Decrease In Proliferative Activity Research Articles

Changes in glucose metabolism during detachment of metastatic cells

The purpose of the work is to investigate changes in survival rates, proliferative potential, glucose metabolism and redox metabolism in metastatically active cells of Lewis lung carcinoma during the transition from anchorage-dependent to anchorage-independent growth. It was shown that the transition from anchorage-dependent growth to anchorage-independent growth is accompanied by a considerable intensification of glucose consumption rate. An increase in glucose consumption during anchorage-independent growth does not cause stimulation of proliferative activity. An increase in glucose consumption and a decrease in proliferative activity indicates a redirection of glucose to the synthesis of enzymes of the antioxidant system in order to prevent apoptotic death during anchorage-independent growth.

СРАВНИТЕЛЬНОЕ ИССЛЕДОВАНИЕ ИЗМЕНЕНИЙ КОЛИЧЕСТВА ФОКУСОВ γH2AX И 53BP1 В МЕЗЕНХИМАЛЬНЫХ СТРОМАЛЬНЫХ КЛЕТКАХ ЧЕЛОВЕКА, ИНКУБИРОВАННЫХ С 3Н-ТИМИДИНОМ ИЛИ ТРИТИРОВАННОЙ ВОДОЙ

Purpose: Comparative study of changes in the number of foci of DNA (DSB) marker proteins (γH2AX and 53BP1) in human mesenchymal stromal cells (MSCs) incubated with 3H-thymidine or HTO for 24, 48, and 72 h. Material and methods: We used the primary culture of human MSCs of passage 5–6, obtained from the collection of LLC “BioloT” (Russia). A sterile solution of 3H-thymidine or HTO with a specific activity of 100 to 400 MBq/l was added to the nutrient medium and incubated under standard conditions of a CO2 incubator for 24, 48, and 72 hours. To quantify γH2AX foci and the proportion of proliferating cells using antibodies to γH2AX, 53BP1 and Ki67 (a marker protein for cell proliferation), were used, respectively. Statistical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). To assess the significance of differences between samples, Student’s t-test was used. Results: Incubation of MSCs with 3H-thymidine with a specific radioactivity of 100-400 MBq/l in the first 24 hours leads to a dose-dependent increase in the number of γH2AX and 53BP1 foci. With a further increase in the incubation time to 48 h and 72 h, a saturation effect is observed ‒ the number of foci reaches a plateau. A statistically significant increase in the number of γH2AX and 53BP1 foci in MSCs incubated with HTO was observed only in actively proliferating cells during the first 24 h of incubation in a medium with specific radioactivity of 300 and 400 MBq/l, after which, with a decrease in proliferative activity, it decreased to control values. Calculations made on the basis of the results of a quantitative analysis of γH2AX and 53BP1 foci after 24 h of incubation of MSCs with tritium compounds obtained in the course of the work show, that under the influence of 3H-thymidine ~ 6 times more DNA double-strand breaks are induced than under the influence of HTO.

Melatonin Can Enhance the Effect of Drugs Used in the Treatment of Leukemia

Melatonin (N-acetyl-5-methoxytryptamine, MEL), secreted by the pineal gland, plays an important role in regulation of various functions in the human body. There is evidence that MEL exhibits antitumor effect in various types of cancer. We studied the combined effect of MEL and drugs from different pharmacological groups, such as cytarabine (CYT) and navitoclax (ABT-737), on the state of the pool of acute myeloid leukemia (AML) tumor cell using the MV4-11 cell line as model. The combined action of MEL with CYT or ABT-737 contributed to the decrease in proliferative activity of leukemic cells, decrease in the membrane potential of mitochondria, and increase in the production of reactive oxygen species (ROS) and cytosolic Ca2+. We have shown that introduction of MEL together with CYT or ABT-737 increases expression of the C/EBP homologous protein (CHOP) and the autophagy marker LC3A/B and decreases expression of the protein disulfide isomerase (PDI) and binding immunoglobulin protein (BIP), and, therefore, could modulate endoplasmic reticulum (ER) stress and initiate autophagy. The findings support an early suggestion that MEL is able to provide benefits for cancer treatment and be considered as an adjunct to the drugs used in cancer therapy.

