WO2003049754A1

WO2003049754A1 - The use of osteogenic growth peptide in the enhancement of haemopoiesis

Info

Publication number: WO2003049754A1
Application number: PCT/CN2002/000772
Authority: WO
Inventors: Wenying Cheng; Honghong Chen; Tongyi Chen; Deyuan Shi; Yi Lu
Original assignee: Shanghai Yizhong Biotechnology Co., Ltd.
Priority date: 2001-11-23
Filing date: 2002-10-31
Publication date: 2003-06-19
Also published as: CN1189209C; CN1421244A; AU2002349719A1

Abstract

The osteogenic growth peptide (OGP) is a kind of polypeptide that has 14 amino acid residues and exists in the animal and human body. According to the present invention OGP has not only the function in the promotion of ossification but the proliferation of haemopoiesis cells as well. The present invention discloses the use of osteogenic growth peptide in the preparation of the pharmaceutical composition for enhancing the proliferation of haemopoietic cells such as granulocytes. It also provides a method for using the OGP in promoting the development of granulocyte progenitor cell in vitro. OGP is different from the granulocyte or granulocyte monocyte colony stimulating factor in that it does not have the function of stimulating leukemia cell such as human TF-1.

Description

Application of osteogenic growth peptide in promoting hematopoiesis

Technical field

The invention relates to the application of Osteogenic Growth Peptide (OGP) in promoting hematopoietic. Background technique

Osteogenic Growth Peptide (Osteogenic Growth Peptide) is a 14-amino acid polypeptide found in Bab and others in 1988 that can promote bone cell growth. When the body is fractured or the bone marrow is injured, in addition to the local osteogenic response in the fractured or damaged bone marrow, there is also a systemic osteogenic response [Bab I, et al. (1985) Calcif Tissue Int. 37: 551-555 Foldes J, et al. (1989) J Bone Miner Res. 4: 643-646]. Intensive experimental studies have shown that the healing bone marrow tissue can release several factors that promote osteogenesis into the blood circulation, resulting in an enhanced osteoblastic response in the whole body; confirmed by research and isolated and purified, there is a factor called Osteogenic Peptide (Osteogenic Growth Peptide, OGP) [Bab I, et al. (1988) Endocrinology 123: 345-352; Bab I, et al. (1992) EMBO J 11: 1867-1873]. Further studies showed that the amino acid sequence of OGP is ALKRQRGTLYGFGG, which is the same as the amino acid sequence of the C-terminal segment of H4 histone, and contains 5 residue sequences of the T-cell receptor β chain V region and Bacillus subtil is) outB region [ Kayne PS, et al., (1988) Cell 55: 27-39; Bab I, et al. (1992) EMBO J 11: 1867-1873], its evolutionary degree is conservative, human and mouse homologous, and the same characteristics .

Under physiological conditions, 0GP exists in the serum of humans and mammals, mainly in the form of binding, that is, the OGP-0GP binding protein (0GPBP) complex, which accounts for about 80% -97% of the total 0GP [Greenberg Z, et al. (1995) JCE & M. 80 (8): 2330-2335]. The molecular structure of 0GPBP is not completely clear at present. It is reported from related literature that it may be α ₂ -macroglobulin, which is similar to other peptide regulators. The role of 0GPBP may be to protect 0GP in serum from being degraded, so it can regulate 0GP. Level of active fraction in serum.

Due to the homology of natural OGP in human and animal serum, evolution is highly conserved, suggesting that OGP may have important physiological functions, and research on its biological activity has mainly focused on its osteogenic activity. From the analysis of the relationship between 0GP and bone marrow hematopoiesis, bone formation, 0GP may have the function of promoting bone marrow hematopoiesis. Present Some experiments suggest that 0GP can promote bone marrow hematopoiesis in normal mice; for bone marrow transplanted mice, it can promote the implantation of exogenous bone marrow and hematopoietic reconstruction, and play an adjuvant treatment role. However, these studies also believe that OGP mainly promotes the proliferation of transplanted stem cells, thereby increasing the availability of hematopoietic cells, including red blood cells and platelets.

At present, the incidence of tumors is high. Clinically, radiotherapy and chemotherapy are used to treat malignant tumors.

