Vol 7, No 3 (2012)


Privetstvennoe slovo professora A.P. Kiyasova

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Genes & Cells. 2012;7(3):10-11
pages 10-11 views

Gene and cell therapy of retinal diseases

Maximov V.V., Lagarkova M.A., Kiselev S.L.


Gene and cell therapy are tightly associated and quickly developing areas of biomedicine, which purpose is development of methods to cure diseases caused by genetic defects and/or death of certain cell types. Current state of methods of retinal diseases gene and cell therapy is analyzed in this review. We reviewed development of gene therapeutic approaches to treatment of the 2-nd form of Lebers congenital amaurosis and other retinal diseases. We also compared therapeutic potential of pluripotent stem cells and human adult retinal stem cells. Therapeutic potential of pluripotent stem cells seems to be better due to their ability for unlimited expansion and organogenesis.
Genes & Cells. 2012;7(3):12-20
pages 12-20 views

Can pancreas be the source of hepatocytes?

Abdulkhakova A.R., Abdulkhakov S.R., Titova M.A., Saifullina K.N., Gumerova A.A., Kiasov A.P.


The possibility of hepatocytes differentiation from pancreatic cells is discussed in the review. Both liver and pancreas develop from endoderm so there could be a common stem cell giving rise to both pancreatic and liver cells. Different experimental models are used to study the possibility of hepatocytes development in pancreas such as influence of peroxisomes proliferation stimulators, hyperexpression of keratinocyte growth factor in pancreatic islets, Cu depletion-repletion model, etc.). There is no enough data to confirm which particular pancreatic cell type can be the source of hepatocytes. Literature data allow supposing that hepatocytes can arise in pancreas from three different sources: acinar, endocrine or ductular cells.
Genes & Cells. 2012;7(3):21-24
pages 21-24 views

Formation of the recombinant adenovirus encoding codon-optimized dysferlin gene and analysisof the recombinant protein expression in cell culture in vitro

Starostina I.G., Solovyeva V.V., Shevchenko K.G., Deev R.V., Isaev A.A., Rizvanov A.A.


Dysferlinopathies belong to neuromuscular diseases associated with aberrant expression and/or function of dysferlin protein in skeletal muscle, which is caused by mutations in the dysf (dystrophy-associated fer-1-like, DYSF) gene. Because of the large size of the codon-optimized dysf coding region (6243 bp), adenoviral vectors are suitable for the creation of genetic constructs, which are capable of delivering a large amount of recombinant genetic information into both dividing and non-dividing cells, as well as provide a high level of transgene expression. We generated a recombinant adenovirus serotype 5 encoding a codon-optimized gene for human dysferlin (Ad5- Dysf) and analysed recombinant protein expression in vitro in HEK-293T cell line.
Genes & Cells. 2012;7(3):25-28
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Gene transfer to mice organs using non-viral systems for targeted delivery with differenthydrophobicity and with lactose addressing group

Bogdanenko E.V., Ibragimova M.Y., Zinnatullina E.T., Shakirova R.I., Khramova A.Y., Zhdanov R.I.


Biodistribution of lipoplexes formed of cholenim substances I-III, containing one, two or three cholesterol moieties, and eukaryotic 14С-DNA and(or) reporter gene into mice organs using a variety of administration routes (intraperitoneally, i.p.; portal vein or left renal artery) is studied in this paper. It is shown that biodistribution doesnt depend on lipoplex lipid composition under i.p. administration, and depends on lipid nature under vein and artery administration. Effective in vivo transfection and reporter gene expression are demonstrated under portal vein administration of lipoplex formed of dicholenim II and lactosylated lipid IV (1 to 1 mass ratio). In the case, the β-Gal gene expression (above 0,3 mcg / g of tissue) is demonstrated in lungs, liver and spleen histochemically and spectrophotometrically. Introduction of cholesterol moieties into oligoethylenimine structure results in optimal hydrophilicity/hydrophobicity ratio, their stabilization, and optimal value of critical constant of micelle formation. There are certain outlooks due to usage of the lipoplexes described for targeted gene delivery.
Genes & Cells. 2012;7(3):29-33
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Changes of the inflammatory activity and fibrosis in patients with alcoholic liver cirrhosis after autologoushematopoietic stem cell transplantation

Burganova G.R., Abdulkhakov S.R., Gumerova A.A., Gazizov I.M., Yylmaz T.S., Titova M.A., Odintsova A.K., Kiyasov A.P., Burganova G.R., Abdulkhakov S.R., Gumerova A.A., Gazizov I.M., Yilmaz T.S., Titova M.A., Odintcova A.K., Kiassov A.P.


Evaluation of treatment results of chronic liver diseases should be made on the basis of morphological analysis of liver biopsies. The aim of our study was to investigate the effect of autologous hematopoietic stem cell transplantation on histology activity index and grade of fibrosis in alcoholic liver cirrhosis patients. The study was performed on liver biopsies of 11 patients with alcoholic liver cirrhosis. Biopsies were taken before the injection of autologous peripheral blood stem cells into celiac trunk, 3 and 12 months after the procedure. Liver biopsy specimens were stained with hematoxylin-eosin and Van Gieson's. Results showed improvement of liver structure and decrease in histology activity index in liver biopsies performed 3 and 12 months after transplantation. Our data suggest that autologous transplantation of hematopoietic stem cell in patients with alcoholic liver cirrhosis is effective method that is capable to reduce inflammation activity in the liver, improve its structure and decrease liver fibrogenesis.
Genes & Cells. 2012;7(3):34-36
pages 34-36 views

Liver pathomorphology of Mus musculus C57BL6 on atherogenic diet

Gaifullina R.F., Katina M.N., Abdulhakov S.R., Kasimova L.R., Abdulhakova A.R., Rizvanov A.A.


