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Rakhmatova Mukaddas Holtaevna, Head of the Department of Histology and Medical Biology of the Tashkent State Dental Institute Uzbekistan Ermatov Nizom Zhumakulovich, Head of the department of hygiene of children and adolescents and food hygiene, Tashkent Medical Academy, Uzbekistan E-mail: evovision@bk.ru
MORPHOLOGICAL FEATURES OF THE DEVELOPMENT OF THE IMMUNE SYSTEM, THE RELATIONSHIP AND INTEGRATION OF EPITHELIAL CELLS AND IMMUNE CELLS OF THE SMALL INTESTINE OF STERILE RATS ASSOCIATED WITH LACTOBACILLI
Abstract. Accelerating the renewal of enterocytes in the crypt-villus system, enhancing the secretion of Paneth cells, fat and eosinophilic cells, enrichment of the villus stroma with plasma cells, formation of structural and functional areas of PB, the appearance of macrophages in the dome zone after the introduction of amicrobial lactobacillus rats, i.e. intestinal microflora indicate morphogenic and immunogenic effects of microorganisms on the functional morphology of the mucous membrane of the small intestine.
Keywords. Immune system, lactobacilli, morphology, cells.
The formation and development of the immune system is key to creating new, more effective ways of dealing with them
the basis of the stability of the child's body, its susceptibility to various diseases of a bacterial, viral, fungal nature, as well as immunodeficiency states and the development of allergies. The process of maturation of this global natural defense lasts for many years, since immunological memory is not inherited, but is acquired by man in the process of development.
The constant presence in the intestine of a sufficient number are attached to its wall resident microorganisms prevents propagation of pathogenic microorganisms, their invasion into enterocytes and passage through the intestinal wall by establishing their biotope unfavorable extraneous microflora pH, generating bacteriostatic low molecular weight metabolites, degradation, bacterial toxins, deconjugation of bile acids, production of a wide range of antimicrobial substances of the bactericin family [2; 3; 4; 5]. Lactobacilli are able to exhibit an inhibitory effect on clostridia, streptococci, entero-bacteria, pseudomonads, listeria, candida, forming lactocins, lactobrevin, lactostrepcin, nisin, diplolocin and helveticin. Bifidobacteria actively suppress the reproduction of putrefactive and pyogenic bacteria, producing bifidine and bifilong.
Gnotobiotic animals are an excellent, if not to say, ideal model for solving the most diverse problems of medical microbiology, a model whose value can hardly be overestimated. After all, in this way, researchers get into the hands of another tool that allows them to penetrate the secrets of the microbial world, and, most importantly, it is the pathogenic microbes. But to know in detail how these microorganisms interact with each other and the highest organism - that means getting the
[1; 6; 7; 8; 9].
Objective: To evaluate the effect of intestinal microbio-cenosis on the formation of integrative and adaptive properties of the mucous membrane of the small intestine and its immune system under experimental conditions.
Materials and research methods. The work was performed on 120 white male rats of the Fisher line at the age of 1, 3, 7, 14, 21 days and 4-6 months.
The morphological, morphometric, and ultramicroscopic parameters, as well as the immunomorphology of the small intestine of sterile rats were studied on the material obtained from the studies conducted in the laboratory of the Gnotobi-ology Research Institute of Epidemiology and Microbiology. N. F. Gamaley with the joint work of the staff of our department. Control animals with normal intestinal microflora were kept under normal vivarium conditions.
Taking into account the goal and objectives set for this work, two series of experiments were carried out.
In the first series, pieces of duodenal tissue, jejunum, ileum and Peyer's patches were studied in microbe free animals at the age of 1, 3, 7, 14, 21 -days and 4-6 months. after birth. The rats with normal intestinal microflora, born and kept in normal vivarium conditions, were selected as controls.
In the second series of experiments, the effect of normal intestinal microflora on the processes of proliferation and differentiation of the epithelium in crypts, their migration and extrusion from the surface of the villi in amicrobial rats, and amicrobic rats associated with lactobacilli associated
MORPHOLOGICAL FEATURES OF THE DEVELOPMENT OF THE IMMUNE SYSTEM, THE RELATIONSHIP AND INTEGRATION OF EPITHELIAL CELLS..,
(Lactobacillus plantarum 8P-A3 and Lactobacillus fermen-tum 90T-90T and Lactobacillus fermentum 90T-90T), was studied.
A light-optical study was carried out after fixation in a mixture of Carnoy, FSU, 12% neutral formalin on paraffin (4-5 ^m) and semi-thin (1 ^m) sections, stained respectively with hematoxylin-eosin and pyronine G-methylene blue.
