Цель: оценить уровень экспрессии генов (TNF, IL6, IL8, IL18, VEGF121, VEGF165 и VEGF189), ассоциированных с опухолеобразованием, в плазме крови больных РМЖ и контрольной группе женщин. Материал и методы. Для определения уровня представленности мРНК исследуемых генов в плазме крови использовали методы обратной транскрипции и количественной полимеразной цепной реакции (наборы реагентов производства ДНКТехнология , Россия). Реакцию амплификации проводили в детектирующем амплификаторе DTprime 4 ( ДНКТехнология , Россия). Результаты. В плазме крови больных РМЖ и женщин контрольной группы относительные количества мРНК генов TNF, IL8, IL18 и VEGF189 были достоверно повышены у больных РМЖ в сравнении со здоровым контролем (p0,01). При сравнении группы контроля с группой больных РМЖ на различных стадиях высокий уровень экспрессии мРНК генов IL8 и IL18 наблюдается преимущественно в группе больных РМЖ с 0 стадией заболевания. Относительное количество мРНК гена IL8 в плазме больных РМЖ всех стадий (0, I и II) существенно превышало относительное количество мРНК этого цитокина в плазме крови женщин здоровой группы (p0,01), при этом различия между группой контроля и группой больных РМЖ III стадии были выявлены при p0,05. Достоверные различия в уровне экспрессии мРНК IL18 были выявлены между группой контроля и группами больных РМЖ 0, I и II стадиями (p0,01), при этом различия в уровне экспрессии мРНК гена IL18 между группой контроля и группой больных РМЖ III стадии заболевания оказались не значимыми (p0,05). Достоверные различия в уровне экспрессии мРНК TNF обнаружены между группой контроля и группой больных РМЖ I стадии заболевания (p0,01) и между группой контроля и группами больных РМЖ с II и III стадиями заболевания (p0,05). Статистически значимые различия в уровне мРНК гена VEGF189 наблюдаются в группах больных РМЖ на 0 и I стадиях по сравнению с группой контроля, где уровень мРНК VEGF189 значительно повышен в этих группах (p0,01). Также достоверные различия наблюдаются между группой контроля и группами больных РМЖ II и III стадий заболевания (p0,05). Заключение. Полученные результаты об уровне представленности мРНК исследованных генов свидетельствуют о том, что мРНК цитокинов (TNF, IL8, IL18) в плазме крови имеют статистически значимый повышенный уровень экспрессии при РМЖ по сравнению с группой контроля. Для доказательства того, что мРНК этих генов возможно использовать в качестве маркеров ранней диагностики рака молочной железы, следует увеличить выборку группы больных РМЖ с 0 стадией заболевания. Для получения статистически достоверных выводов о повышении экспрессии мРНК гена VEGF189 в плазме крови больных РМЖ также необходимы дополнительные исследования с увеличением выборки группы больных РМЖ с 0 стадией заболевания. Purpose: to evaluate the level of gene expression (TNF, IL6, IL8, IL18, VEGF121, VEGF165 and VEGF189) associated with tumor formation in the blood plasma of breast cancer patients and the control group of women. Material and methods. To determine the level of mRNA representation of the studied genes in blood plasma, the methods of reverse transcription and quantitative polymerase chain reaction were used (reagent kits manufactured by DNATechnology, Russia). The amplification reaction was carried out in a DTprime 4 detection amplifier (DNATechnology, Russia). Results. In the blood plasma of breast cancer patients and women in the control group, the relative amounts of mRNA of the TNF, IL8, IL18, and VEGF189 genes were significantly increased in patients with breast cancer compared with the healthy control (p 0.01). When comparing the control group with a group of breast cancer patients at various stages, a high level of IL8 and IL18 mRNA expression is observed mainly in the group of breast cancer patients with stage 0 disease. The relative amount of mRNA of IL8 gene in the plasma of patients with breast cancer of all stages (0, I and II) significantly exceeded the relative amount of mRNA of this cytokine in the blood plasma of women in the healthy group (p 0.01), while the differences between the control group and the group of patients with breast cancer III stages were identified at p 0.05. Significant differences in the level of IL18 mRNA expression were detected between the control group and breast cancer groups of stages 0, I and II (p 0.01), while differences in the level of IL18 mRNA expression between the control group and stage III breast cancer patients the diseases were not significant (p 0.05). Significant differences in the level of TNF mRNA expression were found between the control group and the group of patients with breast cancer stage I disease (p 0.01) and between the control group and the groups of patients with breast cancer with stage II and III disease (p 0.05). Statistically significant differences in the mRNA level of the VEGF189 gene are observed in groups of breast cancer at stages 0 and I compared with the control group, where the level of VEGF189 mRNA is significantly increased in these groups (p 0.01). Significant differences are also observed between the control group and the groups of patients with breast cancer of the II and III stages of the disease (p 0.05). Conclusion The obtained results on the level of mRNA representation of the studied genes indicate that mRNA of cytokines (TNF, IL8, IL18) in blood plasma have a statistically significant increased level of expression in breast cancer compared with the control group. To prove that the mRNA of these genes can be used as markers for early diagnosis of breast cancer, the sample of the group of breast cancer patients with stage 0 disease should be increased. To obtain statistically valid conclusions about the increased expression of the VEGF189 mRNA gene in the blood plasma of breast cancer patients, additional studies with an increase in the sample of the group of breast cancer patients with stage 0 disease are also required.

