Том 12, № 4 (2018)

Two new nonimmortalized human cell lines FRSN-1 and FRSN-2 were established from foreskin of two similarly aged donors (2.5 years). Growth characteristics and differentiation potential of these cell lines studied on the sixth passage confirmed their status as mesenchymal stem cells (MSCs). A number of characteristics have been analyzed during long-term cultivation up to the 26th passage. The dynamics of the process of replicative senescence defined by the activity of β-galactosidase differed between these lines. However, at the 26th passage, the process of replicative senescence was equally enhanced in both lines. The plating efficiency markedly differed between the lines on the sixth passage. In FRSN-1, it was higher than in FRSN-2. The plating efficiency substantially dropped to the 26th passage in FRSN-1 and was lost in FRSN-2 line. Growth curves showed active proliferation of these lines at the 6th passage. The average doubling time did not differ between the lines and was 36.9 and 39.0 h, respectively. Analysis of growth curves on the 26th passage revealed a decline in proliferative activity and increase in average doubling time of cell populations in both lines, more in FRSN-2 than in FRSN-1 lines. The patterns of growth curves differed in these lines. Morphological analysis revealed increased cell size and spreading typical for the phase of the replicative senescence. Numerical and structural karyotypic analysis at the sixth passage showed that both lines have normal karyotype 46, XY. We did not discover interline differences in the frequency of chromosomal aberrations. To determine the status of these cell lines, comparative analysis of the surface markers was performed using flow cytometry. It was revealed that cells of both lines expressed surface antigens characteristic of human MSCs: CD44, CD73, CD90, CD105, and HLA-ABC and did not express CD34, CD45 and HLADR. Cells of both lines displayed SSEA-4 and SOX2, markers of human embryonic stem cells (ESCs). Expression of SSEA-4 was also detected at the 26th passage in both lines. FRSN-1 and FRSN-2 cells expressed the markers of early ESC differentiation into three germ layers. The ability of these cell lines to differentiate into osteogenic, chondrogenic, and adipogenic lineages was shown on the sixth passage. Both lines exhibited substantially reduced adipogenic potential on the 20th passage. These data indicate that in contrast to growth characteristics the adipogenic differentiation potential changes even with an average degree of replicative senescence. It appears that the cell replicative senescence contributed to the change in MSC differentiation potential. Overall, the results demonstrate that cell lines derived from different donors are distinguished in growth characteristics and pattern of replicative senescence. The disparity is due to a direct genetic influence and indirectly by different microenvironment in their donor organisms before cell isolation.

The relevance of our study is due to the unresolved problem of atherosclerosis, a disease that causes a greatest many disabilities and deaths. A definite value in its initiation, progression, and destabilization is assigned to endothelial cells, which are prone to pathological effects of various factors. In patients with atherosclerosis, it is impossible to obtain endothelial cells in vivo and in situ and, accordingly, to characterize their cytological features. Endothelial biopsy in this work was performed by coronary angioplasty in 64 patients with various clinical forms of coronary heart disease. A balloon catheter was used as a probe for biopsy. Preparations of endothelial biopsy were prepared using the principles of liquid-based cytology. Anucleated, polygonal cells with nuclei and their clusters, as well as apoptotic bodies with the immunophenotype CD31⁺, CD34⁺, CD105^+/–, PanCk^+/–, CD45^–, and CD68^–, have been obtained. It is confirmed that they belong to the endothelium, which shows that further cytological studies can be carried out with the purpose of evaluating the etiology and pathogenesis of atherosclerotic processes.

MiR-204-5p is an oncosuppressive microRNA the level of which diminishes in various cancer types. The aim of this study was to evaluate miR-204-5p inhibition of melanoma-cell viability, as well as to determine whether miR-204-5p transfection of melanoma B16 cells with miR-204-5p mimic/inhibitor affected the microRNA expression. Proliferation of transfected cells was slightly, probably nonspecifically, reduced after 96 h. LNA™ inhibitors produced toxic effects in vivo. miR-204-5p-mimic/inhibitor application resulted to a slight, most likely nonspecific, modulation of melanoma-cell proliferation after 96 h. Bioinformatic analysis revealed that miR-204-5p is involved in the estrogen signaling pathway, lysine degradation, signaling pathways regulating pluripotency of stem cells, melanogenesis, transcriptional deregulation in cancer. Injection of miR-204-5p LNA™ inhibitor into mice reduced the level pf miR-204-5p but was not toxic.

A mathematical model of Na⁺/Cl⁻ selectivity in tight junctions (TJs) between epithelial cells was developed. It was demonstrated that Na⁺/Cl⁻ selectivity in TJs depends on the total charge of amino-acid residues of claudin macromolecules within TJs, as well as on the ion-distribution coefficients between TJs and free solution. It was demonstrated that the obtained formulas predict a change of Na⁺/Cl⁻ selectivity in TJs for Cl⁻/Na⁺ selectivity if the sign of the total charge of amino-acid residues in TJ changes. The calculated Na⁺/Cl⁻ selectivity value for MDCK cells coincides with the experimental data of (Colegio et al., 2002). To calculate a change in ion-solvation energy during their transition to TJs, formulas for nonlocal electrostatics with one-, two-, and three-pole models of dielectric function without taking into account the overscreening effect were used.

The vertebrate CRF signaling system consists of corticotropin-releasing factor (CRF), two types of receptors to CRF (CRF-R1 and CRF-R2), and CRF-binding protein (CRF-BP). The aim of this study was to investigate the presence and localization of CRF, CRF-R2, and CRF-BP in the snail atrial neuroendocrine complexes, which include granular cells (GCs) and tightly connected nerve fibers and gliointerstitial cells. Immunofluorescence assay and immunogold electron microscopy using polyclonal antibodies against these proteins revealed immunoreactivity in the granules of all these cells. Western-blot analysis of the snailatria lysate using rabbit anti-CRF-R2 polyclonal antibodies revealed a specific band with a weight of 56 kids. These are the first data on the molecular weight of this receptor in mollusks. Furthermore, to clarify the possible functions of CRF in the neuroendocrine complexes, the hormone was added to GCs isolated from the snail atrium. The proportion of degranulated GCs after CRF addition almost doubled (45.5 vs. 24.5% in control, p < 0.05). The results indicate the presence of all three components of the CRF signaling system in the neuroendocrine complexes of snail atrium. In addition, their presence in both secretory and nervous components of the complexes suggests that the CRF signaling system participates in the nervous regulation of secretory activity of GCs and transfer of information from GCs to the CNS.