RU2330674C1 - Method of intensification of stem cells mobilisation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: in case of extreme affections or pathological conditions accompanied by stem cells mobilisation, or in case of exposure of stem cell functional activity modifiers causing stem cells mobilisation, hyaluronidase is injected intraperitoneally in dose 500-1500 CU/kg within 2 days once a day.

EFFECT: increased level of stem cells mobilisation.

11 ex, 11 tbl

Description

The invention relates to the field of experimental biology and medicine, specifically to cellular technologies and pharmacology.

It is known that extreme effects and the development of certain diseases are accompanied by activation of adaptation mechanisms of the “deep” reserve associated with stem cells (SC) [1, 2, 3, 4, 5]. In this case, mobilization of certain progenitor cells into the blood can often take place, which can subsequently participate in the regeneration of affected target organs. Moreover, the activation of these mechanisms in some cases is insufficient for the "successful" adaptation of the body [1, 4, 5].

Known methods of pharmacological mobilization of various types of stem cells used in the experiment to develop promising methods of treating diseases using various cytokines: granulocyte colony stimulating factor (G-CSF) [1, 3, 6], stem cell factor (FSK) [7] peptide analogue of SDF-1 factor (CCE-0214) [8] and other factors (GM-GSF, IL-2, IL-8, IL-11, MIP-1, 2, erythropoietin, thrombopoietin [6] ) In this case, the most common (due to its effectiveness and safety) is the way to mobilize SC using G-CSF. At the same time, to achieve a more significant mobilizing effect, researchers also use combinations of various cytokines [9]. However, even the complex use of the factors included in the cohort, stimulating the mobilization of SC, gives very mixed results [6].

An adequate prototype was not found by the authors.

In connection with the foregoing, enhancing the mobilization of SC in extreme conditions and certain diseases, as well as under the influence of various pharmacological modifiers of the functional activity of SC, is an urgent task, the solution of which, in turn, can be an important step in improving the effectiveness of treatment of various diseases.

The problem solved by this invention is to increase the level of stem cell mobilization under extreme conditions, pathological conditions and with the introduction into the body of factors that cause stem cell mobilization.

The task is achieved by the fact that in extreme conditions and pathological conditions accompanied by mobilization of stem cells, or when factors causing mobilization of stem cells are introduced into the body, hyaluronidase is administered intraperitoneally once a day for 2 days at a dose of 500-1500 U / kg.

New in the proposed method is the introduction of 1 time per day for 2 days of hyaluronidase at a dose of 500-1500 UE / kg

At present, methods of cell therapy seem to be very promising for the treatment of various diseases, including with the help of pharmacological agents capable of regulating stem cell functions. At the same time, the important role of hyaluronic acid (HA) in the regulation of the functional state of various types of stem cells is known today. HA is the most common glycosaminoglycan (GAG) involved in the formation of the intercellular matrix of various tissues, including bone marrow [10, 11, 12]. In this case, stem cells and progenitor cells of varying degrees of maturity in situ are associated with hyaluronate via CD44 and RHAMM receptors [13, 14]. The participation of hyaluronic acid in the implementation of the process of mobilization of SC is still not studied. A known enzyme that breaks down HA is hyaluronidase, the introduction of which can disrupt the engrafting of exogenous SCs in the bone marrow [13], but the possibility of hyaluronidase to influence the output of cellular elements from hematopoietic organs into the blood is absolutely not known.

The range of doses of hyaluronidase proposed in the claimed invention was determined to be effective and identified in separate series of experiments. The lack of an independent mobilizing effect of hyaluronidase in relation to SC was shown in preliminary experiments.

The fact of the use of hyaluronidase in order to enhance the mobilization of SC in extreme conditions and pathological conditions, as well as under the influence of various pharmacological modifiers of the functional activity of SC for a specialist is not obvious and does not follow explicitly from the prior art in this field. New signs can improve the efficiency of the method of mobilization of stem cells, and the present invention can be used in experimental biology and medicine with access to practical health care to create new effective methods of cell therapy. 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.

