REACTION OF BLOOD TRANSFORMATION OF BLOOD LYMPHOCYTES OF NONLINEAR FEMALES RATS, THEIR OFFSPRING АT THE EARLY STAGES OF POST-EMBRYONIC DEVELOPMENT ON THE BASIS OF THE INFLUENCE OF SALINE EXTRACT HIRUDO VERBANA

  • R. F. Aminov Zaporizhzhya National University
  • A. K. Frolov Zaporizhzhya National University
  • Ye. R. Fedotov Zaporizhzhya National University
Keywords: hirudology, antigens, biologically active substances

Abstract

Hirudotherapy is one of the methods of non-medical, and natural effects on the body. Therapeutic effects of HT due to the composition of the secret salivary glands of medical leeches are more than 100 biologically active substances. Such multicomponent secretion of salivary glands of leech reveals anticosmic, lipolytic, analgesic, immunomodulatory, anticoagulant, thrombolytic, anti-inflammatory and bactericidal action. Therefore, it was important to investigate the reaction of blast transformation of whole blood lymphocytes against the background of the influence of saline extracts from the organs of medical leeches. Female non-linear rats once a week, 2 weeks before the conjugate with males and 2 weeks afterwards, intraperitoneal antigens of the medicinal leech extract Hirudo verbana (Carena, 1820) were obtained by the method proposed by Frolov (2013). Dosage of antigens of the salt extract was carried out on the protein content (determined by Lowry). Fixed animals using a fixing device. Animals were divided into two groups: the first experimental group of animals under the influence of antigens of the salt extract of medical leeches at a dose of 1/10 of LD50 (at the rate of 5 mg / kg of animal weight), 0,5 ml; The second control group of animals, which were intraperitoneally injected with a 0,5 ml saline solution. The females were studied after feeding the offspring and breeding in dynamics on 45 th and 60 th day.

All animals were decapitated under ethereal anesthesia. The proliferative activity of blood lymphocytes was evaluated in the whole blood by the reaction of blast transfection of lymphocytes (RBTL). Blood dissolved in heparin 350 μl and bring to 2,5 ml nutrient medium (1 : 5). The mixture was poured into penicillin vials and saturated with CO2 pipettes. The culture mixture was introduced by mitogen – konkanavalin A (KonA, Germany) at a dose of 10 μg / ml. As antigens (AG), salt extract was used from bodies N. verbana Carena, 1820 at a dose of 5 μg / ml. The concentration of protein in the extract was determined by the Lowry method (Menshikov, 1987). The mitogenic stimulation of lymphocytes by plant lectin ConA was performed in order to determine the conditionality of cell culture conditions, as well as to study the potential proliferative activity of the major populations of T-lymphocytes. The vials were sealed and cultured for 24 hours at a temperature of +37°. After 24 hours of cultivation, the samples were centrifuged (5 min at 1500 rpm), the culture supernatant was selected, and from the cell sieve, preparations were prepared, fixed with methanol, stained with Romanovsky-Gimza, followed by differentiation in acidified hydrochloric acid with distilled water. The level of RBTL was evaluated morphologically, taking into account 300 - 400 lymphocytes from the beginning of the «brush» to the basis of the drug.

Statistical processing of data was carried out using the SPSS v.21,0 computer program. (IBM SPSS Statistics., USA). The sample parameters listed below in the table have the following notation: x is the sample mean, SE is the standard error of the mean. The probability of differences between the average values was estimated according to the Student criterion. The differences were considered reliable at р < 0,05.

As a result of the study in mature female females, the control and experimental groups of animals, when stimulated with plant mitogen and leech antigens, increase the percentage of blast cells p < 0,05. Thus, for stimulation of ConAs by 61,27 %, AG by 37,33 % in the control group, and in the experimental group, KonA by 62,55 % and AG by 64,28 % compared to spontaneous cultures. The same tendency to increase in their litter at all stages of the experiment. Comparing experimental with control groups of spontaneous cultures and stimulating vegetative mitogen almost did not differ from each other, with some tendency to increase in experimental group. And when stimulated by the AG, the experimental group significantly exceeded the figures in comparison with the control group, both in females and in their offspring, p < 0,05. The increased response of lymphocytes to antigens of leeches Hirudo verbana indicates their polyclonal activation, as well as plant lectin, which coincide with our previous results in rats. Thus, in sexually mature females, RBTL in hypertension both in the control group and in the trial significantly exceeded the spontaneous p < 0,05. The same dynamics was noted in their offspring. RBTL at AG was close to the indicators, as on plant lectin ConA. RBTL morphologically differed. Blasters stimulated with plant lectin were typical, indicating a productive lymphogenesis. When stimulated by antigens, blastos had an underdeveloped cytoplasm and reduced basophility, as a result of insufficient development of protein-synthetic system. In cultures in which antigens were added up to 4 % of stimulated lymphocytes, there were signs of apoptosis: cytopic nos, karyopicnose, vacuolation of the nucleus and cytoplasm. In such cultures, necrotic lymphocytes were often found in the form of diffuse eosinophilic hepatitis. Apoptosis and necrosis of lymphocytes may indicate the anti-inflammatory effect of BAR leeches. However, the increase in RBTL in hypertension relative to spontaneous in rats can be explained by the presence of common patterns in the structural organization of all types of kings.

When stimulated by antigens of medical leeches, the percentage of blast cells is significantly increased. In the control group of animals at 37,33 %, and in the experiment at 64,28 % compared with spontaneous ones. Indicators of RBTL in AG were close to indicators, as on plant lectin ConA. In spontaneous and stimulated vegetable lectin cultures, the productive stages of immunogenesis were mainly observed, whereas in the stimulation of hypertension, apoptotic reactions were often observed. An increase in the percentage of blast cells in cultures stimulated by hypertension may be due to heterophilic patterns in their biopolymers.

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Published
2018-07-03
How to Cite
Aminov, R. F., Frolov, A. K., & Fedotov, Y. R. (2018). REACTION OF BLOOD TRANSFORMATION OF BLOOD LYMPHOCYTES OF NONLINEAR FEMALES RATS, THEIR OFFSPRING АT THE EARLY STAGES OF POST-EMBRYONIC DEVELOPMENT ON THE BASIS OF THE INFLUENCE OF SALINE EXTRACT HIRUDO VERBANA. Bulletin of Zaporizhzhia National University. Biological Sciences, (1), 46-52. https://doi.org/10.26661/2410-0943-2018-1-06