Vol 9, No 4 (2014)

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.

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.

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.

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.

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).

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.

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.

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.