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Vol 9, No 4 (2014)


Geneand cell-based therapy of muscle system hereditary disorders: state-of-art

Deev R.V., Mavlikeev M.O., Bozo I.Y., Pulin A.A., Eremin I.I.


Genetic disorders primarily affecting skeletal muscles can be caused by dysfunction of more than 30 genes. To date there is no effective etiotropic and pathogenetic treatment of such disorders. Investigators focus on search for new therapeutic agents based on gene and cell technologies, small molecules as well. There are numerous preclinical and several dozens of clinical studies in the world. Unfortunately tested technologies did not lead to significant advance in treatment of patients with such disorders. At the same time resulting data allow to determine the most feasible directions of future development - combining of genome correction methods with cell delivery of corrected genome to skeletal muscles. This review is intended to give general information about etiology of skeletal muscles genetic disorders, the main directions of biotechnological development and results of the clinical studies.
Genes & Cells. 2014;9(4):6-33
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Features of multipotent mesenchymal stromal cells derived from various intraoral sources

Bozo I.Y., Zorin V.L., Eremin I.I., Deev R.V., Drobyshev A.Y., Korsakov I.N., Pulin A.A.


Multipotent mesenchymal stromal cells (MMSC) derived from various intraoral sources attracts attention of an increasing number of researchers due to availability and some features making them different from MMSC of bone marrow or adipose tissue. The review describes the main intraoral sources, characterizes their anatomical, topographical and histological peculiarities which may influence on MMSC morphofunctional profile. In comparative aspect we systematized the principal data concerning properties of MMSC derived from gingiva, buccal mucosa; periodontal ligament, dental pulp, apical papilla, jaw periosteum. It is not possible to compare MMSC of various intraoral sources under some parameters because of absence appropriate experimental findings. Some additional difficulties are caused by contradictions in published materials that are discussed in the review as well.
Genes & Cells. 2014;9(4):34-42
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Tissue engineering in vascular surgery

Popov G.I., Vavilov V.N.


Tissue engineering is a complex biomedical and technological system of knowledge allowing to make and investigate the artificial tissues and organs. Prevalence of vascular diseases and demand of bypass material in vascular surgery led to a lot of researches, with the ultimate aim to create an artificial artery or vein. This article is dedicated to review the main possible methods of artificial vessel manufacturing, some of which already have been used in a clinic.
Genes & Cells. 2014;9(4):43-49
pages 43-49 views

The role of cytokine system RANKL/RANK/OPG in the regulation of bone metabolism

Aganov D.S., Tyrenko V.V., Tsygan E.N., Toporkov M.M., Bologov S.G.


The review describes the modern data concerning the cellular and molecular mechanisms of mineral metabolism disturbance in bone tissue. Cytokine system consisting of receptor activator of nuclear factor к-b, its ligand and osteoprotegerin (RANKL/RANK/OPG) has a key role in understanding of bone remodeling regulation. We discuss a significance of the cytokine system in osteoblasto- and osteoclastogenesis. Molecular processes of histogenesis and bone resorption have been analyzed.
Genes & Cells. 2014;9(4):50-52
pages 50-52 views

Structural dynamics of adhesive bone marrow cells in cultivation: first passage (part 2)

Omelianenko N.P., Ilyina V.K., Kovalev A.V., Rodionov S.A.


