RÉSUMÉ
We studied the effect of dioxidine on genome-wide methylation in short-term cultures of peripheral blood lymphocytes derived from healthy donors. Methylation was evaluated in lymphocytes before culturing, after 25 h in culture, and 1 h after addition of dioxidine in two concentrations (0.1 and 0.01 mg/ml). The total time in culture was 25 h. The level of methylation was assessed using methyl-sensitive single-cell gel electrophoresis ("comet assay") with additional restriction with HpaII amd MspI. Significant individual differences were found in the levels of methylation in both native cells and in cells treated with dioxidine in both concentrations. Mean group indicators of methylation did not differ before culturing and after 25 h in culture (45.28 and 44.80%, respectively). The mean group rate of methylation increased to 46.14% (p<0.001) after dioxidine treatment in a concentration of 0.01 mg/ml. Dioxidine in 0.1 mg/ml reduced the level of methylation (mean group rate 42.31%; p<0.001).
Sujet(s)
Méthylation de l'ADN/effets des médicaments et des substances chimiques , Lymphocytes/effets des médicaments et des substances chimiques , Mutagènes/pharmacologie , Quinoxalines/pharmacologie , Adulte , Cellules cultivées , Altération de l'ADN/effets des médicaments et des substances chimiques , Femelle , Humains , Mâle , Espèces réactives de l'oxygène , Jeune adulteRÉSUMÉ
The efficacy and safety of gene-engineering recombinant constructions with endothelial growth factor gene and angiogenin for the treatment of the chronic lower limb ischemia were studied. 134 patients were included in prospective controlled study. The main group, who received both traditional treatment and genetic therapy, consisted of 74 patients. The rest 60 patients were included into the control group. Of 74 patients from the main group, genetic therapy was used together with conservative means in 39 patients and with reconstructive vascular operations in 35 patients. The gene-engineering angiogenesis stimulation therapy proved to be effective and safe. The combination of angiogenesis genetic stimulation with reconstructive vascular surgery demonstrated significantly better results, then monotherapy.
Sujet(s)
Agents angiogéniques/usage thérapeutique , Thérapie génétique/méthodes , Ischémie/thérapie , Jambe/vascularisation , Pancreatic ribonuclease/usage thérapeutique , Procédures de chirurgie vasculaire , Adulte , Sujet âgé , Sujet âgé de 80 ans ou plus , Maladie chronique , Femelle , Études de suivi , Humains , Mâle , Adulte d'âge moyen , Néovascularisation pathologique/prévention et contrôle , Études prospectives , Résultat thérapeutiqueRÉSUMÉ
This paper presents the main results of the study on chromosome and genome variability of mesenchymal stem cell cultures from bone marrow and adipose tissue carried out in the Laboratory of Mutagenesis, Research Centre for Medical Genetics, over the last three years. Genome stability was assessed from DNA damage using the DNA comet assay, karyotyping and registration of aneuploidy by the FISH method. We found that DNA damage rate in MSC cultures from bone marrow was 3.9% and 3.8% at the early (2-5) passages and the late (10-15) passages respectively. The cultures were characterized by high dispersion of individual values. Karyotyping showed mosaicism in both types of MSC cultures at the early and late stages of cultivation. The fraction of abnormal cells in some cultures amounted to 80-90%. Evaluation of aneuploidy in interphase cells revealed 1.34% of aneuploid cells (on the average) per one "conventional" chromosome; their overall frequency in the genome amounted to 20-40%. The frequency of aneuploid cells was similar at the early and late passages. Cultures with clones of trisomic and monosomic cells were revealed. The probability of occurrence of abnormal cells may increase by virtue of de novo mutations in the culture and as a result of positive selection of the cells existing in the organism that exhibit a higher reproduction rate in culture. Based on the experimental data on mutational process, selection of mutant cells and clone formation, it is concluded that cytogenetic control of stem cells is necessary to ensure the safety of cellular therapy.