For electron microscopic studies, pieces of tissue of the small intestine, Peyer's plaque were fixed in a buffered 2.5% solution of glutar-aldehyde (20 min), 1% solution of OsO4 (1.5 h) at pH 7.2-7.3. Sections obtained on an ultratome 1 KV-4800 after contrasting in a solution of uranyl acetate and lead citrate were viewed in JEM-7 and JEM-100B electron microscopes.
In all series of radioautographic studies for each term used at least 3 animals. The index of labeled nuclei with single and repeated injections of H3 - thymidine was carried out when viewing 2-3 thousand. epithelial cells. The change in the percentage of labeled mitoses in time after a single injection of H3-thymidine is determined after studying 100 mitoses on average for each term.
In all series of experiments with morphometry, the number of statistical samples was carried out taking into account Avtandilov's criteria, statistical processing was carried out according to Student-Fisher. The difference in values is significant at P < 0.05.
Research results. In order to study the effect of normal intestinal microflora on the morphological, morphometric and ultrastructural features of the immune system of the small intestinal mucosa in the age dynamics, lactobacilli of the series: Lactobacillus plantarum 8P-A3 and Lactobacillus fermentum 90T-S4 were introduced to the microbeless rats. As a control, germ-free rats were taken.
In sexually mature, bacterial-free rats associated with lactobacilli, the lymphoid follicles of the Peyer's patches are
located singly or in groups in their own mucous plate. In PB as well as in intact rats, the following structural and functional zones are clearly defined: follicular, parafollicular, dome. Between each follicle, there is a parafollicular zone, which, without sharp boundaries, passes into the surrounding loose connective tissue of the stroma of the mucous and submucous membranes. A dome extending to the follicle from above, bulging into the hemispheric lumen. Its surface is flat, lined with one layer of prismatic epithelium. On the periphery of the PB is surrounded by crypts and villi.
Detected layer-by-layer arrangement of cells in each area of the PB (table number 1). The separation of one zone from another is carried out by reticular cells. In the bright center of the cell are located loosely. The follicular zone surrounding the bright center is dense due to the dense location of the cells. The follicular and parafollicular zones, as well as the dome, consist mainly of small lymphocytes. In the light center of the follicle lymphoblasts are located, which are in contact with each other using shallow invaginations. In this zone, mitotic dividing lymphoid cells, single differentiated plasma cells, macrophages, in whose cytoplasm polymorphic inclusions are identified, are often detected.
The dome of the PB in ultrastructure differs significantly from other zones. In this zone, macrophages are relatively often detected, while plasma, mast and eosinophilic cells are isolated. Lymphocytes lie in groups, surrounded on the periphery with reticular cells. Occasionally lymphoblasts are found among them. Plasma cells are large, may be located near the epithelial layer, capillaries, on the border with the follicular zone. Everywhere they come in contact with lymphocytes and macrophages. Macrophages are more common, have a characteristic structure: in the cytoplasm of some, varying sizes and forms of lysosomes are detected, inside which digestible cells or their fragments are visible, resembling phagocytosed lymphocytes in structure.
Table 1. - The cellular composition of the structural and functional areas of the lymphatic nodule PB small intestine of mature sterile rats associated with lactobacilli (n = 6)
Cell type Germinal Follicle The parafol zone licular Zone Dome Epithelium
Small lymphocytes 39.9 ± 3.5 61.9 ± 6.1 66.6 ± 6.9 50.0 ± 1.1 43.4 ± 2.6
Large lymphocytes 6.8 ± 0.3 1.2 ± 0.03 1.9 ± 0.06 10.2 ± 1.4 5.9 ± 0.6
Lymphoblasts 16.9 ± 1.2 3.2 ± 0.1 3.0 ± 0.1 5.8 ± 0.6 -
Plasma cells 8.2 ± 0.4 5.1 ± 0.2 1.0 ± 0.02 11.5 ± 1.6 -
Reticular cells 12.6 ± 0.8 12.4 ± 0.8 18.0 ± 1.5 11.3 ± 1.6 -
Macrophages 0.5 ± 0.1 0.2 ± 0.01 - 2.6 ± 0.02 -
Mast cells 0.5 ± 0.01 0.5 ± 0.01 0.3 ± 0.004 1.8 ± 0.1 -
Nediffer- 12.5 ± 0.8 15.5 ± 1.1 9.1 ± 0.6 9.1 ± 1.2 -
cysed cells - - - - 40.8 ± 0.9
Enteric Enterocytes - - - - 8.2 ± 0.1
Fat and eosinophilic cells of PB, as in their own plate of the mucous membrane of the small intestine, are in contact with macrophages, lymphocytes, are located near the blood capillaries, have a characteristic ultrastructure for them.
