RESUMEN
Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.
Asunto(s)
Fibroblastos/citología , Proteómica , Telomerasa/metabolismo , Telómero/fisiología , Adulto , Diferenciación Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Cromosomas Humanos Par 21 , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados , Proteínas del Citoesqueleto/metabolismo , Electroforesis en Gel Bidimensional , Fibroblastos/enzimología , Humanos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/ultraestructura , Neuronas/citología , Estrés Oxidativo , Células Madre/citología , Células Madre/enzimología , Telomerasa/genéticaRESUMEN
Great differences were found in the level of 2',5'-oligoadenylate synthetase 40-46 kDa (OAS1) mRNA in relation to the proliferative state of human diploid fibroblasts at the moment of cytomegalovirus (CMV) (the strain AD169) infection. In the phase of synthesis of cellular cycle DNA (S), CMV induced OAS1 mRNA transcription by 10-100 times stronger than then in phase Go infection. The level of viral induction OAS1 mRNA peaked by hour 12 postinfection. The high gene activity correlated with suppressed DNA synthesis, a slowing-down mitotic cycle, markedly inhibited CMV replication, and delayed cell death. When the cells were infected in phase Go, the stimulation of OAS1 gene activity was less and it was attended by intensive viral replication and rapid cell death. There was a direct relationship between the resistance of cells and the constitutive level of OAS1 gene expression: in the low CMV-sensitive cells, the activity of OAS1 gene was more than 10 times greater than that in the highly sensitive cells. The inhibitors of the enzymatic OAS activity induced by IFN and dsRNA were found in the cytoplasm of the CMV-infected cells.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Regulación de la Expresión Génica , 2',5'-Oligoadenilato Sintetasa/química , 2',5'-Oligoadenilato Sintetasa/metabolismo , Muerte Celular , Línea Celular , Infecciones por Citomegalovirus/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Humanos , Interferón-alfa/metabolismo , Peso Molecular , ARN Bicatenario/metabolismo , ARN Mensajero/genética , Fase de Descanso del Ciclo Celular/fisiología , Fase S/fisiología , Especificidad de la Especie , Replicación Viral/fisiologíaRESUMEN
The study of pathogenetic mechanisms of Peyronie's disease (PD) in respect of raising efficacy of complex conservative therapy was made in 41 patients with PD. The control group consisted of 266 persons. Immunological and virusological investigations were made using enzyme immunoassay. HLA typing of class I antigens was made, antinuclear antibodies and antibodies to DNA were investigated. Effects of intron A and verapamil on proliferation of penile tunica albuginea were studied in vitro. Conservative therapy was given to 32 patients with acute PD. Patients with PD were found to carry chronic infection with type II herpes significantly more frequently. There was a significant association of PD with HLA-antigen B8 with high percentage of diagnostic titers of antinuclear antibodies. In vitro effect of intron A and verapamil was found to be dose- and time-dependent. Verapamil has a narrow range of dose-dependence and cytotoxicity in high concentrations. Combined treatment raised the proportion of good results while that of satisfactory outcomes decreased. Viral infection may be involved in pathogenesis of PD. This infection may alter mechanisms of immune regulation and start of autoimmune process in predisposed patients. Combination of magnetolaser therapy with intron A injections is an effective method of acute PD treatment. The addition of specific antiviral therapy raises treatment efficacy by action on one of pathogenetic mechanisms of the disease.
Asunto(s)
Antivirales/uso terapéutico , Interferón-alfa/uso terapéutico , Induración Peniana/tratamiento farmacológico , Vasodilatadores/uso terapéutico , Verapamilo/uso terapéutico , Adulto , Anciano , Anticuerpos Antinucleares/sangre , Terapia Combinada , Quimioterapia Combinada , Óxido Ferrosoférrico/uso terapéutico , Antígenos HLA/análisis , Herpes Genital/complicaciones , Herpes Genital/virología , Humanos , Interferón alfa-2 , Interferón-alfa/farmacocinética , Terapia por Láser , Masculino , Persona de Mediana Edad , Induración Peniana/inmunología , Induración Peniana/virología , Proteínas Recombinantes , Verapamilo/farmacologíaRESUMEN
Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.
