CN115177715A - Application of recombinant human sDR5-Fc fusion protein in preparation of medicine for preventing and/or treating acute radiation sickness - Google Patents

Abstract

The invention discloses an application of a recombinant human sDR5-Fc fusion protein in preparing a medicament for preventing and/or treating acute radiation sickness. The recombinant human sDR5-Fc fusion protein has good protective effect on bone marrow type acute radiation diseases, intestinal type acute radiation diseases, brain type acute radiation diseases and the like, and is specifically shown in the aspects of improving the levels of peripheral blood leukocytes and platelets of mice after ionizing radiation, promoting the recovery of bone marrow hematopoietic stem cells and progenitor cells, remarkably improving the survival rate of the mice after being damaged by large-dose ionizing radiation and the like; meanwhile, the sDR5 is a human self-protein and has the advantages of low toxicity and no immunogenicity, so the sDR5-Fc fusion protein has good application prospect in the prevention and treatment of severe or extremely severe ARS caused by ionizing radiation, particularly extremely severe myelogenous ARS and the like.

Description

Application of recombinant human sDR5-Fc fusion protein in preparation of medicine for preventing and/or treating acute radiation sickness

Technical Field

The invention relates to the technical field of radioprotection protein medicines and medicines, in particular to application of a recombinant human sDR5-Fc fusion protein in preparation of medicines for preventing and/or treating acute radiation diseases.

Background

Acute Radiation Syndrome (ARS) is a systemic disease caused by a large dose of ionizing radiation in a short time. Ionizing radiation is a direct action such as ionization, excitation and collision on structural molecules in an organism or an indirect action such as generation of free radicals and free radical-like active molecules, so that the structure of a substance in the organism consisting of radiation-targeted structural molecules is damaged, or free radicals generated in the organism due to ionizing radiation compete for electrons in nearby cells, blood vessels, protein molecules, fat compounds and DNA molecules, so that normal tissues are damaged. The ionizing radiation damage is transmitted through different levels of a molecule-cell-tissue system, firstly, normal cell dysfunction and apoptosis are caused, and then, the damage and the dysfunction of normal tissues and organs are caused until visible clinical effects appear.

Research shows that the cell apoptosis induced by ionizing radiation is different from the cell apoptosis induced by chemical drugs (including various prescription or non-prescription chemical drugs, biological agents, traditional Chinese medicines, natural medicines, health care products, dietary supplements, metabolites and auxiliary materials thereof and the like) and other death stimuli (such as autoimmune inflammatory diseases). Ionizing radiation-induced apoptosis is related to radiation dose and radiation sensitivity of cells and presents heterogeneity of different cell types, among which SAPK/JNK (stress activated protein kinase or c2Jun amino terminal kinase) apoptosis signaling pathways, DNA damage-dependent apoptosis signaling pathways, cytoplasmic ionization damage-dependent apoptosis signaling pathways, and plasma membrane damage-dependent apoptosis signaling pathways are mainly involved. Among these ionizing radiation-induced apoptotic signaling pathways, mitochondria can exert central regulatory effects by causing apoptosis through increased mitochondrial membrane permeability, release of Cytc (cytochrome c) and activation of cysteine proteases (caspases) induced by Bcl-2 family proteins (e.g., bcl-2 down-regulation with anti-apoptotic effects and Bax up-regulation, a pro-apoptotic protein molecule) or through cell cycle arrest initiated by p53, the key performers being caspases. In addition, ionizing radiation-induced apoptosis is dependent on the cascade of caspases that can up-regulate the expression of Fas and/or Fasl, mediating normal apoptosis through the death receptor pathway. Therefore, research on the existing ARS prevention and treatment drugs mainly focuses on aspects of oxidation resistance, free radical elimination, anti-inflammation, mitochondrion targeting preparations, hematopoiesis promotion, organism immunity improvement and the like, and the existing ARS prevention and treatment drugs can be divided into sulfur-containing drugs, hormones, natural animals and plants, cytokines, hematopoietic stem cell transplantation, mitochondrion targeting preparations and the like.

(1) Containing sulfur

The sulfur-containing drugs contain free sulfydryl in the structure, and the representative drugs are amifostine (WR-2721) and N-acetylcysteine (NAC). The compounds have good functions of eliminating free radicals of tissues, resisting oxidation and the like due to the reduction characteristics, and are ionizing radiation protective agents with good effects at present. However, although the curative effect of the medicine on acute radiation diseases is definite, the safety of the synthetic medicine is a major hidden trouble in clinical application, for example, WR-2721 has strong free radical scavenging and antioxidant activities, but has high required dosage and more side effects, including hypotension, nausea and vomiting, rash, fever or shock, and the like; at the same time, due to the action mechanism and the characteristics of the drugs, the drugs are generally used before being irradiated by ionization.