Purpose: to evaluate the level of gene expression (TNF, IL6, IL8, IL18, VEGF121, VEGF165 and VEGF189) associated with tumor formation in the blood plasma of breast cancer patients and the control group of women. Material and methods. To determine the level of messenger ribonucleic acid (mRNA) representation of the studied genes in blood plasma, the methods of reverse transcription and quantitative polymerase chain reaction were used (reagent kits manufactured by DNA-Technology, Russia). The amplification reaction was carried out in a DTprime 4 detection amplifier (DNA-Technology, Russia). Results. In the blood plasma of breast cancer patients and women in the control group, the relative amounts of mRNA of the TNF, IL8, IL18, and VEGF189 genes were significantly increased in patients with breast cancer compared with the healthy control (p 0.01). When comparing the control group with a group of breast cancer patients at various stages, a high level of IL8 and IL18 mRNA expression is observed mainly in the group of breast cancer patients with stage 0 disease. The relative amount of mRNA of IL8 gene in the plasma of patients with breast cancer of all stages (0, I and II) significantly exceeded the relative amount of mRNA of this cytokine in the blood plasma of women in the healthy group (p 0.01), while the differences between the control group and the group of patients with breast cancer III stages were identified at p 0.05. Significant differences in the level of IL18 mRNA expression were detected between the control group and breast cancer groups of stages 0, I and II (p 0.01), while differences in the level of IL18 mRNA expression between the control group and stage III breast cancer patients the diseases were not significant (p 0.05). Significant differences in the level of TNF mRNA expression were found between the control group and the group of patients with breast cancer stage I disease (p 0.01) and between the control group and the groups of patients with breast cancer with stage II and III disease (p 0.05). Statistically significant differences in the mRNA level of the VEGF189 gene are observed in groups of breast cancer at stages 0 and I compared with the control group, where the level of VEGF189 mRNA is significantly increased in these groups (p 0.01). Significant differences are also observed between the control group and the groups of patients with breast cancer of the II and III stages of the disease (p 0.05). Conclusion The obtained results on the level of mRNA representation of the studied genes indicate that mRNA of cytokines (TNF, IL8, IL18) in blood plasma have a statistically significant increased level of expression in breast cancer compared with the control group. To prove that the mRNA of these genes can be used as markers for early diagnosis of breast cancer, the sample of the group of breast cancer patients with stage 0 disease should be increased. To obtain statistically valid conclusions about the increased expression of the VEGF189 mRNA gene in the blood plasma of breast cancer patients, additional studies with an increase in the sample of the group of breast cancer patients with stage 0 disease are also required.

In recent decades, nucleic acid sequencing technologies used for metagenomic analysis have become the main methods for assessing the composition of microbiota. At the same time, the use of novel methods of cultivation and identification of microorganisms in microbiological research led to the renaissance of culture-based technologies, because facilitated the discovery and isolation of both new strains of well-known microorganisms as well as uncultivated and unexplored bacterial taxa. The aim of this study was to evaluate the potential of using the culture-based method for the assessment of the qualitative and quantitative composition of the intestinal microbiota in healthy children. Eleven growth media were inoculated with serial dilutions of stool samples in order to analyze the profile of dominant anaerobic bacteria, as well as aerobic bacteria and fungi in 20 healthy children aged 2–4 years. The identification of microorganisms was performed using MALDI TOF MS and 16S rRNA gene fragment sequencing were used. 1,819 isolated and identified strains belong to 7 phyla, 13 classes, 18 orders, 33 families, 77 genera and 149 species in the Bacteria domain. The Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria phyla were most abundant and frequent. The greatest species diversity (more than 85 species) was found in the Firmicutes phylum. Ten new previously uncharacterized bacterial strains were isolated.

