The results revealed that an increase of the immune status in group injected with antibiotics with vaccine than that received the vaccine only and than the control group. There were an increase of lymphocyte transformation test, neutralization test and phagocytosis. It could be concluded that ampicillin and cephalosporin directly increase the immune status in sheep vaccinated with sheep poxvirus vaccine


Subject(s)
Animals , Immunomodulation/drug effects , Ampicillin/pharmacology , Cephalosporins/pharmacology , Capripoxvirus/immunology , Viral Vaccines , Cell Transformation, Viral/drug effects , Colorimetry/methods
15.
Article in Chinese | WPRIM | ID: wpr-349072

ABSTRACT

<p><b>OBJECTIVE</b>Immortalized cell lines of spinocerebellar ataxia type 2 (SCA2) with Parkinson disease symptoms were established in order to provide experimental material for future study.</p><p><b>METHODS</b>The immortalized cell lines were constructed by using Epstein Barr virus and cyclosporine A. Microsatellite markers were detected to see whether there is any change between the cell lines and the original blood samples, and the genetic stability of the cell lines were evaluated.</p><p><b>RESULTS</b>Twenty-five immortalized cell lines were established successfully from the family and the microsatellite markers were unchanged.</p><p><b>CONCLUSION</b>The karyotypes of the immortal cell lines were normal and the cell lines were genetically stable.</p>


Subject(s)
Adult , Asian Continental Ancestry Group , Genetics , Cell Line, Transformed , Cell Transformation, Viral , Female , Herpesvirus 4, Human , Physiology , Humans , Karyotyping , Male , Microsatellite Repeats , Pedigree , Spinocerebellar Ataxias , Genetics , Young Adult
16.
Article in Chinese | WPRIM | ID: wpr-287431

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the potentiality of herpes simplex virus thymidine kinase transduced endothelial progenitor cells (EPC-TK) as angiogenesis-targeting vector in the glioma treatment in vitro and in vivo.</p><p><b>METHODS</b>EPC-TK were mixed with human umbilical vein endothelial cells (HUVECs), U87 or U251 cells at various ratios for ganciclovir (GCV) treatment. The bystander effect was observed by counting the survival cells using MTT assay, and the apoptotic cells were determined by annexin-V and propidium iodide (PI) staining. EPC-TK, EPCs, or phosphate buffered saline (PBS) were injected into the nude mice model of glioma xenograft by tail vein, for the EPC-TK group, EPC group, and PBS group, respectively. And then the changes of tumor volume and tumor vasculature were observed.</p><p><b>RESULTS</b>GCV killed most EPC-TK and reduced the number of other viable cells in a cell:cell ratio-dependent and time-dependent manner. EPC-TK obviously inhibited tumor growth. The tumor volumes on day 21 were 1741.20+/- 576.10 mm(3), 3275.52 +/- 710.86 mm(3) and 3033.09+/-1134.86 mm(3) in the EPC-TK, EPC and PBS group, respectively. EPC-TK also displayed a significant effect on the inhibition of tumor angiogenesis.</p><p><b>CONCLUSION</b>EPC-TK can exert a potent antiglioma effect via inhibiting angiogenesis.</p>


Subject(s)
Angiogenesis Inhibitors , Pharmacology , Animals , Antiviral Agents , Pharmacology , Bystander Effect , Cell Transformation, Viral , Physiology , Endothelial Cells , Virology , Endothelium , Genetic Vectors , Glioma , Therapeutics , Humans , Mice , Mice, Nude , Simplexvirus , Genetics , Thymidine Kinase , Genetics , Transduction, Genetic , Transfection , Xenograft Model Antitumor Assays
17.
Article in Chinese | WPRIM | ID: wpr-229774

ABSTRACT

<p><b>OBJECTIVE</b>To study the genetic stability of an immortalized cell line transformed by Epstein-Barr virus (EBV) after long subculture process.</p><p><b>METHODS</b>In the present study, the genetic stability including chromosome diploidy, karyotypes and microsatellite DNA were evaluated with chromosome banding techniques and microsatellite DNA detection. The telomerase activity of the immortalized cell line was detected by using the telomerase assay kit.</p><p><b>RESULTS</b>From passage 1 to 30, there were no change of the diploidy, karyotypes of chromosome and microsatellite DNA, and the telomerase activity is negative.</p><p><b>CONCLUSION</b>This study indicates that the immortalized cell line remains stable genetically within limited passages.</p>