Atherosclerosis is one of the leading causes of disability and death worldwide. Liver plays a huge role in pathogenesis of atherogenic dislipidemia, development and progression of atherosclerotic lesions. We studied the effect of atherogenic diet on liver morphology in animal model of diet-induced atherosclerosis in mice Mus musculus C57BI6. This strain has a natural ability to develop atherosclerosis, while some other mouse stains has not. After 14 weeks on atherogenic diet a severe hepathomegaly (9% of body mass) and lobular structure deformation was found. We also observed signs of micro- and macrovesicular steatosis, cell apoptosis, fibrosis and inflammatory leukocyte infiltration. So, liver not only plays an important role in dislipidemia, but it is also a target-organ in lipid metabolism imbalance.
Genes & Cells. 2012;7(3):37-40
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Direct observation of «cholesterol - model of biological membrane» complex by NMR spectroscopy

Galiullina, L.F., Blohin, D.S., Aganov, A.V., Klochkov V.V.


Interaction and aggregation of cholesterol and sodium dodecyl sulfate molecules were studied in this paper. Sodium dodecyl sulfate was taken as a model for biological membranes. Cholesterol-sodium dodecyl sulfate complex was described by modern methods of nuclear magnetic resonance spectroscopy. Nuclear magnetic resonance spectra were recorded on «Avance-500» spectrometer (Bruker). To assign 1Н signals of cholesterol, sodium dodecyl sulfate and cholesterol+sodium dodecyl sulfate mixture in nuclear magnetic resonance spectra literature data was used, and 2D homo- end hetero-correlation nuclear magnetic resonance spectra were recorded. To study the formation of sodium dodecyl sulfate micelles and complex of cholesterol-sodium dodecyl sulfate micelles selective nuclear Overhauser effect spectroscopy experiments were carried out. The formation of sodium dodecyl sulfate micelles in dimethyl sulfoxide solution was confirmed by nuclear Overhauser effect spectroscopy data. The presence of a complex between sodium dodecyl sulfate micelles and cholesterol molecules has been proven by selective nuclear Overhauser effect spectroscopy experiments. Nuclear Overhauser effect between OHgroup of cholesterol and «tail» groups of sodium dodecyl sulfate hydrophobic part was observed in the experiment. This observation corresponds to close spatial arrangement of these parts of different molecules and the presence of a complex between cholesterol and sodium dodecyl sulfate micelles. On the basis of the nuclear magnetic resonance experiments was established that molecules of sodium dodecyl sulfate form micelles in dimethyl sulfoxide solution at concentrations above the critical micelle concentration. Cholesterol molecules form an intermolecular complex with sodium dodecyl sulfate micelles by interaction of the OH group of cholesterol and СН3-1 and СН2-2 «tail» aliphatic groups of sodium dodecyl sulfate. This interaction is similar to the behavior of cholesterol in phospholipid bilayer membranes in which cholesterol enters its cyclic part in the hydrophobic tails of phospholipid molecules oriented primarily across the bilayers.
Genes & Cells. 2012;7(3):41-48
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Tromboliticheskaya i fibrinoliticheskaya aktivnost'bakterial'nykh proteaz

Danilova, J.V., Cheremin, A.M., Zamaleeva, A.I., Mardanova, A.M., Zamalutdinova, N.M., Sharipova M.R.


In medical practice is increasingly use proteolytic enzymes of microorganisms. Particular attention of researchers is attracted proteases, which have fibrinolytic properties, and can lyse clots. Previously had been isolated and purified to homogeneity glutamyl endopeptidase, subtilisin-like proteinase and metalloendopeptidase of Bacillus pumilis 3-19, secreted by B. subtilis JB 2036 recombinant strain. The analysis of thrombolytic, fibrinolytic, and anticoagulant properties of the recombinant enzymes was conducted. It is shown that all investigated proteinases are able to efficiently lyse the clot. In an in vitro subtilisin and glutamyl endopeptidase of B.subtilis recombinant strain have anticoagulant activity. Metalloproteinase is not able to influence the process clot formation. Subtilisinlike proteinase and glutamyl endopeptidase possess fibrinolytic activity and the ability of the activator relative to plasminogen. Metalloproteinase shows no fibrinolytic properties. Due to the high incidence of cardiovascular diseases is urgent search for new enzymes with high biological activity, specificity and low toxicity.
Genes & Cells. 2012;7(3):49-51
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Polymer-stabilised magnetic nanoparticles do not affect the viability of magnetically-functionalised cells

Dzamukova M.R., Naumenko E.A., Zakirova E.Y., Dzamukov R.A., Shilyagin P.A., Ilinskaya O.N., Fakhrullin R.F.


Here we report the synthesis of (poly)allylamine-coated superparamagnetic iron oxide nanoparticles for the surface modification of living cells. Magnetic functionalisation of cow embryonic lung cells did not affect the viability of the coated cells, confluent monolayer formation and proliferation, as demonstrated using fluorescence and white light microscopy, flow cytometry and MTT assay. Functionalised cells were magnetically responsive. We believe that the single-step approach described here is a novel and potentially promising way to functionalise mammal cells with magnetic nanoparticles for the subsequent applications in cell therapy, directed cells delivery and spatial positioning in tissue engineering.
Genes & Cells. 2012;7(3):52-56
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Gene transfer using new complexes between cardiolipin-like dicationic lipids and plasmid DNA to tumor cells

Zhdanov R.I., Moskovtsev A.A., Blokhin D.Y., Doynikova A.N., Shakirova R.I., Sebyakin Y.L.