ОСОБЕННОСТИ ОПУХОЛЕВЫХ КЛЕТОК ЧЕЛОВЕКА ЛИНИИ HeLa,ВЫЖИВШИХ И ДАВШИХ УСТОЙЧИВЫЙ РОСТ ПОСЛЕ ОСТРОГО РЕНТГЕНОВСКОГО ОБЛУЧЕНИЯ

Purpose: The evaluation of the repair efficiency of DNA double-strand breaks (DSB), proliferative activity and the yield of cytogenetic disorders in human tumor HeLa cells which survived and gave stable growth after acute irradiation at a dose of 15 Gy. Material and methods: HeLa human tumor cell line (cervical carcinoma) was used. Cells were irradiated on an X-ray biological installation RUST-M1 (Russia), equipped with two X-ray emitters, at a dose rate of 0.85 Gy / min, a voltage of 200 kV, a total current of 10 mA, and a 1.5 mm Al filter. To obtain clones of surviving cells (HeLaRR), after acute irradiation at a dose of 15 Gy, cell cultures were incubated under standard CO2 incubator conditions (37 °C, 5 % CO2) for several weeks until well proliferating cells were obtained. Immunocytochemical staining of the foci of the phosphorylated H2AX protein (γH2AX) was used to quantitatively evaluate the residual foci of DNA DSB repair. The micronuclei number was assessed in cytochalasin-B cytokinesis-blocked binucleated cells stained with acridine orange with luminescence microscopy. The doubling time of the cell population was analyzed by the cell growth curves obtained by daily cell counting for five days. The cell cycle stages distribution was assessed by flow cytometry using the propidium iodide dye. All quantitative indicators of the studies were processed using the Student’s t-test for independent samples and the Kolmogorov – Smirnov test. Results: It was revealed that acute irradiation at a high dose leads to the selection of cells with a higher reparative capacity which is confirmed by the low yield of residual foci of DNA DSB repair and MN after testing irradiation at doses of 5 and 10 Gy. A significant decrease in the proliferative activity of cells that survived after acute X-ray irradiation at a dose of 15 Gy was revealed. The doubling time of the population of unirradiated cells at the stage of exponential growth was ~18 hours while for cells that survived after irradiation at a dose of 15 Gy ~42 hours. A change in the cell cycle phases distribution was observed. Conclusion: Thus, acute irradiation at a high dose leads to the selection of cells with a higher reparative capacity which is confirmed by the low yield of residual γH2AX foci and MN after testing irradiation at doses of 5 and 10 Gy. The decrease in proliferative activity was accompanied by a change in the cell cycle phases distribution.

SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner.

SOX9 is upregulated in the majority of pancreatic ductal adenocarcinoma cases. It is hypothesized that the increased expression of SOX9 is necessary for the formation and maintenance of tumor phenotypes in pancreatic cancer cells. In our research, we studied six pancreatic cancer cell lines, which displayed varying levels of differentiation and a range of oncogenic mutations. We chose the method of downregulation of SOX9 expression via siRNA transfection as the main method for investigating the functional role of the SOX9 factor in pancreatic cancer cells. We discovered that the downregulation of SOX9 expression in the cell lines leads to cell-line-specific changes in the expression levels of epithelial and mesenchymal protein markers. Additionally, the downregulation of SOX9 expression had a specific effect on the expression of pancreatic developmental master genes. SOX9 downregulation had the greatest effect on the expression levels of the protein regulators of cell proliferation. In three of the four cell lines studied, the transfection of siSOX9 led to a significant decrease in proliferative activity and to the activation of proapoptotic caspases in transfected cells. The acquired results demonstrate that the SOX9 protein exerts its multiple functions as a pleiotropic regulator of differentiation and a potential promoter of tumor growth in a cell-specific manner in pancreatic cancer cells.

