Apoptotic body, preparation containing apoptotic body and application of apoptotic body in preparation of related medicines for regulating functions of immune organs and hematopoietic system

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an apoptotic body, a preparation containing the apoptotic body and application of the apoptotic body to preparation of related medicines for regulating functions of immune organs and hematopoietic systems. The apoptotic body is selected from one or a mixture of more than two of a nano-scale apoptotic body, a surface-modified nano-scale apoptotic body and a nano-scale apoptotic body derivative. The formulations include apoptotic bodies and delivery drugs. The apoptotic body or the preparation thereof can target bone marrow, thymus and lymph nodes for delivery of nucleic acid, protein polypeptide and small molecule drugs, can realize collective function regulation of three immune organs and hematopoietic systems, and is suitable for clinical treatment of tumors, autoimmune diseases, myelopathy and blood system diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to apoptotic bodies, preparations containing apoptotic bodies, and applications in the preparation of drugs for regulating immune organ and hematopoietic system functions. Background Art

[0002] In modern medicine, effective treatment for tumors, autoimmune diseases, blood diseases and bone diseases is a major challenge. Because the pathological mechanisms of these diseases are complex and often involve abnormal responses of the immune system, targeted immune system treatment has become a research focus. Immune organs such as bone marrow, thymus and lymph nodes play a key role in the generation, maturation and distribution of immune cells. Targeted delivery of drugs to these immune organs can regulate immune responses at the source, providing an innovative approach to treating immune-related diseases.

[0003] In recent years, vesicles (such as exosomes, liposomes and polymer vesicles) have attracted much attention as drug delivery systems in clinical and scientific research fields. Vesicles have good biocompatibility, targeting and controlled release properties. Through surface modification or genetic engineering, they can carry different drugs, nucleic acids, proteins or small molecule compounds and deliver them to specific tissues or cells. In addition, vesicles have a long circulation time in the body and can enter target cells through the cell membrane, ensuring the effective release of drugs at the target location and reducing side effects on other organs or tissues.

[0004] Drug therapy targeted to immune organs such as the bone marrow, thymus, and lymph nodes offers unique advantages in the treatment of cancer, autoimmune diseases, blood diseases, and bone diseases. In cancer treatment, delivery systems targeting the bone marrow and lymph nodes can enhance anti-tumor immune responses and activate immune cells to recognize and attack tumor cells; in autoimmune diseases, vesicle-delivered immunomodulators can regulate abnormal immune responses and restore immune balance; in the treatment of blood diseases and bone diseases, targeted drug delivery is expected to improve treatment outcomes by regulating the bone marrow microenvironment and blood production.

[0005] In short, the use of vesicles to deliver drugs to immune organs such as the bone marrow, thymus, and lymph nodes not only provides new ideas for the treatment of a variety of diseases, but also greatly reduces the side effects of traditional drugs and improves the efficacy. Future developments will further optimize the stability, targeting, and drug loading of vesicles, making them an important tool for the clinical treatment of immune-related diseases. Summary of the invention

[0006] The purpose of the present invention is to provide an apoptotic body, a preparation containing the same, and the use of the apoptotic body in the preparation of drugs related to regulating the functions of immune organs and hematopoietic systems, so as to achieve efficient and accurate targeting of bone marrow, thymus and lymph nodes for drug delivery, and ultimately regulate the differentiation and function of lymphocytes, monocytes and hematopoietic cells and their upstream cells.

[0007] To this end, the present invention provides the following technical solutions.

[0008] One aspect of the present invention provides an apoptotic body, wherein the apoptotic body is selected from one or a mixture of two or more of a nanoscale apoptotic body, a surface-modified nanoscale apoptotic body, and a nanoscale apoptotic body derivative.

[0009] In some embodiments, the nanoscale apoptotic bodies include but are not limited to mouse-derived cell nanoscale apoptotic bodies and human-derived cell nanoscale apoptotic bodies.

[0010] In some embodiments, the nano-sized apoptotic bodies are nano-sized apoptotic bodies derived from 4T1 cells.