The lipid vesicles of bisamphiphiles cardiolipin-like dicationic lipids (CDL) I-IV were studied for creation of lipoplexes with plasmid DNA of different sizes to obtain stable lipoplexes for gene transfer to gene therapy. Lipoplexes sizes (300±100 nm) and stablity (> 2 hrs) of CDL were sufficient to be used in gene transfer against monolayer and suspension cell cultures. The CDL total cytotoxicity determined by MTT-test was lower compare to lipofectin as a control. Transfection conditions against tumor cells lines were optimized by lipoplexes of CDL and plasmid DNA. The most efficient transfection for lipoplexes CDL-plasmid DNA was at the lipid-DNA (L/D) ratio equal to 5 (for lipofectin, it was 2). For monolayer cell cultures, lipoplexes CDL-I are comparable in terms of transfection efficacy with lipofectin; in the case of suspension culture, their efficiency was lower by one order of magnitude. It permits a usage of lipoplexes suggested as mediators for gene transfer and delivery to human tumor cells.
Genes & Cells. 2012;7(3):57-61
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The cytofluorimetric characteristics of RNAse influence towards pro- and eucariotic cells

Zelenikhin, P.V., Mamedzade, K.R., Ilinskaya O.N.


Cytotoxic ribonucleases (RNAses) are known to be perspective drugs for cancer therapy. The extension of model objects range will give possibility to assess the cytotoxic RNAses selectivity. We estimated the effect of Bacillus intermedius ribonuclease (binase) and bovine RNAse A to E. coli K 12 and lung epithelia of cow embryo (LEC) cells. LEC cells apoptosis was characterized with a double staining with annexin-FITC and propidium iodide (PI). E. coli K12 cells vitality was estimate via PI staning. Binase and RNAse A were not cytotoxic to LEC and E. coli K12 cells in investigated concentrations (100 and 300 ƒg/ml). Low RNAses toxicity for E. coli allows to suppose the binase and RNAse A in concentrations, capable to display antitumor activity, will not to effect the tissues microbial flora during in vivo tests. The lack of apoptosis inducing activity of binase for LEC cells confirms this RNAse selectivity for tumor cells.
Genes & Cells. 2012;7(3):62-65
pages 62-65 views

Influence of serum antibodies to DNA on theMadin-Darby сanine kidney epithelial cells in vitroin depend of immunoreactive regions of the IgG molecule

Ivanov, V.V., Nevzorova T.A.


The data obtained on cell lines MDCK showed that for implementation of the biological function of antibodies to DNA both normal and pathological need the participation as Fab- and Fc-fragments of molecules of IgG. However, antigen-binding region is responsible for the manifestation of the biological function of SLE antibodies to DNA more immunoreactive than antibodies in normal and constant region is probably responsible for the active conformation of antibody for pathobiological function of antibodies to DNA in the cell.
Genes & Cells. 2012;7(3):66-68
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Participation of the mononuclear cells of the cord blood in the physiologic regeneration of the rat kidney

Yilmaz T.S., Mavlikeeva A.V., Gumerova A.A., Andreeva D.I., Fairushina I.F., Kiiasov A.A.


Nowadays ability to use hematopoietic stem cells for treatment of various diseases, including kidney pathology, is widely investigated. Umbilical cord blood as the source of hematopoietic stem cells becomes more and more promising. The purpose of our investigation was to study homing and ways of human umbilical cord blood mononuclear cells differentiation in an intact rat kidney. We transplanted human umbilical cord blood mononuclear cells fraction, enrich with hematopoietic stem cells, into the tail vein of rats. On 2, 5, 7 and 14 days after transplantation paraffin kidney slices were immunohistochemically stained with antibodies against human leukocyte antigen (HLA-ABC) to study migration and differentiation of transplanted cells. Results: HLA-ABC+- cells were revealed in the epithelium of the distal tubule at all experimental dates. But HLA-ABC was expressed not in each distal tubule and not by all tubular cells. We concluded that human umbilical cord blood mononuclear cells transplanted into systemic circulation of rat migrate into the intact kidney and built in the distal tubule epithelium. This data allow to suggest that distal tubule are stem cell «niche» in the kidney.
Genes & Cells. 2012;7(3):69-71
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Antitumor RNase (binase) induces the alteration of cellular permeability

Cabrera-Fuentes H.A., Zelenikhin, P.V., Kolpakov, A.I., Ilinskaya O.N.


Some RNases including ones of microbial origin possess antitumor activity, which mechanisms remains unclear. Here we investigated the first step of RNase action towards eukaryotic cells which is connected with increase of cell permeability for ions and macromolecules. Using radiological analysis of 45Са2+uptake by Candida yeast and fluorescence imaging of human embryo kidney cells HEK stained by Ca2+-specific Fura-2/АМ day the level of intracellular Ca2+ under treatment with the RNase of Bacillus intermedius (binase) was studied. Viability of lung carcinoma epithelial cells A549 treated by binase was measured by WST proliferation kit, stability of erythrocytes was tested by lysis assay. We have shown that binase induces the permeability increase of lower and higher eukaryotic cells for Ca2+as well as the increase of protein permeability of A549 cells. Binase treatment protects erythrocytes from osmotic shock. The protective or cytotoxic binase effect followed by increase of cellular permeability is realized depending on the dell type, where the expression of КСa channels and of certain oncogens, particularly of ras family, is crucial. The obtained data supports the significance of the cell permeability increase as a primary step in the mechanisms of binase-induced biological effects.
Genes & Cells. 2012;7(3):72-76
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С-kit-positive progenitor cells activation in rats pancreas after partial hepatectomy

Kaligin M.S., Plushkina A.S., Gazizov I.M., Sharipova E.I., Gymerova A.A., Kiassov A.A.


One of the most common markers of pancreas stem cells is a stem cell factor receptor C-kit. According to some authors, this marker is presented in islet cells of normal rat pancreas. But it is unknown about the behavior of these cells in disorders of carbohydrate metabolism during liver disease. The aim of our study was to evaluate C-kit expression in pancreas after partial hepatectomy in rats. Partial hepatectomy was performed for 27 white male rats. The expression of C-kit, insulin and glucagon in rats pancreas was studied. The expression of C-kit in islets and interstitial cells was shown in results after 3 days of the experiment, and double staining showed that these cells can express glucagon. Thus, there is the activation of C-kit+ progenitor cells in pancreas after partial hepatectomy and the beginning of there differentiation to ƒ-cells of Langerhance islets.
Genes & Cells. 2012;7(3):77-79
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Isolation, culture and differentiation of rat (Rattus norvegicus) and hamster (Mesocricetus auratus)adipose derived multipotent mesenchymal stromal cells

Katina M.N., Gaifullina R.F., Hayatova Z.G., Emene C.C., Rizvanov A.A.