STRUCTURAL MECHANISMS OF DISTURBANCES IN POSTNATAL MORPHOGENESIS OF THE IMMUNE SYSTEM ORGANS OF THE OFFSPRING BORN UNDER CONDITIONS OF THYROID HYPOFUNCTION IN THE MOTHER

The purpose of this study was to identify the structural and functional features of the postnatal development of the organs of the immune system of the offspring obtained under conditions of experimental hypothyroidism of pregnant women in the mother. The experiments were carried out on rat pups obtained from healthy females and obtained from females with experimental hypothyroidism. Studies have shown that maternal hypothyroidism leads to a disruption in the process of postnatal growth and formation of the immune system organs in offspring, the morphological manifestation of which is a decrease in the areas of the thymus cortex and T-dependent zones of the lymph nodes and spleen compared to the control, a decrease in proliferative activity and an increase in apoptosis T -lymphocytes both in the thymus and in T-dependent zones of peripheral immune organs. The mechanism of disturbances in the postnatal development of immune organs is based on an imbalance between the processes of apoptosis and proliferation of lymphoid cells, caused by a deficiency of thyroid hormones in the mother.

Polycondensed Peptide Carriers Modified with Cyclic RGD Ligand for Targeted Suicide Gene Delivery to Uterine Fibroid Cells.

Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvβ3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.

P02.02 Modeling of response to irradiation in recurrence glioblastoma’s cells culture

Abstract BACKGROUND Radiosensitivity of glioblastoma (GB) cells of local relapses may be markedly different from the primary tumor. Optimal doses and regimes of re-irradiation GB recurrence is not determined yet. MATERIAL AND METHODS GO1 primary GB cell culture was obtained during removal of a recurrent tumor after combined treatment, including irradiation of the surgical bed. Сell’s culture was irradiated by photon beams with energy 6 MeV and dose rate 600 MU/min. Irradiation performed in 1, 3 and 5 fractions, by 10 different doses for each regime. The dose range was determined experimentally for one fraction (5–250 Gy); for other regimes it was calculated according to the biological equivalent dose conception (3 fractions: 5–450 Gy, 5 fractions: 5–550 Gy). The proliferative activity of cells was investigate by MTT test. The results were normalized to the control. Dose-effect curves were plotted for each irradiation regime.The experimental data were approximated by calculated curves obtained by selecting the optimal parameters of the LQ-model and it’s modification. RESULTS Irradiation of GO1 by 1 fraction with the dose 5–250 Gy, causes a slow decrease in proliferative activity, which reaches a minimum value of 23% at 150 Gy and then remains constant. After irradiation by 3 fractions, proliferative activity of the GO1 gradually decrease only at a total dose over 120 Gy and reaches 37% after 450 Gy. When GO1 was irradiated in 5 fractions, a similar dose-effect curve was obtained, gradual decrease was observed to a value of 52% in the range of 250–500 Gy. Thus, the experimental dose-effect curves for irradiation of recurrence GB cells for 3 and 5 fractions have the appreciable “shoulder”, which could be explained by increased radioresistance. When approximating the experimental data by fitting the parameters of the LQ-model, the use of α/β = 8 provided the slope of the curve, close to the experimental data. For reflecting the “shoulder” an additional summand was introduced into the mathematical expression for the number of proliferating cells - a 105 Gy for 3 fractions and 255 Gy for 5 fractions. CONCLUSION Modified LQ-model could be used for an adequate mathematical description of the effectiveness of fractionated irradiation in relapsed GB culture cells in vitro. It’s necessary to introduce a summand into the formula that determines the formation of a “shoulder” on the dose-effect curve for this. The research was supported financially by RFBR (Project No. 18-29-01061).

Response of Human Retinal Pigment Epithelial Cells to the Effect of the Conditioned Media of Newt Retinal Regenerates