(2) Hormones

Hormone drugs mainly have obvious ionizing radiation protection effects on bone marrow nucleated cells, hematopoietic stem cells and progenitor cells and can promote the recovery of the bone marrow nucleated cells, the hematopoietic stem cells and the progenitor cells, and representative drugs comprise '500' injection, '523' tablets, estriol, melatonin and the like. However, the main problem of hormone drugs is their estrogenic activity, which has certain side effects after application.

(3) Natural animal and plant materials

Researches find that various medicinal components and compounds can play a role in protecting ionizing radiation through the effects of resisting oxidation, protecting hematopoietic tissues, improving microcirculation, promoting cell proliferation and the like. The active ingredients such as natural polysaccharides, phenols, alkaloids, flavonoids, etc. have good protective effects on oxidative stress, bone marrow suppression, hypoimmunity, etc. caused by various rays. In addition, some natural medicine compound recipe also has certain therapeutic effect on acute radiation diseases.

(4) Cytokines

The application of cytokines has been a common practice in the treatment of ARS for many years, such as recombinant human granulocyte colony stimulating factor (rhGCSF), IL-12, recombinant human insulin-like growth factor-I (rhIGF-I), recombinant human thrombopoietin (rhTPO), etc. The hematopoietic growth factor is mainly used for treating moderate, severe and light extremely severe bone marrow type patients clinically, the hematopoietic function of the patients can be recovered by themselves at the moment, and the hematopoietic growth factor has certain response to medicaments, such as accelerating the rise of leucocytes, shortening the minimum duration and the like.

(5) Hematopoietic stem cell transplantation

The serious patients with the severe bone marrow type or above have difficult or impossible recovery of the hematopoietic function due to serious illness, and the common treatment is difficult to be effective, so that the hematopoietic stem cell transplantation is required to reconstruct the hematopoietic function. Currently, mesenchymal Stem Cells (MSCs), human umbilical cord blood stem cells, and the like are commonly used.

(6) Mitochondrion targeting formulations

Mitochondria play a central regulatory role in apoptosis signal pathways induced by ionizing radiation, and therefore mitochondrial targeting agents which function in inhibiting release of cytochrome c from mitochondria, inhibiting activation of caspases, down-regulating p53 protein expression, and the like are also ionizing radiation protectants effective in inhibiting apoptosis. For example, isopoxicillin (IF) can inhibit cytochrome c release from mitochondria and reduce ionizing radiation-induced intracellular high Ca ²⁺ The expression of caspase-3 is reduced, fas externalization, caspase-8 activation and the like are reduced, and therefore apoptosis is inhibited; alkene peptide ectopin (JP 4-039), nitric oxide synthase inhibitor-synthase related alkene peptide (MCF 201-89), and P53/MDM2/MDM4 protein complex inhibitor (BEB 55) all reduce ionizing radiation damage.

However, although many of the various ARS prevention and treatment medicines exist at present, much attention is paid to the protection of the injury caused by the ionizing radiation with the smaller dose, and much attention is paid to the prevention and treatment of the injury caused by the ionizing radiation with the larger dose, the fatality rate of the irradiated person with the irradiated ionizing radiation dose less than 6Gy is counted to be about 6.8%, the fatality rate of the irradiated person with the ionizing radiation dose more than 6Gy is counted to be remarkably improved to be 90.0%, and no case of the irradiated person more than 8Gy survives. With the wide application of current nuclear technology in military and civil fields, it is especially important to improve the control capability of ARS, especially for heavy and extremely heavy ARS (such as critical ARS of extremely severe myelotype, intestinal type, brain type, etc.) caused by high dose ionizing radiation. However, to date, there has been limited progress in both domestic and foreign research on the rescue of ARS, particularly heavy and very heavy ARS.