[0011] In some embodiments, the nanoscale apoptotic bodies are obtained by inducing apoptosis in cells and then isolating them.

[0012] In some embodiments, the induction method includes but is not limited to anti-tumor drugs, cytotoxic agents, steroids, cytokines, Fas / FasL, emitted light irradiation, ultrasound, photodynamic therapy, endoplasmic reticulum stress, oxidative stress, nutritional deprivation, mitochondrial damaging agents, acidic / hypoxic / hyperosmotic pressure environments, nanomedicines / nanoparticles, anti-apoptotic gene inhibitors, and pro-apoptotic gene regulatory drugs.

[0013] In some embodiments, the irradiation conditions are: UV irradiation with an irradiation intensity of 100-300 mJ / cm 2 The irradiation time is 15-30min.

[0014] In some embodiments, the irradiation conditions are: irradiation intensity is 150 mJ / cm 2 , the irradiation time is 30min.

[0015] In some embodiments, the separation method includes, but is not limited to, ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, polymer precipitation, immunoaffinity separation, microfluidics, affinity chromatography, and electric field driven separation.

[0016] In some embodiments, the separation method is:

[0017] Take the supernatant of apoptotic cells;

[0018] The supernatant was centrifuged at 300-500 g for 5-10 min to remove dead cells and cell debris, and then centrifuged at 2,000-3000 g for 12-30 min to remove micron-sized apoptotic bodies to obtain a first supernatant;

[0019] The first supernatant obtained is centrifuged at 10,000-20,000 g for 20-30 min, and the precipitate is the nano-scale apoptotic body.

[0020] In some embodiments, the separation method is:

[0021] Take the supernatant of apoptotic cells;

[0022] The supernatant was centrifuged at 300 g for 10 min to remove dead cells and debris, and then centrifuged at 3000 g for 20 min to remove micrometer-sized apoptotic bodies to obtain the first supernatant;

[0023] The first supernatant obtained was centrifuged at 12,000 g for 30 min, and the precipitate was obtained, which was the nano-scale apoptotic body.

[0024] In some embodiments, the surface-modified nanoscale apoptotic bodies include but are not limited to polyethylene glycol-modified nanoscale apoptotic bodies, targeting ligand-modified nanoscale apoptotic bodies, fusion protein-modified nanoscale apoptotic bodies, receptor-ligand interaction-modified nanoscale apoptotic bodies, membrane protein chimeric-modified nanoscale apoptotic bodies, chemically cross-linked modified nanoscale apoptotic bodies, charge-modified nanoscale apoptotic bodies, glycosylation-modified nanoscale apoptotic bodies, and biotin-avidin system-modified nanoscale apoptotic bodies.

[0025] In some embodiments, the targeting ligand includes, but is not limited to, mannose-mannose receptor, glucose / galactose-Asialoglycoprotein receptor, nucleic acid aptamer, and whole antibody / antibody fragment-receptor.

[0026] In some embodiments, the fusion protein includes but is not limited to targeting fusion protein, cell penetrating protein, receptor binding fusion protein, transmembrane protein fusion modification, nucleic acid aptamer fusion protein, GM-CSF fusion protein, integrin fusion protein, antigen fusion protein, Penetratin fusion protein, CD47 fusion protein, receptor binding fusion protein, viral envelope protein fusion, TAT fusion protein, tumor cell membrane fusion protein and immune cell membrane fusion protein.

[0027] In some embodiments, the ligand-receptor interaction modification includes but is not limited to folate-folate receptor, mannose-mannose receptor, transferrin-transferrin-receptor, RGD peptide-integrin, EGF-EGFR, galactose-liver receptor, CD47-SIRPα and tyrosine kinase receptor ligand.

[0028] In some embodiments, the membrane protein chimeric modification includes but is not limited to CD47 protein chimeric, transmembrane glycoprotein chimeric, integrin chimeric, Fc receptor protein chimeric, EGF / HER2 chimeric, LFA-1 and PSGL-1 proteins, T cell receptor (TCR) chimeric, transmembrane chemokine receptor chimeric, viral envelope protein chimeric and CD80 / CD86 co-stimulatory molecule chimeric.