Asunto(s)
Alquilantes/farmacología , Apoptosis , Disparidad de Par Base/fisiología , Daño del ADN , Metilnitrosourea/farmacología , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral/efectos de los fármacos , Etopósido/farmacología , Humanos , Factores de TiempoRESUMEN
Below were demonstrated the differences in the cell reaction of the chromosome loci transference induced by adaptive doses X-radiation in the cells nucleus in norm cells and in cells with repair process defect of the oncological patients and patients with hereditary disease. It was supposed that the transference of the homologous chromosome loci is necessary for the realization of the correct DNA's double strand repair. The chromosome loci transference in normal cells could be induced by different factors such as X-radiation, RNA-polymerize II repression by alpha-amanitin and possible by other factors. In cells with BSCA1 and BRCA2 genes mutations the chromosome loci transference could not be induced by the X-radiation, but it could be induced by the RNA-polymerize II repression by alpha-amanitin. The defect of the chromosome loci transference condition on genetics or the another determinatives and this defect could be the one of the important reasons of the genome instability. There was suggested the new conception of the mechanisms of the cell differentiation and cell chronological senescence. The vector of the cell differentiation mechanisms is the progressive chromatin condensation determined by the physical mechanisms, i.e., transformation from less probable (the stem cells) to more probable cell system state (the differentiated cells) and as a result of it changing of the genes transcription. The continued chromatin condensation (most pronounced in the old cells) decreases the probability of the realization of the DNA repair as the reason of the chromosome loci transference limitation. In this work are presented experimental and theoretical information that proves our conception.
Asunto(s)
Cromatina/química , Reparación del ADN , Células Eucariotas/química , Secuencia de Bases , Núcleo Celular/ultraestructura , Mapeo Cromosómico , Regulación de la Expresión Génica , Modelos Moleculares , Conformación ProteicaRESUMEN
A study was made of the effect of the oxidizing agent potassium chromate (K2CrO4, PC) on cultured dermal fibroblasts of a healthy donor and three patients with rheumatoid arthritis (RA). Characteristics of the rRNA gene (RG) complex-RG copy number, active RG (ARG) dosage, and 18S rRNA content--were determined for each cell line. In cells of the healthy donor, oxidative stress caused by low doses of PC (2-4 microM, 1-4 h) induced an early response, including a 50-80% increase in total RNA and rRNA. An appreciable activation of the nucleolus was observed cytochemically, by silver staining and morphometry. The early response grew considerably lower with the increasing passage number and/or PC concentration. Exposure to 6-12 microM PC for 24 h led to a progressive cell death (late response). The existence and intensity of the early response correlated positively with the cell survival during further culturing. Cells of the RA patients displayed almost no early response even at early passages: total RNA did not increase, and rRNA increased by no more than 10%. Cell disruption (apoptosis) during further culturing was more intense than in the line originating from the healthy donor. The apoptosis intensity characterized by the increase in the content of DNA fragments in the culture medium and in the caspase 3 activity, was inversely proportional to the ARG dosage in the genome. The results provide the first quantitative characterization of the early and late responses of cells to PC-induced oxidative stress and suggest a role of the ARG dosage in cell survival in stress.
Asunto(s)
Artritis Reumatoide/metabolismo , Muerte Celular , Dosificación de Gen , Estrés Oxidativo , ARN Ribosómico 18S/genética , Piel/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Piel/citologíaRESUMEN
The physiological activity of the "recombinant" bradykinin expressed by retrovirus recombinant pPS-3-neo (brd) was tested on cultural atrial (aCMC) and ventricular (vCMC) cardiomyocytes in newborn rats. The "recombinant" bradykinin was shown to have a chronotropic effect on aCMC and an inotropic effect on vCMC. The effects are in line with the action of the synthetic bradykinin preparation at a concentration of around 10(-15) M. A pretreatment of CMC by parmidine, i.e. a bradykinin antagonist, blocked the effect of bradykinin. The contractive CMC activity in the cultural cell medium, transferred by pPS-3-neo without the bradykinin gene, was not different from the control value.
Asunto(s)
Bradiquinina/farmacología , Atrios Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Animales , Células Cultivadas , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Humanos , Ratas , Proteínas Recombinantes/farmacologíaRESUMEN
Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.
Asunto(s)
Línea Celular Transformada/citología , Línea Celular Transformada/fisiología , Telomerasa/metabolismo , Telómero/fisiología , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , División Celular/genética , Senescencia Celular/fisiología , Medio de Cultivo Libre de Suero/farmacología , Proteínas de Unión al ADN , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Genes de ARNr , Humanos , Cariotipificación , Telomerasa/genética , TransfecciónRESUMEN
We studied the subcellular localization of tankyrase in primary and immortalized human cell cultures. In embryonic kidney cell line 293 the enzyme was excluded from the nuclei and distributed in fractions of soluble cytosolic proteins and low-density microsomes. Newly revealed cytosolic tankyrase in its poly(ADP-ribosyl)ated form was passed through a Sepharose 2B column and eluted as an apparently monomeric protein. The cytosolic localization of the enzyme correlated with its relatively high activity in the 293 cell line in comparison to eight other studied cell types.