Disclosure of Invention

In order to solve the problems of the background art, the present invention provides a use of a recombinant human sDR5-Fc fusion protein for preparing a medicament for preventing and/or treating acute radiation sickness, wherein the recombinant human sDR5-Fc fusion protein can achieve the effect of preventing and/or treating acute radiation sickness by inhibiting excessive normal apoptosis caused by ionizing radiation. The invention creatively applies the recombinant human sDR5-Fc fusion protein to the prevention and treatment of ARS caused by ionizing radiation, particularly heavy and extremely heavy ARS caused by large-dose ionizing radiation exposure, and surprisingly discovers that the recombinant human sDR5-Fc fusion protein can really prevent and treat ARS caused by ionizing radiation, particularly heavy and extremely heavy myeloid ARS caused by large-dose ionizing radiation exposure, and particularly shows that the recombinant human sDR5-Fc fusion protein can improve the levels of peripheral blood leukocytes and platelets after ionizing radiation and promote the recovery of hematopoietic stem cells and progenitor cells of bone marrow.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides an application of a recombinant human sDR5-Fc fusion protein in preparing a medicament for preventing and/or treating acute radiation sickness induced by ionizing radiation.

Further, the ionizing radiation-induced acute radiation diseases include bone marrow type acute radiation diseases, intestinal type acute radiation diseases, and brain type acute radiation diseases.

Further, the acute radiation disease induced by ionizing radiation can be acute radiation disease induced by ionizing radiation with a large dose, and the ionizing radiation with the large dose is ionizing radiation with the dose of more than or equal to 6 Gy.

Further, the recombinant human sDR5-Fc fusion protein prevents ionizing radiation-induced acute radiation disease by blocking apoptosis induced by ionizing radiation by blocking or blocking TRAIL signaling pathway.

Further, the recombinant human sDR5-Fc fusion protein treats acute radiation sickness by restoring the level of white blood cells and platelets and the proportion of lymphocytes in peripheral blood of patients with acute radiation sickness, and restoring the number of LK and LSK cells in bone marrow.

Further, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, respectively.

In another aspect, the invention provides an application of a recombinant human sDR5-Fc fusion protein in preparing a medicament for blocking cell apoptosis induced by ionizing radiation.

Furthermore, the recombinant human sDR5-Fc fusion protein can be used for preparing a medicine for blocking apoptosis induced by high-dose ionizing radiation, wherein the high-dose ionizing radiation is ionizing radiation of more than or equal to 6 Gy.

Further, the recombinant human sDR5-Fc fusion protein blocks apoptosis induced by ionizing radiation by blocking or blocking TRAIL signaling pathway.

Further, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, respectively.

In another aspect, the use of the recombinant human sDR5-Fc fusion protein in the preparation of a medicament for restoring the level of leukocytes, platelets and the proportion of lymphocytes in peripheral blood of patients with acute radiation, and for restoring the number of LK and LSK cells in bone marrow.

Further, the amino acid sequence of the recombinant human sDR5-Fc fusion protein is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, respectively.

The beneficial effects of the invention are:

the invention discovers that ionizing radiation causes the up-regulation of DR5 protein expression level in normal tissue cells in organisms for the first time, and proves that TRAIL-DR5 mediated apoptosis signal path plays an important role in normal tissue cell apoptosis caused by ionizing radiation (including high-dose ionizing radiation). The invention provides the application of the recombinant human sDR5-Fc fusion protein in the preparation of the medicine for preventing and/or treating acute radiation sickness for the first time, the recombinant human sDR5-Fc fusion protein takes a TRAIL-DR5 system as a target spot, and inhibits excessive normal tissue apoptosis caused by ionizing radiation by inhibiting an apoptosis signal path of the TRAIL-DR5, so that the recombinant human sDR5-Fc fusion protein can be effectively used for preventing and/or treating ARS.

The recombinant human sDR5-Fc fusion protein has good protective effect on hematopoietic immune tissue (bone marrow type) acute radiation diseases, intestinal acute radiation diseases, brain acute radiation diseases and the like, and is particularly characterized in that the level of peripheral blood leukocytes and platelets of a mouse after ionizing radiation can be improved, the recovery of hematopoietic stem cells and progenitor cells of bone marrow is promoted, the survival rate of the mouse after being damaged by large-dose ionizing radiation is obviously improved, and the like; meanwhile, the sDR5 is a human self-protein and has the advantages of low toxicity and no immunogenicity, so the sDR5-Fc fusion protein has good application prospect in the prevention and treatment of severe or extremely severe ARS caused by ionizing radiation, particularly extremely severe myelogenous ARS and the like.