Cell therapy of various diseases is one of the most perspective fields in modern medicine. Adipose derived autological stem cells can be obtained for therapeutic purposes. Animal model of human diseases are essential for cell therapy research. However, the most frequently used laboratory animals, such as rats and mice, cant suffer the whole rate of common diseases of modern society. At the same time Syrian hamsters can provide scientists with an appropriate animal models of these diseases. Nevertheless, we couldnt find any data on hamsters stem cells isolation and their characteristics. In this study we first isolated Syrian hamsters adipose derived stem cells, characterized their morphology, features and differential potential in several ways. These cells are much alike multipotent mesenchymal stromal cells and can go through osteogenic and, adipogenic differentiation. We have also shown that these cells can differentiate in neurogenic way.
Genes & Cells. 2012;7(3):82-87
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The action of bacterial serine proteases on cultured animal cell lines

Kirillova, J.M., Danilova, J.I., Sharipova M.R.


Our research is direct to determination of serine proteases (subtilisin and glutamyl endopeptidase) action at establish cell lines. We determined that the serine proteases of bacillus are capable cytotoxic action at establish cell lines of animals. The cell lines are differentiating by sensitivity to proteins. The cell lines LEK and NGUK were more sensitive to subtilisin, and line Vero - glutamyl endopeptidase.
Genes & Cells. 2012;7(3):88-91
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Human umbilical cord blood mononuclear cells transfected with dual cassette plasmids(vegf + neurotrophic factor) for the treatment of amyotrophic lateral sclerosis

Kudryashova N.V., Guseva D.S., Salafutdinov I.I., Bashirov F.V., Kiiasov A.P., Rizvanov A.A., Islamov R.R.


To increase the viability of neural cells in neurodegenerative diseases, after neurotraumas and ischemic strokes the most important neurotrophic and neuroprotective factors, which can be used as therapeutic agents were identified in long-term studies in vitro and in vivo. These include brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF). One of the promising ways of the delivery of supporting neuron survival factors is considered to be transplantation of genetically modified cells overexpressing recombinant therapeutic genes. This article describes generation of cellular delivery vectors of therapeutic genes - human umbilical cord blood mononuclear cells genetically modified by dual cassette plasmids, expressing two therapeutic genes. Efficiency of transgene expression was confirmed in vitro using RT-PCR. Analysis of survival, migration, and phenotype of genetically modified cells was performed 2 weeks after transplantation into transgenic mice with amyotrophic lateral sclerosis phenotype.
Genes & Cells. 2012;7(3):92-97
pages 92-97 views

Cellular and receptor mechanisms of impairment of myocardium and aorta contractilityat Alzheimers disease model

Leushina A.V., Gaifullina R.F., Zefirov A.L., Palotas A., Mukhamedyarov M.A.


Introduction. Recent studies certify the existence of link between Alzheimers disease and cardiovascular pathology, however the mechanisms of this phenomenon is unclear. Here we studied the influence of Alzheimers β-amyloid peptide (βAP) on the contractility of rat myocardium and aorta. Material and methods. Contractility of myocardium ventricle strips and transverse fragments of abdominal aorta was measured at Power Lab setup using conventional myographic technique. Contractile responses of aorta strips were evoked by application of receptor agonists, contractile responses of myocardium - by electrical stimulation. Contractile responses of aorta strips after application of carbachol (10-6-10-4 М), histamine (10-6-10-4 М), norepinephrine (10-5-10-3 М) and ATP (10-6-10-4 М) were measured. Results and discussion. We found the impairment of carbachol- and histamine-induced contractility of aorta, appearing as perverse contractile reactions (relaxation instead of contraction) under the action of βAP (10-6 М). Next, we found βAP-induced impairments of ventricle myocardium contractility, appearing as decrease of relaxation phase duration and increase of relaxation speed (positive lusitropic effect). Also, own positive lusitropic effect of norepinephrine was absent in presence of βAP (10-6М). Thus, βAP(25-35) significantly impairs the contractility of rat myocardium and aorta, as well as processes of its regulation. Obtained data significantly broad our understanding of mechanisms of Alzheimers disease pathogenesis and pathophysiology of cardiovascular system.
Genes & Cells. 2012;7(3):98-100
pages 98-100 views

Uluchshenie dostavki kompleksov plazmidnoy DNKs polikationom v kletki chelovekav prisutstvii bloksopolimerov etilen-i propilenoksida

Martynova, A.D., Shevchenko, V.D., Bondar, O.V., Abdullin T.I.


The work is aimed at the study of the effect of novel block copolymers of ethylene oxide and propylene oxide on the delivery of plasmid DNA and its complexes with cationic polymers into human cells. Tri-functional amphiphilic block copolymers on the basis of glycerol (LaprolsTM), polyethyleneimine (25 kDa) and commercial transfection reagent TurboFectTM were tested as delivery systems. TurboFect was found to form more compact and positively charged polyplexes with plasmid DNA (pEGFP-N2) and provided higher expression of GFP in HEK 293 cells compared to polyethyleneimine. Laprols weakly interacted with plasmid DNA and did not improve its intracellular delivery. However they markedly promoted cell transfection by DNA-polyethyleneimine complexes. Results show that Laprols exhibit mild cytotoxicity and produce the interest for gene therapeutics delivery into cells.
Genes & Cells. 2012;7(3):101-104
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Biosafety model of adenovirus infection: effects of bacterial proteases for infection of human cells in vitro

Martinova E.V., Danilova Y.V., Anohin V.A., Khaiboullina S.F., Rizvanov A.A., Sharipova M.R.


