EA019365B1 - The method of boosting stem cell proliferation and increasing their resistance to the adverse effects - Google Patents

Abstract

The invention relates to the field of biotechnology, medicine, pharmacology, cell biology and bioengineering and other fields of biology and medicine which use stem and progenitor cells of different levels of differentiation and differentiated cells of various tissues, particularly for accelerated growth and creation of donor bioimplant or autoimmune human and/or animal stem cells. Technical effect on which the invention is directed is the boosting (activation) of stem cell proliferation and increased resistance to the adverse effects of human and animal stem cells which is not associated with the negative impact of high-frequency electromagnetic fields. Technical result is achieved by treating the cell culture by weak and low-frequency magnetic field collinear with the static magnetic field of the Earth. When using the proclaimed method the following is observed: 1) the increase of the number of stem cells in culture after exposure to a weak magnetic field characteristic for a given cell culture doubling half period, for example, the number of cells increased more than 2.5 times per 24 hours (shown with full doubling of intact cells taken equal to 48 hours); 2) the increase of the intrinsic magnetic radiation amplitude of the primary stem cells culture measured by a SQUID magnetometry that indicates their increased activity; 3) 2 times increased stability of human stem cells to the development of apoptosis and cell synchronization is predominantly in the G1 phase of the cell cycle.

Description

The invention relates to the field of biotechnology, medicine, pharmacology, cell biology and bioengineering and can be used in other branches of biology and medicine, which use stem and progenitor cells of different levels of differentiation and mature differentiated cells of various tissues, in particular, for the accelerated creation of bioimplants from donor or autoimmune stem cells of humans and / or animals.

The proposed method is designed to activate the proliferation of stem cells and increase resistance to adverse effects of human and animal tissues.

Currently, there are a large number of technical solutions that offer various methods of biophysical effects on plant and animal cells (RF patent No. 2332841, published September 10, 2008; RF patent No. 2314844, published January 20, 2008; RF patent No. 2174850, published October 20. 2001; RF patent No. 2049501, published on December 10, 1995; RF patent No. 2158147, published on October 27, 2000). Common disadvantages that combine these inventions are the duration of exposure (at least 3 days), multi-stage methods and exposure to biomaterial with extreme loads.

Closest to our proposed method is a patent of the Russian Federation No. 2405599 (published December 10, 2010). The method according to RF patent No. 2405599 includes irradiating a biological object with an external electromagnetic field with variable parameters. Irradiation is carried out in a living organism in the area of the anatomical location of the red bone marrow by electromagnetic radiation of extremely high frequency in the range of 35-80 GHz, with a surface energy flux density in the range of 0.1-10 mW / cm ² , modulated in amplitude, with a change in the modulation frequency in range of 4-10 Hz. The flux density of 0.1-10 mW / cm ² . The method provides activation of the formation of stem cells of red bone marrow while stimulating the proliferation and differentiation of red bone marrow cells in the body.

The disadvantage of the invention is that electromagnetic radiation of gigahertz frequencies is a dangerous effect in relation to biological objects, in particular, carrying genetic information that is preserved in stem cell nucleoproteins, which is rigidly stored in contrast to cells of a different kind.

The technical result to which the invention is directed is the activation of stem cell proliferation and increased resistance to adverse effects of human and animal stem cells, not associated with the negative effects of a high-frequency electromagnetic field. The technical result is achieved by processing the cell culture with a weak magnetic field of low frequency. In the proposed method, the acting factor is a weak alternating magnetic field collinear to the Earth’s field, magnetic induction B and frequency которого of which is determined by the formula proposed by V.V. Lednev (Lednev V.V. Biological effects of extremely weak alternating magnetic fields: identification of primary meshes. B book Modeling of geophysical processes. 2003, 130-136),

B = Vos + Vls with $ 2 $ 1, where V _ss and B _AC are the values of the magnetic induction of the constant (from the Earth’s field) and the variable field components (specified by the magnetic generator), respectively;

/ is the frequency of the variable component.

In this case, the frequencies / alternating magnetic fields, known as resonant for the Ca ^{2+ ion} in the cell, were selected from the range £ = 25-42 Hz.

The low-frequency magnetic field used does not have resonant-destructive properties, comparable in magnitude to the Earth’s magnetic field and collinear to it.

The achievement of the technical result in the method is confirmed by the fact that in human stem cells subjected to irradiation by our proposed method, a number of intracellular processes are activated, namely, polarization of the cell membrane, change in the total dipole moment of the cell, activation (acceleration) of the process of doubling centrioles, and also a change in the orientation of the mitotic spindle and structural and functional rearrangements of stem factors [Orlova E.V. The influence of structural and functional characteristics of stem factors on asymmetric cell division and regulation of apoptosis. Biomedical journal Meb1te.gi, 2009, v. 10, p. 113-126.], The totality of which prepares stem cells for directed differentiation and accelerated proliferation in a given direction.