In an extreme exposure, or in a pathological condition accompanied by stem cell mobilization, or when factors causing stem cell mobilization are introduced into a laboratory animal (mouse), hyaluronidase is administered intraperitoneally once a day for 2 days at a dose of 500-1500 UE / kg .

The proposed method was studied in experiments on CBA / CaLac mice in the amount of 678 pieces, weighing 18-20 g. Category 1 mice (conventional linear mice) were obtained from the nursery of the experimental biomedical modeling department of the Scientific Research Institute of Pharmacology of the Siberian Branch of the Russian Academy of Medical Sciences (certificate is available).

As extreme impacts, models were selected that are completely different for the reasons that cause them, different in the mechanisms of developing changes at different levels of the organization, as well as effects and outcomes: immobilization stress (IS), which is a “pronounced” hematopoietic stimulating effect, - 12-hour soft binding immobilization in the supine position [15] and cytostatic myelosuppression caused by a single intraperitoneal injection of the maximum tolerated dose of cyclophosphamide (250 mg / kg) [16].

At the same time, in the bone marrow and peripheral blood on the 3rd day with IP and on the 5th day with cytostatic exposure, the content of committed mesenchymal progenitor cells — fibroblast colony forming units (CFU-F) and hematopoietic stem cells ( CCM) - granulocyte-erythro-macrophage-megakaryocytic units (CFU-HEMM) [17], as well as on the 3rd day of the experiment using the method of limiting dilutions — the number of mesenchymal (true) stem cells (MSCs) [3, 18].

As models of pathological conditions, diseases were selected that occupy a major place in the structure of general morbidity and social significance. Moreover, the selected disease models: posthypoxic encephalopathy [1], myocardial infarction [3] and chronic toxic hepatitis [2] differ significantly in etiology and pathogenesis. This circumstance (due to the similarity of many links of pathogenesis in various diseases and the presence of uniform regulatory and adaptive mechanisms of the macroorganism) makes it possible to extrapolate the results obtained on these models to the majority of known pathological conditions.

In these models of pathology in the bone marrow and peripheral blood, in each case, at certain times (indicated in specific examples), the number of MSCs was determined using myocardial infarction with the method of limiting dilutions [3, 18], and the content of MSCs and CFU-F were evaluated in experimental hepatitis [ 9], and in encephalopathy the number of MSCs, CCMs (CFU-HEMM) and CFU-F were determined [3, 9, 18].

For pharmacological mobilization of SC, we used: G-CSF at a dose of 125 μg / kg [1, 3], FSK at a dose of 50 μg / kg [7], peptide-analogue of SDF-1 factor — STCE-0214 at a dose of 10 mg / kg [8] and co-administration of G-CSF and FSK in the indicated doses. The choice of cytokines was based on various spectra of their specific activity, mechanisms of action, biological purpose and ability to cause the mobilization of various types of SC.

In this case, with the introduction of G-CSF, FSK and with their combined administration, the amount of MSC, CFU-F and CCM (CFU-HEMM) in the bone marrow and peripheral blood was determined, and with the introduction of CTC-0214 only the content of CCM (CFU-GEMM) was determined.

The results were processed by the method of variation statistics using the Student t-test and the nonparametric Wil-Coxon-Mann-Whitney U-test.

Example 1

Immobilization stress (IS) in animals was induced by 12-hour immobilization on the back using soft bindings. At the same time, experimental animals immediately after exposure 1 time per day for 2 days were injected intraperitoneally with hyaluronidase (Lidaza, FSUE NPO Microgen, Ministry of Health of the Russian Federation, Russia) at a dose of 500 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline.

During the experiment, immobilization stress on the 3rd day after exposure led to an increase in the number of CFU-F and CCM in the bone marrow. Moreover, an increase in the content of stem hematopoietic cells to 191.7% of the background was observed in peripheral blood. At the same time, the administration of hyaluronidase at a dose of 500 UE / kg significantly increased both CFU-F and CCM in the peripheral blood relative to the control, and also led to an increase in the number of circulating MSCs to 200% of the background (Table 1).

