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Vol 17, No 4 (2022)

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Historical articles

Professor-histologist Nikolay Antonovich Shevchenko (devoted to the 120th anniversary of birth)

Gololobov V.G., Araviiskaia D.D.


The 120th anniversary of professor N.A. Shevchenko, the prominent Soviet scientist which made an worthy impact to the development of evolutionary and experimental histology will held in 2023.

Analyzed his activities in different years at the departments of histology of the S.M. Kirov Military Medical Academy and I.P. Pavlov Leningrad Medical Institute. Professor N.A. Shevchenko conducted a large-scale study of the endothelium of large blood vessels, substantiated the concept of its heteromorphy, established the patterns of reparative regeneration, and studied inflammatory growths of the endothelium. They confirmed the position of the outstanding histologist academician N.G. Khlopin about the endothelium as an angiodermal type epithelium.

The authors shares personal memories of the teacher.

Genes & Cells. 2022;17(4):7-18
pages 7-18 views


Lipopolysaccharide-induced model of inflammation in cells culture

Islaev A.A., Chibirova T.T., Takoeva E.A., Kokaev R.I.


Inflammation is a non-specific process that underlies the pathogenesis of many diseases, including COVID-19. The study of the mechanisms and molecular pathways of this phenomenon is carried out using various models, including in vitro investigations on cell cultures. The most accessible model for reproduction is the lipopolysaccharide-induced one.

This review analyzes the scientific papers in which the model was represented in vitro. It was found that inflammation caused by lipopolysaccharide is realized by activating classical signaling pathways (nuclear factor "kappa-bi", mitogen-activated protein kinase, etc.), changes of expression of circular and non-coding RNAs, etc. Evaluation of the working model of inflammation was carried out by many parameters, the most important of which was the concentration of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α). Inflammation through stimulation with lipopolysaccharide was reproduced in cultures of different cells, however, it was most easily reproduced in the culture of monocytes and macrophages, which is explained by their conversion to the M1 phenotype. The co-cultivation of different cells made it possible to study the mechanisms of the pathological process in the model of inflammation more fully.

Genes & Cells. 2022;17(4):19-30
pages 19-30 views

Molecular mechanisms of obesity: a review of the most relevant gene markers

Karabanov S.Y., Chernukha I.M., Kibitkina A.A., Fedulova L.V.


The development of genome-wide association studies has made it possible to isolate many genes associated with obesity — one of the most common diseases in the world. For a possible correction of this process, there is a need for a more detailed study of certain genes, the expression of which can change during metabolic processes associated with obesity.

The aim of this review is to identify the most promising candidate genes for further studies of metabolic disorders associated with obesity.

Genes & Cells. 2022;17(4):31-45
pages 31-45 views

Features of obtaining and prospects for the use of colorectal tumor organoids

Krasnova M.G., Efremova A.S., Bukharova T.B., Kashirskaya N.Y., Tsukanov A.S., Gorban N.A., Mikhaylenko D.S., Strelnikov V.V., Goldshtein D.V.


The review summarizes innovative advances in the field of organoids as a tool for accurate cancer modeling. The conditions for reproducing the microenvironment in vitro based on organoid technology are generalized, various methods for cultivating the tumoroids are considered, and an analysis of their properties is carried out on the example of colorectal cancer.

The final part of the review summarizes the literature data on the use of tumoroids in predicting the therapeutic response of a tumor to chemotherapeutic drugs, studying the mechanisms associated with resistance, and optimizing strategies and potential treatments for patients with malignant tumors. Currently, the tumoroid model is widely used in personalized medicine, basic research, screening of antitumor drugs, and to create libraries of tumors with various mutational profiles.

Genes & Cells. 2022;17(4):47-62
pages 47-62 views

Neutrophil extracellular traps: structure and biological role

Mirgorodskaya O.E., Mirgorodskaya O.E., Rusakova S.E., Gorbulich A.V., Gololobov V.G.