Sujet(s)
Techniques de culture cellulaire/normes , Clones cellulaires , Instabilité du génome , Hybridation fluorescente in situ , Caryotypage , Transplantation de cellules souches mésenchymateuses , Tissu adipeux/métabolisme , Aneuploïdie , Cellules de la moelle osseuse/métabolisme , Myélogramme/méthodes , Humains , Transplantation de cellules souches mésenchymateuses/effets indésirables , Transplantation de cellules souches mésenchymateuses/normes , Mosaïcisme , Sécurité des patients/normesRÉSUMÉ
The non-random arrangement of chromosomes in the interphase nucleus was observed for the first time in the late XIX century. However, considerable progress in studying chromosome territories became possible only in the end of the XX century mainly due to advances in microscopy and molecular biology. At present, chromosome territories are believed to play an important role in epigenetic regulation of genome activity during various cell processes including but not limited to cell cycle, differentiation, stress response. 3D structure of genome also plays an important role in pathogenesis of various hereditary diseases and cancer. This article describes main provisions of the chromosome territory theory and current trends toward further development of human genetics based on the new knowledge about the role of chromosome territories.
Sujet(s)
Noyau de la cellule , Chromosomes humains/métabolisme , Épigenèse génétique , Interphase/génétique , Noyau de la cellule/génétique , Noyau de la cellule/métabolisme , Noyau de la cellule/ultrastructure , Chromatine/métabolisme , Épigénomique/méthodes , Épigénomique/tendances , Interaction entre gènes et environnement , Génome humain , Humains , Hybridation fluorescente in situ/tendancesRÉSUMÉ
Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.
Sujet(s)
Tissu adipeux/cytologie , Expression des gènes , Cellules souches multipotentes , Cellules stromales , Transfection/méthodes , Facteur de croissance endothéliale vasculaire de type A/génétique , Cellules cultivées , Électroporation , Humains , Cellules souches multipotentes/cytologie , Cellules souches multipotentes/métabolisme , Phosphatidyléthanolamine , Plasmides , Cellules stromales/cytologie , Cellules stromales/métabolisme , Transduction génétiqueRÉSUMÉ
The paper briefly formulates the main regularities of a human mutation process and defines the major topical lines of investigations in this area. It shows it necessary to take into account the known regularities of a mutation process when forming the legislative and legal bases for assuring the genetic safety of environmental factors.
Sujet(s)
Recherche génétique , Mutagenèse , Mutation , Polluants environnementaux/toxicité , Humains , Mutagenèse/effets des médicaments et des substances chimiques , Mutagènes/toxicité , Mutation/effets des médicaments et des substances chimiquesRÉSUMÉ
Immunophenotype, proliferation rate, and genetic stability parameters of bone marrow multipotent mesenchymal stromal cells were studied. Despite the reduction of proliferative activity by passages 11-12, the cells retained the characteristic immunophenotype. The incidence of spontaneous aneuploidy for autosomes 6, 8, 11 and sex chromosomes was evaluated. Two cultures of mesenchymal stromal cells carrying aneuploid cell clones were detected: with chromosome 8 trisomy and X chromosome monosomy. The results indicate the possibility of genetic transformation and selection of mesenchymal stromal cells with abnormal karyotype during in vitro culturing.
Sujet(s)
Cellules de la moelle osseuse/cytologie , Aberrations des chromosomes , Variation génétique , Cellules souches mésenchymateuses/cytologie , Cellules souches multipotentes/cytologie , Aneuploïdie , Différenciation cellulaire , Prolifération cellulaire , Cellules cultivées , Femelle , Humains , Immunophénotypage , Caryotypage , Mâle , PhénotypeRÉSUMÉ
We compared two methods of transfection (lipofection and electroporation) with plasmid containing VEGF121 gene in four cultures of mesenchymal stem cells from the human adipose tissue. The efficacy of transfection after 1 day, the dynamics of plasmid elimination after 3, 6, 9 days, and expression of the target gene were evaluated. Transfection by both methods failed in one of 4 cultures. Analysis of the plasmid elimination dynamics showed that the content of plasmids introduced by both methods decreased by 30-69% in all cultures by day 3 and then remained unchanged from day 3 to day 9. The expression of the target gene did not correlate with the content of plasmids in cells and varied by 2-10 times in control cells and cells transfected by both methods. Fluctuation of VEGF121 expression was not related to methylation.