Thus, the double administration of hyaluronidase at a dose of 500 U / kg against the background of simulating immobilization stress led to an increase in the peripheral blood of both mesenchymal progenitor cells of various maturity levels (including MSCs) and hematopoietic stem cells.

Table 1
The content of various types of stem cells in bone marrow and peripheral blood with immobilization stress and with the introduction of 500 U / kg of hyaluronidase against immobilization stress (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, for 10 ⁵ myelocaryocytes CCM in peripheral blood, per 10 ⁵ mononuclear cells MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood mononuclear cells on June ¹⁰ background 5.4 ± 0.21 0.57 ± 0.1 7.8 ± 0.41 2.4 ± 0.7 12.0 ± 3.0 9.0 ± 3.0 Control (IP) 7.2 ± 0.53 * 0.71 ± 0.2 10.1 ± 0.3 * 4.6 ± 0.2 * 14.0 ± 4.0 13.0 ± 3.0 IP + Hyaluronidase 7.45 ± 0.4 * 1.6 ± 0.21 * # 9.85 ± 1.2 5.87 ± 0.5 * # 12.0 ± 3.0 18.0 ± 3.0 * * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Example 2

Immobilization stress in animals was induced by 12-hour immobilization on the back using soft bindings. At the same time, experimental animals immediately after exposure 1 time per day for 2 days were injected intraperitoneally with hyaluronidase (Lidaza, FSUE NPO Microgen, Ministry of Health of the Russian Federation, Russia) at a dose of 1500 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline.

The introduction of a dose of 1500 UE / kg of hyaluronidase when modeling immobilization stress was accompanied by a significant increase in the content of CFU-F and CCM in the peripheral blood relative to the control (up to 253.5% and 130.7%, respectively) (Table 2).

Example 3

Myelosuppression was caused by a single intraperitoneal injection of cyclophosphamide (CF) into the MTD. At the same time, against the background of the introduction of cytostatic 1 time per day for 2 days, hyaluronidase (Lidaza, FSUE NPO Mikrogen, Ministry of Health of the Russian Federation, Russia) was administered intraperitoneally at a dose of 500 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline.

The introduction of cyclophosphamide led to a decrease in the content of committed mesenchymal progenitor cells in the bone marrow and, on the contrary, an increase in the number of HSCs and ISS in the hematopoietic tissue on the 5th day of the experiment. In the peripheral blood, an increase in the content of CFU-F and hematopoietic stem cells was observed. In this case, the appointment of hyaluronidase was accompanied by a pronounced increase in the yield of committed mesenchymal progenitor cells and SSCs (Table 3).

table 2
The content of various types of stem cells in bone marrow and peripheral blood with immobilization stress and with the introduction of 1500 UE / kg of hyaluronidase against immobilization stress (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, for 10 ⁵ myelocaryocytes CCM in peripheral blood, per 10 ⁵ mononuclear cells MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 5.4 ± 0.21 0.57 ± 0.1 7.8 ± 0.41 2.4 ± 0.7 12.0 ± 3.0 9.0 ± 3.0 Control (IP) 7.2 ± 0.53 * 0.71 ± 0.2 10.1 ± 0.3 * 4.6 ± 0.2 * 14.0 ± 4.0 13.0 ± 3.0 IP + Hyaluronidase 6.3 ± 0.7 1.8 ± 0.4 * # 9.0 ± 1.4 6.0 ± 0.41 * # 12.0 ± 3.0 14.0 ± 4.0 * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Thus, a twofold administration of hyaluronidase at a dose of 500 U / kg against a background of cytostatic exposure led to an increase in certain types of stem cells in the peripheral blood.