Asunto(s)
Citosol/enzimología , Riñón/enzimología , Tanquirasas/metabolismo , Secuencia de Aminoácidos , Ancirinas/química , Ancirinas/genética , Línea Celular , Cromatografía en Gel , Secuencia Conservada , Humanos , Riñón/citología , Riñón/embriología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Microsomas/enzimología , Datos de Secuencia Molecular , Poli Adenosina Difosfato Ribosa/química , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/aislamiento & purificación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Tanquirasas/química , Tanquirasas/genética , Tanquirasas/aislamiento & purificaciónRESUMEN
In the present work it is shown that the phenomenon of interphase chromosome centromeric region displacement, earlier revealed by the authors, is not realized in G0-lymphocytes with heterozygous BRCA1/2 gene mutations. The role of these genes in DNA double strand break (DSB) reparation is known. It is concluded that chromosome locus displacement is necessary for DSB repair, at least in the process of homologous recombination. In accordance with our data, some feature (pericentromeric cluster disintegration and displacement, the nucleus size increasing) characteristic for S- and G0-lymphocytes are observed in normal G0-lymphocytes treated with 3 and 10 cGy. However, the size of nucleus in G0-lymphocytes is restored through 6 hours after irradiation in opposite to the process in dividing cells. It was proposed that some typical for resting cell functions of G0-lymphocytes after inducing by adaptive doze of radiation are stopped as similarly as after stimulation of cells. Interestingly, that the process of the induced chromosome loci displacement is correlated with the decreasing of DNA reparation possibilities under UV-irradiation. The induced apoptosis level also decreases when chromosome loci are displaced. The possible mechanisms of the revealed phenomenon are discussed. This research supported by RFBR grant (No. 01-04-49180).
Asunto(s)
Daño del ADN , Reparación del ADN , ADN , Genes BRCA1/efectos de la radiación , Genes BRCA2/efectos de la radiación , Linfocitos/efectos de la radiación , Mutación , Animales , Apoptosis/efectos de la radiación , Núcleo Celular/efectos de la radiación , Centrómero/efectos de la radiación , ADN/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Interfase/efectos de la radiación , Linfocitos/citología , Ratones , Familia de Multigenes , Dosis de Radiación , Factores de Tiempo , Rayos UltravioletaRESUMEN
The cytotoxicity of four aminoglycoside antibiotics was studied by estimation of the dose-effect relationship using a panel cellular biotest system including cell cultures for test objects. The cultures represented 4 differentiation types: normal human fibroblasts and myoblasts, human or Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells. It was found that three widely used antibiotics gentamicin, kanamycin, and neomycin exhibit similar, but not identical cytotoxicity parameters and differ distinctly from geneticin. Hence, the proposed panel biotest system helps to quantitatively evaluate and differentiate the effects of bioactive substances with similar chemical structure.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Fibroblastos/patología , Mioblastos/patología , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , HumanosRESUMEN
In previous work we demonstrated that various types of cultured cells with a limited life span could not reactivate DNA synthesis in the nuclei of mouse peritoneal macrophages in heterokaryons. We now investigate the role of telomerase in the process of the macrophage nucleus reactivation in heterokaryons with immortal telomerase-positive 3T3 Swiss mouse fibroblasts and human fibroblasts with introduced hTERT gene. We report that introduction of the hTERT gene into human diploid fibroblasts results in emergence of telomerase activity in these cells and the ability to induce the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. Inhibition of telomerase activity in heterokaryons by reverse transcriptase inhibitors (azidothymidine and guanosine polyphosphonate analogues) and by a 2'-O-methyl-RNA oligonucleotide anti-sense to the template region of telomerase RNA, block reactivation of DNA synthesis in macrophage nuclei without inhibiting DNA synthesis in the nuclei of fibroblasts. Our results suggest alterations (shortening or damage) in the macrophage telomere structure. As far as we know, heterokaryons with macrophages are the first cellular model for rapid investigation of the effects of telomerase inhibitors.
Asunto(s)
ADN/biosíntesis , Reactivadores Enzimáticos/metabolismo , Macrófagos Peritoneales/enzimología , Telomerasa/metabolismo , Telómero/enzimología , Células 3T3/enzimología , Animales , Humanos , RatonesRESUMEN
A complex research of cells of a patient with unusual form of premature ageing was made. The clinical picture is not typical for any of known forms of hereditary premature aging--progerias. Skin fibroblasts of the patient AG has limited proliferation capacity in vitro. It was shown by fluorescent-immunochemical hybridization (FISH-method), that the level of stable chromosome aberrations in AG blood lymphocytes was characteristic of aged 55-65 years, though as he was only 26 years old. Some characteristic peculiarities, typical for progerias, were found in the reaction of skin fibroblasts of AG to growth factors addition. Some clinical and biochemical peculiarities are results rather, than reasons of the disease. The conclusion is that the premature ageing in this case is a manifestation of Werner's syndrome--one of hereditary forms of accelerated senescence.