Drawings

FIG. 1 is a TUNNEL-DR5 immunofluorescence image of tissue sections of thoracic glands of BALB/c mice after single total body irradiation with 60Co gamma rays in example 1 of the present invention; wherein A-C: double-label overlay of TUNNEL-DR5 antibody; D-F: the expression level of DR5 protein detected by DR5 antibody marking; G-I: apoptosis rate in the TUNNEL assay;

FIG. 2 is a graph showing survival curves of mice of different treatment groups after ionizing radiation injury in example 2 of the present invention;

FIG. 3 is a graph showing the change in the level of leukocytes in peripheral blood at different times after administration to 6.0Gy ionizing radiation mice in example 3 of the present invention;

FIG. 4 is a graph showing the variation of the platelet level in peripheral blood at different times after administration of 6.0Gy ionizing radiation in example 3 of the present invention;

FIG. 5 is a graph showing the ratio of lymphocytes in peripheral blood at different times after administration of 6.0Gy ionizing radiation mice in example 3 of the present invention;

FIG. 6 is a histogram of the change in the level of LK cells in bone marrow at different times after administration of sDR5-Fc fusion protein to 4.0Gy ionizing radiation mice in example 4 of the present invention;

FIG. 7 is a histogram of the change in the levels of LSK cells in bone marrow at different times after 4.0Gy ionizing radiation administration of sDR5-Fc fusion protein in example 4 of the present invention.

Detailed Description

The term "DR5 (Death Receptor 5)" herein is a member of the Tumor Necrosis Factor (TNF) Receptor family, which is low expressed in normal tissues and high expressed in inflammatory, tumor and ischemic tissues. DR5 specifically binds to tumor necrosis factor-related inducing ligand (TNF-induced apoptosis) to induce apoptosis. DR5 binds to ligand TRAIL to cause DR5 oligomerization and activation, activated death receptor DR5 recruits FADD (Fas-associated death domain) molecules, and FADD recruits Caspase-8 precursor (Pro-Caspase-8) through DED (death-effector domain) to form a death-signaling complex DISC (death-inducing signaling complex). Pro-Caspase-8 is activated to form Caspase-8, caspase-8 triggers apoptosis via both a mitochondria-independent pathway and a mitochondria-independent pathway. The term "soluble DR 5" as used herein is a soluble form of DR5 that does not contain a transmembrane region and is secreted outside the cell due to the absence of the transmembrane region, which is not expressed on the cell membrane. sDR5 is expressed at a low level in normal human peripheral blood, and sDR5, although maintaining its activity of binding to TRAIL ligand, cannot transmit apoptosis signals into cells, and can block TRAIL-DR 5-mediated apoptosis reaction the term "recombinant human sDR5-Fc fusion protein" as used herein, is a recombinant fusion protein obtained by connecting human sDR5 to Fc expression gene and then performing recombinant expression.

The present invention will be described in detail with reference to the following embodiments and drawings.

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: a Molecular Cloning Laboratory Manual (Molecular Cloning: A Laboratory Manual, sambrook, J., russell, david W., molecular Cloning: A Laboratory Manual,3rd edition,2001, NY, cold Spring Harbor).

The various biological materials described in the examples are obtained by way of example only to provide a means for obtaining a test for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

Experimental animals, reagents and instruments and their sources used in the following examples:

1) Laboratory animal

BALB/c mouse, male, 6-8 weeks old, weight 19-21 g, purchased from Beijing Wintolite laboratory animal technology Limited, animal quality qualification No: SCXK- (Jing) 2016-0006.

2) Reagent and apparatus

sDR5-Fc fusion protein: SEQ ID NO:4, test code AS1501, 25 mg/bottle, from shenzhen zhongke Ai Shen pharmaceuticals, ltd;

electronic analytical balance: ME235S, available from Sartorius, germany;

an electronic balance: EPS-2001, available from Changshan Xiangping science and technology development Inc.;

bolingman full-automatic blood analyzer: model BM860 available from beijing trealmann sunshine technology ltd.

Example 1: detection of DR5 protein expression level in ionizing radiation induced apoptosis

In this example, an experimental mouse is irradiated with ionizing radiation to detect the expression of DR5 protein level in an immune organ (e.g., thymus) of the irradiated mouse, and the inhibition of ionizing radiation-induced apoptosis by sDR5-Fc fusion protein (AS 1501) is preliminarily examined, which specifically includes the following steps.