Исследована морфология и структурная динамика адгезивных клеток костного мозга человека после первого рассева первичной культуры (культивированных в виде ад-гезированного монослоя на плотном субстрате стеклянного или пластикового дна культурального флакона). Выявлены визуализируемые структурные изменения, которые приобретают клетки в искусственных условиях in vitro, прослежена их динамика при переходе от первичной культуры к клеточной линии. Охарактеризованы адгезивные клетки на разных этапах культивирования: 1) во взвеси перед рассевом, после их открепления от подложки и округления; 2) при прикреплении к дну флакона и распластывании; 3) при образовании монослоя. Наиболее выраженными структурными изменениями являются выпячивания или складчатость клеточной мембраны, образующиеся у культивируемых клеток после открепления от дна культуральных флаконов и помещения в питательную среду во взвешенном состоянии. Наблюдалось значительное обводнение клеточного матрикса. В больших выпячиваниях мембраны клеточный матрикс отсутствовал. Динамика структурных изменений культивируемых клеток оценивалась методом светооптической микроскопии с помощью дифференциального интерференционного контраста по Номарскому и документировалась автоматической цифровой цейтра-ферной съемкой в условиях культурального инкубатора, интегрированного с инвертированным микроскопом. Для визуализации клеточных структур вне пределов разрешающей способности светового микроскопа использовалась трансмиссионная электронная микроскопия. Выявленное многообразие структурных форм адгезивных клеток на дне культурального флакона может быть связано с присутствием в составе костного мозга разных соединительнотканных дифферонов.
Genes & Cells. 2014;9(4):56-62
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Non-human primate oesophagus decellularization

Gubareva E.A., Sjoqvist S., Sotnichenko A.S., Mei L.L., Torres N.F., Danilenko K.A., Orlov S.V., Chvalun S.N., Grigoriev T.E., Krasheninnikov S.N., Porhanov V.A., Polaykov I.S., Kuevda E.V., Gumenyuk I.S., Macchiarini P.


Transplantation is an effective treatment option for patients suffering from different end-stage diseases; however it is associated with a constant shortage of donor organs and lifelong immunosuppressive therapy. Obtainment of tissuengineered scaffolds using decellularization with the following recellularization may become an alternative treatment option due to restoration, replacement and regeneration of damaged cells, tissues and organs. The main objective of this research is obtainment of decellularized esophagus matrices in non-human primate models followed by pathomorphological evaluation. A modified detergent-enzymatic method involving sodium deoxycholate and DNAse was used for esophagus decellularization. The conducted morphological investigation proved preservation of tissue architectonics as well as absence of cells and nuclear material. Evaluation of biomechanical properties of the scaffold revealed similar mechanical strength characteristics in native and decellularized samples; however, rupturing deformation was higher. The obtained results allow continuing research on the possibility of using decellularized esophagus matrix in non-human primate models for recellularization with the following differentiation, revascularization and reinnervation for hetero- and orthotopic transplantations.
Genes & Cells. 2014;9(4):64-69
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Hematopoietic stem cells with induced apoptosis effectively inhibit glioma cell growth in vitro, but started new mechanism of tumor stem cells

Bryukhovetskiy I.S., Mischenko P.V., Tolok E.V., Khotimchenko Y.S., Zaitsev S.V., Bryukhovetskiy A.S.


The presence of hematopoietic stem cells in culture inhibits proliferation of tumor cells. We hypothesized that pretreatment of stem cell apoptosis inducers increase their anti-tumor efficacy. The aim of the study was to explore the possibility of stem cells to induce apoptosis effectively inhibit the growth and induce the death of tumor cells in vitro. Use line C6 glioma cultures and hematopoietic (CD34+/CD45 + ) stem cells. We used dexamethasone as the apoptosis inducer. It's known that hematopoietic stem cells with apoptosis induced more effectively inhibit growth and induce a death of C6 glioma cells than native stem cells. In the process of intercellular interaction we observed phenomenon of merging stem and tumor cells, which apparently is one of the mechanisms of new tumor stem cells formation.
Genes & Cells. 2014;9(4):70-75
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Effects of Dalarginum on proliferation of multipotent mesenchymal stromal cells, dermal fibroblasts, and human osteosarcoma cells in vitro

Vasilyev A.V., Bukharova T.B., Volkov A.V., Vikhrova E.B., Bolshakova G.B., Goldstein D.V.