The development of the retina and the signaling pathways involved in this process are conserved among vertebrates. Therefore, it was hypothesized that active factors stimulating the retinal regeneration in newts can cause transdifferentiation of human retinal pigment epithelial (RPE) cells. To verify this, we studied the effect of conditioned media (CM) obtained from in vitro regenerating retinas of newts on the phenotype, expression of a number of genes, and localization of cell proteins of human RPE cells of the ARPE-19 line. Within 120 h of cultivation, a change in the morphology of human RPE cells was observed, which manifested in a change in shape and an increase in cell size, which was accompanied by a decrease in proliferative activity. 24 hours after exposure to CM, ARPE-19 showed a short-term increase in the expression of TUBB3 (a panneuronal marker) and a decrease in the expression of BMP2 and BMP4 (neural diffe-rentiation inhibitors). Using immunocytochemical (ICC) methods, we observed an increase in the intensity of staining of cells with antibodies to βIII-tubulin protein and a decrease in the intensity of staining with antibodies to the Cx43 protein of gap junctions (compared to the control) during the period between 24 and 72 h after exposure. At the same time, we observed a decrease in the levels of mRNA expression of the RPE markers OTX2 and KRT18, as well as the KLF4 gene, which is responsible for proliferation and differentiation. Additionally, the shift in the distribution of β-catenin to cytoplasmic localization was recorded using the ICC method. To exclude mesenchymal (and osteogenic in particular) differentiation under the effect of CM, we studied expression of the SPP1 and RUNX2 genes by qPCR. No SPP1 transcription was found in ARPE-19 cells in the control and under the effect of CM, and a decrease in the expression level of RUNX2 mRNA under the effect of CM was observed. Therefore, an attempt to shift to neuronal differentiation was observed in ARPE-19 cells during the period between 24 and 72 h after exposure to the CM of newt retinal regenerates, as indicated by an increase in transcription and translation of βIII-tubulin, a weakening of intercellular adhesion, and a decrease in the expression of inhibitors of neural differentiation BMP2 and BMP4, as well as RPE markers OTX2 and KRT18. However, after that, due to the short-term effect of CM, during the period between 72 and 120 h after exposure, the cells returned to the initial differentiation, as evidenced by a decrease in the expression of neuronal differentiation markers (NES, TUBB3, PAX6) and an increase in the expression of RPE markers (OTX2, MITF).

The features of proliferative processes in the thyroid gland of the Wistar rat’s offspring after intrauterine action of dexamethasone

The features of proliferative processes in the thyroid gland of the Wistar rat’s offspring after intrauterine action of dexamethasone were studied. Animals were divided into 3 groups: I – intact rats; II – control – animals, which on the 18th day of the dated pregnancy transuterine, transdermal, subcutaneously in the interscapular area was injected with 0.9 % saline in the amount of 0.05 ml; III – experimental group – animals, which during laparotomy by intrauterine, transdermal subcutaneous injection in the interscapular area was injected with a solution of dexamethasone at a dose of 0.05 ml at a dilution of 1:40 intrauterinely on the 18th day of pregnancy (Ukrainian patent No.112288). In the experimental subgroups used the allowable, generally accepted number of animals for statistical processing and obtaining reliable results – 6 animals. The thyroid gland with the tracheal area was removed on the 21st, 30th, 45th, 60th, 90th, 120th days of life. Immunohistochemical study was performed according to the protocol recommended for a particular antibody manufacturer. Monoclonal antibodies ki-67 (Ki-67), Fox-1 Antibody (A-12) were used to assess proliferative activity, the company Santa Cruz Biotechnology, Inc. (USA). The study found that the thyroid gland of rats of infantile period, which prenatally exposed to dexamethasone, is structurally represented by chaotically located follicles of different diameters with a predominance of large with desquamated cells in the lumen, and proliferative changes aimed at forming extrafollicular which is confirmed immunohistochemically by the presence of Ki-67-positive cells. Intracellularly, protein- synthesizing organelles of thyrocytes also proliferate, to which there is a clear cytoplasmic and nuclear reaction with Fox-1 antibodies. Dining the juvenile period, proliferative processes in the thyroid gland of animals of the experimental group are stabilized while maintaining the morphological structure of the hypofunctional type, and remain lower compared to the control and intact groups. Morphological signs of functional tension of the thyroid gland animals exposed prenatally to dexamethasone, which correlate with a decrease in proliferative activity, indicate a functional compensatory response of synthetic and hormone-producing function, but suppression of proliferative processes, despite the slight manifestations. The thyroid gland of morphological hypofunctional type after prenatal action of dexamethasone in young rats, indicates an adaptogenic compensatory response and morphofunctional immaturity of the organ during this period, which may be the basis for provoking the preservation of such morphogenetic factors under the influence of stressors. Keywords: thyroid gland, proliferation, dexamethasone, experiment, rats.

The expression of macrophage migration inhibitory factor in the non-small cell lung cancer.

