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 the application of a recombinant human sDR5-Fc fusion protein in preparing a medicine for preventing and/or treating acute radiation sickness. Recombinant human sDR5-Fc fusion protein has a good protective effect on bone marrow-type acute radiation sickness, intestinal-type acute radiation sickness and cerebral-type acute radiation sickness, etc. The specific manifestation is that it can increase the levels of peripheral blood leukocytes and platelets in mice after ionizing radiation, and promote bone marrow hematopoiesis Stem and progenitor cells recover, and significantly improve the survival rate of mice after high-dose ionizing radiation injury; at the same time, since sDR5 itself is a human body protein, it has the advantages of low toxicity and no immunogenicity. It can have a good application prospect in the prevention and treatment of severe or extremely severe ARS caused by ionizing radiation, especially in extremely severe myeloid ARS.

Description

Recombinant human sDR5-Fc fusion protein in the preparation of prevention and/or treatment of acute radiation sickness application in medicine

technical field

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

Background technique

Acute radiation syndrome (ARS) is a systemic disease caused by a large dose of ionizing radiation in a short period of time. Ionizing radiation is a direct effect on the ionization, excitation and collision of structural molecules in the organism or indirect effect on the generation of active molecules such as free radicals and free radical-like molecules, resulting in structural damage to the material in vivo composed of structural molecules targeted by radiation or due to ionization. The free radicals generated in the living body by radiation will cause damage to normal tissues by competing for electrons in nearby cells, blood vessels, protein molecules, fatty compounds, and DNA molecules. This ionizing radiation damage is transmitted through different levels of molecule-cell-tissue-system, first leading to normal cell dysfunction and apoptosis, and then causing normal tissue and organ damage and dysfunction until visible clinical effects occur.

Studies have shown that ionizing radiation-induced apoptosis is different from chemical drugs (including various prescription or non-prescription chemical drugs, biological agents, traditional Chinese medicines, natural medicines, health products, dietary supplements and their metabolites and excipients, etc.) and Apoptosis induced by other death stimuli such as autoimmune inflammatory diseases. Ionizing radiation-induced apoptosis is related to radiation dose and the radiation sensitivity of cells, and exhibits heterogeneity across different cell types, among which SAPK/JNK (stress-activated protein kinase or c2Jun N-terminal kinase) apoptosis is mainly involved Signaling pathway, DNA damage-dependent apoptosis signalling pathway, cytoplasmic ionization-damage-dependent apoptosis signalling pathway, and plasma membrane damage-dependent apoptosis signalling pathway. In these ionizing radiation-induced apoptosis signaling pathways, mitochondria can pass through the mitochondrial membrane induced by Bcl-2 family proteins (such as Bcl-2 down-regulation with anti-apoptotic effect and up-regulation of pro-apoptotic protein molecule Bax). Increased permeability, release of Cytc (cytochrome c) and activation of caspases or apoptosis through p53-initiated cell cycle arrest play central regulatory roles, of which the key executors are caspases. In addition, ionizing radiation-induced apoptosis also depends on the cascade transmission of caspases, which can upregulate the expression of Fas and/or Fasl and mediate normal apoptosis through the death receptor pathway. Therefore, the current research on ARS prevention and treatment drugs mainly focuses on anti-oxidation, elimination of free radicals, anti-inflammatory, mitochondrial targeting agents, promotion of hematopoiesis, and improvement of body immunity. , natural animals and plants, cytokines, hematopoietic stem cell transplantation, mitochondrial targeting agents, etc.

(1) Sulfur-containing

Sulfur-containing drugs contain free sulfhydryl groups, and representative drugs are amifostine (WR-2721) and N-acetylcysteine (NAC). The reducing properties of these compounds make them have good effects such as scavenging free radicals in tissues and anti-oxidation, and are currently a class of ionizing radiation protection agents with good effects. However, although these drugs are effective in treating acute radiation sickness, the safety of these synthetic drugs is the main hidden danger in clinical application. For example, although WR-2721 has strong free radical scavenging and antioxidant activities, its required dose Higher, more side effects, including hypotension, nausea and vomiting, rash, fever or shock, etc. At the same time, due to the mechanism of action and drug properties, these drugs are usually effective before being exposed to ionizing radiation.

