RU2639769C1 - Method for direct impact on tissues cells of odd-toed animals - Google Patents

Abstract

FIELD: veterinary medicine.SUBSTANCE: effect on the cell suspension is provided by a continuous ultrasonic wave at a frequency of 0.88 MHz, intensity of 0.05-1.0 W/cmfor 15-60 s - for nucleate cells larger than 7 μ, and for anucleate cells up to 6 μ - intensity of 0.7-1.0 W/cmfor 20-40 s; as well as by pulsed ultrasound with a generation frequency of 2.64 MHz with an intensity range of 0.4-1.0 W/cmfor 20-60 s - on nucleate cells larger than 7 μ, and for anucleate cells up to 6 μ - intensity of 0.7-1.0 W/cmfor 25-60 s with subsequent preparation of smears, their staining with trypanic blue and differential dyes, analysis of the cytoplasmic membranes state and cell viability.EFFECT: directed change in the permeability of the cell cytoplasmic membrane, inhibition or activation of the cells transport systems, regulation of the membrane-bound enzymes activity.7 dwg, 2 tbl

Description

The invention relates to medicine, biotechnology and veterinary medicine, in particular to the use of acoustic waves for non-invasive effects on target cells of biological tissues in order to control vital processes, proliferative activity of cells / cultures of cells and tissues and the possibility of selective suppression or activation of their functions. The invention can also be used in cell and molecular biology, is of interest for developing methods of experimental medicine, pharmacology, diagnosis of malignant neoplasms, individual selection of drugs and gerondoprotectors, with tactics of radio, chemo and ozone therapy.

When exposed to acoustic waves due to compression in the wave of cell membranes and the implementation of the piezoelectric effect, the effect of a change in the surface charge and the functional state of the membranes is possible. Thus, the membrane can be a target at the level of which a chain of cytological, biochemical, and biophysical effects is realized. Compared to the wavelength, the cells located in the acoustic field are punctate, experiencing compression and expansion of the volume, reaching 20%, which, in turn, can reduce the number of active channels during lateral diffusion of lipid bilayer molecules, and change the permeability of the cytoplasmic membrane (CPM) and the functional state of the cell [1-9].

The prior art method for immunocorrection in the autoimmune process (patent for invention RU 2098139, publ. 10.12.1997). After premedication, the patient's blood is perfused in the veno-venous version through xenosplenic sections previously activated with ultrasound of low intensity 0.3-0.4 W / cm ² in a pulse mode of 50 pulses / s for 8-10 minutes. The method is used to simplify the process of hemoperfusion and increase the sorption capacity of the spleen in the treatment of psoriasis. This method is effective for perfusion with a bulk velocity of 75-80 mm / min for 40-45 min for 2-3 sessions with an interval between them of 3-5 days at the beginning of the course of traditional complex therapy.

However, this method is costly due to the use of expensive stationary equipment and material. The method requires, when it is implemented, the work of specially trained personnel, is complicated in the execution of the methodology, is time-consuming, is feasible when the ultrasound equipment is operating in pulsed mode and at the cut of one type of tissue.

A known method of non-invasive destruction of biological tissues located behind the bones of the chest (patent for invention RU 2472545 dated January 20, 2013, bull. No. 2), selected as the closest analogue. This method is based on the action of a focused ultrasound beam on biological tissue for local destruction of cells only at the location of the main focus, without damage to the side foci.

This method is used in ultrasound surgery only for both thermal and mechanical effects, accompanied by a strong heating of the tissue. The limitation in the application of the method of acoustic destruction of cells is determined by the use of high-intensity ultrasound, the possibility of affecting only one type of tissue of the human body - bone, and on the entire tissue at the same time, and not on individual cells (osteoblasts), by generating a local excess peak positive pressure of 30-80 MPa place of exposure.

The objective of the invention is to develop an effective method of non-invasive directed exposure to animal tissue cells, safe to implement and not requiring expensive stationary equipment, specially trained personnel and specially equipped rooms; the implementation of the method without additional technical means and chemicals; minimum time consumption (15-60 s); complete safety of the method for medical personnel and researchers with maximum effect.