• The larger the amount, the stronger the lethality to the tumor cells and the higher the cure rate. However, it is accompanied by serious side effects, which mainly lead to bone marrow suppression or injury, resulting in low hematopoietic function and decreased immunity, which limits the increase of radiotherapy and chemotherapy doses. Therefore, promoting the recovery of hematopoietic function in cancer patients is very important to improve the cure rate of tumors and reduce the incidence of infection, tumor recurrence and mortality.

Recombinant human granulocyte colony stimulating factor (rhG-CSF, Wheel's blood) and / or recombinant human granulocyte colony stimulating factor (rhGM-CSF, whitening energy) are mainly used clinically to promote the recovery of hematopoietic function. They directly stimulate the proliferation of granulocytic hematopoietic progenitor cells, shorten the recovery time of leukocytes and neutrophils, and have high efficacy, but they are expensive and overburdened by the working class.

In addition, there are still some problems in the clinical use of rhG-CSF and rhGM-CSF: (1) Tumors and leukemia cells have normal receptors for CSF. After treatment, it may increase the tumor recurrence rate by increasing the proliferation of host residual tumor cells; (2) ) CSF directly stimulates the proliferation of hematopoietic progenitor cells without self-renewal ability, leading to the depletion of hematopoietic progenitor cells; (3) selectively promotes the proliferation of granulocyte progenitor cells, but has no promotion effect on red blood cells and megakaryocytes; (4) clinical The use of white blood cells needs to be closely monitored to prevent juvenile cell proliferation.

Therefore, there is an urgent need in the art to develop drugs that can effectively promote the proliferation of hematopoietic cells without stimulating the proliferation of tumor cells. Summary of the Invention

The object of the present invention is to provide a new hematopoietic factor that promotes the proliferation of hematopoietic cells, mainly granulocytes, without stimulating the proliferation of some tumor cells.

Another object of the present invention is to provide a pharmaceutical composition for promoting the proliferation of hematopoietic cells mainly composed of granulocytes. In a first aspect of the present invention, the use of an osteogenic growth peptide (OGP) is provided, which is used to prepare a pharmaceutical composition that promotes the proliferation of hematopoietic cells, mainly granulocytes. In a preferred example, the pharmaceutical composition treats the following diseases or conditions:

(1) Hematopoietic dysfunction caused by radiation injury;

(2) Treatment of hypo-hematopoiesis caused by chemotherapy drugs;

(3) promote the proliferation of human bone marrow granulocyte hematopoietic progenitor cells;

(4) Treatment of leukopenia.

In another preferred example, the pharmaceutical composition contains an osteogenic growth peptide and a hematopoietic factor selected from the group consisting of G-CSF, GM_CSF, TP0, or a mixture thereof.

In another preferred embodiment, OGP is used to prepare a pharmaceutical composition that promotes granulocyte proliferation.

In another preferred example, the pharmaceutical composition is used before, during, or after radiotherapy or chemotherapy.

In a second aspect of the present invention, a method for promoting the growth of granulocyte progenitor cells in vitro is provided, comprising the steps of: culturing granulocyte progenitor cells in a medium suitable for the growth of granulocyte progenitor cells, wherein the medium contains 10- ¹⁴ -10- ⁵ mol / L osteogenic growth peptide.

In a preferred embodiment of the present invention, said medium containing ^{^{l (T 13 -l (T 5}} m 0 l / L of osteogenic growth peptide, more preferably the medium containing l (T ¹² -l ( T ⁵ m ₀ l / L osteogenic growth peptide.

Figure 1 shows the effect of sOGP on the number of white blood cells (WBC) in mice at different times after 4Gy irradiation. Figure 2 shows the effect of different doses of sOGP on the number of white blood cells (WBC) on the 8th day after 4Gy irradiation in mice.

Figure 3 shows the effect of different doses of sOGP on the number of bone marrow nucleated cells on the 8th day after 4Gy irradiation in mice.

Figure 4 shows the effect of different doses of sOGP on the CFU-S and spleen coefficient of mice after 7.5Gy irradiation. Figure 5 shows the effect of different doses of sOGP on the number of white blood cells (WBC) in normal mice

Figure 6 shows the effect of different doses of sOGP on the number of bone marrow nucleated cells in normal mice.

Figure 7 shows the effects of different doses of sOGP on colony formation of isolated human bone marrow granulocyte progenitor cells in vitro.

