RU2589288C1 - METHOD FOR STIMULATING PRODUCTION OF ERYTHROPOIETIN IN A CELL CULTURE in vitro - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to experimental biology and medicine, specifically to a method for erythropoietin production stimulation of bone marrow cells in vitro. Method for stimulating production of erythropoietin bone marrow cells in vitro, consists in that non-adhesive fraction of myelocytes is cultivated in a CO₂-incubator in a complete culture medium of following composition: 90 % RPMI-1640 medium, 10 % ETS, 280 mg/l L-glutamine, 50 mg/l gentamycin, addition of inhibitor of protein kinase p38 in concentration 300 mcM under certain conditions.

EFFECT: method described above enables to efficiently stimulate development of erythropoietin with non-adhesive myelocariocytes in vitro.

1 cl, 1 tbl, 1 ex

Description

The invention relates to the field of experimental biology and medicine and relates to a method of stimulating the production of erythropoietin in vitro.

The closest in technical essence, mechanism of action and the achieved result (prototype) is a method of stimulating the production of erythropoietin using concavalin-A (Con-A), which is added in the required concentration to the tissue culture and incubated at 37 ° C, 5% CO ₂ and 100% air humidity within 24 hours [1].

The disadvantage of this method is a slight stimulation of hematopoietin production under the influence of this mitogen.

The problem solved by this invention is to increase the efficiency of the method.

The problem is solved in that the non-adherent fraction of bone marrow, which is cultivated in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity for 24 hours in a complete culture medium of the following composition: 90% RPMI-1640 medium , 10% ETS, 280 mg / l L-glutamine, 50 mg / l gentamicin, add a p38 protein kinase inhibitor at a concentration of 300 μM, which determines the functional activity of hematopoietic cells.

New in the present invention is the use of an inhibitor of protein kinase p38 as a stimulator of erythropoietin production.

Today, among the various pharmacological agents used as haemostimulants, the most significant group are drugs based on endogenous hemopoietic stimulants (erythropoietin and colony stimulating factors), which, when associated with specific receptors, trigger numerous signaling mechanisms involved in the implementation of key cell functions - differentiation, proliferation, aging, tumor transformation. Recombinant erythropoietin preparations deserve special attention, the hemostatic effects of which are comparable to the activity of natural erythropoietin [2]. Given the promise of developing drugs with targeted action for modulating the production of endogenous hematopoietins by activating / inhibiting intracellular signaling, the study of the synthesis and specific biological properties of this hematopoietin in the in vitro system will form the basis of the methodology for creating this group of pharmaceuticals.

It is known that along with classical STAT pathways, MAP and PI3K / Akt signaling cascades can be involved in the conduction of signals from growth factors into the cell [3, 4, 5]. It should be noted that these pathways also play an important role in controlling the production of hematopoietins by the cells of the hematopoietic microenvironment [6, 7]. However, studies whose results would reveal the specific role of these signaling pathways (their activation or inhibition) in the production of erythropoietin in the literature we studied were not found. Thus, studies aimed at studying ways to control the level of hematopoietins are undoubtedly relevant and will help in the development of new drugs to stimulate hematopoiesis.

The fact of the use of a p38 protein kinase inhibitor with the achievement of a new technical result, which consists in effectively stimulating the production of erythropoietin in vitro, is not obvious to a specialist.

The claimed essential features in the aggregate showed new properties that are not explicitly derived from the prior art in this field. The present invention can be used in experimental biology and medicine with access to practical health care. An identical set of features was not found in the study of the state of the art in patent and medical literature.

Based on the foregoing, the claimed technical solution should be considered relevant criteria: "Novelty", "Inventive step", "Industrial applicability".

The method is as follows.

A suspension of bone marrow cells of mice (5 × 10 ⁶ cells / ml) is divided into adhesive and non-adhesive fractions. The resulting non-adherent cells are incubated in a complete culture medium of the following composition: 90% RPMI-1640 medium, 10% ETS, 280 mg / L L-glutamine, 50 mg / L gentamicin, 300 μM p38 protein kinase inhibitor, in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity for 24 hours. At the end of the incubation, conditioned media are collected after centrifugation at 1500 rpm for 10 minutes and stored for no more than a month at -26 ° C.