The aim of the invention is a planned non-invasive effect on cells of different types and sizes.

The technical result of the claimed invention is: directed change in the permeability of the CPM; inhibition or activation of cell transport systems, phonophoresis of drugs at the cellular level; regulation of membrane-bound enzyme activity; directed suppression of cell growth, including abnormal, due to a violation of the apparatus of intercellular interaction and cellular contacts mediated by CPM, which will make it possible to arrest diseases of various etiologies at the cellular level.

The claimed technical result is achieved by the fact that the effect on the cell suspension is produced by a continuous ultrasonic wave with a frequency of 0.88 MHz, an intensity of 0.05-1.0 W / cm ² for 15-60 s - on nucleated cells larger than 7 μ, and on nuclear-free cells up to 6 μ in size - with an intensity of 0.7-1.0 W / cm ² for 20-40 s; as well as pulsed ultrasound with a generation frequency of 2.64 MHz with an intensity range of 0.4-1.0 W / cm ² for 20-60 s - on nucleated cells larger than 7 μm, and on nuclear-free cells up to 6 μm in intensity 0 , 7-1.0 W / cm for 25-60 s followed by preparation of smears, their staining with trypan blue and differential dyes, analysis of the state of cytoplasmic membranes and cell viability.

The claimed invention is carried out by ultrasonic exposure to tissue, leading to a selective change in cytomorphology or to the destruction of cells / cell structures of animals of the order of artiodactyls.

Studies have been carried out to determine the characteristics of the behavior of horse blood cells in the ultrasound field, and ranges of intensities causing reversible and irreversible changes have been identified. The following cytomorphological effects of ultrasound (carrier frequency of 880 kHz and 2.64 MHz) on erythrocytes were found: change in shape, the formation of symmetrical groups around the cell and chains of red blood cells without cytolysis, the appearance of cell shadows. White blood cells changed earlier than red blood cells, 15-30 seconds after the onset of insonation. The effect on granulocytes, leading to a change in CPM, and then the cells as a whole, began earlier than on agranulocytes. In all animals in the range of 0.4-1.0 W / cm ² , in the pulsed and continuous mode of ultrasonic treatment, the same effects occurred: deformation and violation of boundaries, destruction and aggregation of cells, CPM rupture and nuclear explosion, lysis.

Disclosure of the invention. Blood was drawn from the jugular vein of horses during the annual monitoring of fasting health in the morning.

Ultrasound treatment was subjected only to the blood of healthy animals. The group of animals is riding horses (n = 10). A blood volume of 5 to 10 ml was voiced under absolutely identical conditions according to the method of the author [4]. The control was intact cells of the same animals. The source of ultrasound can be devices for ultrasound therapy operating at the generation frequencies of 2.64 MHz and 0.88 MHz - UZT-3.06 UZT - 1-01 F; UZT-1.02 C, or any other device of domestic production. The intensity of ultrasound ranged from 0.4 W / cm ² to 1.0 W / cm ² that was controlled using a differential thermocouple calibrated by intensity. The intensity of ultrasound transmitted into tissue in vitro was 90% of the nominal intensity. The action of ultrasonic waves was carried out in a continuous (frequency of 0.88 MHz) and a pulsed mode of 10 ms (2.64 MHz). Blood samples were processed in a thermostatic cuvette at a temperature of 20 ° С (thermostat U-7 s).

The duration of exposure was varied in different series from 15 s to 1 minute. The effect of ultrasound on blood cells was judged by quantitative and qualitative changes. Changes in the CPM permeability of all cells were determined by trypan blue test [10]. Reversibility of changes was checked after 20 minutes. In case of loss of viability, the cells remained stained blue. Smears were made, stained by the method of rapid differential staining of biological products with the Diffkvik set and the leukogram was determined by the standard method [7]. The result of exposure was observed under a light microscope (immersion, LOMO microscope, 100 ^x / 1.25 objective, 10 ^x / 18 eyepiece). Reference row of cell sizes according to N.A. Lyubin [11]. Statistical data processing is available in the Statistica 6.0 application package. The differences were considered significant at p <0.05.