The aim of this work was to assess in vitro the effect of various doses of Dalarginum and its blocker Naloxone on the proliferation of multipotent mesenchymal stromal cells (MMSC), dermal fibroblasts (FD) and human osteosarcoma cell (line HOS). We used MMSC and FD derived from three donors. The cells were cultured for 3 days with solutions of the test substances at following concentrations: Dalarginum 10 jg/l, 100 jg/l, 1000jg/l; combination of naloxone 0.5 mg/l and Dalarginum, 100 jg/l; combination of Naloxone 3 mg/l and Dalarginum, 100jg/l. Saline was added in the culture medium in the control group. The cells were counted by the end of day 3 of culturing. Dalarginum at 100 jg/l significantly increased the number of MMSC by 19-34 % and decreased the count of cells (line HOS) by 22-34 % compared with the control values. There were no significant differences in cell numbers between the groups with addition of dalarginum at 10jg/l and 1000jg/l as well as significant changes in the number of FD under the influence of the test substances. Dalarginum combined with Naloxone, opioid receptor antagonist, had no impact on the number of cells, which confirmed its receptor-mediated action. The optimum effect of Dalarginum on opioid receptors was observed at100jg/l.
Genes & Cells. 2014;9(4):76-80
pages 76-80 views

Delivery of nerve growth factor (NGF) gene via recombinant plasmid vector induces angiogenesis in murine ischemic hind limb

Boldyreva M.A., Makarevich P.I., Rafieva L.M., Beloglazova I.B., Dergilev K.V., Kostrov S.V., Parfyonova Y.V.


The development of therapeutic angiogenesis that can stimulate the formation of mature vessels is a valuable prospect for treatment of ischemic disease, and the combination of well-known angiogenic factors with other growth factors is now beginning to show promise in therapy. In our efforts to identify possible targets for therapeutic intervention using combinations of growth factors, nerve growth factor (NGF) seems to be a possible candidate. In this study we analyzed the possibility to stimulate angiogenesis via local delivery of a plasmid encoding human nerve growth factor (hNGF). We used a murine hind-limb ischemia model to assess plasmid angiogenic potential in vivo. Plasmid DNA was diluted in saline and injected into ischemic m. tibialis anterior. Blood flow restoration was analyzed by laser Doppler imaging every 7 days after surgery, and throughout the experiment we assessed total hind-limb necrosis. After animals were sacrificed, muscle samples were frozen for histological analysis. Tissue sections were stained with antibodies against endothelium marker CD31 to assess vascular density. Blood perfusion by day 7 was higher in the NGF-treated group compared to control (p = 0.01), and by day 14 animals in the NGF-treated group had perfusion 2.8 fold higher than control animals (NGF 44.62±7.68; control 16.74±5.85; р = 0.005). Vascular density in tissue samples by day 14 in NGF-treated animals was about 2-fold higher than in the control group (р<0.05).
Genes & Cells. 2014;9(4):81-87
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Effect of cryopreserved nucleated cord blood cells on homeostasis state in animals of different age groups under stress-induced arterial hypertension

Babiychuk L.V., Babiychuk V.G., Sirotenko L.A., Malova N.G., Koval S.N.


Arterial hypertension (AH) might be entirely referred to the socially important pathologies by its prevalence, impact on quality of life and contribution to mortality rate. Nowadays there is accumulated a great number of data testifying to a positive therapeutic effect of cord blood-derived preparations on both different organs and systems, and the whole body as well. This research was aimed to study the dynamics of changes in the indices of heart rate variability (HRV) spectral analysis and lipid homeostasis in blood serum of animals from different age groups under neurogenic stress-induced AH prior to and after administering cryopreserved preparation of cord blood nucleated cells (CB NCs). Neurogenic stress-induced AH caused by emotional and painful stresses was established to be accompanied by a significant decrease in all the studied HRV parameters, as well as disorders in lipid composition of blood serum in rats irrespective of their age. There was observed a decreased activity of autonomic regulation of cardiac rhythm, as well as the tendency to reduction of humoral and metabolic effects. Administration of CB NCs cryopreserved preparation was shown to result in an increased tonus of sympathetic and parasympathetic divisions of autonomic nervous system, as well as humoral link of regulation, that testified to the presence in experimental animals of developed balanced autonomic regulation. In addition the application of CB NCs preparation leveled the state of lipid profile indices, observed at the background of stress-induced AH, and the preparation itself had an antiatherogenic effect and might significantly improve blood rheology.
Genes & Cells. 2014;9(4):88-94
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The effects of the male sex hormone (testosterone) on the functional activity of T-lymphocytes different degrees of differentiation