To determine the antiviral activity of various biologically active compounds, the model of adenovirus infection on the basis of cell cultures of human HEK293A and recombinant adenovirus Ad-EGFP, expressing green fluorescent protein EGFP. Adenoviruses have a capsid size of 70-90 nm and are able to infect dividing and nondividing cells in vitro and in vivo. Recombinant adenoviruses are the replicative defect in the cells of humans and animals. The developed model allowed us to determine the effect of bacterial proteases in the infected cell cultures with adenovirus. This model can also be used for screening drugs with potential protivivovirusnoy activity.
Genes & Cells. 2012;7(3):105-107
pages 105-107 views

Effect of binase on phorbol myristate acetate-induced apoptosis of human peripheral bloodgranulocytes and monocytes

Mironov V.A., Shirshikov F.V., Kalacheva N.V., Cherepnev G.V.


Effect of binase (RNAse of Bacillus intermedius) on phorbol myristate acetate-(PMA)-induced apoptosis of human peripheral blood granulocytes and monocytes was studied in vitro by flow cytometry. Both toxic (400 μg/ml) and nontoxic (40 μg/ml) binase concentrations were tested. The binase end-point effect was dependent on the target cell population and the binase concentration. In a granulocyte subset, the 400 μg/ml concentration resulted in strongly pronounced stimulation of PMA-induced apoptosis. In a monocyte subset, the 40 μg/ml concentration developed a protective effect as judged by an increase in a percantage of viable cell subset and by slowing-down cells transtion from an early to late PMAinduced apoptotic phase.
Genes & Cells. 2012;7(3):108-111
pages 108-111 views

Isolation and cultivation of myofibroblasts from rats liver using explantation method

Mijanovic О., Shafigullina А.К., Rizvanov А.А., Kiasov А.P.


During liver fibrosis development connective tissue is produced by myofibroblasts that could originate from two hepatic populations: hepatic stellate cells and portal fibroblasts. A marker of myofibroblasts is the expression of ƒ-smooth muscle actin (ƒ-SMA). Distinctive feature of myofibroblasts, derived from hepatic stellate cells, is the preservation of the hepatic stellate cells marker expression - desmin. The processes of activation, proliferation and cells trans-differentiation into myofibroblasts are closely related to the activity of transcription factor NF-kB and its inhibitor IkBƒ. The aim of our work was to obtain a culture of hepatic myofibrobasts, to study their origin, phenotype, relations between NF-kB and IkBƒ expression and the processes of activation and cells trans-differentiation into myofibroblasts. For this purpose we isolated heterogeneous population of cells from rat liver by the method of explantation. Almost all the cells had desmin and ƒ-SMA expression. On this basis, we suppose that these myofibroblasts were hepatic stellate cells derivatives, and singular desmin-negative cells originated from portal fibroblasts. Thus, hepatic stellate cells have major potential to activation, growth, proliferation and transdifferentiation into myofibroblasts in comparison to portal fibroblasts. Activated state of the cells was confirmed by stable expression of NF-kB and its inhibitor IkBƒ in all the cells throughout the whole experiment.
Genes & Cells. 2012;7(3):112-115
pages 112-115 views

Efficiency and mechanism of antitumor activity of cardiolipin-like lipid/thymidine kinase gene HSV-tklipoplexes in the presence of gancyclovir

Moskovtsev A.A., Sokolovskaya A.A., Ibragimova M.Y., Dojnikova A.N., Sebyakin U.L., Zhdanov R.I.


The herpes simplex virus thymidine kinase/gancyclovir (HSV-tk/GCV) system is studied as cytotoxic lipoplex based on cardiolipin-like dicationic lipid CDL-I. It is proposed to be used as nonviral gene transfer system in cancer gene therapy protocols. Аn efficient transfection of MCF7 and HEC293 cell lines with this lipoplex was earlier demonstrated. Non-viral system based on the CDL-I/HSV-tk lipoplex and ganciclovir treatment causes efficiently death of tumor cells with an involvement of apoptosis key stages. It was proved that depolarization of mitochondrial membrane and increased level of NF-kB transcription factor take place as a response to CDL-I / HSV-tk lipoplex action followed by gancyclovir treatment. It suggests an early involvement of apoptosis mitochondrial way to an action of this certain «suicide» system, delivered using dicationic lipid.
Genes & Cells. 2012;7(3):116-120
pages 116-120 views

Preparation and reversible aggregation of human cells encased in biocompatible polysaccharide shell

Mukhamadeeva, R.A., Mavlyutova, I.I., Bondar O.V.


A deposition of cationic (chitosan) and anionic (alginic acid) polysaccharides onto the surface of normal and cancer human cells was studied with the use of dynamic light scattering. A method for preparation of multilayer polymeric shell by means of electrostatic adsorption of polysaccharides onto cell plasma membrane has been proposed. According to confocal microscopy, the polymeric shell evenly covers the cell and is 1-5 μm in thickness. Under experimental conditions, the modification with polysaccharides inhibits human skin fibroblasts growth but does not exhibit cytotoxicity to HeLa cells. We developed an approach to controllable and reversible aggregation of modified cells by their cross-linking in the presence of calcium ion. Resulting aggregates have spherical shape and the size of 100-500 μm. Proposed approaches and methods represent an alternative to cell microencapsulation technique and are of interest for the development of three-dimensional cell models and their delivery in vivo.
Genes & Cells. 2012;7(3):121-124
pages 121-124 views

Survival and differentiation of endogenous Schwann cells migrating into spinal cordunder the influence of neurotrophic factors

Mukhamedshina Y.O., Shaymardanova G.F., Muhitov A.R., Salafutdinov I.I., Zarubina V.N., Rizvanov А.А., Chelyshev Y.A.