ObjectiveThe objective was to investigate the expression of macrophage migration inhibitory factor (MIF) in non-small cell lung cancer (NSCLC), as well as the effects of macrophage MIF on tumor cells.MethodsThe human NSCLC cell strains H358 and H524 were selected as research objects. The Real-Time Polymerase Chain Reaction (RT-PCR) and Western Blot were utilized to detect the expression levels of MIF in human NSCLC cell strains. The lentiviral plasmid was utilized for MIF-mRNA interference. The expression levels of MIF before and after transfection were compared. The cell strains were cultured and proliferated for cell count and comparison.ResultsH358 showed MIF high expression while H524 showed MIF low expression. Once the H358 cells were constructed as silent MIF expression, compared with the original H358 cells, the difference was statistically significant. Once the H524 cells were constructed as high MIF expression, compared with original H524 cells, the difference was statistically significant. Being cultured for respective 3, 5, and 7 days, the transfected H358 cells showed a significant decrease in proliferative activity compared with original H358 cells, while the transfected H524 cells showed a significant increase in proliferative activity compared with original H524 cells.ConclusionMIF has high expression in H358 cells while low expression in H524 cells. The expression of MIF could enhance the proliferative activity of NSCLC tumor cells.

The effect of low doses ionizing radiation on microscopic and morphometric characteristics of tooth germs in fetal rats

The aim of the study.To reveal tooth germs structural changes induced by low doses of ionizing radiation in the experiment using light-microscopic examination. Objects and methods. The experiments were carried out on albino rats mongrel gregarious breeding. Gravid female rats were exposed to chronic irradiation on “Gammarid 192/120 unit” at 110 mR/h power of exposition for 18 days at the Institute of Radiobiology of the National Academy of Sciences of the Republic of Belarus. The total dose absorbed by the fetuses was 0.42 Gy. Results and discussion. The experiment showed a significant decrease of proliferative activity of tooth germ cells. It resulted in the decrease in size and in the number of cell layers, shape change up to the appearance of deformed tooth germs. Reducing the intensity of mitotic division also led to the lack of “concentric structures” and cell cushion-shaped thickenings in the enamel epithelium in the experiment. Conclusion. These series of experiments revealed such odontogenesis disorders that cannot be explained only by the decrease of proliferative activity.

Effect of erythropoietin on bone marrow mononuclear cells

Stem/progenitor cells are considered an alternative method of heart failure therapy by promoting regeneration of damaged myocardium in myocardial infarction. Effectiveness of cell therapy depends on the population composition and functional activity of the cell graft, and, in turn, it depends on the conditions of microenvironment. Cultivation of stem/progenitor cells with erythropoietin stimulates proliferative potential causing in vitro resistance to hypoxia, and in vivo stimulation of angiogenesis. We aimed for assessing effects of erythropoietin upon hematopoietic cells. We studied some effects of short-term incubation of bone marrow mononuclear cells (BM-MNCs) in patients with coronary heart disease (CHD) with erythropoietin upon cellular phenotype, cell cycle, apoptosis and their proliferative potential. BM-MNCs were isolated from bone marrow aspirate from patients with CHD in a density gradient, then incubated for 60 minutes with erythropoietin (33.4 IU/ml). Using flow cytometric assay of the total BM-MNCs pool, we have shown there endothelial progenitor cells at different stages of maturation and differentiation, mesenchymal stem cells are. Their total number did not exceed 30%. Short-term incubation of BM-MNCs with erythropoietin reduces expression of CD184 “homing receptor” molecules on CD34+ cells, and causes increase of CD184 on CD31+ cells in the BM-MNCs pool (p < 0.05). In addition, erythropoietin has been shown to cause a delay of CD34+ cells in the resting phase (G0G1), reduce a proportion of cells in the synthetic phase (S) and mitosis (G2/M) (p<0.05), and does not affect apoptosis, as shown by Annexin V-FITC Apoptosis Detection Kit. Erythropoietin had no significant effects on expression on BM-MNCs surface molecules involved in providing adhesion, such as CD18, CD29, CD44, CD49a, CD54, CD62E, CD146, and CD202b. MTT-method has shown that the short-term preincubation of BM-MNCs with erythropoietin contributed to a significant decrease in proliferative activity of BM-MNCs (p < 0.05). However, there was a tendency towards increased resistance of erythropoietin-pretreated BM-MNCs to oxidative stress induced by hydrogen peroxide. We have also revealed a correlation between the numbers of endothelial progenitor cells at different stages of differentiation, and numbers of hematopoietic stem cells in the total BM-MNCs pool. The number of CD34+/CD133+, CD34- / CD31+, CD45+/EpoR+, and CD34+/EpoR+ in BM-MNCs pool are dependent on the age of patients. Hence, a short-term incubation of BM-MNCs with erythropoietin promotes the cells to be retained in resting phase of the cell cycle, thus, in turn, helping to reduce proliferative potential of BM-MNCs.