Subject(s)
Cell Transformation, Viral , Genetics , Herpesvirus 4, Human , Genetics , Humans , Lymphocytes , Cell Biology , Metabolism , Virology , Microsatellite Repeats , Genetics , Polymerase Chain Reaction
18.
Article in Chinese | WPRIM | ID: wpr-298207

ABSTRACT

<p><b>OBJECTIVE</b>To detect bcl-2 gene expression in Epstein-Barr virus (EBV)-infected human gastric epithelial cell line GES-1 for understanding the role of bcl-2 gene in the carcinogenesis of EBV-associated gastric carcinoma.</p><p><b>METHODS</b>Akata 1061 cells producing recombined EBV carrying neomycin resistance gene (NEOr) was used to mediate the EBV infection of human gastric epithelial cell line GES-1 via close contact, with the empty plasmid pcDNA3-transfected GES-1 cells via lipofectamine method as a control. The EBV-infected and pcDNA3-transfected cells were cloned by limited dilution and the positive clones selected with G418. Immunocytochemical staining was performed to detect the expressions of EBNA1 and Bcl-2 protein.</p><p><b>RESULTS</b>Bcl-2 protein expression was detected in EBV-infected cells but not in the control cells.</p><p><b>CONCLUSION</b>EBV infection can increase Bcl-2 expression in gastric epithelial cells, and such cell transformation effect of EBV is related to the overexpression of bcl-2 gene.</p>


Subject(s)
Cell Line, Transformed , Cell Transformation, Viral , Epithelial Cells , Cell Biology , Metabolism , Virology , Herpesvirus 4, Human , Humans , Immunohistochemistry , Proto-Oncogene Proteins c-bcl-2 , Stomach , Cell Biology
19.
Article in Chinese | WPRIM | ID: wpr-282932

ABSTRACT

<p><b>OBJECTIVE</b>To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97.</p><p><b>METHODS</b>V60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay.</p><p><b>RESULTS</b>DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant.</p><p><b>CONCLUSION</b>Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.</p>


Subject(s)
B-Lymphocytes , Cell Biology , Virology , Base Sequence , Blotting, Western , Cell Line, Transformed , Cell Transformation, Viral , DNA, Viral , Genetics , Eukaryotic Cells , Metabolism , Gene Expression Regulation, Viral , Genetic Vectors , Genome, Viral , Genetics , Hepatitis B Core Antigens , Genetics , Metabolism , Hepatitis B virus , Genetics , Metabolism , Humans , Molecular Sequence Data , Point Mutation , Reverse Transcriptase Polymerase Chain Reaction , Transfection
20.
Article in Chinese | WPRIM | ID: wpr-234204

ABSTRACT

<p><b>OBJECTIVE</b>To investigate a method for establishing immortalized lymphoblastoid cell bank of keloid pedigree so as to provide a long-term source of specimens for keloid research.</p><p><b>METHODS</b>With Epstein-Barr virus transformation, fresh and frozen blood samples collected from all members of the keloid pedigree were used respectively to establish the immortalized lymphoblastoid cell lines of B lymphocytes.</p><p><b>RESULTS</b>Twenty-seven immortalized lymphoblastoid cell lines of the keloid pedigree were obtained successfully, and all cell lines survived cryopreservation in liquid nitrogen.</p><p><b>CONCLUSIONS</b>The immortalized lymphoblastoid cell bank of the keloid pedigree can preserve the whole gene information and provide long-term DNA sources for keloid research. Preparation of the cell lines with fresh blood is more efficient than that with frozen blood.</p>


Subject(s)
Adolescent , Adult , Aged , Cell Line, Transformed , Cell Transformation, Viral , Child , Child, Preschool , Cryopreservation , Female , Herpesvirus 4, Human , Physiology , Humans , Keloid , Blood , Genetics , Lymphocytes , Pathology , Virology , Male , Middle Aged , Pedigree
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