Neutrophilic granulocytes make up the majority of blood leukocytes and realize their functions in tissues. Highly differentiated cells of neutrophilic granulocytic differon are characterized by the ability to form neutrophilic extracellular traps (NEТs) — network-like structures formed mainly from the intracellular components of the neutrophil. NEТs are an important factor in the body's defense against infectious pathogens, aimed at the destruction of viruses, bacteria, fungi and protozoa. Analyzed the composition of NEТs, methods of detection, mechanisms and main stages of their formation: 1) the effect of the inductor on the neutrophilic granulocyte and the activation of the respiratory burst with the release of reactive oxygen species (oxygen superoxide); 2) loss of the characteristic segmentation of the nucleus, decondensation of chromatin; 3) the disintegration of the karyolemma into many small vesicles, the movement of chromatin strands (DNA with histone proteins) into the cytoplasm; 4) the formation of NEТs due to the connection of chromatin with numerous biologically active substances of the cytoplasm (mainly from lysed granules); 5) destruction of the plasmolemma and the release of NEТs into the intercellular environment. Data on the participation of NEТs in protective reactions and pathological processes are presented.

Genes & Cells. 2022;17(4):63-74
pages 63-74 views

Autosomal recessive congenital ichthyosis: diagnosis, modeling and approaches to therapy

Ponomarev A.S., Chulpanova D.S., Shaimardanova A.A., Rizvanov A.A., Solovyeva V.V.


Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of diseases caused by mutations in at least ten genes. ARCI is characterized by varying degrees of hyperkeratosis and the presence of scales on the surface of the patients’ skin since birth. Despite the variety of mutations and phenotypic manifestations of ARCI, from 32 to 68% of cases are due to a mutation in the transglutaminase-1 gene.

Currently, ARCI therapy is aimed at reducing the symptoms of the disease. To alleviate clinical symptoms, symptomatic therapy with moisturizers, keratolytics, retinoids and other cosmetic substances that improve the condition of the patients' skin is used.

There is a great need for the development of therapy aimed at the root cause of the development of the ARCI. Graft transplantation is usually used to correct eyelid defects in ARCI. Gene and cell therapy are developing as promising methods for the treatment of ARCI, with the help of which it is possible to correct the functional activity of mutant genes, in particular, transglutaminase-1. The review discusses current research on gene and cell therapy approaches and their future prospects in the treatment of patients with various forms of ichthyosis.

Genes & Cells. 2022;17(4):75-90
pages 75-90 views

Original Study Articles

The optimization of methods for the establishment of heterogeneous three-dimensional cellular models of breast cancer

Abdurakhmanova M.M., Ermakov M.S., Richter V.A., Koval O.A., Nushtaeva A.A.


BACKGROUND: Spheroids are self-assembled clusters of cells mimicking a tissue-like architecture. Since the structure of complex three-dimensional cellular models is not stable, the formation of core spheroids and further maintenance are crucial stages within the cultivation process. There are a lot of options described for the establishment of 3D cell models. A wide range of reagents is presented from simple hydrogels to complex natural and synthetic composites. However, cultivation of 3D models is still a technically challenging task requiring adaptations of protocols for particular purposes.

AIM: To compare methods of the formation of gomogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and/or stromal cells of breast cancer using hydrogels such as agarose, gelatin and Matrigel™, as well as using ultra-low-adherent plates.

MATERIALS AND METHODS: Breast cancer cell lines MCF7, MDA-MB-231 SK-BR-3 and stromal fibroblasts BrC4f, BrC120f, BN120f were used as a models for 3D and 3D-2 cultures. Spheroids were obtained on a substrate of simple hydrogels or when cultured on a low-adhesive plastic. The processes of formation and growth of spheroids, as well as "crushed preparations" were visualized using a Nikon Eclipse Ti-S series fluorescent inverted microscope (Nikon, Japan).

RESULTS: We demonstrated, that gelatin-based hydrogel is not suitable as a substrate for obtaining 3D and 3D-2 spheroids for any of the cell lines used in the work. The use of only one type of hydrogels does not allow to obtain the entire repertoire of tumor, stromal, and heterogeneous 3D models. Agarose exhibited high output for stromal spheroids and Matrigel™ for tumor cells, and the use of ultra-low-adherent Nunclon™ Sphera™ plates was preferable for 3D-2 models combining both cell types. We also revealed that the application of cooled cultivation plastic and solutions is a technological advantage for handling spheroids growing in low-adherent plates.

CONCLUSION: The proposed approaches for the formation of both homogeneous (3D) and heterogeneous (3D-2) spheroids from tumor and (or) stromal breast cancer cells are a generalized guide to the most effective production of spheroids of various cellular composition.

Genes & Cells. 2022;17(4):91-103
pages 91-103 views

About cases of fibrodysplasia ossification progressive

Deev R.V., Presnyakov E.V., Kopylov E.D., Podluzhny P.S., Kurilin I.S., Zhemkov N.I.