[0029] In some embodiments, the chemical cross-linking modification includes but is not limited to thiol-maleimide cross-linking, NHS ester-amine cross-linking, glutaraldehyde cross-linking, carbodiimide cross-linking, copper-catalyzed azide-alkyne cycloaddition reaction, thiol-ene addition reaction, strain-promoted azide-alkyne cycloaddition reaction (SPAAC), boronate formation reaction, Diels-Alder reaction, sulfhydryl and disulfide bond cross-linking, hydrophilic polymer modification and phospholipid-cholesterol cross-linking.

[0030] In some embodiments, the charge modification includes but is not limited to polyethyleneimine PEI, poly-L-lysine PLL, chitosan, hyaluronic acid HA, alginate, polyacrylic acid, phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidic acid, cholesterol, pH-sensitive cationic polymers and metal ions.

[0031] In some embodiments, the glycosylation modification includes but is not limited to mannose modification, galactose modification, glucose modification, hyaluronic acid modification, sialic acid modification, chitosan modification, arabinose modification, lactose modification and polysaccharide modification.

[0032] In some embodiments, the modified biotin-avidin system includes but is not limited to a biotin-avidin system, a biotin antibody / ligand-avidin system, and a modified biotin antibody / ligand-avidin system.

[0033] In some embodiments, the derivatives of the nanoscale apoptotic bodies include but are not limited to those obtained by using nanoscale apoptotic bodies as base materials through any of the following methods: cell membrane extraction, modification, packaging, adsorption, and extrusion.

[0034] In some embodiments, the particle size of the nanoscale apoptotic body is 10-1000 nm, the particle size of the surface-modified nanoscale apoptotic body is 10-1000 nm, and the particle size of the derivative of the nanoscale apoptotic body is 10-1000 nm.

[0035] The second aspect of the present invention provides a preparation, which comprises the apoptotic body as described above, and a delivery drug.

[0036] In some embodiments, the drug includes but is not limited to nucleic acid drugs, protein drugs, polypeptide drugs, small molecule drugs and gene editing drugs.

[0037] In some embodiments, the nucleic acid drug includes, but is not limited to, antisense oligonucleotides, small interfering RNA, mRNA, gene vaccines, aptamers, deoxyoligonucleotides, small nucleolar RNA, ribozymes, and DNA enzymes.

[0038] In some embodiments, the protein drugs include, but are not limited to, therapeutic monoclonal antibodies, hormone proteins, cytokines, vaccine proteins, and enzymes.

[0039] In some embodiments, the polypeptide drugs include but are not limited to hormone polypeptides, anti-inflammatory polypeptides, vaccine polypeptides and anti-tumor polypeptides.

[0040] In some embodiments, the small molecule drugs include, but are not limited to, kinase inhibitors, receptor antagonists, antimetabolites, antibiotics, antifungal drugs, immunomodulatory drugs, osteoporosis drugs, and antiviral drugs.

[0041] In some embodiments, the gene editing drug includes but is not limited to CRISPR-Cas system, TALEN (transcription activator-like effector nuclease), ZFN (zinc finger nuclease), base editing, in situ gene insertion, guide editing, DNA editing and suicide gene editing.

[0042] In some embodiments, the formulation is nanosized apoptotic bodies containing OVA.

[0043] The third aspect of the present invention provides a use of the apoptotic body or preparation as described above in the preparation of drugs related to regulating the functions of immune organs and hematopoietic system.

[0044] In some embodiments, the immune organs and hematopoietic system include but are not limited to bone marrow, thymus, and lymph nodes.

[0045] In some embodiments, the lymph nodes include, but are not limited to, inguinal lymph nodes, axillary lymph nodes, cervical lymph nodes, popliteal lymph nodes, mesenteric lymph nodes, and tertiary lymph nodes in pathological settings.

[0046] The fourth aspect of the present invention provides a drug for regulating the functions of immune organs and hematopoietic system, including: the apoptotic body or preparation as described above.