Schwann cells are a major figure in the process of regeneration in the peripheral nervous system. They migrate into the injury region of spinal cord, which are involved in remyelination and are regarded as the source of numerous molecular signals that could potentially support the growth of axons in the central nervous system. In the present work we describe the behavior of migrating into the injury dosed region spinal cord Schwann cells under the influence of neurotrophic factors - vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), delivered by direct introduction of «naked» plasmid DNA and by transplantation of genetically modified human umbilical cord blood mononuclear cells. Using immunohistochemical detection of markers of S100, GFAP, Krox20 and HSP25 identified different phenotypes of migrating into the spinal cord of endogenous Schwann cells. Found that greatest influence on their numbers in the injury region provides local delivery of genes vegf and fgf2 by human umbilical cord blood mononuclear cells. However, the direct introduction of the same plasmid may also be promising in the case of synthetic platforms that enhance its transfection activity.
Genes & Cells. 2012;7(3):125-129
pages 125-129 views

Analysis of recombinant vegf gene expression by genetically modified umbilical cord bloodmononuclear cells in experiment in vivo

Mukhamedshina Y.O., Solov'eva V.V., Salafutdinov I.I., Cherenkova E.E., Fedotova V.Y., Safiullov Z.Z., Izmaylov A.A., Sharifullina G.A., Abdulkhakov S.R., Kaligin M.S., Bashirov F.V., Mukhamed'yarov M.A., Shmarov M.M., Naroditskiy B.S., Kiyasov A.P., Rizvanov A.A., Islamov R.R., Mukhamedshina Y.O., Solovieva V.V., Salafutdinov I.I., Cherenkova E.E., Fedotova V.Y., Safiullov Z.Z., Izmailov A.A., Sharifullina G.A., Abdulhakov S.R., Kaligin M.S., Bashirov F.V., Muhamediarov M.A., Shmarov M.M., Naroditskii B.S., Kiiasov A.P., Rizvanov A.A., Islamov R.R.


To obtain a significant therapeutic effect transplanted genetically modified cells should have an enhanced ability to survive and active expression of the therapeutic gene. In this paper, by using immunofluorescent staining we investigated the functional activity of the gene-cell formulation designed to deliver a therapeutic gene into the area of regeneration. As a model we used transgenic SOD1- G93A mice with amyotrophic lateral sclerosis phenotype which received xenotransplantation of human umbilical cord blood mononuclear cells, genetically modified with adenoviral expression vector encoding vascular endothelial growth factor (VEGF) and the reporter green fluorescent protein (EGFP). Results of the study allowed to establish not only the duration of survival of transplanted cells, but also the efficiency of expression of recombinant genes in genetically modified cells in vivo. Double immunofluorescent staining with antibodies against human nuclear antigen HNA and VEGF detected HNA+/VEGF+ cells in the terminal stage of disease 15 weeks after transplantation. These data suggest that genetically modified umbilical cord blood mononuclear cells, transplanted into SOD1-G93A transgenic mice, are able to penetrate the blood-brain barrier and migrate into the area of degeneration of nerve tissue and survive from the time of transplantation until the death of animals at the terminal stage of disease. At that time adenoviral expression vector encoding therapeutic gene is functionally active in transplanted cells, and secretory products of recombinant gene act on target cells by a paracrine mechanism.
Genes & Cells. 2012;7(3):130-134
pages 130-134 views

The mechanism of ƒ-amyloid peptide influence on the retrograde axon transport

Mukhamed'yarov M.A., Safiullov Z.Z., Utyasheva R.P., Rizvanov A.A., Zefirov A.L., Islamov R.R., Mukhamedyarov M.A., Safiullov Z.Z., Utyasheva R.P., Rizvanov A.A., Zefirov A.L., Islamov R.R.


Impairment of axon transport is widespread and early event in a number of neurodegenerative diseases. The goal of study is to investigate the mechanisms of retrograde axon transport impairment in mouse spinal motoneurons after application of ƒ-amyloid peptide (ƒAP) (25-35) on the central stump of transected sciatic nerve. Retrograde fluorescent tracer Fluorogold (5%), ƒAP (25-35) (10-6 М), or mix was applied to the proximal stump of the transected sciatic nerve of mouse under the general anesthesia. At 24 hours after surgery lumbar spinal cord was processed for morphometric and immunohistochemical analysis. The amount of Fluorogold-positive motoneurons at control was 1223,7162,7 (n = 7), whereas after application of ƒAP(25-35) - 393,285,3 (n = 5, p < 0,01), which certifies pronounced inhibition of retrograde axonal transport. Staining with polyclonal antibodies against caspase-3 did not reveal motoneurons in apoptotic state. Staining with monoclonal antibodies against the ƒAP (25-35) was negative both at operated and intact sides of spinal cord. Thus, revealed inhibitory action of ƒAP (25-35) on the retrograde axon transport is not related to apoptotic death of neurons or accumulation of ƒAP (25-35) inside the neuronal soma, but, evidently, is mediated by intraaxonal effects. Obtained data has great importance for understanding of mechanisms of Alzheimers disease pathogenesis.
Genes & Cells. 2012;7(3):135-137
pages 135-137 views

C-kit-positive pancreas islets cell of rats pancreas as a endocrine cells progenitor during alloxan diabetes

Plyushkina A.S., Kaligin M.S., Andreeva D.I., Titova A.A., Valeeva I.K., Dem'yanov A.V., Gumerova A.A., Kiyasov A.P., Plushkina A.S., Kaligin M.S., Andreeva D.I., Titova A.A., Valeeva I.H., Demyanov A.V., Gumerova A.A., Kiassov A.P.