(2) Hormones

Hormone drugs mainly have obvious ionizing radiation protection effect on bone marrow nucleated cells, hematopoietic stem cells and progenitor cells, and can promote their recovery. Representative drugs include "500" injection, "523" tablet, estriol and Melatonin etc. However, the main problem of hormonal drugs is that they have estrogenic activity and have certain side effects after application.

(3) Natural animals and plants

Studies have found that a variety of drug components and compounds can exert their protective effects on ionizing radiation through their antioxidant, hematopoietic tissue protection, microcirculation, and cell proliferation-promoting effects. Such as natural polysaccharides, phenols, alkaloids, flavonoids and other active ingredients have a good protective effect on oxidative stress, bone marrow suppression, and immunosuppression caused by various rays. In addition, some natural medicine compounds also have a certain therapeutic effect on acute radiation sickness.

(4) Cytokines

The application of cytokines has been a common method 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) and recombinant human thrombopoietin (rhIGF-I). rhTPO) and so on. Clinically, hematopoietic growth factor is mainly used for the treatment of moderate, severe and mildly severe myeloid patients. At this time, the patient's hematopoietic function can still recover by itself, and it can respond to drugs to a certain extent. For example, the recovery of white blood cells is accelerated, and the minimum duration is shorten etc.

(5) Hematopoietic stem cell transplantation

Severely severe patients with myeloid type or above are severely ill and the hematopoietic function is difficult to recover or cannot recover on their own, and the general treatment is not effective. At this time, hematopoietic stem cell transplantation is required to rebuild the hematopoietic function. At present, mesenchymal stem cells (MSCs) and human cord blood stem cells are commonly used.

(6) Mitochondrial targeting preparations

Mitochondria play a central regulatory role in the ionizing radiation-induced apoptosis signaling pathway, so mitochondria-targeted agents that inhibit the release of cytochrome c from mitochondria, inhibit the activation of caspases, and downregulate p53 protein expression are also effective in inhibiting apoptosis. Ionizing radiation protectant. For example, isoflurane (IF) can inhibit the release of cytochrome c from mitochondria, reduce the high intracellular Ca ²⁺ induced by ionizing radiation, down-regulate the expression of caspases-3, reduce the externalization of Fas and the activation of caspases-8, etc., thereby inhibiting apoptosis alkene peptide ectopic protein (JP4-039), nitric oxide synthase inhibitor-synthase-related alkene peptide (MCF201-89) and P53/MDM2/MDM4 protein complex inhibitor (BEB55), etc. Reduce ionizing radiation damage.

However, although there are many types of ARS prevention and treatment drugs mentioned above, they focus more on the protection of small doses of ionizing radiation damage, and less attention on the prevention and treatment of large doses of ionizing radiation damage. According to statistics, exposure to ionizing radiation The case fatality rate was about 6.8% in the doses <6Gy, and the case fatality rate was significantly increased to 90.0% in the doses ≥6Gy, and none of the subjects with ≥8Gy survived. With the wide application of current nuclear technology in military and civilian fields, the prevention and treatment of ARS, especially for severe and extremely severe ARS (such as severe bone marrow type, intestinal type, brain type, etc.) caused by high-dose ionizing radiation, should be improved. Ability is particularly important. But so far, domestic and foreign research on the treatment of ARS, especially heavy and extremely heavy ARS, has been limited.