1.1, irradiating a BALB/c mouse by 60Co gamma rays on the whole body once, wherein the irradiation dose is 6.0Gy, the irradiation dose rate is 68.7cGy/min, and the irradiation distance is 90cm;

1.2, randomly dividing the irradiated BALB/c mice into a normal control group (non-irradiated group), a model control group and an AS1501 administration group, wherein the AS1501 administration group is continuously administered with 10mg/kg of tail vein every day on the day of irradiation, the first day, the second day and the third day; the model control group is given physiological saline blank solvent;

1.3, taking off the neck of each group of mice to euthanasia 24 hours after administration, taking thymus tissues, making sections, detecting the apoptosis rate by an immunohistochemical TUNNEL method, and simultaneously detecting the change of the expression level of the DR5 protein by a fluorescence labeling DR5 antibody.

The results are shown in FIG. 1, which is a TUNNEL-DR5 immunofluorescence image of tissue sections of the thoracic gland of a single total body irradiation BALB/C mice with 60Co gamma rays, wherein A-C: TUNNEL-DR5 antibody double-label overlay; D-F: the expression level of DR5 protein detected by DR5 antibody marking; G-I: apoptosis rate measured by TUNNEL. Therefore, after BALB/c mice are irradiated by 6.0Gy of single total 60Co gamma ray, the expression level of DR5 protein and the apoptosis level in thymus tissues are obviously increased, which shows that the DR5 protein level of normal tissues of the mice is obviously up-regulated after the mice are irradiated by large dose of ionizing radiation, and the TRAIL-DR5 mediated apoptosis is also proved to have an important role in a wound-causing mechanism of ionizing radiation damage. On the other hand, after the mice are irradiated on the whole body by 60Co gamma rays once, compared with a model control group, the DR5 protein expression level of the thymus tissue of the mice damaged by ionizing radiation in the AS1501 administration group has no obvious change, but the apoptosis quantity is reduced, further proving that TRAIL-DR5 mediated apoptosis signal channel plays an important role in the apoptosis induced by ionizing radiation, and preliminarily proving that the sDR5-Fc fusion protein can really inhibit the excessive apoptosis of the thymus cells of the mice caused by ionizing radiation damage.

Example 2: effect of sDR5-Fc fusion protein on mouse survival Rate after Large dose ionizing radiation

In this embodiment, the sDR5-Fc fusion protein is used to administer drug to a mouse after a large dose of ionizing radiation, and the survival rate of the mouse after administration is statistically analyzed to detect the influence of the sDR5-Fc fusion protein on the survival rate of the mouse after the large dose of ionizing radiation, which specifically includes the following steps.

2.1, randomly dividing BALB/c mice into a radiation model control group, an amifostine (AS a positive drug) group, an AS1501 high, medium and low dose group and a normal control group, and totally 6 groups, wherein 9 mice in each group are AS follows:

radiation model control group: on the day after radiation and on days 1-3, a physiological saline blank solvent control is given by tail vein injection every day;

amifostine group: 30 minutes before radiation, 150mg/kg of intraperitoneal injection of amifostine is given;

AS1501 high dose group: on the day after irradiation and on days 1-3, 15mg/kg of AS1501 solution was administered by tail vein injection every day;

AS1501 dose group: on the day after irradiation and on days 1-3, the AS1501 solution was administered at a dose of 10mg/kg by tail vein injection every day;

AS1501 low dose group: on the day after irradiation and on days 1-3, 5mg/kg of AS1501 solution was administered by tail vein injection every day;

normal control group: normal BALB/c mice that did not receive radiation.

2.2, radiation dose: mice in a radiation model control group, an amifostine group and AS1501 high, medium and low dose groups all receive 60Co gamma rays for whole body irradiation once, the irradiation dose is 8.5Gy, the irradiation dose rate is 68.68cGy/min, and the irradiation distance is 90cm.

2.3, index detection: after the mice of different experimental groups are irradiated, the death data of the mice are observed, and survival curves at different time after administration are drawn.

The results are shown in fig. 2, which is a survival curve of mice in different experimental groups, and it can be seen that, compared to a radiation model control group (NS), BALB/c mice subjected to single systemic 60Co γ ray ionizing radiation of 8.5Gy can significantly improve the survival rate of mice subjected to ionizing radiation after administration of high, medium and low doses of sDR5-Fc fusion protein through continuous tail vein injection, and exhibit significant dose dependence, which indicates that the sDR5-Fc fusion protein has the effect of significantly improving the survival rate of mice damaged by ionizing radiation, the survival state of mice in a normal control group is good, the survival rate is 100%, and the survival curve is not shown in fig. 2.