Asunto(s)
Envejecimiento/patología , Síndrome de Werner/diagnóstico , Adulto , Antiinflamatorios/efectos adversos , División Celular , Cutis Laxo/diagnóstico , Diagnóstico Diferencial , Fibroblastos/patología , Hepatitis Autoinmune/complicaciones , Hepatitis Autoinmune/tratamiento farmacológico , Humanos , Hibridación Fluorescente in Situ , Cirrosis Hepática/prevención & control , Masculino , Prednisolona/efectos adversos , Progeria/diagnóstico , Piel/patología , Síndrome de Werner/genética , Síndrome de Werner/patologíaAsunto(s)
Metilación de ADN , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Fibroblastos/fisiología , Telómero/genética , División Celular/genética , Transformación Celular Neoplásica/genética , Senescencia Celular/genética , Sondas de ADN/genética , Proteínas de Unión al ADN , Fibroblastos/citología , Dosificación de Gen , Humanos , Secuencias Repetitivas de Ácidos Nucleicos , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismo , TransfecciónRESUMEN
The most important stage in the making of mutations is a reparation of different DNA damage, including the more deleterious double-strand DNA breaks (DSB). The first stage of adaptive response--fundamental antimutagenic cell reaction, purposeful to reparation for induced DSB repair--is investigated in present work. Non-radioactive in situ hybridization of biotin-labeled DNA probe was used to mark chromosome 1 pericentromeric regions (PR) in G0 human lymphocytes. It was shown that under 3-10 cGy (X-radiation, 160 kV) PR become displaced from a nucleus periphery to inner territory of a nucleus. The moving process realizes during several hours after an irradiation. As far as some non-specific gene repressors are co-localized with chromosome centromeric regions it is possible hypothesizes that the displacement cause changing expression of some genes. It is possible to propose that an absence of radiation induced chromosome locus displacement may be one of causes DSB repair disturbance. This hypothesis was tested by the model. It is assumed that one consequence of the underlying defect may be inappropriate involvement of cell's recombination machinery in the repair of DSB. We studied lymphocytes of patients with hereditary BRCA2 mutation. It is thought that this gene takes part in DSB repair. The significant differences of the PR moving between control samples and the cases were revealed under 10 cGy. Similar results were observed on lymphocytes of patients with Fanconi syndrome. Thus, abnormal moving of interphase nucleus chromosomes conditioned by low-dose irradiation may suggest on imperfect machinery of DSB repair, i.e. genetic risk. We realize that further investigations are needed for definitive conclusion.
Asunto(s)
Cromosomas Humanos Par 1/efectos de la radiación , Interfase/efectos de la radiación , Daño del ADN , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Humanos , Hibridación in Situ , Linfocitos/efectos de la radiación , Linfocitos/ultraestructuraRESUMEN
Cultures of human and mammalian cells presenting 4 types of differentiation (normal human fibroblasts and myoblasts, human and Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells) were used in a panel biotest system. This system allowed to evaluate the cytotoxic and stimulatory effect of bioactive compounds by determining the dose-effect relationships and some quantitative parameters including LD(50). Examination of some biolactive compounds of different nature (sangviritrin, escin, deltostim, cycloheximide, dexamethasone) confirmed high efficacy of this biotest system.
Asunto(s)
Bioensayo , Técnicas de Cultivo de Célula , Monitoreo de Drogas/métodos , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetinae , Humanos , RatonesRESUMEN
Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used.
Asunto(s)
Trasplante de Células , Expresión Génica , Músculo Esquelético/trasplante , Distrofias Musculares/genética , Antígenos de Superficie/análisis , Ensayos Clínicos como Asunto , Distrofina/genética , Humanos , Masculino , Músculo Esquelético/citología , Distrofias Musculares/inmunología , Distrofias Musculares/terapiaRESUMEN
The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds.
Asunto(s)
Músculos/citología , Distrofia Muscular de Duchenne/terapia , Mioblastos/fisiología , Células Madre/fisiología , Células Cultivadas , Humanos , Distrofia Muscular de Duchenne/genética , Mioblastos/citologíaRESUMEN
In CHO cell line and primary human diploid fibroblasts culture an incorporation of protein, RNA and DNA biosyntheses precursors was investigated under different conditions of inhibition of translation by cycloheximide (CHM). Both CHO and human fibroblasts transitory treatment by CHM in the serumfree medium resulted in inhibition of protein and DNA syntheses during S-period while RNA synthesis increased up to 130% (CHM concentration from 0.003 to 2 Mg/ml), as well as in Go--an incorporation of 3H-U increased to 200% (CHM concentration-100 Mg/ml). Long-term treatment (48 hours) in the serum-free medium resulted in decreased uptake of 3H-T and 3H-L during first 6 hours of experiment, while incorporation of 3H-U increased to 160%. By 16-th hour of treatment characters of protein, RNA and DNA syntheses came back to control levels.