Shupletsova V.V., Khaziakhmatova O.G., Gutsol A.A., Sokhonevich N.A., Yurova K.A., Litvinova L.S.


The aim of the study was a comprehensive assessment of testosterone effects on the functional activity of T lymphocytes of different differentiation degrees (naive -CD45R + and primed -CD45RO+). Material and methods. CD45RA+ and CD45RO+ T cells obtained from a suspension of mononuclear cells from healthy donors (by immunomagnetic separation) were used as a study material (n = 48). The activation model which reflects the interaction of T lymphocytes of different differentiation degrees with antigen-presenting cells (CD2/CD3/CD28-complex activated T cells) was used to assess dose-dependent effects of testosterone on functional activity of T memory cells of different differentiation degrees. Viability assessment and identification of surface molecules CD25, CD71, CD95 on T cells of different differentiation degrees were performed by flow cytometry; the concentration of IL-2 in supernatant cell cultures was performed by enzyme immunoassay; assessment of the relative mRNA expression level of the telomerase catalytic subunit hTERT gene was performed by polymerase chain reaction. Statistical analysis was made using IBM SPSS Statistics 20 (Statistical Package for the Social Sciences). Results. The proapoptotic effect of testosterone on CD2/ CD3 / CD28-activated primed (CD45RO+) T cells has been established that may be due to nongenomic effects of the male sex hormone. Testosterone-induced changes of the system parameters IL-2/IL-2Ra induced by activated T-cells different degrees of differentiation is unidirectional, have different rates and depend on concentration of the hormone.. Suppressive effects of testosterone largely affect naive (CD45RA+) T cells. Dose-dependent effects of testosterone on the telomerase catalytic subunit (hTERT) gene expression in the background of antigen-independent activation are multidirectional and determined by the degree of T cells differentiation.
Genes & Cells. 2014;9(4):95-102
pages 95-102 views

Experimental and Morphological study of the xenogenic biological membranes

Ryabov A.Y., Fadeeva I.S., Deev R.V., Vezhnina N.O., Yurasova Y.B., Fesenko N.I., Guriev V.V., Sklyanchuk E.D., Lekishvili M.V., Akatov V.S.


In the last decade barrier the application of different types of membranes in the oral and maxillofacial surgery has been increasing. Their main reliability features include resorption times, the lack of toxic and antigenic effects, the absence of negative effects from the membrane on the structure of the surrounding soft tissues, as well as a high degree of biointegration and a potentiality of steady stabilization in the wound. Considering the abovementioned parameters, graft barrier membranes are a material of choice in the modern reconstructive oral and maxillofacial surgery. The present study includes an experimental and morphological assessment of biologic xenogenic barrier membranes for the evaluation of their immunological potency and the time of biodegradation using a model test system in vivo. Three groups of barrier membranes were assayed - M-D (decellu-larized diaphragm), M-P (pericardium), M-DM (dura mater), prepared from the corresponding tissues of a mature ox, in accordance to the method developed in the Priorov Central Research Institute of Traumatology and Orthopaedics. The experiments were carried out on a model of 6-week heterotopic subcutaneous implantation in rats and included a complex histological and immunohistochemical analysis, with quantitative estimation of biograft mineralization by adsorption spectroscopy. It was found that: the material of M-DM has the highest and fullest (while retaining the strength properties) resorption; a relatively long bi-odegradation time, expressed conductive effect and constraint function are specific for the М-Р material; M-D membranes have faster resorption, enhanced thinning and relative biological inertness. The results of the conducted study indicate that all the implanted materials are biocompatible and efficient, and thus may be used in various medical cases.
Genes & Cells. 2014;9(4):103-109
pages 103-109 views

Features of bone tissue regeneration after gunshot fractures of human long bones

Gololobov V.G.