Example 3: effect of sDR5-Fc fusion protein on ionizing radiation damage mouse peripheral hemogram

The embodiment utilizes the sDR5-Fc fusion protein to carry out administration treatment on a mouse after ionizing radiation and measure peripheral hemogram of the mouse after administration so as to detect the influence of the sDR5-Fc fusion protein on the peripheral hemogram of the mouse damaged by ionizing radiation.

3.1, randomly dividing BALB/c mice into a normal control group, a normal saline control group and an sDR5-Fc fusion protein administration group, wherein:

saline control group: on the day after radiation and on days 1-3, a physiological saline blank solvent control is given by tail vein injection every day;

sDR5-Fc fusion protein administration group: on the day after irradiation and on days 1-3, 5mg/kg of AS1501 solution was administered by tail vein injection every day;

normal control group: normal BALB/c mice that did not receive radiation.

3.2, radiation dose: mice of a normal saline control group and an sDR5-Fc fusion protein administration group both receive 60Co gamma rays for whole body irradiation once, the irradiation dose is 6Gy, the irradiation dose rate is 68.7cGy/min, and the irradiation distance is 90cm.

3.3, sample collection: on the day of and 1, 3, 5, 7, 11, 15, 19, 24, 30d after irradiation, 3 mice were randomly collected from the normal control group, the normal saline control group, and the group administered with the sDR5-Fc fusion protein, 30 μ L of blood was collected from the orbital venous plexus in an anticoagulation tube, and peripheral blood samples including White Blood Cell (WBC) level, platelet (PLT) level, and lymphocyte ratio (LYM%) in the peripheral blood sample were detected using a fully automatic hematology analyzer.

The results of detecting the level of white blood cells in peripheral hemograms of mice in each group are shown in fig. 3, and it can be seen that WBC in peripheral blood of mice in an sDR5-Fc fusion protein administration group and a physiological saline control group are significantly reduced after 6.0Gy irradiation relative to mice in a normal control group, and are most significant 3-7 days after irradiation, which indicates that the level of WBC in peripheral blood of mice can be reduced in the process of inducing apoptosis by ionizing radiation. Mice in the group to which the sDR5-Fc fusion protein was administered were treated with AS1501 after irradiation, and the level of leukocytes in peripheral blood was significantly higher (p < 0.05) on days 7 to 13 relative to those in the normal saline control group, indicating that the sDR5-Fc fusion protein had a tendency to promote the recovery of the level of leukocytes in peripheral blood at an early stage after ionizing radiation-induced injury.

The detection results of the platelet levels in the peripheral hemogram of each group of mice are shown in fig. 4, and it can be seen that, compared with the normal control group of mice, after 6.0Gy radiation, PLT in the peripheral blood of mice in the sDR5-Fc fusion protein administration group and the normal saline control group is significantly reduced, and is most significant about 7 days after radiation, which indicates that ionizing radiation can cause the reduction of PLT level in the peripheral blood in the process of inducing apoptosis. The mice of the sDR5-Fc fusion protein administration group are subjected to AS1501 administration treatment after irradiation, and compared with the mice of a normal saline control group, the mice of the sDR5-Fc fusion protein administration group show a more obvious function of promoting the recovery of the platelet level 2-4 weeks after irradiation, and quickly reach the level of the platelets in the peripheral blood of the mice of a normal control group, so that the sDR5-Fc fusion protein can effectively promote the quick recovery of the level of the platelets in the peripheral blood after ionizing radiation induced damage.

The detection results of the lymphocyte proportion in the peripheral hemogram of each group of mice are shown in fig. 5, and it can be seen that, compared with the normal control group of mice, after 6.0Gy radiation, the lymphocyte proportion of mice in an sDR5-Fc fusion protein administration group and a normal saline control group of mice is obviously reduced, which indicates that the lymphocyte proportion in peripheral blood can be obviously reduced in the process of inducing apoptosis by ionizing radiation. As1501 administration treatment is carried out on mice in an sDR5-Fc fusion protein administration group after irradiation, and compared with mice in a normal saline control group, the lymphocyte level can be remarkably increased, which indicates that the sDR5-Fc fusion protein can effectively recover the lymphocyte level after ionizing radiation induced damage.

Example 4: influence of sDR5-Fc fusion protein on bone marrow hematopoietic function of mice damaged by ionizing radiation

The embodiment utilizes the sDR5-Fc fusion protein to carry out administration treatment on a mouse subjected to ionizing radiation and detect the bone marrow hematopoietic function of the mouse subjected to the administration so as to detect the influence of the sDR5-Fc fusion protein on the bone marrow hematopoietic function of the mouse subjected to ionizing radiation damage.