Fibrodysplasia ossificans progressive is a uniquely rare autosomal dominant disease with complete penetrance that develops as a result of a spontaneous mutation in the type IA activin gene (ACVR1, ALK2), which is a bone morphogenetic protein receptor. The main manifestation of the disease is the development of heterotopic osteogenesis in "soft tissues" — subcutaneously, inter- and intramuscularly.

This report summarizes the results of an intravital pathoanatomical study of erroneously taken biopsy specimens in children of different ages. Correspondence was shown to previously published data that the development of heterotopic ossification is associated with an increase in local tissue manifestations of inflammation — infiltration by CD45, CD68, CD163 cells, immune responses — accumulation of CD3 cells. At the same time, it has been suggested that the development of bone tissue can be associated not only with the development of the process of enchondral osteogenesis, but also in a direct way — due to the direct differentiation of paravascular (adventitial) osteochondrogenic cells (stem stromal cells, multipotent mesenchymal stromal cells) into osteoblasts. The typical structure of ossificates consisting of reticulofibrous bone tissue produced by active osteoblasts and resorbed by CAII-positive giant multinucleated osteoclast cells is shown.

Genes & Cells. 2022;17(4):105-114
pages 105-114 views

An assessment of physiological and biochemical characteristics of cells based on the respiratory activity under substrates and toxic substances impact

Iliasov P.V., Guseva O.S., Kuricyna A.P., Limareva L.V.


BACKGROUND: Metabolic profiling of cells is an essential aspect of studies of intracellular metabolism. One of the parameters which can be used for this purpose is a rate of oxygen consumption during degradation or transformation of substrates by cells.

AIM: This study was aimed at the development of a method for assessment of cell physiology and biochemistry based on the metabolic profiling via registration of the cell respiratory activity, and use of this method for evaluation of inhibition and recovery of liver parenchyma cells affected by different compounds.

MATERIALS AND METODS: The method used a Clark-type oxygen electrode with the cells located at its working area, in order to measure dissolved oxygen concentration in the immediate surrounding of living intact cells thereby ensuring their metabolic profiling and physiological status assessment. The experiments were performed using Wistar rat liver parenchyma fragments. Three experimental series were performed: first (series A) was aimed at metabolic profiling of the liver parenchyma; the second (series B) determined the stability of parenchyma’s response to sodium citrate over time; and the third (series C) ensured real-time recording and assessment of the effect of hepatotoxicants on the liver tissue. Solutions of glucose, fructose, sucrose, sodium citrate, sodium pyruvate, L-glutamine, ethanol and hydroquinone were used as the substrates for liver tissue metabolic profiling, and solutions of methanol and clarithromycin as the toxic agents.

RESULTS: During the experiments, a metabolic profiling of tissue and real-time monitoring of toxicity effect on liver parenchyma cells have been demonstrated.

CONCLUSION: The developed method for assessing the physiological and biochemical characteristics of cells can be used to track the metabolic activity, inhibition and restoration of liver parenchyma cells when exposed to substrates and toxic compounds.

Genes & Cells. 2022;17(4):115-124
pages 115-124 views

Melatonin role in formation of T helper subpopulations co-expressing Th17 and Treg markers

Kuklina Е.М., Glebezdina N.S., Nekrasova I.V.


BACKGROUND: One of the main pineal hormones, melatonin, has multifunctional activity including ability to effectively regulate immune cells. Subpopulations of Th17 (T helpers producing interleukin 17) and Treg (regulatory T cells) are under the hormone direct control. It has now been established that both subpopulations are heterogeneous, have high plasticity, and non-classical Th17/Treg variants play leading role in the pathogenesis of various diseases.

AIM: To evaluate exogenous melatonin effects on classical Th17 and Treg subpopulations, as well as non-classical population of cells co-expressing Th17 and Treg markers.

METHODS: The objects of the study were leukocytes of healthy donors. Differentiation of Th17 and Treg was assessed in vitro in response to polyclonal activation (anti-CD3/CD28) by the presence and level of corresponding markers using flow cytometry. The role of specific receptors in melatonin-dependent regulation of Treg and Th17 was determined by inhibitory analysis.

RESULTS: It has been shown that the hormone in concentration corresponding to its level in peripheral blood during pharmacological usage is able not only to regulate formation of classical Th17 and Treg populations by suppressing the differentiation of CD4+FOXP3+ cells, but also to induce formation of T lymphocytes that simultaneously express Th17 and Treg cells’ markers.