In FIG. 1 shows an increase in the number of human mesenchymal stem cells (MSCs) after irradiation in an alternating magnetic field B _AC = 89.4 μT; frequency £ = 37.1 Hz; the temperature of the thermostatically controlled cell is 37 ° C for 24 hours (half of the standard period of the doubling period) - (A - medium is oxidized, has a light brown color), compared with cells cultured without exposure to a low-frequency magnetic field (B - medium is conditioned, has color standard for α-MEM).

In FIG. 2 shows the results of flow cytometry of a stem cell culture processed according to the proposed method, A - control, B - culture after treatment by the proposed method.

In FIG. 3 presents the results of electrophoresis and PCR products.

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An example implementation of the method.

The experiment was conducted on mesenchymal stem cells (MSCs) isolated from human fat (provided by Biolot, St. Petersburg). The doubling period of these cells is initially about 48 hours. The cells were taken in passage 3 and placed in T ₂₅ culture bottles with α-MEM medium standard for this type of cell. The following experiments were performed: experience and control, in each of which about 200 thousand cells / ml were used.

In the experiment, the Earth’s constant magnetic field was measured, which amounted to V _SS = 48.6 μT; and with the help of a magnetizing system, an alternating magnetic field B _AC = 89.4 μT was established; frequency 1 = 37.1 Hz, collinear to the Earth's field according to the above formula

B- Vos + Vas soztf, where VAc is the amplitude of the external magnetic field, T;

- its frequency, T;

irradiation time 24 h; temperature of thermostatic cell 37 ° С.

In the above experiment, cells were transplanted into experimental cells under extreme conditions at an increased density close to the maximum for this type of cell. At the same time, microscopy reveals the effect of reducing the number of dendritic processes, and the cells line up in columns, the appearance of which is the first visual indicator of the readiness of cells for differentiation into fibroblasts (i.e., adverse conditions were created that increased the likelihood of cultured cells losing their stem cell properties). After 24 hours of exposure in a weak magnetic field (outside the CO ₂ incubator, without stabilizing the pH of the medium), an increase in the number of dendritic processes in cells was observed to the basal level of the standard for this stem cell culture, i.e. the maximum branching of dendrites and the spreading of cells were observed even in the presence of an oxidized medium (Fig. 1A). Moreover, in the experiment there was an increase in the number of cells by 2.8 times, i.e. there was an acceleration in the growth of culture (Fig. 1), despite the substandard cultivation conditions.

Thus, the processing of cells using a low-frequency magnetic field in the selected range with the above input values not only accelerates the growth of human stem cell culture by 2.8 times, but also minimizes the effect of the negative pH shift, supporting proliferation and differentiation. In other words, in this system, a certain selection condition is realized for the system’s own stability (the total enthalpy of the system (cell) is minimized), and, as a result, spontaneous disturbances in the system are minimized, caused, for example, by a non-optimal cultivation mode. An increase in the amplitude of the intrinsic radiation of the primary stem cell culture, recorded with a SQUID magnetometer, indicates their increased activity.

Treated stem cells undergo apoptosis to a lesser extent and make more synchronous transitions in the phases of the cell cycle, as illustrated in FIG. 2.

The results of gene expression after activation of proliferation in an alternating magnetic field were also analyzed during the half-period of the doubling of human stem cells characteristic of a given cell culture. In control and experimental cells, mRNA levels were compared to study the expression of genes involved in the regulation of the cell cycle, proliferation, differentiation, and cell death processes: cus11pE1 (official symbol: CCIO1), cus11pE1 (CCIE1), p21 / \\ aH (SOKMA), ErB3 (EBBB3), k167 (ΜΚΙ67), ΜΌΚ.1 (ABCB1), p16 (SPKY2A), p27 / k1p (SOKMV), ΥΒ1 (UVKh1), bax (BAC), bak (BAK1), bc1Xb (BK2B1), bc12 ( BC2), Go8 (PO8), tus (ICC), gas (NKA81), bad (BAO1).

Isolation of total RNA.