4.1, randomly dividing BALB/c mice into a normal control group, a normal saline control group and an sDR5-Fc fusion protein administration group, wherein:

saline control group: on the day after radiation and on days 1-3, a physiological saline blank solvent control is given by tail vein injection every day;

sDR5-Fc fusion protein administration group: on the day after irradiation and on days 1-3, the AS1501 solution is administered at a dose of 10mg/kg by tail vein injection every day;

normal control group: normal BALB/c mice that did not receive radiation.

4.2, radiation dose: mice of a normal saline control group and an sDR5-Fc fusion protein administration group both receive 60Co gamma rays for whole body irradiation once, the irradiation dose is 4Gy, the irradiation dose rate is 68.7cGy/min, and the irradiation distance is 90cm.

4.3, sample collection: on the day (2 h) after irradiation and on the 1 st, 4 th, 7 th, 11 th, 14 th, 19d after irradiation, 3 mice were randomly selected from the normal control group, the normal saline control group and the group administered with the sDR5-Fc fusion protein, respectively, anesthetized and sacrificed, bone marrow cells were isolated and counted, and after incubation with the CD16/32 antibody, 1 st blocking with BSA was performed; the subsequent labeling with the linkage antibody, sca-1 antibody and c-kit was performed at 4 ℃ for 30min. After 0.4% paraformaldehyde is fixed, the percentage of LinSca-1-c-Kit + (LK) and Lin-Sca-1+c-Kit + (LSK) cells in bone marrow nucleated cells is detected by a flow cytometer and the number of cells is calculated.

The results of detecting the numbers of the bone marrow LK cells and the LSK cells of the mice in each group are respectively shown in fig. 6 and fig. 7, and it can be seen that after 4.0Gy radiation, the numbers of the bone marrow LK cells and the LSK cells of the mice in the sDR5-Fc fusion protein administration group and the normal saline control group are both significantly reduced, which indicates that the ionizing radiation can cause the significant reduction of the numbers of the bone marrow LK cells and the LSK cells in the process of inducing apoptosis. Compared with a normal saline control group, the sDR5-Fc fusion protein administration group can obviously increase the levels of the marrow LK and LSK cells at a plurality of time points, and the sDR5-Fc fusion protein has a certain effect of promoting the repair of the hematopoietic function of the marrow in the treatment of ionizing radiation damage.

As can be seen from the above results, ionizing radiation can induce apoptosis of normal tissue cells, and in the case of ionizing radiation-induced apoptosis of normal tissue (e.g., immune organs, hematopoietic system, etc.), expression level of DR5 protein in the tissue is detected to be significantly up-regulated, and based on the result that sDR5-Fc fusion protein can effectively reduce apoptosis rate, it is demonstrated that TRAIL-DR 5-mediated apoptosis signaling pathway plays an important role in ionizing radiation-induced apoptosis, at least inducing apoptosis of leukocytes, platelets and lymphocytes in peripheral blood, as well as LK and LSK cells in hematopoietic system such as bone marrow, resulting in change of blood image balance and reduction of the number of hematopoietic stem and progenitor cells, and further triggering severe acute radiation disease ARS. Based on the above, the inventor creatively uses the sdR5-Fc fusion protein in the prevention and/or treatment of ARS caused by ionizing radiation, and utilizes the principle that the sdR5-Fc fusion protein can block TRAIL-DR 5-mediated apoptosis signal pathway, effectively protects normal tissue cells from radiation in the ionizing radiation process and promotes the recovery of cell level in normal tissues after the ionizing radiation damage, and particularly can remarkably improve the levels of leukocyte, platelet and lymphocyte which are key indexes of hemogram in peripheral blood after the ionizing radiation damage, promote the rapid recovery of the hemogram to normal state, and promote the recovery of bone marrow hematopoietic stem cells and progenitor cells. Therefore, the sDR5-Fc fusion protein has good application prospect in the prevention and/or treatment of severe or extremely severe ARS caused by ionizing radiation, especially extremely severe myelogenous ARS and the like, and provides a new method for the treatment of ARS.