Continuous ultrasound

The features of the effect of continuous ultrasound on the cell membranes and cells as a whole are shown in table 1 and in photographs of blood smears of horses (Fig. 1-3). Some examples of cytomorphological effects.

0.05 W / cm ² 40-50 s. The leukogram without features, but from 5% (treatment 40-45 s) to 20% (50 s) of leukocytes with altered CPM. CPM lysis after 60 s of treatment.

0.2 W / cm ² 15-45 s. The leukogram is without features, but there is a change in the volume of the cytoplasm of the lymphocytes compared with the control.

0.2 W / cm ² 60 s. In 20% of segmented neutrophils and eosinophils, CPM lysis. Other types of leukocytes without features. Leukogram changes within the reference series.

0.4 W / cm ² 30 s. (Fig. 1) Leukogram without features. The beginning of the destruction of white blood cells. Segmented neutrophils (Fig. 1, top) and lymphocytes with signs of cell lysis and destruction. After 45 s, changes in the nuclei of lymphocytes and the volume of their cytoplasm.

1.0 W / cm ² 20 s. Change in the permeability of the CPM of red blood cells and platelets, the destruction of the CPM of leukocytes. 30-40 s and platelet aggregation and leukocyte lysis (Fig. 2). 60 s - platelets in large groups. Only lymphocytes are preserved, but 90% of them are without cytoplasm. There are no other white blood cells in the smear. Under the influence of continuous ultrasound in the blood of a horse, changes in red blood cells were recorded. So, after irradiation with an intensity of 1.0 W / cm ^{2 for} more than 30 s, inclusions in cells and aggregation were detected. In this case, degenerative changes in white blood cells occurred. In FIG. Figure 3 shows the aggregation of cells: platelets and a lymphocyte surrounded by red blood cells.

Pulse ultrasound

Changes in blood cells after insonation by pulsed ultrasound began to be recorded at high ultrasound intensities (Table 2 and Fig. 5-7) due to a decrease in the thermal component at the time of the action of the emitter.

0.4 W / cm ² , 20 s. Degenerative changes in neutrophils and eosinophils (Fig. 4). 30 s - lysis of the cytoplasm and degenerative changes in the nuclei of leukocytes of all types. Platelets and red blood cells without features. 60 s — complete leukocyte lysis, change in the permeability of the platelet CPM, deformation of the red blood cell CPM (lymphocyte lysis in Fig. 5).

0.7 W / cm ² , 25 s. Lysis of the largest types of leukocytes-monocytes, degenerative changes in neutrophils and eosinophils (Fig. 6).

Reversible changes in the permeability of the platelet CPM while maintaining their viability. The beginning of cytomorphological changes in red blood cells: single cells have club-shaped thickenings.

0.7 W / cm ² , 60 s. White blood cell lysis. Changing the shape of red blood cells. The appearance of cytochains (Fig. 7) and cell shadows. Hemolysis is possible.

1.0 W / cm ² , when irradiated for more than 20 s, it is difficult to identify cells. Irreversible changes in white blood cells occur. 30 s - foaming of the cytoplasm and nuclear rupture. Platelet Aggregation 60 s - lysis of nuclei and cytoplasm of leukocytes.

findings

1. Ultrasonic treatment of samples of cell suspensions in vitro by ultrasonic waves of a generation frequency of 2.64 MHz in a pulsed mode, I _sata 0.7-1.0 W / cm ² for a duration of 25-60 s successively changes the permeability and then the structure of the CPM nuclear-free cells of a size of 4.5-5.8 microns.

2. The action of pulsed ultrasound (2.64 MHz) with an intensity range of 0.4-1.0 W / cm ² for 20-60 s on nucleated cells larger than 7 μ in size changes the permeability, the structure of the CPM, and then causes their destruction.