Figure 8 shows the effect of different doses of sOGP on the proliferation of erythroleukemia TF-1 cells. detailed description

After extensive and in-depth research, the inventors have shown in vitro experiments that sOGP can promote the proliferation of normal human bone marrow granulocyte progenitor cells, and that the colony formation rate of granulocyte progenitor cells in vitro cultures increases with increasing sOGP concentration within a certain concentration range , Showing a clear dose-response relationship.

In addition, the in vivo experimental research of the present invention also shows that sOGP can promote the recovery of hypopoietic function caused by radiation damage and chemotherapy drugs. Subcutaneous injection of sOGP after 4Gy irradiation in mice can accelerate the recovery of peripheral blood leukocytes and bone marrow nucleated cells. It is significantly higher than the irradiation control group, showing a dose-response relationship within a certain dose range. SOGP can also make the CFU-S and corresponding spleen coefficient of 7.5Gy irradiation mice significantly higher than the irradiation control group, and promote extramedullary hematopoietic. In addition, sOGP can promote the recovery of bone marrow nucleated cells and peripheral blood leukocytes in mice injected with the chemotherapy drug cyclophosphamide, which is significantly different from the cyclophosphamide control group.

The animal experimental study of the present invention also found that sOGP can promote the increase of the number of nucleated cells in bone marrow of normal mice by 15-20%, increase the number of peripheral blood WBC by 30-40%, and increase platelets and red blood cells by about 10%, indicating that sOGP can promote Granulocytes are mainly hematopoiesis, and red and platelet lines are also increasing. These studies of the present invention show that OGP can be regarded as an effective hematopoietic factor, and can have clinical application prospects.

In addition, one of the prerequisites for the restoration of normal hematopoietic and bone marrow transplantation is the existence of functional stromal cells and tissues that make up the hematopoietic microenvironment, which determines the proliferation of residual hematopoietic stem cells and the injection of hematopoietic stem cells from peripheral blood circulation into bone marrow tissue. And support hematopoiesis. In long-term in vitro bone marrow culture, bone marrow containing stromal tissue can maintain the survival of hematopoietic stem cells. Adding an appropriate amount of sOGP that promotes hematopoietic effects in this culture system will help the expansion of hematopoietic stem cells in vitro and provide more hematopoietic stem cells for transplantation. Combining in vivo and in vitro methods will provide a more effective solution for bone marrow transplantation.

Studies have suggested that sOGP stimulates the proliferation of bone marrow mesenchymal stem cells in the body, improves the hematopoietic microenvironment (mainly including fibrous tissue, bone and osteocytes), promotes the recovery of hematopoietic function that occurs naturally or induced bone marrow suppression or injury, and can stimulate the bone marrow Hematopoietic reconstruction after transplantation.

The definition of OGP used herein includes natural peptides, synthetic peptides, all homologues, isomers or genetic variants, and all other variants of OGP.

Experimental data show that OGP is a single polypeptide with a defined sequence (Ala-Leu- Lys-Arg- Gln- Gly- Arg- Thr- Leu- Tyr- Gly- Phe- Gly_Gly). 0GP homologs, isomers Or genetic variant refers to a conserved sequence that contains at least about 40% of the native OGP amino acid sequence, a polypeptide that contains at least about 60% of the conserved sequence is preferentially protected, and a polypeptide that contains at least about 75% of the conserved sequence is more preferred. Preferably, the osteogenic growth peptide used in the present invention has the following amino acid sequence: Ala-Leu- Lys- Arg- Gln-Gly-Arg- Thr-Leu-Tyr_Gly_Phe- Gly-Gly.

It should be noted that other variants of OGP are also included in the scope of the present invention. This includes, in particular, any variants other than the native OGP obtained by substitution of only conservative amino acids. The present invention also includes various OGPs and fragments thereof, as long as the purified polypeptide exhibits osteogenic and hematopoietic effects in vitro and in vivo. OGP fragments may be small peptides containing 6 or more amino acids. Polypeptides larger than 0GP and having osteogenic and hematopoietic effects are also included in the scope of the present invention.