Suspensions of control and experimental cells (~ 10-12 × 10 ⁶ ) were presented for analysis. Cells were centrifuged for 10 min at 4000 d, cell pellets were resuspended in 2 ml of a lysing solution containing 4 M guanidine isothiocyanate, 0.02 M sodium citrate, 0.5% sarcosyl, 0.1 M mercaptoethanol. Then 1/10 part of 1M sodium acetate pH 4.4 was added. After stirring, an equal volume of phenol balanced with water and 1.5 volumes of a mixture of chloroform and isoamyl alcohol (24: 1) were added. The mixture was vortexed and incubated for 15 minutes at 2-8 ° C. Next, the tubes were centrifuged in a bucket rotor for 20 min at 4000 d with cooling. The upper phase was transferred to another tube. An equal volume of a phenol-chloroform mixture (2: 1) was added to it, stirred on a vortex and centrifuged again for 20 min at 4000 d. An equal volume of isopropyl alcohol was added to the upper phase and left overnight at -20 ° С. Then it was centrifuged for 20 min at 4000 d, the precipitate was washed with 80% ethanol and dissolved in 100 μl of water treated with diethyl pyrocarbonate. 1/20 volume of 4M LiCl and 2 volumes of ethanol were added to the RNA solution. The isolated RNA was stored at -20 ° C. The concentration of the isolated RNA was measured using a 8Tigres p1iz spectrophotometer (WuWab).

Conducting a reverse transcription reaction.

The RNA pellet (10 μg) under ethanol was washed with 1 ml of 80% ethanol and dissolved in 12 μl of water. 1 μl of 10 tM idobT was added to the tube and incubated at 70 ° C for 5 min. After cooling on ice for 5 minutes, the mixture was left at room temperature for 15 minutes. Then, 4 μl of 5X reverse transcription buffer, 2 μl of 10 tM 6ΝΤΡ, 1 μl of Weuei reverse transcriptase were added

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Άίά (Regshep1a8). The reaction was carried out for one hour at 42 ° C. Samples were stored at -20 ° C. Polymerase chain reaction:

Amplification of the studied genes was carried out in a mixture of the following composition: 10XTAC] buffer (Regshep1az) - 2 μl tM MDS1 ₂ - 1.6 μl tM 6ΝΤΡ - 0.4 μl tM primer 1 - 0.2 μl tM primer 2 - 0.2 μl

Tad polymerase (5i / μl) (Reptep1az) - 1 μl

DNA (reverse transcription product) 3 μl of water - 1.6 μl in a Maz1egSue1eg dgaSep amplifier! (Errepbog!) In the following conditions:

Operations Temperature ° С Time Number of cycles Initial denaturation 95 5 minutes one Denaturation 95 20 s Annealing 60 20 s 40 Synthesis 72 30 s Final synthesis 72 2 minutes one

The selection of oligonucleotide primers and conditions for PCR was performed using the program Θ1ί§ο 4.0. The following primers were used for PCR:

SusnpO 1 5 ’STSSSSASSAAS ASAASTSK'S ACC 3’

Сус11пО2 5’6ОАТ0ОА0ТТ0ТСССТ0ТА6АТСКА 3 ’

SusipE 1 5'ACSTSTTTTTGTSSASSASSATSSAOAT6 3 ’

SusNpE2 5 ’SATSSTSS A ATA ATCC6 AStSSTTS 3’

P211 5’STTSbSSSSASTASTSASASSO 3 ’

P212 5 ’SetSKYUAASOTA6A6STta <US 3’

EGB1 5 SSTSAOOTOTOASSOOOAT6C 3 ’

EGV2 5 ’AS ASAATTSATTSATSSSS ASSASS 3’

Κ.Ϊ671 5 GOTSTASATSSOTATSSASSGTSSTO 3 ’

ΚΪ672 5 ’C ATTTTS AGASSTSAASSAASSATS AATAA 3’

IOC I 5 ’TTTSAATSTTTSSSTATTS AAATTSS 3’

ΜϋΚ2 5 'STTTS AS ATS ASATSTTSTAAATTTSSTSS 3 ’

P161 5’SSSTSSASSSSSSSSSSASAAS 3 ’

P162 5’CSTASASOST6CSTSSTSASTASZS 3 ’

P271 5 SSSUOASTT6SA6AA6SAST6S 3 ’

P272 5 SSSASSTT6SA0SSASSTTT6 3 ’

ΥΒ1 5’TSSSSASSTTASTASATSSSCZA <ZASS 3 ’

ΥΒ2 5’TASSST6TSTTTSSSSASSASSASS 3 ’

Sun121 5 ’SSSSTSTSOATOASTSASTASS G6LLS 3’

Sun122 b'OSSAAASTSSASSASASGSGGSASASASA 3 ’

Vakh1 5’GTASSA'GSSSSSSLSSLSSSSASASZ ·

Вах2 ЗПТААССГГСЛССТСТССССССССАСССТ 3 ’

Vak 1 5 ’AT АСС АГССТСССТТС6СККК ЛАОС 3’