Table 3
The content of various types of stem cells in bone marrow and peripheral blood with cytostatic effects and with the introduction of 500 UE / kg of hyaluronidase against the background of myelosuppression (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, for 10 ⁵ myelocaryocytes CCM in peripheral blood, per 10 ⁵ mononuclear cells MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 5.4 ± 0.21 0.57 ± 0.1 7.8 ± 0.41 2.4 ± 0.7 12.0 ± 3.0 9.0 ± 3.0 CF 0.2 ± 0.03 * 0.99 ± 0.14 * 14.7 ± 1.1 * 3.9 ± 0.09 * 24.0 ± 4.0 * 8.0 ± 2.0 CF + Hyaluronidase 0.4 ± 0.08 * 2.4 ± 0.6 * # 12.8 ± 0.7 * 8.0 ± 0.31 * # 22.0 ± 6.0 12.0 ± 4.0 * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Example 4

Myelosuppression was caused by a single intraperitoneal injection of cyclophosphamide (CF) into the MTD. At the same time, against the background of cytostatic administration, once a day for 2 days, hyaluronidase (Lidaza, FSUE NPO Mikrogen, Ministry of Health of the Russian Federation, Russia) was administered intraperitoneally at a dose of 1500 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline.

The introduction of a dose of 1500 UE / kg of hyaluronidase led to an increase in the number of CFU-F and CCM in the bone marrow. In this case, however, only CCM was mobilized into the peripheral blood (up to 194.9% of the control), but at a similar level with the introduction of hyaluronidase at a dose of 500 U / kg (Table 4).

Thus, a twofold administration of hyaluronidase at a dose of 1500 U / kg against a background of cytostatic exposure led to an increase in hematopoietic stem cells in the peripheral blood.

Table 4
The content of various types of stem cells in bone marrow and peripheral blood with cytostatic effects and with the introduction of 1500 UE / kg of hyaluronidase against myelosuppression (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, for 10 ⁵ myelocaryocytes JCC in peripheral blood mononuclear cells per 10 ⁵ MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 5.4 ± 0.21 0.57 ± 0.1 7.8 ± 0.41 2.4 ± 0.7 12.0 ± 3.0 9.0 ± 3.0 CF 0.2 ± 0.03 * 0.99 ± 0.14 * 14.7 ± 1.1 * 3.9 ± 0.09 * 24.0 ± 4.0 * 8.0 ± 2.0 CF + Hyaluronidase 1.4 ± 0.04 * # 1.7 ± 0.47 * 14.5 ± 1.2 * 7.6 ± 0.4 * # 18.0 ± 4.0 10.0 ± 3.0 * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Example 5

Posthypoxic encephalopathy was modeled using a 500 ml pressure chamber. The mice were placed in a pressure chamber, the lid was closed, and the mice remained there until an agonal seizure or respiratory arrest, as determined visually, for 10-15 seconds. After extracting from the pressure chamber and restoring spontaneous breathing, after 5-10 minutes, the mice were again placed in the pressure chamber also before the onset of the agonal state (generalized seizure or respiratory arrest). At the same time, experimental animals immediately after encephalopathy modeling 1 time per day for 2 days were injected intraperitoneally with hyaluronidase (Lidaza, FSUE NPO Microgen, Ministry of Health of the Russian Federation, Russia) at a dose of 500 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline.

The development of encephalopathy on the 5th day of the experiment led to an increase in the number in the bone marrow of mesenchymal progenitor cells of varying degrees of maturity, and even more of the number of CCMs. Moreover, statistically significant mobilization occurred only in relation to stem hematopoietic cells (up to 327.5% of the background). There was also a clear tendency towards an increase in the content of MSCs in peripheral blood (up to 166.7% of the background), however, these changes did not reach statistical significance. The introduction of hyaluronidase increased the content of CFU-F in the hematopoietic tissue, and also contributed to their enhanced release into the blood. In addition, the enzyme significantly increased the amount of CCM in peripheral blood and led to the development of the phenomenon of mobilization of MSCs (Table 5).

