Том 11, № 1 (2017)

miRNAs are regulators of gene expression at the post-transcriptional level. The multifunctional properties of these molecules make them potential targets for controlling the behavior of tumor cells. Thousands of types of microRNAs have been identified. Their expression profiles significantly alter during the malignant transformation of cells. In this study, we investigated the effect of miR-106a functional inhibition on the growth, viability and death of melanoma cells. The miR-106a level of expression monitored with a microarray was enhanced in melanoma cells and melanocytic nevi. Its inhibition did not affect melanoma cell proliferation, viability, and migration, but increased tumor cell invasive activity and clonogenicity. miRNA alterations in melanoma and other malignant tumors and the functional role of miR-106a in melanoma, as well in other pathologies, are discussed.

Fluorescent proteins (FPs) are widely used as genetically encoded markers for quantitative and noninvasive study of biological processes. Development of biomarkers that are fluorescent in the near-infrared spectral range allows the tissues of animals to be studied at a deeper level because they are more permeable to the light of this wavelength range than that of visible range. Such properties as low molecular weight and monomeric state are important for widespread use of FPs. In this paper, we managed to obtain FP based on the chromophore-binding domain of bacterial phytochrome (BphP) from Rhodopseudomonas palustris (RpB-phP1), named GAF-FP, with a molecular weight of ~19 kDa, which is half that of other FP based on BphP and 1.4 times lower than that of commonly used GFP-like proteins, which are fluorescent in the near-infrared range. In contrast to most other near-infrared FPs, GAF-FP is a monomer, which has a high photostability, and its structure is stable to the incorporation of small peptide inserts. Moreover, GAF-FP is capable of covalent attachment of two different tetrapyrrole chromophores: phycocyanobilin (PCB) and biliverdin (BV), which is contained in mammalian tissues. GAF-FP with attached BV as a chromophore (GAF-FP–BV) has the main absorption band with a maximum at 635 nm. The fluorescence maximum falls at 670 nm, whereby GAF-FP has a high ratio of the fluorescence signal to the background signal even if FP is localized at a depth of several mm below the tissue surface. Together with the near-infrared absorption band, GAF-FP–BV also has an absorption band in the violet region of the spectrum with a maximum at 378 nm. We used this property to design a chimeric protein consisting of modified luciferase from Renilla reniformis (RLuc8) and GAF-FP. We showed resonance energy transfer from the substrate, the excited state of which occurs when oxidized by luciferase, to the chromophore GAF-FP–BV in the designed fusion protein. In the absence of an energy acceptor, RLuc8 catalyzes the cleavage of the substrate with the emission of the light with a maximum at 400 nm. At the same time, the energy from the substrate is transferred to the FP chromophore and then emitted in the near-infrared range corresponding to the spectrum of GAF-FP fluorescence in the GAF-FP–RLuc8 chimeric protein. These results open the way for the development of new small near-infrared FPs based on various natural BphPs with a view to their widespread use in cell and molecular biology.

The aim of this study was to monitor the genetic stability of endometrial mesenchymal stem cells (eMSCs) by G-banding and molecular karyotyping. We evaluated the sensitivity of each method to assess the genetic stability of eMSCs. G-banding karyotyping performed on passages 6 and 15 showed that more than 80% cells had normal karyotype. Random karyotypic changes were found in a small part of the cell population: aneuploidy, isochromosomes, chromosome breakages, interchromosomal association. Molecular karyotyping carried out on the 6th and 14th passages revealed genomic stability, except for in the case of chromosomes 7 and 14. Microduplications 7q36.3 (62 kb) and 14q11.2 (165kb) were found in these chromosomes. We interpreted these aberrations as being derived from the donor of these cells. The morphological and molecular karyotyping complemented each other. Using these methods, we can analyze karyotypic stability at different levels of the genomic organization.

The structural organization of mitochondria reflects their functional status and largely is an index of cell viability. The indirect parameter to assess the functional state of mitochondria in cells is the degree of their fragmentation, i.e., the ratio of long or branched mitochondrial structures to round mitochondria. Such evaluations requires an approach that allows to create an integral pattern of the three-dimensional organization of mitochondrial reticulum using confocal images of mitochondria stained with a fluorescent probe. In the present study, we tested three approaches to analyzing the structural architecture of mitochondria under normal conditions and fission induced by oxidative stress. We revealed that, while the most informative is a three-dimensional reconstruction based on series of confocal images taken along the Z-dimension, with some restrictions it is plausible to use more simple algorithms of analysis, including one that uses unitary twodimensional images. Further improvement of these methods of image analysis will allow more comprehensive study of mitochondrial architecture under normal conditions and different pathological states. It may also provide quantification of a number of mitochondrial parameters determining the morphofunctional state of mitochondria—primarily, their absolute and relative volumes—and give additional information on threedimensional organization of the mitochondrion.

We investigated the effect of mexicor on functional indices of erythrocytes and the structure of myocardial microcirculation in rats suffering from traumatic brain injury (TBI). At 3, 7, and 12 days after TBI, we measured the concentration of 2,3-diphosphoglycerate (2,3-DPG) and the degree of erythrocyte aggregation and their electrophoretic mobility (EPME) in the blood of rats, as well as analyzing sections of the left ventricular myocardium. The first day after the TBI, we observed a decrease in EPME, an increase of erythrocyte aggregation, and an increase of 2,3-DFG concentration in erythrocytes as compared with intact animals. Intraperitoneal injection of mexicor led to an increase of EPME and 2,3-DPG level and reduced the aggregation of erythrocytes, which was most pronounced during the 3–7 days of the post-traumatic period. Improved functional parameters of erythrocytes were accompanied by the dynamics of regenerative processes in the heart. Intraperitoneal injection of mexicor restrained architectonic damage of microvasculature and cardiomyocytes ultrastructure of the left ventricular myocardium of the heart.

The engineering of blood vessels that could ensure efficient transport of various nutrients and metabolites is a challenge in tissue engineering. The creation of cell-seeded bioconstructs using modified natural polymers, in particular, PEGylated fibrin is under investigation, which will help overcome this problem. Therefore, the purpose of this study was to determine the optimal ratio of the hydrogel components of modified fibrin to provide favorable conditions for the vascular development of endothelial and mesenchymal stem cell coculture. We have shown that PEGylated fibrin gels are capable of maintaining three-dimensional growth of HUVEC and hASC cells. Hydrogel with a filamentous microporous structure obtained from PEGylated 5: 1 fibrinogen and thrombin at a concentration of 0.2 U per 1 mg ensured optimal conditions for spreading, growth, and development of cocultured cells as well as the expression of proteins involved in angiogenesis.