Sujet(s)
Expression des gènes , Cellules souches mésenchymateuses/métabolisme , Plasmides/génétique , Transfection/méthodes , Facteur de croissance endothéliale vasculaire de type A/génétique , Tissu adipeux/cytologie , Tissu adipeux/métabolisme , Cellules cultivées , Électroporation , Humains , Lipides/composition chimique , Cellules souches mésenchymateuses/cytologie , Facteur de croissance endothéliale vasculaire de type A/métabolismeRÉSUMÉ
We performed a statistical analysis of clone formation from aneuploid cells (chromosomes 6, 8, 11, X) in cultures of bone marrow-derived human multipotent mesenchymal stromal cells by spontaneous level of aneuploidy at different terms of culturing (from 2 to 19 cell cycles). It was found that the duration of cell cycle increased from 65.6 h at passages 2-3 to 164.5 h at passage 12. The expected ratio of aneuploid cells was calculated using modeled 5, 10, 20 and 30% selective preference in reproduction. The size of samples for detecting 10, 25, and 50% increased level of aneuploidy was calculated. The presented principles for evaluation of aneuploid clone formation may be used to distinguish clones of any abnormal cells.
Sujet(s)
Clones cellulaires/cytologie , Cellules souches mésenchymateuses/cytologie , Aneuploïdie , Cellules cultivées , HumainsRÉSUMÉ
We carried out a comparative analysis of DNA damage (percentage of DNA in comet tail) and frequencies of comets in apoptotic cells in BM samples and cultures of BM multipotent mesenchymal stromal cells at different terms of culturing (passages 3-11). The levels of DNA damage in mesenchymal stromal cells remained unchanged during culturing (3.5 ± 0.9 and 4.4 ± 1.2%) and did not differ from those in BM cells (3.6 ± 0.8%). In BM samples, 10-28% atypical cells with high level of DNA damage were detected. In mesenchymal stromal cells, 2.8 ± 0.9 and 3.6 ± 1.8% apoptotic cells were detected at early and late passages, respectively.
Sujet(s)
Cellules de la moelle osseuse/ultrastructure , Altération de l'ADN , Cellules souches mésenchymateuses/ultrastructure , Cellules souches multipotentes/ultrastructure , Cellules stromales/ultrastructure , Cellules cultivées , HumainsRÉSUMÉ
Clinical study of intracoronary transplantation of allogenic multipotent bone marrow stromal cells was carried out in patients with severe chronic cardiac failure against the background of dilatated cardiomyopathy. The results indicate that intracoronary injection of allogenic multipotent stromal precursors is a safe procedure. No complications and side effects directly or indirectly related to cell transplantation were recorded during the immediate and delayed postoperative periods. The positive effect of cell transplantation developed from week 1 after transplantation and persisted for 6 months. It manifested in reduction of the level of brain natriuretic peptide and improvement of patient's functional status and quality of life. No appreciable changes in the main echocardiographic values were noted. Transplantation of allogenic multipotent stromal cells is effective as a component of combined therapy for chronic cardiac failure at the stage of preparation to surgery as a "bridge to surgical treatment".
Sujet(s)
Transplantation de moelle osseuse/méthodes , Cardiomyopathie dilatée/complications , Défaillance cardiaque/chirurgie , Cellules souches multipotentes/transplantation , Adulte , Sujet âgé , Cardiomyopathie dilatée/chirurgie , Défaillance cardiaque/étiologie , Humains , Mâle , Adulte d'âge moyen , Peptide natriurétique cérébral/sang , Statistique non paramétrique , Cellules stromales/transplantation , Transplantation homologue , Résultat thérapeutiqueRÉSUMÉ
Cytogenetic analysis of 13 mesenchymal stem cell cultures isolated from normal human adipose tissue was carried out at different stages of culturing. The incidence of chromosomes 6, 8, 11, and X aneuploidy and polyploidy was studied by fluorescent in situ hybridization. During the early passages, monosomal cells were more often detected than trisomal ones. A clone with chromosome 6 monosomy was detected in three cultures during late passages.
Sujet(s)
Tissu adipeux/cytologie , Aneuploïdie , Clones cellulaires , Cellules souches/cytologie , Cellules cultivées , Chromosomes humains , HumainsRÉSUMÉ
A combination of karyotyping and aneuploidy analysis by interphase fluorescent in situ hybridization is a sensitive method for evaluation of genetic stability of stem cell cultures. The methodology and specific features of preparing and analyzing the cytogenetic preparations are described as exemplified by human multipotent mesenchymal stromal cells.