Table 5
The content of various types of stem cells in bone marrow and peripheral blood with the development of encephalopathy and with the introduction of 500 UE / kg of hyaluronidase against the background of modeling encephalopathy (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, for 10 ⁵ myelocaryocytes JCC in peripheral blood mononuclear cells per 10 ⁵ MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood mononuclear cells on June ¹⁰ background 8.7 ± 0.68 1.34 ± 0.1 9.1 ± 0.63 1.4 ± 0.6 31.0 ± 4.0 12.0 ± 3.0 encephalopathy (EP) 17.2 ± 1.3 * 2.4 ± 1.0 29.8 ± 3.4 * 8.87 ± 1.2 * 59.0 ± 9.0 * 20.0 ± 4.0 EP + hyaluronidase 28.9 ± 2.7 * # 4.6 ± 1.2 * # 25.3 ± 1.7 * 16.0 ± 2.6 * # 47.0 ± 8.0 28.0 ± 4.0 * * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Example 6

Posthypoxic encephalopathy was modeled using a 500 ml pressure chamber. The mice were placed in a pressure chamber, the lid was closed, and the mice remained there until an agonal seizure or respiratory arrest, as determined visually, for 10-15 seconds. After extracting from the pressure chamber and restoring spontaneous breathing, after 5-10 minutes, the mice were again placed in the pressure chamber also before the onset of the agonal state (generalized seizure or respiratory arrest). At the same time, experimental animals immediately after encephalopathy modeling 1 time per day for 2 days were injected intraperitoneally with hyaluronidase (Lidaza, FSUE NPO Mikrogen, Ministry of Health of the Russian Federation, Russia) at a dose of 1500 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline. The introduction of hyaluronidase at a dose of 1500 UE / kg was accompanied by an increase in hematopoietic tissue on the 5th day, not only CFU-F, but also MSCs, and, as with a lower dose, contributed to their enhanced release into the blood (Table 6).

Table 6
The content of various types of stem cells in the bone marrow and peripheral blood during the development of encephalopathy and with the introduction of 1500 UE / kg of hyaluronidase against the background of modeling encephalopathy (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, for 10 ⁵ myelocaryocytes CCM in peripheral blood, per 10 ⁵ mononuclear cells MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 8.7 ± 0.68 1.34 ± 0.1 9.1 ± 0.63 1.4 ± 0.6 31.0 ± 4.0 12.0 ± 3.0 encephalopathy (EP) 17.2 ± 1.3 * 2.4 ± 1.0 29.8 ± 3.4 * 8.87 ± 1.2 * 59.0 ± 8.0 * 20.0 ± 4.0 EP + hyaluronidase 26.2 ± 2.0 * # 5.1 ± 0.87 * # 30.4 ± 3.0 * 18.2 ± 2.45 * # 86.0 ± 9.0 28.0 ± 4.0 * * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Thus, hyaluronidase led to an increase in the degree of mobilization of all the studied types of stem cells against the background of the development of ambiguous changes in their content in the bone marrow.

Example 7

Myocardial infarction was modeled by the intravenous administration of adrenaline at a dose of 0.25 mg / kg [3] in its own modification. At the same time, experimental animals immediately after encephalopathy modeling 1 time per day for 2 days were injected intraperitoneally with hyaluronidase (Lidaza, FSUE NPO Microgen, Ministry of Health of the Russian Federation, Russia) at a dose of 1000 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline.

Simulation of myocardial infarction led to an increase in the number of MSCs in bone marrow and peripheral blood. However, the introduction of hyaluronidase more significantly increased the release of MSCs in the peripheral blood against the background of the absence of changes in the bone marrow pool of MSCs (Table 7).

Table 7
The content of various types of stem cells in bone marrow and peripheral blood with myocardial infarction and with the introduction of 1000 UE / kg of hyaluronidase against the background of modeling myocardial infarction (M ± m) MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 31.0 ± 4.0 12.0 ± 3.0 Myocardial infarction (MI) 78.0 ± 10.0 * 46.0 ± 8.0 * MI + Hyaluronidase 74.0 ± 12.0 * 82.0 ± 12.0 * # * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Thus, the introduction of hyaluronidase enhanced the mobilization of mesenchymal stem cells in peripheral blood.