One of the most common markers for stem cells in pancreas is the stem cell factor receptor C-kit (CD117) that plays a main role in differentiation of progenitor endocrine cells of pancreas islets in prenatal development and persists after birth. But still the role of C-kit positive cells in islet ƒ-cells regeneration during the diabetes mellitus type I has not been studied. Thats why the aim of our work was to study the dynamic of C-kit expression in the pancreas islets during the experimental alloxan diabetes in rats. The work was made on 33 rats with the experimental diabetes. Blood glucose levels, levels of insulin and glucagon were measured. And also we studied the expression of C-kit, insulin and glucagon in rat pancreas. The results of the study showed the C-kit expression after one day of the experimental hyperglycemia. These cells were also expressed insulin and glucagon. We suppose that C-kit+-cells, which produce insulin, were enable to correct disrupted carbohydrate metabolism during alloxan diabetes.
Genes & Cells. 2012;7(3):138-141
pages 138-141 views

Chromatographic purification of plasmid DNA for clinical applications (gene therapy)

Romanova J.J., Salafutdinov I.I., Zamalyutdinova N.M., Rizvanov A.A.


For the successful application of plasmid vectors in gene therapy protocols it is necessary to develop methods for purification of highly homogeneous preparations of recombinant DNA that do not contain contaminants, primarily chromosomal DNA, bacterial proteins, RNA and endotoxins. In the course of our study we performed optimization of the purification of plasmid supercoiled DNA by three chromatographic steps from an alkaline lysate of bacterial strain of E. coli. We determined an optimal conditions for alkaline lysis step in order to increase the yield and minimize the duration of the plasmid purification by gel filtration.
Genes & Cells. 2012;7(3):142-145
pages 142-145 views

Autophagy induction in peripheral blood T-lymphocytes of atopic asthma patients

Skibo Y.V., Ponomareva A.A., Reshetnikova I.D., Abramova Z.I.


Autophagy is a fundamental process that ensures the regulation of T-cell homeostasis. In case of apoptosis induction disruption in the cell it could be single mechanism of the cell death. Previously was shown inhibition of lymphocyte apoptosis in patients with bronchial asthma, so the main study of this work has focused on the study development process of autophagy in T-lymphocytes of patients with bronchial asthma. The article presents the main morphological changes in cells associated with activation of autophagy (formation autophagosome). In addition to morphological changes in lymphocytes, we have shown the expression of autophagy marker protein (LC3B). We found that in T-lymphocytes of patients with severe asthma are simultaneous activation of both autophagy and apoptosis, and autophagy is a stimulus to cell death.
Genes & Cells. 2012;7(3):146-150
pages 146-150 views

Influence of recombinant histone H1.3 on the efficiency of lentiviral transduction of human cells in vitro

Solovyeva V.V., Isaev A.A., Genkin D.D., Rizvanov A.A.


Lentiviral vectors are widely used in genetic modification of human and animal cells (lentiviral transduction) to enhance their therapeutic potential by expression of recombinant protective and trophic factors. Genetic modification of cells in vitro or ex vivo achieves the specificity of viral transduction, as modified are just cells that have been manipulated in the laboratory. In addition, the introduction of genetically modified cells, but not pure virus, helps to avoid introduction of viral particles into the body of the recipient. This approach allows us to control the expression of therapeutic genes, the immunogenicity of viral vectors and viral transduction. To date, different approaches are used to improve the lentiviral transduction (polycations, protamine sulfate, etc.), but these methods suffer from limited efficacy or high toxicity. For the first time we demonstrated that the recombinant histone N1.3 increases the efficiency of lentiviral transduction by more than 2 times and has no toxic effect on target cells in a wide range of concentrations studied.
Genes & Cells. 2012;7(3):151-154
pages 151-154 views

Endogenous secretion of vascular endothelial growth factor by multipotent mesenchymal stromal cellsderived from human third molar dental follicles

Solovyeva V.V., Blatt N.L., Shafigullina A.K., Rizvanov A.A.


Human stem cells secretome is currently a very hot area of research. We report that multipotent mesenchymal stromal cells isolated from human third molar dental follicles (MMSCTMDF), are able to secrete high levels of vascular endothelial growth factor (VEGF) when cultured in vitro. Due to the fact that VEGF is a well known angiogenic and neuroprotective factor, the use of MMSC-TMDF is promising for the development of stem cell therapy of various degenerative human diseases.
Genes & Cells. 2012;7(3):155-158
pages 155-158 views

Influence of Trichoderma asperellum metabolites on tissue regeneration against pyrene

Tukhbatova, R.I., Abd El-Rahman A.A., Mukhametzyanova, A.S., Nguen, T.T., Hoang, T.L., Fattahova, A.N., Alimova F.K.


We studied the influence of the culture fluid of fungi of the genus Trichoderma on Swiss Webster CFW mice after exposure to pyrene - polycyclic aromatic hydrocarbons, which can cause pathological changes in the body. Beneficial effect of Trichoderma metabolites on haematological parameters, the functioning of liver and nephros was shown, the trend toward regeneration of the structure of skin and liver after the damages, caused by the introduction of pyrene, was identified.
Genes & Cells. 2012;7(3):159-163
pages 159-163 views

Generation of recombinant adenoviruses and lentiviruses expressing angiogenic and neuroprotectivefactors using Gateway cloning technology

Cherenkova E.E., Fedotova V.Y., Borisov M.A., Islamov R.R., Rizvanov A.A.


The critical aspect in gene and gene-cell therapy is to find an optimal vector - a carrier of genetic information. Viruses represent a natural biological system for gene transfer into eukaryotic cells. One of the most effective and proven vectors for delivery of recombinant nucleic acids into mammalian cells are adenoviruses and lentiviruses. In this study using the Gateway cloning we have constructed adenoviral and lentiviral vectors encoding angiogenic and neuroprotective factors: various isoforms of vascular endothelial growth factor vegf121, vegf165, vegf189; basic fibroblast growth factor fgf2; glial cell-derived neurotrophic factor gdnf. The efficiency of transduction of HEK293A cell line with generated recombinant viruses and expression of recombinant proteins were confirmed by immunofluorescent analysis.
Genes & Cells. 2012;7(3):164-168
pages 164-168 views

Transplanted Hepatic Stellate Cells participate in liver regeneration after partial hepatectomywithout risk of hepatic fibrosis

Shafigullina A.K., Gumerova A.A., Trondin A.A., Titova M.A., Gazizov I.M., Burganova G.R., Kaligin M.S., Andreeva D.I., Rizvanov A.A., Muhamedov A.R., Kiassov A.P.