SUMMARY OF THE INVENTION

In order to solve the problems raised in the above-mentioned background art, the purpose of the present invention is to provide the application of a recombinant human sDR5-Fc fusion protein in the preparation of a medicine for preventing and/or treating acute radiation sickness, the recombinant human sDR5-Fc fusion protein can be Preventive and/or therapeutic effects on acute radiation sickness are achieved by inhibiting excessive normal cell apoptosis caused by ionizing radiation. The present invention creatively applies the recombinant human sDR5-Fc fusion protein to the prevention and treatment of ARS caused by ionizing radiation, especially severe and very severe ARS caused by exposure to large doses of ionizing radiation. Fc fusion protein can indeed prevent and treat ARS caused by ionizing radiation, especially severe and very severe myeloid ARS caused by exposure to large doses of ionizing radiation. The levels of white blood cells and platelets can promote the recovery of bone marrow hematopoietic stem and progenitor cells.

In order to achieve the above purpose, the technical solutions adopted in the present invention are as follows: On the one hand, the present invention provides the application of a recombinant human sDR5-Fc fusion protein in the preparation of a medicine for preventing and/or treating acute radiation sickness induced by ionizing radiation.

Further, the acute radiation sickness induced by ionizing radiation includes acute radiation sickness of bone marrow type, acute radiation sickness of intestinal type and acute radiation sickness of brain type.

Further, the acute radiation sickness induced by ionizing radiation may be acute radiation sickness induced by high-dose ionizing radiation, and the high-dose ionizing radiation is ionizing radiation of ≥6 Gy.

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

Further, the recombinant human sDR5-Fc fusion protein treats acute radiation sickness by restoring the level of leukocytes, platelets and lymphocyte ratio 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 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

In another aspect, the present invention provides the use of a recombinant human sDR5-Fc fusion protein in the preparation of a drug for blocking apoptosis induced by ionizing radiation.

Further, the recombinant human sDR5-Fc fusion protein can be used to prepare a drug for blocking apoptosis induced by high-dose ionizing radiation, where the high-dose ionizing radiation is ionizing radiation of ≥6 Gy.

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

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

In another aspect, the recombinant human sDR5-Fc fusion protein is used in the preparation of a drug for restoring the level of leukocytes, platelets and lymphocyte ratio 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 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

The beneficial effects of the present invention are:

The present invention finds for the first time that ionizing radiation leads to the up-regulation of the expression level of DR5 protein in normal tissue cells in vivo, and proves that TRAIL-DR5-mediated apoptosis signaling pathway plays an important role in the apoptosis of normal tissue cells caused by ionizing radiation (including high-dose ionizing radiation). play an important role in. The present invention proposes for the first time the application of recombinant human sDR5-Fc fusion protein in the preparation of medicines for preventing and/or treating acute radiation sickness. Therefore, it can effectively prevent and/or treat ARS by inhibiting excessive normal tissue apoptosis caused by ionizing radiation.

Recombinant human sDR5-Fc fusion protein has a good protective effect on hematopoietic immune tissue (bone marrow type) acute radiation sickness, intestinal type acute radiation sickness and cerebral acute radiation sickness, etc. The specific manifestation is that it can increase the peripheral blood leukocytes and platelets of mice after ionizing radiation. At the same time, because sDR5 itself is the human body’s own protein, it has the advantages of low toxicity and no immunogenicity. Therefore, The sDR5-Fc fusion protein can have a good application prospect in the prevention and treatment of severe or very severe ARS caused by ionizing radiation, especially the very severe myeloid ARS.

Description of drawings

Figure 1 is the TUNNEL-DR5 immunofluorescence image of the thymus tissue section of BALB/c mice after a single whole body irradiation of 60Coγ rays in Example 1 of the present invention; A-C: TUNNEL-DR5 antibody double-labeled overlay images; D-F: DR5 antibody labeling detection DR5 protein expression level; G-I: apoptosis rate detected by TUNNEL;

Fig. 2 is the survival curve of mice in different treatment groups after ionizing radiation injury in Example 2 of the present invention;

Fig. 3 is the change curve of leukocyte level in peripheral blood at different times after administration of 6.0 Gy ionizing radiation to mice in Example 3 of the present invention;

Fig. 4 is the variation curve of platelet level in peripheral blood at different times after administration of 6.0 Gy ionizing radiation to mice in Example 3 of the present invention;

Fig. 5 is the change curve of the proportion of lymphocytes in peripheral blood at different times after administration of 6.0 Gy ionizing radiation to mice in Example 3 of the present invention;

Figure 6 is a histogram of changes in the level of LK cells in the bone marrow at different times after administration of sDR5-Fc fusion protein to 4.0 Gy ionizing mice in Example 4 of the present invention;

Figure 7 is a histogram of changes in the level of LSK cells in the bone marrow at different times after administration of sDR5-Fc fusion protein to mice with 4.0 Gy ionizing radiation in Example 4 of the present invention.