The goal of the study was to analyse the features of tissue reactions during reparation of the bone wound following gunshot long bone fracture in human. Analysis of biopsy specimens, i.e. bone splinters, edges of bones fragments and surrounding them tissues taken from 9 male patients undergoing surgical treatment due to gunshot fracture of long bones has been carried out. Histological study using light and electron microscopy has been performed on the 1st, 2d, 3d, 4th, 5th, 14th, 23d and 34th days following the injury. It has been shown that newly developed blood vessels are capable for transportation of osteogenic cambial cells into the zone of the fracture thus optimizing osteogenesis. Regenerative endossal osteogenesis found out in this study proved to promote reconstructed osteons formation missing a stage of reticulofibrous bone tissue. It has been shown also that cells carrying osteogenic properties introduced into retained splinters during re-vascularization are responsible for the bone tissue construction. Such fragments served as additional and considerable source of bone regenerate formation. They are considered to be so-called «post-injury organ culture» in vivo. Difference between development of regeneration zone from distal and proximal edges of fractured bones was documented. Ultrastructural changes within osteoblasts, osteocytes and extracellular matrix reflected the intensity of bone tissue formation. Our results have been taken into consideration in routine clinical management and methods of treatment among such patients have been proposed.
Genes & Cells. 2014;9(4):110-115
pages 110-115 views

Gastrointestinal stromal tumor cell lines - the mechanisms of chemosensitivity in vitro

Boichuk S.V., Ramazanov B.R., Galembikova A.R., Mustafin I.G., Duensing A.


This research was aimed to study the sensitivity of gastrointestinal stromal tumors (GISTs) to chemotherapeutic drugs. We studied the chemosensitivity of GISTs by using several GIST cell lines. Cytotoxicity of chemotherapeutic drugs was assessed by colorometric MTS-assay. The expression level of DNA damage markers and repair proteins was assessed by immunoblotting. Apoptosis analysis was conducted by measuring the amount of hypodiploid cells by using a propidium iodide DNA staining procedure and flow cytometry analysis. We found that GISTs are sensitive to certain chemotherapeutic drugs - topoisomerase II inhibitors (etoposide and doxorubicine). DNA damage induction (in particular, DNA double-strand breaks formation) in GISTs induced cell apoptotic cell death despite the activation of DNA repair pathways. Of note, this effect was observed in imatinib-sensitive and resistant GIST cell lines, as well. We also found an increase accumulation phosphorylated form of histone 2A in GISTs after imatinib treatment. This effect was associated with the significant reduction of Rad51 recombinase expression, known as a key factor of homologous recombination. Taken together, this might explain the possible mechanisms of cell death in GISTs after targeted therapy. We found the chemosensitivity of GIST cell lines to some certain chemotherapeutic agents. Thus, the current point of view indicating about GISTs resistance to chemotherapy is needed to be re-evaluated.
Genes & Cells. 2014;9(4):116-120
pages 116-120 views

Characteristics of neoosteogenesis in the model of critical defect of rats' parietal bone using traditional and three-dimensional morphometry

Vasilyev A.V., Volkov A.V., Bolshakova G.B., Goldstein D.V.


We modified the techniques of 3D-morphometry using histological sections based on computer-generated ЗЮ reconstruction and mathematical modeling. We compared the results of measurements obtained with the use of above methods and those taken with traditional methods based on the analysis of X-ray pictures. It was shown that the methods of 3D-morphometry we developed provided more precise evaluation of the size of newly formed bone tissue. Basis for technical recommendations to perform the 3D-morphometry at every stage, from surgery to morphological analysis, were proposed.
Genes & Cells. 2014;9(4):121-127
pages 121-127 views

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