[0047] The fifth aspect of the present invention provides a drug for treating diseases related to disorders of immune organs and hematopoietic system, comprising: the apoptotic body or preparation as described above.

[0048] In some embodiments, the disease includes, but is not limited to, tumors, autoimmune diseases, bone marrow diseases, and blood system diseases.

[0049] In some embodiments, the tumor includes but is not limited to lymphoma, sarcoma, liver cancer, lung cancer, gastric cancer, colorectal cancer, breast cancer, cervical cancer, pancreatic cancer, small intestine tumor, biliary tract tumor, multiple myeloma or melanoma cells.

[0050] In some embodiments, the autoimmune disease includes, but is not limited to, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, and vasculitis.

[0051] In some embodiments, the bone marrow disease includes but is not limited to osteomyelitis, primary bone marrow cancer, and bone marrow metastatic cancer.

[0052] In some embodiments, the hematological disease includes but is not limited to aplastic anemia, polycythemia vera, essential thrombocythemia, and myelofibrosis.

[0053] By means of the above technical solution, the present invention has at least the following advantages:

[0054] The apoptotic bodies and preparations thereof provided by the present invention have natural high targeting properties and can accurately deliver drugs to the hematopoietic system and / or immune organ tissues such as the bone marrow, thymus and lymph. Not only do they achieve efficient targeting of central immune organs such as the bone marrow and thymus for the first time, but they also achieve joint regulation of the immune organs and the hematopoietic system.

[0055] The apoptotic bodies of the present invention and preparations containing the same are suitable for the joint regulation of the growth and development of the immune system and the hematopoietic system. The targeted delivery of apoptotic bodies allows the delivered drugs to be used not only for the treatment of diseases of the mature immune system (such as tumors, autoimmune diseases, infectious diseases, etc.), but also for the early growth and development of the immune system and the hematopoietic system, as an auxiliary treatment for diseases such as congenital immunodeficiency and hematopoietic dysfunction.

[0056] The apoptotic bodies and preparations containing the same provided by the present invention can be loaded with different drugs or gene regulatory molecules as needed to achieve precise regulation of specific immune pathways or hematopoietic factors, have personalized and diversified therapeutic potential, and provide direction for the development of a drug for regulating and / or treating immune organs and hematopoietic system functions.

[0057] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1The characteristic identification of the nano-scale apoptotic bodies prepared in Example 1 is shown, wherein: (A): the secretion morphology of the nano-scale apoptotic bodies; (B): the particle size distribution of the nano-scale apoptotic bodies collected at different times; (C): the purity of the nano-scale apoptotic bodies collected at different times;

[0059] Figure 2 The distribution of fluorescently labeled nanoscale apoptotic bodies in various tissues and organs of mice over time is shown;

[0060] Figure 3 The figure shows the regulatory effect of nano-scale apoptotic bodies that deliver OVA information on mouse thymic T cells, including: (A): the proportion of CD4 T cells, CD8 T cells and DP T cells in the thymus of mice in the OVA-encapsulated nano-scale apoptotic body injection group and the control group; (B): statistical analysis of the proportion of CD8 T cells in the thymus of the two groups of mice; (C): statistical analysis of the proportion of DP T cells in the thymus of the two groups of mice. DETAILED DESCRIPTION

[0061] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] As used in the present disclosure, “preferred” is only used to describe an implementation method or example with better effects, and it should be understood that it does not constitute a limitation on the scope of protection of the present invention.

[0063] As used in the present disclosure, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unrecited elements or method steps.