Detailed ways

The term "DR5 (Death Receptor 5)" herein is a member of the tumor necrosis factor (TNF) receptor family, which is lowly expressed in normal tissues and highly expressed in inflammatory, tumoral and ischemic tissues. DR5 can specifically bind to tumor necrosis factor-related inducing ligand (TNF-related apoptosis inducing ligand, TRAIL) to induce apoptosis. DR5 is activated by oligomerization of DR5 after binding to the ligand TRAIL. The activated death receptor DR5 recruits FADD (Fas-associated death domain) molecules, and FADD recruits the precursor of Caspase-8 through DED (deatheffector domain). (Pro-caspase-8), forming a death signaling complex DISC (death-inducing signalingcomplex). Pro-caspase-8 is activated to form Caspase-8, which triggers apoptosis through mitochondria-independent and mitochondria-independent pathways. The term "sDR5 (soluble DR5, soluble DR5)" as used herein is a soluble form of DR5 that does not contain a transmembrane domain, and is secreted outside the cell due to the lack of a transmembrane domain that cannot be expressed on the cell membrane. sDR5 is expressed at a low level in normal human peripheral blood. Although sDR5 maintains the activity of binding to TRAIL ligands, it cannot transmit apoptosis signals into cells and can block the apoptosis response mediated by TRAIL-DR5. "Recombinant human sDR5-Fc fusion protein" is a recombinant fusion protein obtained by linking human sDR5 and an Fc expression gene and then recombinantly expressing.

The content of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art would realize, 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 restrictive.

The methods used in the following examples are conventional methods unless otherwise specified, and the specific steps can be found in: "Molecular Cloning: A 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 ways of obtaining various biological materials described in the examples are only to provide a way to obtain experimentally to achieve the purpose of specific disclosure, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the tips in the examples.

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

1) Experimental animals

BALB/c mice, male, 6-8 weeks old, weighing 19-21 g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal quality certificate number: SCXK-(Beijing) 2016-0006.

2) Reagents and instruments

sDR5-Fc fusion protein: SEQ ID NO: 4, test code AS1501, 25mg/bottle, from Shenzhen Zhongke Aishen Pharmaceutical Co., Ltd.;

Electronic analytical balance: ME235S, purchased from Sartorius, Germany;

Electronic balance: EPS-2001 type, purchased from Changsha Xiangping Technology Development Co., Ltd.;

Baolingman automatic blood analyzer: model BM860, purchased from Beijing Baolingman Sunshine Technology Co., Ltd.

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

In this example, experimental mice were irradiated with ionizing radiation to detect the expression of DR5 protein in immune organs (such as thymus) of irradiated mice, and to preliminarily test the effect of sDR5-Fc fusion protein (AS1501) on ionizing radiation-induced apoptosis. The inhibition of death includes the following steps.

1.1. The whole body of BALB/c mice was irradiated with 60Co gamma rays for a single time, the irradiation dose was 6.0Gy, the irradiation dose rate was 68.7cGy/min, and the irradiation distance was 90cm;

1.2. The irradiated BALB/c mice were randomly divided into normal control group (non-irradiated group), model control group and AS1501 administration group. On the day, the first day, the second day and the third day after irradiation, AS1501 The administration group was administered 10 mg/kg through the tail vein every day; the model control group was administered with normal saline blank solvent;

1.3. Twenty-four hours after administration, the mice in each group were euthanized by de-neck, thymus tissue was taken, and slices were made. Immunohistochemical TUNNEL method was used to detect the apoptosis rate, and fluorescently labeled DR5 antibody was used to detect the change of DR5 protein expression level.