3. The intensity of 0.05-1.0 W / cm ² continuous ultrasonic waves with a frequency of 0.88 MHz when irradiated for 15-60 with a suspension of nucleated cells larger than 7 μ affects the permeability and structure of the CPM, and then on the whole cell altogether changing its morphology.

4. Influence on the membranes of nuclear-free cells up to 6 μ in size of a continuous ultrasonic wave with a generation frequency of 0.88 MHz can be directed with an intensity of 0.7-1.0 W / cm ² for 20-40 s.

Bibliography

1. Oleshkevich A.A. The effect of continuous and modulated ultrasound on animal blood cells in vitro / V Congress of Russian Biophysicists. Materials of reports: in 2 volumes - Rostov-on-Don: SFedU. - T. 2: 2015 .-- S. 107.

2. Maksutova D.Zh. The use of focused ultrasound under the control of magnetic resonance imaging // Problems of reproduction / Russian Journal of Human Reproduction. 2009. No2. S. 30-36.

3. Oleshkevich A.A., Nosovsky A.M., Kaminsky E.V. Experimental and theoretical substantiation of methods for increasing the production of cells of various etiologies after treatment with acoustic (ultrasonic) waves. Part 1. A method of intensifying the processes of bacterial cell metabolism in suspension // Biomedical Radioelectronics, 2014. - No. 2. - S. 53-57.

4. Oleshkevich A.A., Kaminskaya E.V., Nosovsky A.M. Experimental and theoretical substantiation of methods for increasing the production of cells of various etiologies after treatment with acoustic (ultrasound) waves. Part 2. Methods of acoustic stimulation of animal cells // Biomed. radioelectric. 2014. - No. 3. - S. 33-39.

5. Botyan N.I., Akopyan V.B., Oleshkevich A.A. Cytostatic effect of ultrasound combined with medicine on photobacteria // V National conference on biomedical physics & engineering, Bulgaria, 1989, p. 109-110.

6. Shary N.I., Kovaleva B.M., Kryukova G.V. The influence of the repeated action of ultrasound on the culture of human cells // Hygiene and sanitation. 1979. No. 2. S. 48-50.

7. Oleshkevich A.A., Pashovkin T.N. The possibility of changing animal leukograms under the action of continuous ultrasound of the therapeutic range of intensities // Agrarian Russia. - No. 6 (2015). S. 13-17.

8. Pashovkin T.N., Sadikova D.G. Stratification, separation and concentration of cells in the field of standing ultrasonic waves // Acoustic. l T. 55. 2009. No. 4-5. S. 575-585.

9. Akopyan VB, Ershov Yu.A. The basics of the interaction of ultrasound with biological objects MSTU. N.E. Bauman. M., 2005.223 s.

10. Skibo Yu.V., Abramova Z.I. Research Methods for Programmed Cell Death: A Teaching Aid “Apoptosis Theory” / Yu.V. Skibo, Z.I. Abramova. - Kazan: Federal State Autonomous Educational Institution of Higher Professional Education KFU, 2011. - 61 p.

11. Methodological recommendations for the determination and removal of hemograms in agricultural and laboratory animals with pathologies // N.A. Lyubin, L.B. Konova. Ulyanovsk, 2005 - 112 s.

Claims (1)

A method of directed exposure to cells of tissues of animals of the order of artiodactyls, including exposure to a cell suspension of a continuous ultrasonic wave with a frequency of 0.88 MHz, an intensity of 0.05-1.0 W / cm ² for 15-60 s - on nucleated cells larger than 7 μ and on nuclear-free cells up to 6 μ in size - with an intensity of 0.7-1.0 W / cm ² for 20-40 s; as well as pulsed ultrasound with a generation frequency of 2.64 MHz with an intensity range of 0.4-1.0 W / cm ² for 20-60 s - on nucleated cells larger than 7 μm, and on nuclear-free cells up to 6 μm in intensity 0 , 7-1.0 W / cm ² for 25-60 s followed by preparation of smears, their staining with trypan blue and differential dyes, analysis of the state of cytoplasmic membranes and cell viability.

Images