[0064] As used in this disclosure, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals or humans, as appropriate. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

[0065] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0066] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0067] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0068] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0069] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0070] Example 1: Extraction and identification of nanoscale apoptotic bodies

[0071] 1. Extraction of Nanoscale Apoptotic Bodies

[0072] In this example, 4T1 cells (ATCC, CRL-2539) were used as the cell source to prepare nano-scale apoptotic bodies. The specific process was as follows: 4T1 cells were cultured to 90% confluence and then exposed to 150 mJ / cm 2 The cells were irradiated with UV rays for 30 minutes to induce cell apoptosis. After irradiation, the cells were washed with PBS, and the obtained apoptotic cells were added to a serum-free culture medium (Gibco RPMI 1640, C11875500BT) containing H2O2 (mother solution is 30%, working concentration is 400nM), and then placed in an incubator at 37°C, 5% CO2 for continued culture. The cell supernatant was collected at 24h, 36h, 48h and 72h of culture. The obtained supernatant was centrifuged at 300g for 10min and 3000g for 20min in turn to remove cell debris and micron-sized apoptotic bodies to obtain the first supernatant. The obtained first supernatant was centrifuged at 12,000g for 30min, and the precipitate was taken, which was the nano-sized apoptotic body derived from 4T1 cells.

[0073] According to the above method, the cell supernatants collected at 24h, 36h, 48h and 72h of culture were centrifuged to obtain nano-scale apoptotic bodies, which were named 4T1-naABs-24, 4T1-naABs-36, 4T1-naABs-48 and 4T1-naABs-72, respectively.

[0074] 2. Characterization of Nanoscale Apoptotic Bodies

[0075] The four 4T1 cell-derived nano-sized apoptotic bodies 4T1-naABs-24, 4T1-naABs-36, 4T1-naABs-48, and 4T1-naABs-72 were resuspended in phosphate buffer, and the particle size distribution and purity were detected by nanosight and flow cytometry. Figure 1 .

[0076] Figure 1 The secretion morphology of nano-scale apoptotic bodies at different induction times ( Figure 1 A) Particle size distribution ( Figure 1 B) and purity ( Figure 1 C) characterization; Figure 1 A shows that nanoscale apoptotic bodies are formed by direct blebbing of the cell membrane and are bright round in shape with a size of less than 1 micron; Figure 1 B shows that the particle sizes of the four nano-sized apoptotic bodies are distributed between 10 and 700 nm; Figure 1 C shows that the purity of the four nanosized apoptotic bodies is above 90%.

[0077] Example 2: Targeted regulation of multi-level immune organs by nanoscale apoptotic bodies

[0078] In this example, the targeted regulatory effect of nano-scale apoptotic bodies on multi-level immune organs was investigated, and the specific operations included:

[0079] (1) Staining of nano-scale apoptotic bodies: The nano-scale apoptotic bodies 4T1-naABs-36 derived from 4T1 cells prepared in Example 1 and obtained 36 h after apoptosis induction were resuspended in sterile phosphate buffer, and then 2 mg / ml of DII dye (C1036, Bio-Tech) was added at a ratio of 1:1000, and incubated at 4°C for 30 min with rotation. After the incubation, the cells were washed twice with phosphate buffer to obtain DII-labeled nano-scale apoptotic bodies 4T1-naABs-36 derived from 4T1 cells.

[0080] (2) Tail vein injection of nano-sized apoptotic bodies: The obtained DII-labeled 4T1 cell-derived nano-sized apoptotic bodies 4T1-naABs-36 were adjusted to a concentration of 1×10 10 / ml to obtain a suspension for later use.

[0081] First, 7 6-week-old male mice (C57BL / 6JGpt, purchased from Jicui Yaokang) weighing 18-20g were randomly divided into an injection group and a control group, of which 6 mice were injected at different times and 1 was injected into the PBS control group. Then, each group of mice was treated as described below:

[0082] Injection group: The suspension was injected into mice via tail vein injection at a volume of 100ul per mouse;

[0083] Control group: PBS was injected into the mice via tail vein injection at a volume of 100ul per mouse.

[0084] (3) Isolation and preparation of mouse tissues: Based on the injection time, the tissues of mice in each group were collected at 2h, 4h, 8h, 12h and 24h after injection. Before collection, the mice were anesthetized and cardiac perfusion was performed with phosphate buffer to remove blood from the tissues to prevent blood from interfering with the test results.