The results are shown in Figure 1, which are the TUNNEL-DR5 immunofluorescence images of thymus tissue sections of BALB/c mice after a single whole body irradiation of 60Coγ-rays, in which A-C: TUNNEL-DR5 antibody double-labeled overlay; D-F: DR5 antibody labeled detection DR5 protein expression level; G-I: apoptosis rate detected by TUNNEL. It can be seen that after BALB/c mice were irradiated with 6.0 Gy of whole body 60Coγ rays, the expression level of DR5 protein and the level of apoptosis in thymus tissue were significantly increased, indicating that the DR5 protein in normal tissues of mice after high-dose ionizing radiation was significantly increased. The levels were significantly up-regulated, and it was also confirmed that TRAIL-DR5-mediated apoptosis has an important role in the injury mechanism of ionizing radiation injury. On the other hand, after a single whole-body irradiation of mice with 60Coγ-rays, compared with the model control group, the expression level of DR5 protein in the thymus tissue of ionizing radiation-injured mice in the AS1501 administration group did not change significantly, but the number of apoptotic cells decreased. , further proved that TRAIL-DR5-mediated apoptosis signaling pathway plays an important role in ionizing radiation-induced apoptosis, and also preliminarily proved that sDR5-Fc fusion protein can indeed inhibit the excessive apoptosis of mouse thymocytes induced by ionizing radiation injury. Death.

Example 2: Effect of sDR5-Fc fusion protein on survival rate of mice after high-dose ionizing radiation

In this example, the sDR5-Fc fusion protein was used to administer the mice after high-dose ionizing radiation, and the survival rate of the mice after administration was statistically analyzed to detect the effect of the sDR5-Fc fusion protein on the small amount of mice after high-dose ionizing radiation. The influence of mouse survival rate includes the following steps.

2.1. BALB/c mice were randomly divided into radiation model control group, amifostine (as a positive drug) group, AS1501 high, medium and low dose groups and normal control group, a total of 6 groups with 9 mice in each group, including:

Radiation model control group: on the day after radiation and on the 1st to 3rd day, a normal saline blank solvent control was given by tail vein injection every day;

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

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

AS1501 medium-dose group: on the day after radiation and from 1 to 3 days, 10 mg/kg AS1501 solution was administered by tail vein injection every day;

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

Normal control group: normal BALB/c mice that were not irradiated.

2.2. Radiation dose: The mice in the radiation model control group, the amifostine group and the AS1501 high, medium and low dose groups were all irradiated with 60Coγ ray once, the irradiation dose was 8.5Gy, and the irradiation dose rate was 68.68cGy/min. is 90cm.

2.3. Index detection: After radiation administration of mice in different experimental groups, the death data of mice were observed, and the survival curves at different times after administration were drawn.

The results are shown in Figure 2, which are the survival curves of mice in different experimental groups. It can be seen that compared with the radiation model control group (NS), BALB/c mice after a single 8.5Gy whole-body 60Coγ-ray ionizing radiation were injected by continuous tail vein. After administration of high, medium and low doses of sDR5-Fc fusion protein, the survival rate of mice after ionizing radiation could be significantly improved, and the dose-dependent manner showed that sDR5-Fc fusion protein could significantly improve the survival rate of mice damaged by ionizing radiation. The mice in the normal control group survived well, with a survival rate of 100%. The survival curve is not shown in Figure 2.

Example 3: Effect of sDR5-Fc fusion protein on peripheral blood of mice injured by ionizing radiation

In this example, the sDR5-Fc fusion protein was used to administer the mice after ionizing radiation, and the peripheral blood of the mice after administration was measured to detect the effect of the sDR5-Fc fusion protein on the peripheral blood of mice damaged by ionizing radiation. Specifically include the following steps.

3.1. BALB/c mice were randomly divided into normal control group, normal saline control group and sDR5-Fc fusion protein administration group, among which:

Physiological saline control group: On the day after radiation and on the 1st to 3rd day, a normal saline blank solvent control was given by tail vein injection every day;

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

Normal control group: normal BALB/c mice that were not irradiated.