CONCLUSION: The effects of melatonin on both classical Th17 and Treg populations and non-classical one lead to a shift in the balance towards Th17 cells, which may contribute to inflammation development in various pathological situations.

Genes & Cells. 2022;17(4):125-132
pages 125-132 views

Evaluation of the properties of osteogenic gene-activated matrices based on hydrogels impregnated with polyplexes with the BMP2 gene

Meglei A.Y., Nedorubova I.A., Mokrousova V.O., Basina V.P., Vasilyev A.V., Makhnach O.V., Goldshtein D.V., Bukharova T.B.


BACKGROUND: Currently, to effective bone regeneration, biocompatible matrices containing plasmid constructs with osteoinductor genes are developed.

AIM: To study in vitro the properties of gene-activated matrices based on collagen and platelet-rich plasma impregnated with polyplexes with the bone morphogenetic protein 2 gene.

METHODS: The methods of fluorescence microscopy, flow cytofluorimetry, ELISA, real-time PCR and MTT test were used in the study.

RESULTS: Using the MTT test and fluorescence microscopy to detect live and dead cells, it was shown that the obtained matrices have high cytocompatibility. Impregnation of plasmid constructs in hydrogel matrices based on collagen and fibrin ensures their prolonged release within 25 days. Using flow cytometry and fluorescence microscopy it was shown that polyplexes released from matrices are able to effectively transfect multipotent mesenchymal stromal cells derived from rat adipose tissue. 3 weeks after incubation of cells with matrices impregnated with polyplexes with BMP2 gene, it was shown a 25-fold increase in BMP-2 protein production by enzyme-linked immunosorbent assay. BMP-2 secreted by transfected cells induced osteogenic differentiation of multipotent mesenchymal stromal cells, as evidenced by increased expression of the osteopontin gene and extracellular matrix mineralization.

CONCLUSION: The developed matrices in the future can be used in gene therapy for damaged bone.

Genes & Cells. 2022;17(4):133-141
pages 133-141 views

A comparative study on morphology of corneal endothelium and Descemet membrane, TGF-β1 aqueous humor level in Fuchs endothelial corneal dystrophy and pseudophakic bullous keratopathy

Yusef .N., Subbot A.M., Fisenko N.V., Osipyan G.A., Demura T.A.


BACKGROUND: Chronic corneal edema is often associated with endothelial cell loss due to Fuchs endothelial dystrophy and pseudophakic bullous keratopathy.

AIM: to compare the changes of Descemet membrane/endothelium complexes and aqueous humor TGF-β1 level in Fuchs endothelial dystrophy and pseudophakic bullous keratopathy.

MATERIALS AND METODS: The study included 35 patients (36 eyes) with chronic corneal edema caused by irreversible dysfunction of endothelial cells: 1a group — 12 patients (12 eyes) with Fuchs endothelial dystrophy, 1b group — 10 patients (10 eyes) with pseudophakic bullous keratopathy; 2a group — 8 patients (9 eyes) with Fuchs endothelial dystrophy, 2b group — 5 patients (5 eyes) with pseudophakic bullous keratopathy. Control groups — 5 (for groups 1a, 1b) and 7 (for groups 2a, 2b) donor corneoscleral discs.

TGF-β1 level in patients of groups 1a, 1b was determined by enzyme immunoassay of intraocular fluid. Descemet membrane/endothelium çomplexes (1a, 1b groups and control group (5 of 12 preserved human corneas) were stained with hematoxylin/eosin. Descemet membrane/endothelium çomplexes (2a, 2b groups and control group (7 of 12 preserved human corneas) were stained with ZO-1, α-SMА antibodies, nuclei were counterstained (Hoechst 33342).

RESULTS: TGF-β1 level was elevated in 1a group (p <0.05). In 1a group Descemet membrane was thickened by guttae. Descemet membrane/endothelium çomplexes of 2a, 2b groups presented enlarged endothelial cells with lower levels of ZO-1 expression. There was an increase in nuclear areas in 2a, 2b groups compared to controls (p <0.05). Nuclear aspect ratio was lower in 2a, 2b groups compared to the controls (p <0.05). In 2a group endothelial cells showed high level of α-SMА expression.

CONCLUSION: Fuchs endothelial dystrophy and pseudophakic bullous keratopathy are conditions of different structural changes in Descemet membrane/endothelium çomplexes. TGF-β1 may play a pivotal role in guttae formation in Fuchs endothelial dystrophy.

Genes & Cells. 2022;17(4):143-152
pages 143-152 views

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