[0085] (4) In vivo imaging detection of the distribution of nanoscale apoptotic bodies: The obtained tissues were neatly arranged on the detection plate, and the fluorescence intensity of each tissue was detected under the conditions of excitation light of 530nm and emission light of 580nm. The results are shown in Figure 2 .

[0086] like Figure 2 As shown, compared with the control group (PBS group), after tail vein injection of mice in each injection group, the nano-scale apoptotic bodies 4T1-naABs-36 were significantly distributed in the bone marrow, thymus and lymph node tissues of mice, and with the extension of injection time, the distribution amount of nano-scale apoptotic bodies in the bone marrow, thymus and lymph node tissues of mice showed a significant increasing trend.

[0087] Example 3: Nano-scale apoptotic bodies delivering antigen drugs to the thymus regulate central immune tolerance and its effects on the peripheral immune system

[0088] In this example, the regulation of central immune tolerance and the effect on the peripheral immune system by delivering antigen drugs to the thymus by nanoscale apoptotic bodies were investigated. The specific operations include:

[0089] (1) Preparation of chicken ovalbumin (OVA) expression plasmid: A synthetic OVA expression plasmid was designed and named pSLenti-EF1-EGFP-P2A-Puro-CMV-MCS-3×FLAG-WRPE as the target plasmid.

[0090] (2) Lentivirus preparation: 293T cells (ATCC, ACS-4500) were cultured with 10% FBSDMEM medium (Gibco, 11965092) until the density was expanded to 70%, and the plasmid (Cas9 / sgRNA-lentiGuid plasmid) and tool plasmid were transfected into the cells by Lipofectamine 2000 to prepare lentivirus. The specific operation is as follows:

[0091] ① Add OVA expression plasmid (10ug) and tool plasmid (PSPAX2: 7.5g; PMD2.G: 5g) to lipo-MEM serum-free culture medium (3ml), mix well to obtain the plasmid system.

[0092] In another system, 25 ul of Lipofectamine 2000 was added to the lipo-MEM serum-free culture medium (3 ml), and the culture was allowed to stand at room temperature for 5 minutes to obtain the Lipofectamine 2000 system.

[0093] ② Gently add the obtained plasmid system to the Lipofectamine 2000 system and let it stand at room temperature for 20 minutes to obtain a mixture.

[0094] ③Discard the original culture medium of 293T cells, wash with PBS, add the mixture in step ② and perform cell transfection.

[0095] ④ 6 hours after transfection, discard the cell supernatant, add 10% FBSDMEM culture medium again, and continue culturing at 37°C, 5% CO2.

[0096] ⑤ After 48 hours of culture, collect the supernatant and extract the lentivirus, that is, centrifuge the collected supernatant at 2000rpm for 10 minutes, discard the cells and debris, and obtain the first centrifugation supernatant. Transfer the obtained first centrifugation supernatant to a 15ml, 100KD ultrafiltration centrifuge tube, centrifuge at 4000g for 30 minutes at 4°C to obtain a concentrated virus solution. If not used in time, the concentrated virus solution can be stored at -80°C for future use.

[0097] (3) Lentivirus infection: 4T1 cells were cultured to a density of 30-50% using 1640 medium (Gibco RPMI 1640, C11875500BT) containing 10% FBS, and then infected. That is, the original medium was discarded and the cells were washed several times with PBS, and then fresh medium containing concentrated virus solution was added and cultured for 24 h for infection. After the infection, the old medium was discarded and new medium was added for 72 h.

[0098] (4) Cell screening: Puromycin was selected for positive cell screening based on plasmid resistance. The concentrations of puromycin screening were 1 mg / ml, 2 mg / ml, 5 mg / ml and 10 mg / ml, respectively. The normal proliferation of the first cells at the highest concentration indicated that the target gene was stably expressed in the cells. Finally, 4T1 cells expressing OVA protein were obtained and named OVA-4T1 cells.

[0099] (5) Preparation of nanoscale apoptotic bodies containing OVA: OVA-4T1 cells were induced to apoptosis according to the method described in Example 1. After 36 h, the cell supernatant was collected and separated to obtain nanoscale apoptotic bodies derived from 4T1 cells containing OVA, which were named 4T1-naABs-OVA.