Вак2 5САСААССТТСТАСТСАТАСССАТТСТСТСКСС 3 ’

Vs1X1 Z'GAGSSLTSSASSTTTSSSSLSTAAASSAGASAS 3 ’

Vs1X2 5 SOSAASSTTSSTSTS AT ATSSTSTSSS 3 ’

In hell! 5’ATSSSTSYUSSTTSATSAS 3 ’

Va§2 5 ’<EASASTOST6 OSSATSU 3’

GO81 Z’LSATSTSTSSSTSTSSSTTSATSTS 3 ’

Rob2 5’AASTSAGSAAASSSSSTSSSTSTTS 3 ’

Mus! 5’LASAAATAAAASSSSSSSSAASSTA 3 ’

Mus2 Z’GSSSTASK'TSTTSLLSYYYYGSG'P'SAA 3 ’

How! 5 “SASSAATATSASSSSASASAATASASSATTS 3’

K; q2 5 ATTATTOATSYUSAAATASASASA6CAA6C 3 ’

To confirm an equal amount of nucleic acids in the studied samples,

- 3 019365 amplification of the actin gene with the corresponding primers

Αοίίηΐ CCAASASASTASSTOTSTSSSSYUS

Αοίίη2 TASTSST6STT0STSATSSASATSTS

Electrophoresis and quantification of PCR products. PCR reaction products were separated in a 6% polyacryamide gel (composition: 1.4 ml of a 30% AA solution, 1.4 ml of 5X TBE buffer, 4.2 ml of dist. Water, 30 μl of 10% ammonium persulfate (AP8), 20 μl TEMED.) In 1XTVE buffer (0.089 M Tris, 0.089 M Boric acid, 0.002 m ΕΌΤΆ pH 8.3) 40 min at 20 mA. After staining with ethidium bromide solution (1 μg / ml), the gels were photographed and quantified using the To1a1EaB2.01 program. in comparison with standard samples. For this, standard samples with a known concentration of complementary DNA were titrated on a polyacrylamide gel. After scanning the gel, the intensity of the bands was quantitatively measured using the program. Then, a calibration graph was constructed, according to which the relative amounts of the studied amplicons were determined.

Quantification of the results shown in FIG. 3, (A) - relative to control (1) in experimental cells (2), a change in the amount of mRNA of the following genes was detected: k167 - 4x increase; p27 - 2x magnification; Bax - 1.5x magnification; Bc1X - 1.5x magnification; Bc12 - 2x magnification; 1oz - 2x increase; LAD - 1.7x magnification.

Thus, the activation of anti-apoptotic genes is more pronounced than that of pro-apoptotic (Lax), but it should be borne in mind that the apoptotic proteins Bax and Bax can form stable, apoptotic-blocking conglomerates with anti-apoptotic proteins, for example, B1X and Bc12.

In addition, in the experimental cells, the synthesis of mRNA of the ΜΌΚ and Lb genes was shown (see Fig. 3B). Increased expression of ΜΌΚ is associated with drug resistance of mammalian cell cultures [Сорор ΙΜ 1993. P-§1usorgo1esh 81gis1ige apy eνο1иΐ ^ οηа ^ in Yosho1o§1e8. Su1o1esipo1ou 12: 1-32].

At the same time, the expression of the genes of bacillum, bacillus, p21 (SHAN), Ebb3, p16, b1, tcu, gas did not change (see Fig. 3B), which indicates the absence of the ability of the stem cells activated in the above way to transform into tumor cells (in the presence of a similar possibility is the activation of the above series of cyclins).

Thus, the achievement of the technical result is confirmed:

1) an increase in the number of human stem cells in a culture after exposure to a weak magnetic field during a doubling half-life characteristic of a given culture of cells, for example, over 24 hours the number of cells increases by more than 2.5 times (with a full doubling period of intact cells taken equal to 48 h);

2) an increase in the amplitude of the intrinsic magnetic radiation of the primary stem cell culture, as measured by a SQUID magnetometer, indicates their increased activity;

3) human stem cells, after exposure in a weak magnetic field during the doubling half-life characteristic of the given cell culture, are 2 times more resistant to the development of apoptosis and are synchronized mainly in the O1 phase of the cell cycle.

Claims (1)

CLAIM A method of activating proliferation and increasing resistance to adverse effects of cultured stem cells, which consists in exposing the stem cells to an alternating magnetic field, characterized in that the alternating magnetic field is applied collinear to the Earth's magnetic field, while the frequency of the alternating magnetic field is set in the range from 25 to 42 Hz , and its amplitude is set in the range from 75 to 110 μT.