We isolated and characterized cell cultures from eutopic endometrium and endometriotic lesions of women with malformations of the internal reproductive organs. The cells had fibroblast-like shape and intensively expressed CD90, CD73, CD105, CD44, CD146, and CD117 and were capable of induced adipogenic and osteogenic differentiation in vitro . The obtained cultures exhibited properties of multipotent mesenchymal stromal cells; at the same time, they demonstrated in vitro immunophenotypic differences from cell cultures of eutopic and ectopic endometrium of women without developmental abnormalities, which suggests their functional difference. The cells from eutopic endometrium and from ectopic endometriotic lesions can be used as the model for studying of the etiology and pathogenesis of endometriosis and for testing new drugs for this specific group of patients. Markers CD90 and CD117 were identified as promising molecules for the development of minimally invasive diagnostics of endometriosis based on cell cultures from eutopic endometrium.

Cell cultures isolated from endometriosis lesions by enzymatic dissociation consisted of fibroblast-like cells expressing CD90, CD73, and CD105; cell viability in these cultures was >90%, but this parameter decreased by passage 3. Zero passage cultures contained 10-25% epithelial cells expressing cytokeratin-7, but by passage 2, the cultures became more homogeneous and epithelial cells disappeared. The proportion of proliferating cells and population doubling level increased from passage 1 to passage 3. The cultures from the endometrium were induced to adipogenic and osteogenic differentiation in vitro. The cultures derived from ectopic endometrium have properties of multipotent mesenchymal stromal cells that exhibited in vitro similarities and differences from cell cultures from eutopic endometrium, which allows using this cell model for the search and testing of new drugs and technologies aimed at suppression of the growth and spread of endometriosis lesions.

Proliferative activity of mesenchymal stromal cells isolated from five sources (chorionic villi, Wharton’s jelly, amnion, endometrium, and adipose tissue) was compared by flow cytometry and real-time PCR (by the content of mRNA of genes encoding of cell cycle regulators). Mesenchymal stromal cells derived from the endometrium demonstrated maximum stability and high proliferative potential.

We studied the interaction of neural stem cells and dental pulp-derived mesenchymal stem cells with lymphocytes from autologous and heterologous donors. Flow cytometry analysis with the use of CFSE-labeled lymphocytes demonstrated an increase in the content of proliferating CD8, CD16 and CD56 cells, but not CD4 cells in cultures of HLA-DR-negative mesenchymal stromal cells from the dental pulp co-cultured with lymphocytes. In neural cultures expressing HLA-DR, all subpopulations of T cells and NK cells were activated. No differences between the autologous and heterologous cultures were revealed.

mRNA of genes ( tgfβ , il6 , il10 , il1β , and vegf165 ) involved in cell proliferation, inflammatory, anti-inflammatory, and angiogenic processes were detected and quantified in cultures of mesenchymal stromal cells of different passages derived from human extraembryonic tissues (amniotic sac, umbilical cord, chorionic villi and trophoblast of the placenta). The concentrations of IL-10, IL-6, IL-1β, and TGFβ were measured.

The expression of mRNA of 36 genes involved in implantation was studied by reverse transcription and real-time PCR. Signifi cant differences in mRNA expression during the early and middle stages of the secretion phase were detected for genes mmp7 , vegf , il2m , il1β , il8 , il18 , tnfα , il10 , tgfβ , igfbp2 , etc .

Cell cultures isolated by the enzymatic method from the terminal placenta amnion consist mainly from epithelial cells, expressing cytokeratin-7, CD90, and CD73, are characterized by high viability and low proliferative potential. Adhesive cultures of umbilical (Wharton's jelly) cells, despite the fibroblast-like shape of the cells and expression of surface markers, intrinsic to mesenchymal stromal cells, are also characterized by high heterogeneity during the initial stages of culturing, judging by an appreciable share of cytokeratin-expressing cells. The terminal placenta chorionic villi can be a source of cells with the most typical morphology and immunophenotypical profile of the resident multipotent mesenchymal stromal cells, which retain high viability in vitro and have a high proliferative potential.

The expression of mRNA of cytokines and immunoregulatory molecules characterizing the interaction of mesenchymal stromal cells from chorionic villi of postpartum placenta and allogenic mononuclear blood cells was studied during 3-day co-culturing of these cells. The expression of foxp3 , il2ra , and il10 mRNA in floating mononuclear cells increased from day 1 to 3 in co-culture, which can refl ect the process of induction of regulatory T cells in the lymphocyte population under the action of mesenchymal stromal cells.

We propose a method of quantitative functional activity assessment in cells isolated via sorting on a flow cytometer. We show that cell populations vary in the mRNA expression of cytokine genes immediately after isolation and sorting, while the maintenance of homogenous populations in culture without stimulation results in an increase in these gene mRNA expression. Using the original system, it is now possible to detect mRNA cytokine genes with high sensitivity, starting from 90 cells per specimen. This approach permits genetic and immunogenetic analysis of gene expression with the goal of determining their functions in the in vitro studies.