3.2. Radiation dose: The mice in the normal saline control group and the sDR5-Fc fusion protein administration group were all irradiated with 60Coγ rays once, the irradiation dose was 6Gy, the irradiation dose rate was 68.7cGy/min, and the irradiation distance was both 90cm.

3.3. Sample collection: on the day after radiation and on the 1st, 3rd, 5th, 7th, 11th, 15th, 19th, 24th and 30th days after radiation, samples were collected from the normal control group, the normal saline control group and the sDR5-Fc fusion protein administration group, respectively. Three mice were randomly selected, and 30 μL of blood was collected from the orbital venous plexus in an anticoagulant tube, and an automatic hematology analyzer was used to detect the peripheral blood picture, including the white blood cell (WBC) level, platelet (PLT) level and lymphocyte ratio ( LYM%).

The detection results of leukocyte levels in the peripheral blood of the mice in each group are shown in Figure 3. It can be seen that, compared with the normal control group mice, after 6.0Gy radiation, the peripheral blood of the mice in the sDR5-Fc fusion protein administration group and the normal saline control group The WBC levels in blood were significantly decreased, and the most obvious was 3-7 days after radiation, indicating that ionizing radiation can lead to the decrease of WBC levels in the peripheral blood of mice in the process of inducing apoptosis. The mice in the sDR5-Fc fusion protein administration group were treated with AS1501 after irradiation. Compared with the mice in the normal saline control group, the level of leukocytes in peripheral blood was significantly higher on days 7-13 (*p<0.05) , indicating that the sDR5-Fc fusion protein has a tendency to promote the recovery of leukocyte levels in peripheral blood in the early stage after ionizing radiation-induced injury.

The detection results of platelet levels in the peripheral blood of the mice in each group are shown in Figure 4. It can be seen that, compared with the normal control group mice, after 6.0Gy irradiation, the peripheral blood of the mice in the sDR5-Fc fusion protein administration group and the normal saline control group PLT in the blood decreased significantly, and the most obvious was about 7 days after radiation, indicating that ionizing radiation can lead to the decrease of PLT level in peripheral blood during the process of inducing apoptosis. The mice in the sDR5-Fc fusion protein administration group were treated with AS1501 after irradiation. Compared with the mice in the normal saline control group, the mice in the sDR5-Fc fusion protein administration group showed a more pronounced promotion of platelet level recovery function at 2-4 weeks after irradiation, and quickly reached the normal control. The platelet level in the peripheral blood of the mice in the group of mice showed that the sDR5-Fc fusion protein could effectively promote the rapid recovery of the platelet level in the peripheral blood after ionizing radiation-induced injury.

The detection results of the proportion of lymphocytes in the peripheral blood of the mice in each group are shown in Figure 5. It can be seen that, compared with the normal control group mice, after 6.0Gy irradiation, the sDR5-Fc fusion protein administration group and the normal saline control group mice The proportion of lymphocytes was significantly decreased, indicating that ionizing radiation can significantly reduce the proportion of lymphocytes in peripheral blood in the process of inducing apoptosis. The mice in the sDR5-Fc fusion protein administration group were treated with AS1501 after irradiation. Compared with the mice in the normal saline control group, the level of lymphocytes was significantly increased, indicating that the sDR5-Fc fusion protein can effectively treat ionizing radiation-induced injury. Restore lymphocyte levels.

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

In this example, the sDR5-Fc fusion protein was used to administer the mice after ionizing radiation, and the bone marrow hematopoietic function of the mice after administration was tested to detect the effect of the sDR5-Fc fusion protein on the bone marrow hematopoietic function of the mice damaged by ionizing radiation. impact, including the following steps.