Sujet(s)
Cytogénétique/méthodes , Hybridation fluorescente in situ/méthodes , Caryotypage/méthodes , Transplantation de cellules souches mésenchymateuses , Ploïdies , Techniques de culture cellulaire/méthodes , Cellules cultivées , Recommandations comme sujet , Humains , Transplantation de cellules souches mésenchymateuses/effets indésirables , Transplantation de cellules souches mésenchymateuses/méthodes , Cellules souches multipotentes/cytologie , Cellules souches multipotentes/physiologie , Cellules stromales/cytologie , Cellules stromales/physiologieRÉSUMÉ
Two-year experiments were performed to evaluate the neurotrophic effect of hypoxia-inducible factors (vascular endothelial growth factor and angiogenin) expressed in recombinant human adenoviruses in amyotrophic lateral sclerosis. Randomized placebo-controlled trial demonstrated safety and good tolerability of the recombinant antiviral drugs. The life span of patients under conditions of hypoxia increased after treatment with the test drug, which was probably related to improved resistance of motoneurons. The presence of virus-neutralizing antibodies decreases the effectiveness of adenoviral vectors, which necessitates differential approach to the selection of patients and continuous monitoring of gene therapy.
Sujet(s)
Sclérose latérale amyotrophique/thérapie , Angiotensinogène/administration et posologie , Thérapie génétique/méthodes , Facteur de croissance endothéliale vasculaire de type A/administration et posologie , Adenoviridae/génétique , Adulte , Sclérose latérale amyotrophique/génétique , Sclérose latérale amyotrophique/mortalité , Angiotensinogène/effets indésirables , Angiotensinogène/génétique , Cellules cultivées , Femelle , Thérapie génétique/effets indésirables , Vecteurs génétiques/administration et posologie , Vecteurs génétiques/effets indésirables , Humains , Injections musculaires , Mâle , Adulte d'âge moyen , Placebo , Transgènes/génétique , Résultat thérapeutique , Facteur de croissance endothéliale vasculaire de type A/effets indésirables , Facteur de croissance endothéliale vasculaire de type A/génétiqueRÉSUMÉ
The authors have studied therapeutic outcomes in a total of 38 patients diagnosed with occlusions of the femoropopliteal segment. In the Study Group patients (n = 19), the operation of femoropopliteal bypass grafting was supplemented by using gene stimulators of angiogenesis (gene constructions with the genes of vascular endothelial growth factor, and angiogenin). The Control Group patients (n = 19) were subjected to a reconstructive vascular operation alone. The remote results were followed up from six to twenty-six months, having shown reliably better therapeutic outcomes obtained in the Study Group patients, as judged by the distance of pain-free walking, the time of restoration of the baseline parameters of blood flow during the treadmill test, muscular perfusion, and the quality of life indices. A conclusion was made that the use ofangiogenesis-stimulating methods combined with reconstructive vascular operations improves the long-term outcomes in patients presenting with lower limb chronic ischaemia.
Sujet(s)
Prothèse vasculaire , Adhésion aux directives , Ischémie/thérapie , Jambe/vascularisation , Néovascularisation physiologique/physiologie , Ingénierie tissulaire/méthodes , Procédures de chirurgie vasculaire/méthodes , Sujet âgé , Agents angiogéniques/administration et posologie , Maladie chronique , Association de médicaments , Épreuve d'effort , Femelle , Études de suivi , Humains , Injections musculaires , Ischémie/anatomopathologie , Ischémie/physiopathologie , Mâle , Adulte d'âge moyen , Qualité de vie , Pancreatic ribonuclease/administration et posologie , Facteurs temps , Résultat thérapeutique , Facteur de croissance endothéliale vasculaire de type A/administration et posologieRÉSUMÉ
The incidence of autosomes 6 and 8 aneuploidy in stem cell cultures derived from adipose tissue was evaluated at different stages of culturing. Monosomy was more incident than trisomy during the early passages. Distribution of cultures by the incidence of aneuploidy in different chromosomes was virtually the same. Clones with chromosome 6 monosomy were detected in two cultures during late passages.