[0100] (6) Animal testing

[0101] According to the method described in Example 2, a concentration of 1×10 10 / ml of 4T1-naABs-OVA suspension.

[0102] Six 6-week-old male OT1 mice (The Jackson Laboratory, C57BL / 6-Tg(TcraTcrb)1100Mjb / J, 003831) weighing 18-20 g were randomly divided into an injection group and a control group, with 3 mice in each group. The mice in each group were then treated as described below:

[0103] Injection group: The obtained suspension was injected into OT1 mice via tail vein injection at an injection volume of 100ul per mouse;

[0104] Control group: Following the same method, an equal volume of OVA (2ug / mouse) solution was injected into mice.

[0105] The thymus of each group of mice was collected 48 hours after injection, and the number and proportion of T cells in the thymus were detected by flow cytometry. Figure 3 .

[0106] like Figure 3 As shown, the flow cytometry results showed that compared with the control group (OVA group), the proportion of CD8 T cells and DP T cells in the thymus of mice injected with nano-scale apoptotic bodies 4T1-naABs-OVA derived from 4T1 cells loaded with OVA protein (naABs group) was significantly decreased ( Figure 3 A). Statistical analysis results showed that the proportion of DP T cells decreased by about 75%, and the proportion of CD8 T cells decreased by about 50% ( Figure 3 B and Figure 3 C).

[0107] The above results indicate that nanoscale apoptotic bodies containing OVA protein can utilize the negative selection mechanism in the thymus to specifically induce apoptosis of CD8 T cells and DP cells, resulting in a significant decrease in the proportion of CD8 T cells and DP cells, ultimately affecting the maturation and efflux of T cells.

[0108] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The use of apoptotic bodies or their preparations in the preparation of drugs related to regulating the functions of immune organs and hematopoietic systems.

2. The use according to claim 1, characterized in that: The apoptotic body is selected from one or a mixture of two or more of a nano-scale apoptotic body, a surface-modified nano-scale apoptotic body, and a nano-scale apoptotic body derivative; The nanoscale apoptotic bodies include but are not limited to mouse-derived cell nanoscale apoptotic bodies and human-derived cell nanoscale apoptotic bodies.

3. The use according to claim 2, characterized in that: The method for preparing the nanoscale apoptotic body comprises the following steps: S1: Inducing cell apoptosis by irradiation with emitted light; S2: The supernatant of apoptotic cells is separated by gradient centrifugation to obtain nanoscale apoptotic bodies.

4. The use according to claim 3, characterized in that: In step S1, the irradiation conditions are: UV irradiation with an irradiation intensity of 100-300 mJ / cm 2 The irradiation time is 15-30min.

5. The apoptotic body according to claim 3, characterized in that In step S2, the gradient centrifugation method comprises the following steps: S21: Take the supernatant of apoptotic cells; S22: centrifuging the supernatant at 300-500 g for 5-10 min to remove dead cells and cell debris, and then centrifuging at 2,000-3000 g for 12-30 min to remove micron-sized apoptotic bodies to obtain the first supernatant; S23: Centrifuge the obtained first supernatant at 10,000-20,000 g for 20-30 min, and collect the precipitate, which is the nano-scale apoptotic body.

6. The use according to claim 1, characterized in that: The particle size of the apoptotic body is 10-1000 nm.

7. The use according to claim 1, characterized in that: The formulation includes the apoptotic body and a delivery drug.

8. The use according to claim 7, characterized in that: The preparation is nano-scale apoptotic bodies containing OVA.

9. The use according to claim 1, characterized in that: The immune organs and hematopoietic system include but are not limited to bone marrow, thymus and lymph nodes; The lymph nodes include, but are not limited to, inguinal lymph nodes, axillary lymph nodes, cervical lymph nodes, popliteal lymph nodes, mesenteric lymph nodes, and tertiary lymph nodes in pathological settings.

10. A drug for regulating the functions of immune organs and hematopoietic system, characterized in that: Including the apoptotic body or its preparation.

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