Compared to other zones, the dome is relatively rich in blood and lymphatic capillaries, in the lumen of which lymphocytes with processes occur, which are in contact with each other and endothelial cells. In the parafollicular zone, small and medium lymphocytes of typical structure are detected. Single macrophages, large, polymorphic, in contact with mast cells and lymphocytes own plate of the small intestine. Capillaries with flattened or high endothelium.
The surface of PB is lined with a single-layer prismatic epithelium, where poorly differentiated, limbate and single goblet cells differ. Between them in large numbers at different levels of the epithelium are lymphocytes.
The limbate cells have a highly prismatic shape, an oval elongated nucleus, which is located in the basal part of the cell, sometimes in the middle part of the cell, if they are displaced by interepithelial lymphocytes, so they look multi-core. The layer of mucus on the surface of the epithelial cells of the dome is thinned in its direction from the base to the top, sometimes fragmented. In the same direction, the number of interepithelial lymphocytes increases. They practically do not occur between epithelial cells located in the base area of the dome dome PB.
Among the modified epithelial cells in the area of the dome are M-cells. They differ from typical epithelial cells by the presence of microvilli on the apical surface and by invaginations of the plasmolemma in the region of the microvilli, which probably form pinocytosis vesicles. At the base of the M-cells often 1-2 lymphocytes.
Thus, a comparative study of the structure of the mucous membrane of the small intestine of rats without microbes and with normal microflora made it possible to establish a number of significant differences indicating the morphogenic influence of microorganisms. Normal microflora causes the acceleration of epithelial renewal in the villus-crypt system, reduces the linear parameters of the villi and increases the -crypt, the proximal-distal gradient becomes more distinct. In the lamina propria, the number of cells of loose connective tissue increases. Particularly noticeable increase in plasma cells and macrophages, their cooperation with other cells of the stroma, the number of infiltrating lymphocyte epithelial layer. Significantly reduced the number of secretory granules in them.
A comparative analysis of the morphological and functional rearrangements of the small intestinal mucosa of gno-
tobiont rats during contamination with representatives of the normal and pathogenic intestinal microflora suggests that pathogenic microbes have a more pronounced immunogenic effect than representatives of normal microflora.
In germ-free rats, there is a slow migration of cells in the crypt-villus system, a longer lifespan than in normal ones. Lactobacilli accelerate the migration of epithelial cells, but not as intensively as with normal microflora (in animals living under normal vivarium conditions). The life cycle of enterocytes in the crypt-villus system is shortened. This is accompanied by a deepening of the crypts and a decrease in the height of the villi. This is a universal mechanism of adaptation to changes in the microbiocenosis of the intestine, which is observed in various infectious and allergic enterocolitis, resection of the small intestine, aimed at maintaining the established relationship in the crypt-villus system, the architectonics of the small intestinal mucosa.
Thus, the study of the mucous membrane of the small intestine in early postnatal ontogenesis, in germ-free animals, after the introduction of various microbial associations, allowed the influence of intestinal microflora on the epithelium kinetics, linear parameters of the crypt-villus system, change in proximal-distal gradients, structure of the lamina propria and structurally - functional zones PB. Without touching upon the various complex, intensively developed aspects of their functioning, it was noted that under physiological conditions, the integration of the activity of PB cells, the lamina and the interepithelial lymphocytes ensures the homeostasis of the internal environment, protection from microorganisms, products of their vital activity, food antigens, intestinal microflora. The interaction of fat and eosinophilic cells in germ-free animals during all periods of postnatal life, various types of cooperation of connective tissue cells in the lamina of the small intestinal mucosa of adult rats with normal microflora expands the existing ideas about the immune function of the organ, its innate ability to form protective mechanisms.
Conclusion. 1. Structural and functional zones of PB of adult conventional rats differ in their cellular composition and lymphocyte kinetics. Lymphocytes synchronously, synchronously, with high speed, migrate from all areas of the PB to the dome area, from where they quickly, within a day, decrease.
2. Changes in the structure and function of the mucous membrane of the small intestine at different periods of ontogenesis in the dynamics of the process of absorption reflect its ability, respectively, to slow and fast adaptations.
MORPHOLOGICAL FEATURES OF THE DEVELOPMENT OF THE IMMUNE SYSTEM, THE RELATIONSHIP AND INTEGRATION OF EPITHELIAL CELLS...
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