Sujet(s)
Tissu adipeux/cytologie , Aneuploïdie , Cellules souches/ultrastructure , Cellules cultivées , Chromosomes humains de la paire 6 , Chromosomes humains de la paire 8 , Humains , Hybridation fluorescente in situ , CaryotypageRÉSUMÉ
This analytical review of the literature on cellular therapy of hereditary diseases summarizes results of their treatment by stem cell transplantation. It describes the main sources of stem cells and considers prospects of and limitations on the use of cell therapy for the management of hereditary disorders. Cell technologies are considered to be promising for the treatment of a variety of inherited diseases including metabolic disturbances, hemopathies, hematopoietic disorders, pulmonary diseases in children, bone and nervous diseases. Special attention is given to the use of genetically modified stem cells. An international register of patients is deemed necessary for the evaluation of outcomes of cellular therapy.
Sujet(s)
Maladies génétiques congénitales/thérapie , Transplantation de cellules souches , Adrénoleucodystrophie/thérapie , Adulte , Transplantation de moelle osseuse , Dysplasie bronchopulmonaire/thérapie , Enfant , Mucoviscidose/thérapie , Syndrome de Fanconi/thérapie , Transplantation de cellules souches hématopoïétiques , Hémoglobinopathies/thérapie , Humains , Nouveau-né , Leucodystrophie à cellules globoïdes/thérapie , Mucopolysaccharidose de type I/thérapie , Cellules souches multipotentes/transplantation , Maladies du système nerveux/thérapieRÉSUMÉ
We elaborated a method of preparing cytogenetic preparations of cultured multipotent mesenchymal stromal cells from the adipose tissue. It was found that karyotypic changes (monosomy, translocations) appear in some samples during culturing. Clones with changed karyotype were detected in 11-14-passage cultures from 2 of 7 individuals. The percent of aberrant cells in cultures from different individuals varied from 1.5 to 5.95 per 100 cells, which attested to karyotype instability. These data substantiate the need for cytogenetic control of cells before their transplantation into donor organism and further investigation of chromosome variability in stem cells.
Sujet(s)
Aberrations des chromosomes , Chromosomes humains , Cellules souches mésenchymateuses/cytologie , Cellules souches multipotentes/cytologie , Adulte , Techniques de culture cellulaire , Cellules cultivées , Analyse cytogénétique , Humains , Caryotypage , Adulte d'âge moyen , Cellules stromales/cytologieRÉSUMÉ
Peripheral blood leucocytes of healthy donors, both intact and mutagen-processed with cadmium chloride, dioxidine, and bleomycin in vitro, were studied with cytogenetic methods in G2 cell cycle period before, during, and after 14- and 30-day oral administration of vitamin and vitamin-mineral complexes. The study found that intake of complexes containing vitamins in total doses exceeding day requirements did not increase the rate of spontaneous mutations and decreased the sensitivity of human cells to the cytogenetic activity of chemical mutagens. At the same time, vitamin complexes of certain quantitative and qualitative composition were shown to be capable of giving rise to comutagenic modification of sensitive cells to the action of separate mutagens in certain concentrations in vitro. The above effects of anti- and comutagenic modification depend on the duration of intake, the quantitative and qualitative composition of the vitamin complex, the nature of mutagens, and the level of induced cytogenetic lesions.
Sujet(s)
Leucocytes/effets des médicaments et des substances chimiques , Mutagenèse/effets des médicaments et des substances chimiques , Vitamines/usage thérapeutique , Cellules cultivées , Aberrations des chromosomes/effets des médicaments et des substances chimiques , Études de suivi , Humains , Leucocytes/cytologie , Mutation/effets des médicaments et des substances chimiques , Valeurs de référenceRÉSUMÉ
We carried out a pilot study on intracoronary transplantation of prenatal allogenic skeletal myoblasts and multipotent bone marrow stromal cells to patients with dilated cardiomyopathy. Intracoronary transplantation of allogenic cells is a feasible and safe procedure: there were no life-threatening rhythm and conduction disturbances, symptoms of hemodynamic instability, and thromboembolic complications. Positive clinical effect of cell transplantation persisted for 6 months and consisted in decreased content of brain natriuretic peptide and improved tolerance to physical exercises. We observed no reliable dynamics of the major echocardiographic parameters and their correlations with patient's status.