The amino acid sequence is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 has the amino acid sequence shown as SEQ ID NO:4 similar effect of sDR5-Fc fusion protein.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Zhongke Ai Shen medicine Limited

Application of recombinant human sDR5-Fc fusion protein in preparation of medicine for preventing and/or treating acute radiation sickness

<130> CP121010180C

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 370

<212> PRT

<213> Artificial sequence

<400> 1

Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln

1 5 10 15

Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile

20 25 30

Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr

35 40 45

Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys

50 55 60

Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr

65 70 75 80

Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu

85 90 95

Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val

100 105 110

Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Gly

115 120 125

Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

130 135 140

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

145 150 155 160

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

165 170 175

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

180 185 190

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

195 200 205

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

210 215 220

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

225 230 235 240

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

245 250 255

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

260 265 270

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

275 280 285

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

290 295 300

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

305 310 315 320

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

325 330 335

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

340 345 350

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

355 360 365

Gly Lys

370

<210> 2

<211> 359

<212> PRT

<213> Artificial sequence

<400> 2

Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln

1 5 10 15

Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile

20 25 30

Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr

35 40 45

Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys

50 55 60

Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr

65 70 75 80

Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu

85 90 95

Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val

100 105 110

Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Glu

115 120 125

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

130 135 140

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

145 150 155 160

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

165 170 175

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

180 185 190

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

195 200 205

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

210 215 220

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

225 230 235 240

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

245 250 255

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

260 265 270

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

275 280 285

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

290 295 300

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

305 310 315 320

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

325 330 335

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

340 345 350

Leu Ser Leu Ser Pro Gly Lys

355

<210> 3

<211> 348

<212> PRT

<213> Artificial sequence

<400> 3

Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro

1 5 10 15

Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys

20 25 30

Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu

35 40 45

Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr

50 55 60

Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu

65 70 75 80

Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg

85 90 95

Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys

100 105 110

Val His Lys Glu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

115 120 125

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

130 135 140

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

145 150 155 160

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

165 170 175

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

180 185 190

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

195 200 205

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

210 215 220

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

225 230 235 240

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

245 250 255

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

260 265 270

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

275 280 285

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

290 295 300

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

305 310 315 320

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

325 330 335

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345

<210> 4

<211> 341

<212> PRT

<213> Artificial sequence

<400> 4

Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser Glu

1 5 10 15

Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr

20 25 30

His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser

35 40 45

Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr Val Cys

50 55 60

Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Met Cys

65 70 75 80

Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val Gly Asp

85 90 95

Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Glu Pro Lys

100 105 110

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

115 120 125

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

130 135 140

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

145 150 155 160

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

165 170 175

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

180 185 190

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

195 200 205

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

210 215 220

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

225 230 235 240

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

245 250 255

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

260 265 270

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

275 280 285

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

290 295 300

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

305 310 315 320

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

325 330 335

Leu Ser Pro Gly Lys

340

Claims (10)

1. Application of the recombinant human sDR5-Fc fusion protein in preparing a medicament for preventing and/or treating acute radiation sickness induced by ionizing radiation.

2. The use of claim 1, wherein the ionizing radiation-induced acute radiation disease comprises acute radiation disease of the myeloid type, acute radiation disease of the intestinal type and acute radiation disease of the brain type.

3. The use of claim 1, wherein the recombinant human sDR5-Fc fusion protein prevents ionizing radiation-induced acute radiation sickness by blocking or blocking TRAIL signaling pathway to block ionizing radiation-induced apoptosis.

4. The use of claim 1, wherein the recombinant human sDR5-Fc fusion protein treats acute radiation sickness by restoring leukocyte, platelet levels and lymphocyte ratios in peripheral blood of patients with acute radiation, and restoring the numbers of LK and LSK cells in bone marrow.

5. The use of any one of claims 1-4, wherein the amino acid sequence of the recombinant human sDR5-Fc fusion protein is as set forth in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, respectively.

6. Application of recombinant human sDR5-Fc fusion protein in preparing medicine for blocking cell apoptosis induced by ionizing radiation.

7. The use of claim 6, wherein the recombinant human sDR5-Fc fusion protein blocks apoptosis induced by ionizing radiation by blocking or blocking a TRAIL signaling pathway.

8. The use of any one of claims 6-7, wherein the amino acid sequence of the recombinant human sDR5-Fc fusion protein is as set forth in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, respectively.

9. The recombinant human sDR5-Fc fusion protein is applied to the preparation of medicines for restoring the level of leukocytes and platelets and the proportion of lymphocytes in peripheral blood of patients with acute radiation and restoring the number of LK and LSK cells in bone marrow.

10. The use of claim 9, wherein the amino acid sequence of the recombinant human sDR5-Fc fusion protein is as set forth in SEQ ID NO: 1. SEQ ID NO: 2. the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, respectively.

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