Предложен способ количественной оценки функциональной активности клеток, выделенных методом сортировки на проточном цитометре. Показано, что сразу после выделения и сортировки популяции клеток различались по уровню экспрессии мРНК генов цитокинов, а при культивировании гомогенных популяций без стимуляции наблюдалось увеличение экспрессии мРНК исследованных генов. С помощью разработанной системы можно детектировать количество мРНК генов цитокинов с высокой чувствительностью, от 90 клеток в образце, что позволяет проводить генетический и иммуногенетический анализ экспрессии генов для определения их функции в экспериментах in vitro.

We describe a method of isolation of human mesenchymal stromal cells from the umbilical cord (Wharton’s jelly) and human placenta: amnion, placental villi, and trophoblast. Morphology, immunophenotypic characteristics, and differentiation potencies of isolated cells were studied. The capacity of mesenchymal stromal cells from extraembryonic tissues to osteogenic, adipogenic, and chondrogenic differentiation was demonstrated and the dynamics of this process was described. The isolated cells met the criteria for multipotent mesenchymal stem cells.

Kunitz-type proteinase inhibitor proteins of group A (KPI-A) are involved in the protection of potato plants from pathogens and pests. Although sequences of large number of the KPI-A genes from different species of cultivated potato (Solanum tuberosum subsp. tuberosum) and a few genes from tomato (Solanum lycopersicum) are known to date, information about the allelic diversity of these genes in other species of the genus Solanum is lacking. In our work, the consensus sequences of the KPI-A genes were established in two species of subgenus Potatoe sect. Petota (Solanum tuberosum subsp. andigenum--5 genes and Solanum stoloniferum--2 genes) and in the subgenus Solanum (Solanum nigrum--5 genes) by amplification, cloning, sequencing and subsequent analysis. The determined sequences of KPI-A genes were 97-100% identical to known sequences of the cultivated potato of sect. Petota (cultivated potato Solanum tuberosum subsp. tuberosum) and sect. Etuberosum (S. palustre). The interspecific variability of these genes did not exceed the intraspecific variability for all studied species except Solanum lycopersicum. The distribution of highly variable and conserved sequences in the mature protein-encoding regions was uniform for all investigated KPI-A genes. However, our attempts to amplify the homologous genes using the same primers and the genomes of Solanum dulcamarum, Solanum lycopersicum and Mandragora officinarum resulted in no product formation. Phylogenetic analysis of KPI-A diversity showed that the sequences of the S. lycopersicum form independent cluster, whereas KPI-A of S. nigrum and species of sect. Etuberosum and sect. Petota are closely related and do not form species-specific subclasters. Although Solanum nigrum is resistant to all known races of economically one of the most important diseases of solanaceous plants oomycete Phytophthora infestans aminoacid sequences encoding by KPI-A genes from its genome have nearly or absolutely no differences to the same from genomes of cultivated potatoes involved by P. infestans.

Group A Kunitz-type protease inhibitors (KPI-A) are involved in protecting potato plants from microorganisms and pests. While the nucleotide sequence is known for many KPI-A genes of various potato cultivars ( Solanum tuberosum subsp. tuberosum ) and a few genes of tomato ( Solanum lycopersicum ), there are no data on their allelic diversity in other species of the genus Solanum. KPI-A fragments were cloned, amplified, sequenced, and analyzed from plants of the subgenera Potatoe sect. Petota (five genes from S. tuberosum ssp. andigenum and two genes from S. stoloniferum ) and Solanum (five genes from S. nugrum ), and their consensus sequences were established. An identity of 97–100% was observed among these sequences and the KPI-A sequences of the sections Petota (cultivated potato Solanum tuberosum ssp. tuberosum ) and Etuberosum ( S. palustre ) The interspecific variation of KPI-A did not exceed its intraspecific variation for all but one species ( S. lycopersicum ). The distribution of highly variable and conserved sequences in the mature protein-coding region was the same in all of the above species. The same primers failed to amplify the homologous genes from Solanum dulcamara , S. lycopersicum , and Mandragora officinarum . Phylogenetic analysis of the KPI-A sequences showed that S. lycopersicum clustered separately from all of the other species examined, that S. nigrum clustered together with species of the sections Etuberosum and Petota , and that these species produced no species-specific clusters. Although S. nigrum is resistant to all known races of the oomycete Phytophthora infestans , which causes one of the most economically important diseases of Solanaceae, the amino acid sequences encoded by S. nigrum KPI-A differed slightly, if at all, from their counterparts of cultivated potato, which is susceptible to P. infestans infection.