Example 8

In mice, chronic hepatitis (CG) was modeled by intragastric administration of tetrachlorocarbon (TCA) in the form of a 20% solution in olive oil in a volume of 0.2 ml / mouse live weight for three weeks twice a week [2]. At the same time, experimental animals immediately after HG modeling 1 time per day for 2 days were injected intraperitoneally with hyaluronidase (Lidaza, FSUE NPO Mikrogen, Ministry of Health of the Russian Federation, Russia) at a dose of 1000 UE / kg (in 0.5 ml of physiological saline). Control animals were intraperitoneally injected 1 time per day for 2 days with 0.5 ml of physiological saline.

In the control group, an increase in the number of CFU-F in bone marrow tissue was detected on the 7th day after the last TCA administration, which included both precursors of stromal elements and true SC, which was confirmed by an increase in the number of MSCs in hematopoietic tissue at the same observation time . In addition, there was an increase in the content of fibroblast and MSC progenitor cells in peripheral blood, indicating mobilization of stem cells of varying degrees of maturity with this type of exposure, which is essentially an extreme effect. In this case, the introduction of hyaluronidase was accompanied by an increase in hematopoietic tissue in the studied period of CFU-F against the background of a sharp increase in the number of CFU-F and MSC in peripheral blood (Table 8).

Table 8
The content of various types of stem cells in the bone marrow and peripheral blood during the development of chronic hepatitis and with the introduction of 1000 UE / kg of hyaluronidase against the background of hepatitis modeling (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 15.17 ± 1.3 6.5 ± 0.99 24.0 ± 4.0 31.0 ± 7.0 Hg 28.7 ± 3.4 * 9.1 ± 0.48 * 66.0 ± 8.0 * 64.0 ± 10.0 * HCG + hyaluronidase 39.2 ± 2.1 * # 14.09 ± 0.7 * # 84.0 ± 18.0 * 88.0 ± 4.0 * # * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Thus, the introduction of hyaluronidase in experimental hepatitis led to an increase in the release into the blood of mesenchymal progenitor cells of varying degrees of maturity.

Example 9

Hyaluronidase was administered intraperitoneally to experimental experimental intact animals on the background of the introduction of granulocyte colony stimulating factor (Neupogen, Hoffman-la-Roche, Switzerland) at a dose of 125 μg / kg 1 time per day for 3 days an additional 1 time per day for 2 days ( “Lidaza”, FSUE “NPO Microgen” of the Ministry of Health of the Russian Federation, Russia) at a dose of 500 UE / kg (in 0.5 ml of physiological saline). The control animals were injected subcutaneously with a granulocyte colony stimulating factor (Neupogen, Hoffman-la-Roche, Switzerland) at a dose of 125 μg / kg 1 time per day for 3 days in an equivalent volume (0.2 ml) and intraperitoneally 1 time per day for 2 days, 0.5 ml of saline.

During the experiment, the administration of rhG-CSF led to a decrease in the number of committed progenitor cells in the bone marrow on the 4th day after the start of exposure. At the same time, the content of MSCs in the bone marrow remained at the level of background values. These changes were accompanied by a significant increase in the number of MSCs in peripheral blood to 311.1% of the background, CCM to 201.6% of the background, and CFU-F to 152.4% of the background. Additional administration of hyaluronidase led to a significant accumulation of MSCs and CFU-F in the bone marrow. However, the combined use of rhG-CSF and hyaluronidase was accompanied by a significantly more pronounced increase in the number of MSCs (up to 1000.0% of the background), CCM (up to 386.6% of the background) and CFU-F (up to 211.9% of the background) in peripheral blood (table 9).

Example 10

Hyaluronidase was administered intraperitoneally to experimental experimental animals on the background of the introduction of a granulocyte colony-stimulating factor (Neupogen, Hoffman-la-Roche, Switzerland) at a dose of 125 μg / kg 1 time per day for 5 days for an additional 1 time per day for 2 days ( Lidaza ”, Federal State Unitary Enterprise“ NPO Microgen ”of the Ministry of Health of the Russian Federation, Russia) at a dose of 1500 UE / kg (in 0.5 ml of physiological saline). Control animals were injected subcutaneously with a granulocyte colony-stimulating factor (Neupogen, Hoffman-la-Roche, Switzerland) at a dose of 125 μg / kg 1 time per day for 5 days in an equivalent volume (0.2 ml) and intraperitoneally 1 time per day for 2 days, 0.5 ml of saline.