Micronuclei in blood lumphocytes of existing and former coal miners: evaluation of the effect of anthracosilicosis

Background. The purpose of this study was to investigate the genotoxic risk in anthracosilicosis patients and in those with occupational exposure to coal dust.
 Materials and methods. We studied micronuclei (MN) and other cytogenetic lesions in blood lymphocytes in three groups of men comparable in age: 74 coal miners suffering from anthracosilicosis (AS), 41 healthy miners, and 70 control donors.
 Results. A significant increase in the frequency of MN was revealed with a simultaneous decrease in proliferative activity in samples of healthy and sick miners compared with the control. The level of MN in the lymphocytes of patients with AS significantly exceeded the corresponding indicator in the sample of healthy miners (1.22 0.05% versus 1.03 0.07%; p 0.01). The age of the subjects and the status of smoking did not have a significant effect on the frequency of cytogenetic parameters.
 Conclusion. AS in miners makes an additional contribution to the formation of DNA damage in lymphocytes. This contribution is probably due to oxidative stress accompanying inflammatory processes in pulmonary fibrosis. The results of the study also indicate the absence of differences in the frequency of MN when comparing subgroups of current and former miners. This means that the genotoxic effects in the lymphocytes of miners are able to persist for a long time after the termination of exposure by adverse factors in coal mining.

P11.48 Dose-dependent effects of radiation exposure on the cell culture of glioblastoma G01, obtained from the patient with a long-term survival

Abstract Background: The predictors of a favorable prognosis regarding radiation exposure in patients with glioblastoma have not been studied enough, therefore, the search for factors determining a tumor response to the combined chemoradiation treatment is an actual task at present. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for homologous recombination and its inhibition has been shown to sensitize glioma stem cells to irradiation. Materials and Methods: G01 primary glioblastoma cell culture was obtained from tumor tissues from patient with long term survival (42 month).An irradiation with the energy of 6 MeV was carried out in 1 fraction 20,40,60,80,100,125,150,200,250 Gy with Linac. mRNA expression of the RAD51 gene, XRCC6, XRCC5 genes, relative amount of RAD51 protein and Ku70, Ku80 proteins, proliferative activity of cell culture was evaluated by RT-PCR, Western blotting, MTT-test, respectively.Results: When G01 cells were irradiated with doses from 5 to 180 Gy there was a uniform linear decrease in the population of proliferating cells compared to the control, but at a dose of 200 Gy a rise to 24% was observed. The mRNA expression of the RAD51 gene increased 2 times after irradiation at a dose of 20 Gy compared with control and then increased, reaching a peak at the level of 100,250 Gy. Minimal expression was observed after irradiation at a dose of 20 and also decreased at 200 Gy. The mRNA expression of the XRCC5 gene increased 2-fold after irradiation at a dose of 20 Gy compared with control and then decreased, remaining at the same level. Minimal expression was observed after irradiation at a dose of 200Gy. The relative amount of Ku80 protein reached a peak at a dose of 100 Gy, followed by a decrease. Maximum expression level of mRNA of the XRCC6 gene was observed after irradiation at a dose of 250Gy. The relative amount of Ku70 protein also decreased at 200 Gy and reached a peak at a dose of 250 Gy. Conclusions: After irradiation transplantable G01 cell culture from patient with long term survival a linear decrease in proliferative activity depending on the dose was observed with an decrease in mRNA expression of Rad51 gene and the relative amount of RAD51,Ku70 in dose level of 200Gy that could potentially indicate sensitivity point to radiation exposure in these cell culture. Further studies using cell cultures from patients may help in understanding the mechanisms of radioresistance and radiosensitivity in glioblastoma.The research was supported financially by RFBR (Project No. 18-29-01061).