OGP can be prepared by isolation, recombination, and artificial methods. In a preferred example of the present invention, the sOGP artificially synthesized by the biochemical method is consistent with the structure of the naturally occurring OGP in the serum, and has the effect of promoting bone formation and hematopoiesis.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned OGP polypeptide, said pharmaceutical composition comprising the OGP polypeptide as a basic active ingredient and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of the OGP polypeptide is preferably non-toxic and dosage-stable.

The pharmaceutical composition of the present invention has the ability to improve the proliferative activity of human and mammalian hematopoietic stem / progenitor cells, and promotes hematopoietic reconstruction, and can be used to treat hypoxia caused by bone marrow injury caused by radiotherapy, chemotherapy or spontaneous, and promote bone marrow transplantation in China and abroad. Implantation of derived hematopoietic cells shortens recovery time and has no stimulating proliferation effect on some tumor cells.

Compared with the recombinant human granulocyte colony-stimulating factors rhG-CSF and rhGM-CSF commonly used in clinical practice, the 0GP pharmaceutical composition of the present invention can promote the proliferation of bone marrow mesenchymal stem cells, improve the hematopoietic microenvironment, and benefit hematopoietic stem / progenitors. Cell proliferation, speeding up the recovery of peripheral blood cells, has the advantages of mitigating effects, two-way regulation, easy control, etc., and has no effect on promoting proliferation of leukemia cells such as human TF-1. In addition, the main component that exerts a medicinal effect in the pharmaceutical composition of OGP of the present invention has the same structure as the naturally occurring human OGP, thereby significantly avoiding immunogenicity that may be caused after long-term use.

In addition, OGP peptides prepared by biochemical synthesis or by recombinant DNA technology can also be prepared into pharmaceutically acceptable salts, especially base addition salts, by various known methods. For example, these peptides can be treated with a suitable base in accordance with methods well known to those skilled in the art to prepare base addition salts of acidic amino acids.

OGP polypeptides can be made to suit specific clinical administration methods according to conventional methods known in the pharmaceutical field Formula of a drug compound. For example, an appropriate carrier or diluent such as water, physiological saline, isotonic glucose solution can be added to the OGP to prepare solutions, injections, emulsions, nasal drops, eye drops that can be administered by routes other than the gastrointestinal tract. Excipients or carriers such as starch, lactose, talc, sucrose, glucose or glycerin, liquid paraffin, liposomes, or gelatin can also be added to make OGP into suppositories, tablets, and powders that can be administered via the gastrointestinal tract. , Granules, capsules or liposome encapsulants. In addition to the active ingredients and appropriate carriers or excipients, these preparations can also be supplemented with other auxiliary ingredients as needed, such as one or more diluents, fillers, emulsifiers, preservatives, surfactants, absorption Accelerators, buffers, fragrances and colorants.

The OGP pharmaceutical composition of the present invention can be administered by various conventional administration routes, for example, it can be administered via the gastrointestinal tract, subcutaneously, intradermally, intranasally, intravenously, intramuscularly, intrarectally, intraocularly, etc., but Among them, the preferred route of administration is intramuscular injection, subcutaneous injection, nasal spray or oral administration. In addition, the OGP pharmaceutical composition of the present invention can also be administered at any time, for example, before, during, or after radiotherapy or chemotherapy.

In summary, based on the new findings of the present invention, it is expected that the OGP peptides or their salts or pharmaceutical compositions containing these peptides or their salts of the present invention can be used to treat hematopoietic diseases caused by radiation, chemotherapy, or naturally occurring bone marrow injury Low function, speed up the implantation of bone marrow transplantation, and promote hematopoietic reconstruction. Specifically, 0GP can be applied in the following areas:

1. Accelerate the recovery of granulocyte-based blood cells when hypoxia is caused by radiation injury.

2. Promote the recovery of granulocyte-based blood cells when hematopoiesis is caused by chemotherapy drugs. 3. Promote the proliferation of human bone marrow granulocyte hematopoietic progenitor cells.

4. Accelerating hematopoietic reconstruction after bone marrow transplantation.

5. Play the role of hematopoietic reconstruction by stimulating the proliferation of bone marrow mesenchymal stem cells and stromal cells;

6. Increase the number of donor peripheral blood hematopoietic stem cells and hematopoietic progenitor cells.

7. Promote the proliferation of hematopoietic stem cells in long-term bone marrow culture in vitro to facilitate bone marrow transplantation. 8. Combine with or cooperate with other hematopoietic factors (such as G-CSF, GM-CSF, TP0, etc.) to improve the efficacy.