The combined use of rhG-CSF and 1,500 UE / kg of hyaluronidase was accompanied by a more pronounced increase in the number of MSCs, CCMs and CFU-F in peripheral blood relative to control animals receiving only rhG-CSF (but less significant compared to the group of mice that were additionally administered 500 UE / kg hyaluronidase) (see table 9).

Thus, the introduction of hyaluronidase against the background of the administration of rhG-CSF to intact animals significantly increases the degree of mobilization of various types of bone marrow stem cells in peripheral blood.

Table 9
Dynamics of stem cell content in bone marrow and peripheral blood upon administration of rhG-CSF (1) and co-administration of rhG-CSF with 500 UE / kg (2) and 1500 UE / kg (3) hyaluronidase (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, for 10 ⁵ myelocaryocytes CCM in peripheral blood, per 10 ⁵ mononuclear cells MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 3.17 ± 0.48 7.0 ± 0.37 14.3 ± 2.1 7.0 ± 0.98 28.0 ± 4.0 9.0 ± 5.0 one 2.0 ± 0.26 * 10.67 ± 0.7 * 12.6 ± 1.2 14.1 ± 2.3 * 22.0 ± 4.0 28.0 ± 4.0 * 2 7.5 ± 0.76 * # 14.8 ± 0.7 * # 16.31 ± 4.5 27.2 ± 1.4 * # 51.0 ± 7.0 * # 90.0 ± 26.0 * # 3 7.2 ± 0.78 * # 15.1 ± 0.84 * # 14.3 ± 3.9 24.2 ± 2.3 * # 37.0 ± 12.0 62.0 ± 18.0 * * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with indices in the group of animals receiving only G-CSF was noted at p <0.05

Example 10

Intact animals on the background of the introduction of granulocyte colony stimulating factor (Neupogen, Hofrman-la-Roche, Switzerland) at a dose of 125 μg / kg 1 time per day for 3 days in conjunction with intraperitoneal injection of stem cell factor (Sigma, USA) in a dose of 50 μg / kg 1 time per day for 2 days an additional 1 time per day for 2 days was injected intraperitoneally with hyaluronidase (Lidaza, FSUE NPO Microgen, Ministry of Health of the Russian Federation, Russia) at a dose of 1000 UE / kg (at 0, 5 ml of physiological saline). Only rhG-CSF and FSK were introduced into control animals according to similar schemes.

During the experiment, on the 4th day after the administration of rhG-CSF and FSK, a regular increase in the number of SCs in the peripheral blood was observed with changes in the ambiguous nature of their bone marrow pools. Moreover, an increase in the number of circulating committed progenitor cells (CFU-F to 344.5% and CCM to 347% of the background) was observed to a greater extent than true (mesenchymal) stem cells (to 266.7% of the background). The additional administration of hyaluronidase to the animals had practically no effect on the content of various types of SC in the hematopoietic tissue compared to the control. However, there was an even more pronounced increase in the number of parent cells in peripheral blood. At the same time, the maximum level of rise was observed, on the contrary, for the most primitive ones - MSC (up to 1088.2% of the background) than CFU-F (up to 401.4% of the background) and CCM (up to 502.0% of the background) ( table 10).