FEATURES OF IMMUNOCORCTION IN COMPLEX TREATMENT FOR PATIENTS WITH EXTERNAL GENITAL ENDOMETRIOSIS

Despite the centuries-old history of studying various aspects of endometriosis, this disease remains one of the central medical and social problems.The aim of the study: to improve the results of complex treatment of patients with external genital endometriosis by the use of a metabolic immunomodulator.Materials and methods. At the first stage, we conducted an experimental part of the work, which was performed on 50 white, sexually mature nonlinear female rats. The second stage of the work was the clinical studies of the effect of low-molecular metabolic immunomodulator on the results of treatment of endometriosis.Results and discussion. It has been experimentally proved that subskills injections to female rats 5-amino-1,2,3,4- tetrahydrophthalazine-1,4-dione sodium salt in a dose of 2 mg/kg / day (for 15 days) lead to a decrease in proliferative activity of cells ectopic endometrium and reduction of the area of the endometrioid glands: the height of the epithelial layer in the glands of the “red” centers of heterotopy decreased by 17,8 %, the area of nuclei - by 10 %, the area of the cytoplasm - by 6,6 %. The height of the epithelium of the glands of the “black” foci of heterotopy decreased by 35.2 %, the area of epithelial cells was 19 %, the area of nuclei of epitheliocytes was 46.8 %, which led to a sharp decrease in the nuclear-cytoplasmic ratio by 35 %. Significant mobilization of the immune system in the complex treatment of patients with external endometriosis using immunomodulator, shows an increase in the concentration of CD 16 by 1.4 %, CD 22 by 3.8 %.Conclusions. The inclusion of low molecular weight immunomodulator in the complex therapy of external genital endometriosis provides restoration of the menstrual cycle, reduction of the severity of the pain syndrome and the frequency of relapses of the disease, which significantly affects the quality of life.

THE RESEARCH OF ANTIMICROBIAL EFFICACY OF ANTISEPTICS DECAMETHOXIN, MIRAMISTIN AND THEIR EFFECT ON NUCLEAR DNA FRAGMENTATION AND EPITHELIAL CELL CYCLE

Introduction: Nowadays, the study of biological safety of modern cationic surface-active antiseptics with a wide antimicrobial spectrum has acquired particular importance. The aim was to study antimicrobial effectiveness of antiseptics decamethoxin, miramistin and their influence on nuclear DNA fragmentation and cellular cycle. Materials and methods: A comparative microbiological study of antimicrobial efficacy and a cytometric study of the effect of decamethoxin 0,02% and miramistin 0,01% on the cellular cycle were carried out. Antimicrobial activity of decamethoxin and miramistin was estimated by their minimal inhibitory and minimal microbicidal concentrations against opportunistic microorganisms using serial double dilution technique. Decamethoxin and miramistin cytotoxicity on anterior corneal epithelial cells, after their two-week daily instillation into the eyes of a Vistar line male rats was studied using flow cytometry. The parameters of epithelial cellular cycle, nuclear DNA fragmentation and apoptosis under the influence of antiseptics were registered. Results: High antimicrobial effect of decamethoxin and miramistin against Gram-positive, Gram-negative bacteria with the significant advantages of decamethoxin were found (р<0,001). Decamethoxin caused minimal influence on anterior corneal epithelial cells, the insignificant decrease of their proliferation index, low increase of apoptosis (0.68%), no difference of mitotic activity (p>0.05). But the use of miramistin resulted in the significant increase of nuclear DNA fragmentation, decrease of proliferative activity (р<0.05). Conclusions: Higher antimicrobial effect against a wide range of opportunistic pathogens is proved in decamethoxin 0,02% comparably to miramistin 0,01% (р<0,001). In prolonged antiseptic use of the first one there were found no cytotoxic and no pro-apoptotic effects on the epithelium (р<0,05).