9. Play a synergistic role in gene therapy of blood diseases;

10. Promote hematopoiesis while treating osteoporosis, promoting fracture healing and cartilage repair. In addition, with the progress of society and economic development, people's lifespans have been extended, the world is entering an aging society, osteoporosis accounts for a high proportion of the elderly, and the hematopoietic function of the elderly is reduced.

0GP can treat osteoporosis, promote fracture healing and cartilage repair, at the same time can promote hematopoiesis, improve immunity, and improve the quality of life of patients. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, for example, Sambrook et al., Molecular Cloning: The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions Conditions recommended by the manufacturer. Example 1

Hematopoietic effect of osteogenic growth peptide in radiation injured animal models

The purpose of this embodiment is to observe the therapeutic effect of sOGP on leukocytopenia, and to find a safe and effective drug for the treatment of tumor patients with low blood production caused by bone marrow injury after radiotherapy and chemotherapy. Method:

Clean-grade ICR mice were used, weighing 18 to 20 g, and randomly divided into groups of 10 mice. Two irradiation doses were used, and the experiments were performed in two batches. The experiments are grouped as follows:

Group A: normal control group, without irradiation;

Group B: irradiation control group;

Group C: Huer blood positive drug control group, 10 g / kg / day;

Group D: 0GP medication group, divided into 9 dose points,

(1) 0.0078 nmol / rat / day; (2) 0.00156 nmol / rat / day; (3) 0.000625 nmol / rat / day; (4) 0,025 nmol / rat / day; (5) ) 0. 10 nmol / rat / day; (6) 0. 40 nmol / rat / day; (7) 1. 60 nmol / rat / day; (8) 6. 40 nmol / rat / day; (9) 12 80 nmol / rat / day. Experiment 1.

4. After 0Gy ¹³⁷ Cs Y-rays were irradiated to mice at one time, different doses of sc were injected subcutaneously (sc) for 14 consecutive days. sOGP, detects the following indicators:

①Bone marrow nucleated cells (BMNC);

② White blood cell (WBC) count: measured on days 5, 8, 12, and 15 after irradiation; experiment 2.

7. After 5Gy ¹³⁷ Cs y-rays were irradiated to mice at one time, different doses of sOGP were sc for 7 consecutive days. The detection indicators are as follows:

① endogenous spleen nodules (CFU-S);

② Spleen coefficient; Statistical processing:

Each data is expressed as ± s. After the experimental data are tested for homogeneity of variance, analysis of variance or t-test is used. Results:

1. Effect of sOGP on the number of peripheral blood leukocytes in mice at different time after 4Gy irradiation

It can be seen from FIG. 1 that on the 5th day after irradiation, 100 g / kg Wheeler blood (ie rhG_CSF) only slightly increased the WBC number of the mice compared with the irradiation control group, with no significant difference, sOGPO. 10 and 0.50 nmol / rat / There were no effects in the 2 dose groups. On the 8th day after irradiation, the effects of sOGPO. 10 and 0.50 nmol / mouse dose group and Wheel's blood group were similar, and the WBC numbers in peripheral blood were significantly higher than those in the irradiation control (P <0.001). ); On the 12th day after irradiation, the effect of Wheeler's blood was still very significant (P <0.001), and sOGP also had a significant effect (P <0.05); On the 15th day after irradiation, sOGP increased the WBC number although still It is higher than the irradiation control, but the difference is not significant.

It can be seen that the measurement of peripheral blood leukocytes on the 8th day after 4Gy irradiation in mice can better reflect the role of sOGP in promoting hematopoiesis.

2. Effects of different doses of sOGP on peripheral blood leukocytes in 4Gy-irradiated mice

It can be seen from FIG. 2 that on the 8th day after 4. OGy irradiation in mice, WBC in peripheral blood decreased significantly (P <0.001). After continuous subcutaneous injection of 0.00000-12.80 nmol / rat / day with different doses of sOGP on the 8th day, starting from the 0.00156nmol / rat / day dose group, as the dose of sOGP increased, peripheral blood WBC Recovery was significantly accelerated (P <0. 01 ~ 0. 001), and showed a dose-response relationship within a certain dose range.