The proposed method was studied in experiments on male mice of the C57B I / 6JY line in the amount of 2 pcs., Weight 20-22 g. Animals were obtained from the department of experimental biological models of the Research Institute of Pharmacology and Regenerative Medicine named after E.D. Goldberg (certificate available).

Example 1

A suspension of bone marrow cells obtained under sterile conditions (a box equipped with a laminar) was incubated for 45 min in RPMI-1640 medium (Sigma, USA) containing 10% ETS (Sigma, USA) in 90 mm plastic cups Petri (Corning, USA) with a diameter of 35 mm for 40-50 minutes at 37 ° C, 5% CO ₂ and 100% humidity to isolate a non-adherent fraction of myelocaryocytes. Then non-adherent myelokaryocytes were collected with a pipette and incubated for 24 hours in a complete culture medium of the following composition: 90% RPMI-1640 medium (Sigma, USA), 10% ETS (Sigma, USA), 280 mg / l of L-glutamine (Sigma, USA), 50 mg / l of gentamicin (Sigma, USA), 300 μM p38 protein kinase inhibitor (Calbiochem, USA), at 37 ° С, 5% CO ₂ and 100% humidity. At the end of the incubation, conditioned media were collected and the level of erythropoietin was determined in them by ELISA using a kit of R&D systems (USA) according to the manufacturer's guidelines.

In this case, the control and comparison group were conditioned media from non-adherent bone marrow cells without the addition of an inhibitor and with the addition of concavalin-A, respectively, obtained by the method of the prototype: similar to the method described above.

The experiment showed that the addition of a p38 protein kinase inhibitor to the culture of non-adherent myelocaryocytes significantly increased the level of erythropoietin in conditioned media compared to that in the supernatants obtained by culturing non-adherent myelocaryocytes without an inhibitor and with the addition of concavalin-A (Table 1).

Table 1 The level of erythropoietin (EP, mU / mL) in conditioned media from non-adherent myelocaryocytes of CBA / CaLac mice cultured without an inhibitor (1), with the addition of concavalin-A (2) or a protein kinase inhibitor p38 (3), (X ± m) Groups EP one 12.69 ± 0.45 2 14.25 ± 0.53 * 3 26.33 ± 0.83 * #

* - the significance of differences with the indices in the 1st group was noted at p <0.05;

# - the significance of differences with the indicators in the 2nd group at p <0.05 was noted.

Thus, incubation of the non-adherent fraction of the hematopoietic-inducing microenvironment with the p38 protein kinase inhibitor stimulates the production of erythropoietin in vitro.

Proposed as an invention, the method allows you to effectively stimulate the production of erythropoietin non-adherent myelokaryocytes in vitro.

Cited literature

1. Goldberg E.D., Dygay A.M., Shakhov V.P. Methods of tissue culture in hematology, Tomsk, 1992, p. 64-65.

2. Digay A.M., Zhdanov VV, Udut U.V. Pharmacological regulation of erythropoiesis, Moscow, 2009, p. 58-71.

3. Fischer O.M., Hart S., Gschwind A., Ullrich A. EGFR signal transactivation in cancer cells. // Biochemical Society Transactions, 2003, 31, p. 1203-1208.

4. Grimberg A. Mechanisms by which IGF-I may promote cancer. // Cancer Biol Ther, 2003, 2, p. 630-635.

5. Whitmarsh A.J., Cavanagh J., Tournier C. et al. A mammalian scaffold complex that selectively mediates MAP kinase activation // Science. 1998, 281, p. 1671-1674.

6. Digay A.M., Zhdanov VV, Miroshnichenko L.A. et al. // Bull. ex-per. biol. 2014. No9. S. 282-286.

7. Digay A.M., Zhdanov VV, Zyuzkov G.N. // Bull. expert. biol. No. 1. S. 39-49.

Claims (1)

A method of stimulating the production of erythropoietin by bone marrow cells in vitro, which consists in the fact that the non-sticking fraction of myelokaryocytes is cultured in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity for 24 hours in a complete culture medium of the following composition: 90 % RPMI-1640 medium, 10% ETS, 280 mg / l L-glutamine, 50 mg / l gentamicin, with the addition of a stimulant, characterized in that a p38 protein kinase inhibitor at a concentration of 300 μM is used as a stimulant.