4.1. BALB/c mice were randomly divided into normal control group, normal saline control group and sDR5-Fc fusion protein administration group, among which:

Physiological saline control group: On the day after radiation and on the 1st to 3rd day, a normal saline blank solvent control was given by tail vein injection every day;

sDR5-Fc fusion protein administration group: on the day after radiation and on days 1-3, 10 mg/kg AS1501 solution was administered by tail vein injection every day;

Normal control group: normal BALB/c mice that were not irradiated.

4.2. Radiation dose: The mice in the normal saline control group and the sDR5-Fc fusion protein administration group were all irradiated with 60Coγ rays once, the irradiation dose was 4Gy, the irradiation dose rate was 68.7cGy/min, and the irradiation distance was 90cm.

4.3. Sample collection: On the day (2h) after radiation and on the 1st, 4th, 7th, 11th, 14th, and 19th days after radiation, 3 samples were randomly selected from the normal control group, the normal saline control group and the sDR5-Fc fusion protein administration group, respectively. Each mouse was sacrificed by anesthesia, and the bone marrow cells of the mice were isolated and counted. After incubation with CD16/32 antibody, 1% BSA was used to block them; then lineage antibody, Sca-1 antibody and c-kit were used for labeling at 4°C for 30 min. After fixation with 0.4% paraformaldehyde, the percentage of LinSca-1-c-Kit+(LK) and Lin-Sca-1+c-Kit+(LSK) cells in bone marrow nucleated cells were detected by flow cytometry and the cell number was calculated.

The detection results of the number of LK cells and LSK cells in the bone marrow of mice in each group are shown in Figure 6 and Figure 7, respectively. The numbers of LSK cells were significantly decreased, indicating that ionizing radiation can significantly decrease the numbers of LK and LSK cells in bone marrow during the induction of apoptosis. Compared with the normal saline control group, the sDR5-Fc fusion protein administration group could significantly increase the levels of bone marrow LK and LSK cells at multiple time points, indicating that the sDR5-Fc fusion protein has a certain role in promoting bone marrow hematopoiesis in the treatment of ionizing radiation injury. Functional repair.

From the results of the above examples, it can be seen that ionizing radiation can cause apoptosis of normal tissue cells, and when ionizing radiation induces apoptosis of normal tissue (such as immune organs, hematopoietic system, etc.), it is detected that the expression level of DR5 protein in the tissue is significantly increased. , and based on the results that the sDR5-Fc fusion protein can effectively reduce the apoptosis rate, it is proved that the TRAIL-DR5-mediated apoptosis signaling pathway plays an important role in ionizing radiation-induced apoptosis, at least it can induce apoptosis in peripheral blood. The apoptosis of leukocytes, platelets and lymphocytes, as well as LK and LSK cells in the hematopoietic system such as bone marrow, leads to changes in blood balance and a decrease in the number of hematopoietic stem and progenitor cells, which leads to severe acute radiation sickness (ARS). Based on this, the inventors creatively used the sDR5-Fc fusion protein in the prevention and/or treatment of ARS caused by ionizing radiation, and the principle that the sDR5-Fc fusion protein can block the TRAIL-DR5-mediated apoptosis signaling pathway It can effectively protect normal tissue cells from radiation effects during ionizing radiation and promote the recovery of cell levels in normal tissues after ionizing radiation injury. Specifically, it can significantly improve the key indicators of blood in peripheral blood after ionizing radiation injury. Leukocytes, platelets and lymphocytes At the cellular level, it can promote its rapid recovery to normal state, and can promote the recovery of bone marrow hematopoietic stem and progenitor cells. Therefore, the sDR5-Fc fusion protein can have a good application prospect in the prevention and/or treatment of severe or very severe ARS caused by ionizing radiation, especially severe myeloid ARS, etc., and provides a new method for the treatment of ARS.

The sDR5-Fc fusion proteins whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 have similar effects to the sDR5-Fc fusion proteins whose amino acid sequences are shown in SEQ ID NO: 4.

Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, the The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Zhongke Aishen Pharmaceutical Co., Ltd.

<120> Application of recombinant human sDR5-Fc fusion protein in preparing 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 sequences

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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 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 sequences

<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 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 sequences

<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 sequences

<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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