Hepatic stellate cells are considered as one of the potential stem cells candidates in the liver. The aim of our work was to study the probability of hepatic stellate cells transplantation to rats after partial hepatectomy, their further homing, the ways of differentiation and hepatocytes repopulation in the recipient liver. For this reason fresh isolated rat`s hepatic stellate cells were transplanted into portal vein of intact rats (control group) and rats immediately after partial hepatectomy (experimental group). Before transplantation cells were labeled by adenovirus expressing green fluorescent protein. Our results showed that it was possible to detect 2 types of donor cells in the recipient liver of control and experimental groups: 1) hepatocyte-like cells in liver parenchyma; 2) small, spindle-shaped, rounded and triangular cells in liver sinusoids and portal areas. Transplantation after partial hepatectomy leads to significant increase of transplanted cells homing and stimulation of their differentiation into hepatocytes. Over the whole experiment there was no hepatic stellate cells transdifferentiation into myofibroblasts, thus there is no risk of liver fibrosis development after this cell type transplantation. In summary hepatic stellate cells after being transplanted are able to differentiate into hepatocytes and do not induce liver fibrosis, that confirms their role in organ regeneration and probable belonging to hepatic progenitor cells.
Genes & Cells. 2012;7(3):169-172
pages 169-172 views

Comparative study of an activity of rat spinal ganglion cells and PC12 cells on the surfacesmodified with bioadhesive polymers

Yakunina, L.D., Kurbanov, R.A., Bondar, O.V., Abdullin T.I.


We studied the adsorption of bioadhesive polymers (polyornithine, gelatin, laminin) on polystyrene surface by the use of dynamic light scattering. The contribution of biopolymers to resulting zeta potential of the modified surface was assessed. PC12 cells do not exhibit selective adhesion in the presence of foetal bovine serum. Polystyrene with adsorbed polyornithine promotes primary adhesion of PC12 cells cultured in serum-free medium with nerve growth factor. Subsequently adsorbed laminin induces spreading and differentiation of the cells into neuronal direction. Primary neurons isolated from rat spinal ganglion adhere preferentially on the polyornithine-modified surface. On the polyornithine-laminin surface neurons intensively form neuritis that correlates with proliferation of glial cells positive for S100 protein. The results show that PC12 cells and primary neurons exhibit similar response to surface material with the latter cells being more sensitive to this factor. Isolated cell culture can be used to study the relationship between neurite outgrowth and Schwann cells proliferation on different biomaterials.
Genes & Cells. 2012;7(3):173-176
pages 173-176 views

Correction of the face soft tissue defect using autologous fat tissue enriched by cellsof stromal-vascular fraction

Mastgutov R.F., Rizvanov A.A., Salafutdinov I.I., Hannanova I.G., Mullin R.I., Bogov jr A.A., Gallyamov A.R., Bogov A.A.


The article presents a clinical case of successful surgical treatment of a patient with idiopathic progressive hemifacial atrophy. We have used lipofilling with autologous fat tissue enriched by stromal-vascular fraction cells. Observation of the patient within two years suggests that this method of treatment is safe and highly effective method of face soft tissue defect substitution of such pathology.
Genes & Cells. 2012;7(3):177-179
pages 177-179 views

The first clinical experience of direct gene therapy using VEGF and bFGF in treatmentpatients with critical lower limb ischemia

Plotnikov M.V., Rizvanov A.A., Masgutov R.F., Mavlikeev M.O., Salafutdinov I.I., Gazizov I.M., Romanova Y.D., Shamsutdinova I.I., Bogov A.A., Maksimov A.V.


In this paper we present the clinical observation of successful treatment of distal form of peripheral artery disease of the lower extremity with symptoms of critical ischemia in a 60 years old patient. Intramuscular injection of dual expression plasmid, encoding vascular endothelial growth factor VEGF and basic fibroblast growth factor FGF2, was performed to the affected lower extremity. The effect of treatment was evaluated by functional tests: measurement of ankle-brachial index, treadmill test, the recovery time, shoulder-ankle index after strain and temporary occlusion. Performed immunohistochemical examination of biopsy samples of the affected lower extremity muscles.
Genes & Cells. 2012;7(3):180-184
pages 180-184 views

Life without water: cryptobiosis of invertebrates as a model for next generation techmologyof biomaterials preservation

Shagimardanova, E.I., Sharipova, M.R., Rizvanov, A.A., Zaharov, I.S., Gusev O.A.


To date, advances in the field of tissue engineering, cell transplantation and genetic engineering have made the biological materials of different origin an important therapeutic tool in clinical medicine. Currently, cells preservation is achieved by freezing at -80°С or in liquid nitrogen. Cryopreservation technology is expensive and has considerable limits during transportation. Preservation of viable biological material in dry state under ambient temperature is considered as attractive, but yet fully achieved alternative. There are organisms which are able to survive complete water loss. Understanding of mechanisms underlying dehydration tolerance will allow the development of dry preservation technology for molecules, cells and organs, and further use of these methods in medicine, pharmacology and biotechnology.
Genes & Cells. 2012;7(3):185-189
pages 185-189 views


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В июне этого года отметила свой юбилей профессор Нина Андреевна Онищенко - заведующая лабораторией биотехнологии стволовых клеток ФГУ «ФНЦ Трансплантологии и искусственных органов им. В.И. Шумакова» МЗ СР, участник редакционного совета журнала «Клеточная трансплантология и тканевая инженерия».
Genes & Cells. 2012;7(3):190-191
pages 190-191 views

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