3. Effect of sOGP on the number of bone marrow nucleated cells in 4Gy-irradiated mice

As can be seen from Figure 3, mice were subcutaneously injected with SOGP 14 days after 4Gy irradiation, and the number of bone marrow nucleated cells increased significantly from 0. 025 to 12.80 nmol / mouse dose group compared with the irradiation control (P <0. 01 ~ 0. 001) , Similar to the role of Wheeler blood.

4. Effect of sOGP on CFU- S and spleen coefficient of 7.5Gy-irradiated mice

As shown in Figure 4, the spleen nodules on the 8th day after irradiating the mice with a sublethal dose of 7.5 Gy showed that the number of CFU-S in the control group was significantly increased compared with the control group, and the spleen coefficient was significantly reduced. For 7 consecutive days, different doses of sOGP were given. The CFU-S in the dose group of 0. 02-2. 5nmol / rat / day was significantly higher than that in the irradiation control group (P <0. 05 ~ 0. 01), and the spleen coefficient also increased significantly. (P <0. 05), similar to the effect of Wheel's blood.

The results suggest that sOGP can protect the remaining hematopoietic stem cells and promote extramedullary hematopoietic. Example 2

Effect of sOGP on Hematopoietic System in Normal Mice

The purpose of this example is to observe whether sOGP can promote the hematopoietic function of normal mice, and provide a basis for the future clinical treatment of osteoporosis and fractures while improving the hematopoietic function, which is conducive to enhancing the immunity and improving the therapeutic effect. Method:

Clean-grade ICR mice, weighing 18-20 g, were randomly divided into groups of 10 mice each. The experiments are grouped as follows:

Group A: blank control group;

Group B: sOGP medication group, divided into 3 dose points,

(1) 0.02 nmol / rat / day;

(2) 0.10 nmol / rat / day; (3) 0.50 nmol / rat / day.

Normal mice were sc sOGP continuously for 14 days, and the following indicators were detected: ① Peripheral blood: WBC, Pit, RBC ② BMNC number, ③ Bone marrow cell classification count. Results:

1. Effects of sOGP on the numbers of peripheral blood leukocytes, red blood cells and platelets in normal mice

Normal mice sc s0GP 3 different doses 0.02, 0.10, and 0.50 nmol / mouse 14 days later, the three dose groups significantly increased peripheral blood WBC numbers by 30 to 40% compared to the blank control group (Figure 5). Platelets And red blood cells increased by about 5-10%, indicating that sOGP mainly promotes granulocytic hematopoiesis, and the red and platelet lines also have an increasing trend.

2. Effect of sOGP on the number and classification of bone marrow nucleated cells in normal mice

As shown in Figure 6, the B-C number of the three dose groups of sOGP increased significantly by 15-20% compared with the blank control group. Bone myeloid cell counts showed that the proportion of bone marrow cells in the sOGP group was the same as that of the blank control mice, but the sOGP group division index was 2 / 6≥2, indicating that the bone marrow cell proliferation of the 0GP group was more active than that of the blank control group. Compared with the control group, the particle red ratio of the drug group was 5/6> 1, and the latter was 3/6> 1. The granulocyte proliferation was more significant. 0GP makes the changes of bone marrow consistent with the peripheral blood, and promotes hematopoietic hematopoiesis. Example 3

Hematopoietic activity of osteogenic growth peptide in vitro

The purpose of this example is to observe whether sOGP can promote the colony formation of human bone marrow granulocyte progenitor cells in vitro and provide a basis for sOGP to promote the proliferation of hematopoietic cells. Method:

Bone marrow cell semi-solid colony culture method was used. Bone marrow cell suspension was first prepared, and the cell concentration was adjusted to 1 X 10 ⁶ cells / ml with RPMI-1640. Different concentrations of sOGP and Hueyer blood were added to the culture system, and cultured at 37 ° C and 5% C0 ₂ 11 On the day, the number of CFU-G colonies was counted under a microscope (1 colony above 50 cells). The experiment was divided into three groups, that is, the negative control group (excluding sOGP and G-CSF), the Huey blood (G-CSF) positive control group, and the sOGP experimental group with different concentrations, and each experimental point was repeated 3-4 dishes. result: It can be seen from FIG. 7 that the number of CFU-G colonies formed by in vitro cultured bone marrow cells in each concentration gradient group of sOGP was significantly higher than that in the negative control group (P <0.05), and the number of colonies increased with increasing concentration (P <0. 05), showing a significant dose-response relationship. At a concentration of 1.33 X 10- ⁴ nmol / ml in Huer blood, the number of granulocyte progenitor cell colonies formed was about 60, which had a strong effect. Corresponding to the results of in vivo experiments, it showed that the mechanism of action between the two may be different. Huer Blood directly stimulates the proliferation of granulocyte progenitor cells, and OGP may exert its hematopoietic activity by stimulating bone marrow mesenchymal stem cells, improving the hematopoietic microenvironment, and other mechanisms. Example 4

Effect of Osteogenic Growth Peptide on the Proliferation of GM-CSF (Whitening) -dependent Human Red Leukemia Myeloma Cells The purpose of this example is to observe the effect of sOGP on GM-CSF-dependent red leukemia myeloma cells (TF-

1 cell line) with or without proliferation.

Method:

釆 Set the negative control group (excluding sOGP and GM- CSF), whitening positive control and sOGP experimental group by MTT colorimetry. The specific steps are as follows:

1. Preparation of cell suspension: After cell washing, adjust the concentration to 4 X 10 ⁵ cells with 1640 + 20% FBS

/ ^ml , spare;

2. Dosing in a 96-well plate: Appropriately diluted sOGP and raw white can be added to each well in a half-dilution method, with a volume of 50 μ 1;

3. Add cells to a 96-well plate: add 50 μl of cell suspension to each well;

4. Negative control well: Add 1640 + 20% FBS, 100 μ1.

5. Cultivate for 48 hours;

6. MTT solution: add 5mg / ml MTT solution 10 μ 1 to each well, and continue to culture for 4-6 hours;

7. Add cell lysate to each well and incubate overnight;

8. Colorimetry: Use a blank hole to zero, and measure the A value at a wavelength of 570nm. Results:

As shown in Figure 8, the 0D value of the whitening-positive control group increased significantly with increasing concentration, and in the low concentration of whitening-energy range, the 0D value was very close to the negative control group, indicating that the whitening energy was within a certain range. Within the concentration range, it can significantly promote the proliferation of human erythroleukemia TF-1 cells and has a significant dose-effect relationship. The OD value of each concentration gradient group of sOGP was similar to that of the negative control group, indicating that within this concentration range, sOGP did not promote the proliferation of CSF-dependent TF-1 red leukemia cells. All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

Rights request

1. Use of an osteogenic growth peptide, characterized in that it is used to prepare a pharmaceutical composition that promotes the proliferation of hematopoietic cells, mainly granulocytes.

2. The use according to claim 1, wherein the pharmaceutical composition treats the following disorders or conditions:

(1) Hematopoietic dysfunction caused by radiation injury;

(2) Treatment of hypo-hematopoiesis caused by chemotherapy drugs;

(Promote the proliferation of human bone marrow granulocyte hematopoietic progenitor cells;

(4) Treatment of leukopenia.

3. The use according to claim 1, wherein the pharmaceutical composition comprises an osteogenic growth peptide and a hematopoietic factor selected from the group consisting of G-CSF, GM-CSF, TP0 or a mixture thereof.

4. The use according to claim 1, wherein the pharmaceutical composition is used to prepare a pharmaceutical composition for promoting granulocyte proliferation.

5. The use according to claim 1, wherein the pharmaceutical composition is used before, during, or after radiotherapy or chemotherapy.

The use according to claim 1, wherein the osteogenic growth peptide has the following amino acid sequence:

Ala-Leu-Lys-Arg-Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-Gly.

7. A method for promoting the growth of granulocyte progenitor cells in vitro, characterized in that the granulocyte progenitor cells are cultured in a medium suitable for the growth of granulocyte progenitor cells, wherein the medium contains 1 (T ¹⁴ -l (T ⁵ m ₀ l / L of osteogenic growth peptide.

8. The method according to claim 7, wherein said medium containing ^{^{10 · 13 - 10- 5 mol /}} L of osteogenic growth peptide.

9. The method according to claim 7, wherein said medium containing ^10-12--

10- ⁵ mol / L osteogenic growth peptide.