A multi-centre study on the role of the thioredoxin system in breast cancer cell proliferation

Redox proteins (thioredoxin, glutaredoxin) are key macromolecules capable of modulating intracellular processes. This determines research choices in the field of redox-dependent cell proliferation management. The study of the molecular mechanisms of the onset, development and progression of malignant neoplasms underlies the search for tumor-associated markers and potential targets for personalized antitumor therapy.Purpose.To establish the role of the “thioredoxin – thioredoxin-reductase” system in the impaired proliferation of mammary adenocarcinoma cells under the action of the cyclin-dependent protein kinase roskovitin blocker.Materials and methods.The study was carried out using the culture of mammary adenocarcinoma cells of the MCF-7 line incubated in the presence and absence of roskovitin at a final concentration of 20 μM for 18 h. The intracellular content of thioredoxin and protein regulators of proliferation (cyclin E and cyclin-dependent protein kinase 2) were determined by Western blotting technique, the expression level of thioredoxin mRNA was determined by real-time polymerase chain reaction and the activity of thioredoxin-reductase was measured by a spectrophotometric method.Results.It was established that the decrease in proliferative activity of MCF-7 tumor cells incubated in the presence of roskovitin was accompanied by a decrease in the content of cyclin E and cyclin-dependent kinase on the background of a decrease in the expression level of thioredoxin mRNA and an increase in the activity of thioredoxin-reductase.Conclusion.The involvement of the components of the thioredoxin system (thioredoxin, thioredoxinreductase) in disrupting the proliferation of MCF-7 tumor cells was detected under the action of the cyclindependent protein kinases of roskovitin.

Type-1 astrocyte-like stem cells harboring Cacna1d gene deletion exhibit reduced proliferation and decreased neuronal fate choice.

In the central nervous system, CaV 1.2 and CaV 1. 3 constitute the main L-type voltage-gated calcium channels (LTCCs) coupling membrane depolarization to gene transcription. We have previously demonstrated that inducible disruption of Cav1.2 in type-1 astrocyte-like stem cells of the adult dentate gyrus (DG) impairs hippocampal neurogenesis in a cell-autonomous fashion. To address the role of Cav1.3 channels (encoded by the Cacna1d gene), we here generated TgGLAST-CreERT2 /Cacna1dfl/fl /RCE:loxP mice which facilitate inducible deletion of Cacna1d in tandem with induction of EGFP expression in type-1 cells, allowing tracking of recombined cells and their descendants. Neurosphere cultures derived from fluorescence-activated cell sorting sorted Cacna1d-deficient (Cacna1d-/- /EGFP) hippocampal neural precursor cells (NPCs) exhibited a significant decrease in proliferative activity. Further, under differentiation conditions, Cacna1d deficiency conferred an increase in astrogenesis at the expense of neurogenesis. In like manner, type-1 cells lacking Cacna1d showed reduced proliferation in the dentate gyrus (DG) in vivo. Moreover, Cacna1d deficiency resulted in a significant decrease in the number of newly born cells adopting a neuronal fate. Finally, massive excitation induced by repeated electroconvulsive seizures rescued the proliferation defect of Cacna1d-/- /EGFP type-1 cells. Together, the effects of Cacna1d gene deletion closely recapitulate our earlier findings on the role of Cav1.2 channels expressed by type-1 cells. Similar to Cav1.2 channels, Cav1.3 channels on type-1 cells boost type-1 cell proliferation and promote subsequent neuronal fate choice.

Features of the immune status of patients with communityacquired viral pneumonia which was complicated by development of the acute respiratory distress syndrome

30 patients with community-acquired viral pneumonia with laboratory confirmed diagnoses virus of an influenza A H1N1 (n=17) and a parainfluenza (n=13), complicated by the development of the acute respiratory a distress syndrome are examined. At these patients was established: decrease of proliferative activity of T lymphocytes, of quantity of serum immunoglobulins of a class M and G and active formation of immune complexes. At the died patients direct correlation dependence of quantity of the mononuclear cells which entered an apoptosis with the relative number of natural killers and absolute quantity of CD8+ of cytotoxic T-lymphocytes is established (R=0,71 and R=0,67 respectively, р < 0,05). It confirms a role of cellular immune reactions in development of the activation induced death of lymphocytes at serious viral pneumonia