Table 10
Dynamics of stem cells in bone marrow and peripheral blood with the introduction of rhG-CSF together with FSK (1) and the additional introduction of 1000 UE / kg of hyaluronidase (2) (M ± m) CFU-F in the bone marrow, for 250 thousand myelokaryocytes CFU-F in the peripheral blood, for 250 thousand myelokaryocytes CCM in the bone marrow, per 10 ⁵ myelokaryocytes CCM in peripheral blood, per 10 ⁵ mononuclear cells MSCs in the bone marrow, by 10 ⁶ myelocaryocytes MSCs in peripheral blood, per 10 ⁶ mononuclear cells background 3.17 ± 0.48 7.0 ± 0.37 14.3 ± 2.1 7.0 ± 0.98 28.0 ± 4.0 9.0 ± 6.0 one 2.64 ± 0.14 * 24.12 ± 1.0 * 19.6 ± 3.4 24.3 ± 1.8 * 34.0 ± 6.0 24.0 ± 4.0 * 2 6.4 ± 0.72 * # 28.1 ± 1.09 * 14.4 ± 0.78 36.4 ± 2.3 * # 44.0 ± 8.0 * 98.0 ± 22.0 * # * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with control was noted at p <0.05

Thus, the introduction of hyaluronidase when used together with rhG-CSF and FSK led to increased mobilization of SC, and to a greater extent the most differentiated ones.

Example 11

Intact animals on the background of a single intravenous administration of CTCE-0214 protein (peptide analogue of SDF-1 factor) at a dose of 10 mg / kg (Sigma, USA) an additional 1 time per day for 2 days was injected intraperitoneally with hyaluronidase (Lidase) , FSUE NPO Microgen, Ministry of Health of the Russian Federation, Russia) at a dose of 1000 UE / kg (in 0.5 ml of physiological saline). Control animals in an equivalent volume (0.2 ml) intravenously and 0.5 ml were injected intraperitoneally with saline.

On the 3rd day after the start of the experiment, an increase in the number of hematopoietic stem cells (CFU-HEMM) was recorded in the bone marrow. At the same time, there was an increase in the number of these elements in peripheral blood (up to 433.3% of the background). The introduction of hyaluronidase abolished the influence of the factor on the content of CCM in the bone marrow, but led to a significantly more pronounced increase in the number of these parent cells in peripheral blood, reaching 747.6% of the background (Table 11).

Table 11
Dynamics of the content of hematopoietic stem cells in the bone marrow and peripheral blood with the administration of CTCE-0214 (1) and with the additional administration of 1000 UE / kg of hyaluronidase (2) (M ± m) CCM in the bone marrow, for 10 ⁵ myelocaryocytes CCM in peripheral blood, per 10 ⁵ mononuclear cells background 5.8 ± 0.97 2.1 ± 0.24 one 8.4 ± 0.36 * 9.1 ± 2.3 * 2 7.62 ± 1.2 15.7 ± 1.07 * # * - the significance of differences with background values is noted at p <0.05 # - the significance of differences with indices in the group of animals receiving only G-CSF was noted at p <0.05

Thus, hyaluronidase enhanced the mobilizing effect of the substance CTCE-0214 (peptide analogue of SDF-1 factor) in relation to hematopoietic stem cells.

In general, based on the data obtained, it follows that when hyaluronidase is introduced against the background of modeling extreme effects and with pathological conditions of various origins, as well as when substances causing the release of SC into the blood are introduced into the body, a significant increase in the level of mobilized stem cells is observed. Moreover, there is a greater degree of mobilization effect in relation to the earliest parent elements - mesenchymal (true) stem cells. At the same time, the mechanism of the specified specific action of this enzyme is obviously the destruction / weakening of SC bonds with one of the main components of the intercellular matrix - hyaluronic acid, bound in situ to stem cells via specific receptors.

The proposed method allows to significantly increase the level of mobilization of various types of stem cells under extreme influences and pathological conditions, as well as to increase the degree of mobilizing effect of substances that can stimulate the release of SC into the blood by additional administration of hyaluronidase.

Claims (1)

A method of enhancing stem cell mobilization, characterized in that under extreme conditions or in pathological conditions accompanied by stem cell mobilization, or under the influence of modifiers of stem cell functional activity on the body that cause stem cell mobilization into the blood, hyaluronidase is administered intraperitoneally at a dose of 500- 1500 UE / kg for 2 days 1 time per day.