Vol 13, No 3 (2019)

Sheep are often used as preclinical large animal models for testing the therapeutic potential of multipotent mesenchymal stem cells (MMSCs). A cell culture with characteristics typical for MMSCs was obtained due to optimization of the method of cell isolation from sheep bone marrow (BM) in a Ficoll density gradient. The advantage of using SepMate-15 tube compared to the isolation by the classical method has been demonstrated. The obtained cells demonstrated a strong adhesion to the culture plastic, fibroblast-like morphology, and, being induced in vitro, the ability to differentiate into cells of adipose, bone, and cartilage tissues. Adipogenic differentiation was confirmed on day 14, when adipocytes with lipid vesicles positive for Oil Red O specific staining had been formed. On day 14, specific alkaline phosphatase activity appeared in cells in osteogenic medium. Von Kossa staining revealed the presence of insoluble calcium salts in the intercellular space. Chondrogenic differentiation appeared on day 14 and was accompanied with the formation of multilayer structures with an abandon matrix, in which the isogenic groups that resemble hyaline cartilage lacunae were visualized. On day 21, the cells formed tight microgranules with glycosaminoglycan matrixes. Cells were cryopreserved in three different media at the zero passage. A comparative analysis of cell viability was then performed, which revealed a relatively good cell state (more than 70% of live cells). The MMSCs culture obtained from sheep BM was deposited at two to three passages in the specialized Collection of Somatic Cell Cultures of Agricultural and Industrial Animals at the All-Russian Research Institute of Experimental Veterinary Medicine of Russian Academy of Science and could be used in preclinical studies.

Normal human CD4⁺ T-lymphocytes can undergo malignant transformation during prolong cultivation in conditions of high endonuclease G (EndoG) expression or after DNA damage. The aim of this work was to study biochemical and cytogenetic features of transformed ex vivo human malignant CD4⁺ T- lymphocytes, as well as biochemical and morphological characteristics of tumors that develop in athymic mice after transplantation of these cells. The telomerase activity was higher and telomere length was shorter in tumor cells than in control cells. Transformed malignant cells exhibited a high level of chromosomal aberrations. Expression of genes regulating the cell cycle changed in transformed malignant CD4⁺ T-lymphocytes and tumor cells. The tumors that developed were classified as multicomponent Т-cell lymphomas and panniculitis-like T-cell lymphomas. Thus, transformed CD4⁺ T-lymphocytes can generate malignant tumors of various histogenesis.

T-lymphocytes, their subpopulations, and natural killer cells (NCs) were characterized according to the distribution of membrane molecules involved in intercellular interactions and signaling (CD45, CD3, CD4, CD8, CD16, CD56) in patients with chronic lymphocytic leukemia at the stage of disease diagnosis. Current methodological approaches to determine the studied parameters were used, taking into account the characteristics of the lymphoid pool of blood in patients with chronic lymphocytic leukemia. It has been established that the population of T-lymphocytes of the patient peripheral blood is characterized by decreased content of CD45 and CD3 surface receptors. The average level of the membrane CD4 receptor was reduced in lymphocytes belonging to the T-helper subpopulation. The CD8 receptor remained unchanged in subpopulation of cytotoxic T-lymphocytes. CD8 and CD16 molecules were distributed on the surface of NC cells of patients with chronic lymphocytic leukemia at a normal level, while the expression of CD56 was significantly less. The data facilitate understanding of the energy of immune system cells and can be used to predict the course of chronic lymphocytic leukemia.

The fertility of female mammals depends on the quality of oocytes, which decreases in the process of biological aging. We found an inhibitory effect of two related pituitary hormones (prolactin (PRL) and growth hormone (GH)) on destructive modifications of metaphase chromosomes in the eggs of domestic cow (Bos taurus taurus), aging in vitro. The involvement of different NO synthase (NOS) isoforms in realization of the effect of PRL and GH on age-related changes of MII chromosomes with prolonged cultivation of in vitro matured cow oocytes was studied in the present work. In the absence of hormones NPLA, neuronal NOS (nNOS) inhibitor had no effect on the frequency of chromosomal abnormalities in aging oocytes. At the same time, 1400 W (inducible NOS (iNOS) inhibitor) and L-NAME (efficient endothelial NOS (eNOS and nNOS ) inhibitor) decreased this frequency, while the blocking of all three NO synthase isoforms led to the opposite effect. The inhibitory effect of PRL on destructive chromosome modifications in aging eggs increased in the presence of L-NAME, but was not expressed with the inhibition of nNOS, iNOS, or all NO synthase isoforms simultaneously. Neither NPLA nor L-NAME modulated the inhibitory effect of GH on abnormal chromosome changes in oocytes. On the contrary, the blocking of iNOS, as well as all three NO synthase isoforms, led to an increase in the portion of oocytes with destructive changes of MII chromosomes. At the same time, the level of total activity of NO synthase in oocytes did not depend on the presence of the studied hormones or NOS inhibitors during the prolonged cultivation of oocyte–cumulus complexes. Data obtained suggest that the inhibitory effect of PRL and GH on abnormal changes of metaphase chromosomes in aging cow eggs is associated with a decrease in the activity of endothelial NO synthase, as well as (in the case of PRL) with an increase in the activity of neuronal NO synthase in the cumulus cells surrounding the oocytes.

The rigid skeleton formed by networks of A- and B-type lamins is responsible for maintaining the nucleus shape. Moreover, the change in the ratio of lamin proteins in its composition, apparently, is a key factor determining mechanical properties of the cell nucleus, in particular plasticity. In this paper, the effect of the component composition of the nuclear lamina on the resistance of cells to mechanical stress and on the cell motility was considered. Expression of mutant forms of lamin A is accompanied by changes in the distance between microdomains of lamins within the nuclear membrane, and its increased bubbling relative to wild-type cells and cells overexpressing lamin A is observed. An increased number of deformed nuclei are noted when exposed to osmotic shock. The assessment of the effect of a change in the molecular composition of the nuclear lamina due to an increase (decrease) in expression of lamin A, as well as the introduction of progerin in an experimental wound model, showed that there are no differences under conditions of unlimited space.