Stem cell exosomes and their application in the preparation of drugs for the prevention and treatment of pet bone and joint diseases

Abstract

The present invention addresses the technical field of pet bone and joint disease prevention and treatment, specifically providing stem cell exosomes and their use in the preparation of medications for the prevention and treatment of pet bone and joint diseases. This invention, for the first time, combines the RANKL monoclonal antibody 5F16 with bone marrow mesenchymal stem cell exosomes for the prevention and treatment of pet bone and joint diseases. Experimental analysis shows that the formulation can effectively reduce serum levels of IL-1β, TNF-ɑ, and IL-6 in pets with bone and joint injuries, inhibit the secretion of IL-1β, TNF-ɑ, and IL-6, repair bone and joint injuries, and improve and promote cartilage tissue repair, demonstrating promising application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of pet bone and joint prevention and treatment, and in particular to stem cell exosomes and their application in the preparation of drugs for preventing and treating pet bone and joint diseases. Background Art

[0002] Bone and joint diseases are common health issues in pets, particularly among older pets. These diseases, including osteoarthritis, hip dysplasia, and patellar luxation, primarily present with joint pain and limited mobility, severely impacting quality of life. These conditions can be caused by genetic predisposition, trauma, or aging, making early detection and intervention crucial to slowing the progression of the disease.

[0003] Currently, the most common treatment for pet bone and joint diseases primarily focuses on anti-inflammatory and analgesic measures. Oral nonsteroidal anti-inflammatory drugs (such as meloxicam tablets), glucosamine tablets, and vitamin B1 can be used to reduce inflammation and pain. Joint supplements, such as Gulijian beef tablets, can also be used to promote joint repair. Alternatively, treatments such as infrared therapy and acupuncture can promote blood circulation and relieve joint pain and stiffness. However, these methods are not effective in fundamentally addressing pet bone and joint diseases, leading to the need for a cure suitable for the prevention and treatment of pet bone and joint diseases. Summary of the Invention

[0004] To address the technical issues existing in the prior art, the present invention provides stem cell exosomes and their use in the preparation of a drug for the prevention and treatment of pet bone and joint diseases. This invention, for the first time, combines the RANKL monoclonal antibody 5F16 with bone marrow mesenchymal stem cell exosomes for the prevention and treatment of pet bone and joint diseases. Experimental analysis shows that this preparation can effectively reduce serum levels of IL-1β, TNF-ɑ, and IL-6 in pets with bone and joint injuries, inhibit their secretion, repair bone and joint injuries, and improve and promote cartilage tissue repair, demonstrating promising application prospects.

[0005] Specifically, the present invention first provides a stem cell exosome preparation for preventing and treating pet bone and joint injuries. Each 1000 mL contains 10-40 mL of bone marrow mesenchymal stem cell exosomes, 20-50 μg of RANKL monoclonal antibody 5F16, 5-20 mg of chondroitin sulfate, and the balance is normal saline.

[0006] Preferably, the stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 10 mL of bone marrow mesenchymal stem cell exosomes, 20 μg of RANKL monoclonal antibody 5F16, 5 mg of chondroitin sulfate per 1000 mL, and the balance is normal saline.

[0007] Preferably, the stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 20 mL of bone marrow mesenchymal stem cell exosomes, 30 μg of RANKL monoclonal antibody 5F16, 10 mg of chondroitin sulfate per 1000 mL, and the balance is normal saline.

[0008] Preferably, the stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 40 mL of bone marrow mesenchymal stem cell exosomes, 50 μg of RANKL monoclonal antibody 5F16, 20 mg of chondroitin sulfate per 1000 mL, with the remainder being normal saline.

[0009] Preferably, the heavy chain variable region sequence of the RANKL monoclonal antibody 5F16 is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO:2.

[0010] Preferably, the heavy chain variable region of the RANKL monoclonal antibody 5F16 includes CDR-H1, CDR-H2, and CDR-H3, and the sequences of CDR-H1-3 are shown in SEQ ID NO.3-5.

[0011] Preferably, the light chain variable region of the RANKL monoclonal antibody 5F16 includes CDR-L1, CDR-L2, and CDR-L3, and the sequences of CDR-L1-3 are shown in SEQ ID NOs. 6-8.

[0012] Preferably, the method for extracting exosomes from bone marrow mesenchymal stem cells comprises the following steps:

[0013] 1) Bone marrow tissue was obtained and digested with trypsin to prepare single cell suspension, and CD29 was isolated by flow cytometry. + CD90 + CD105 + CD14 - CD34 - CD45 - bone marrow mesenchymal stem cell exosomes;

[0014] 2) The bone marrow mesenchymal stem cell exosomes obtained in step 1) were placed in DMEM / F12 medium for continuous subculture;

[0015] 3) After the cells have stabilized at the passage level, collect the cell supernatant by centrifugation;

[0016] 4) Filtering the supernatant from step 3) through a membrane filter, performing ultrahigh-speed centrifugation, and collecting the precipitate;

[0017] 5) Resuspend the pellet from step 4) in 1× PBS and perform ultracentrifugation again;

[0018] 6) Resuspend the precipitate from step 5) in 1× PBS, collect the filtrate through a filter membrane, and adjust the density to 1×10 4 -1×10 6 Exosomes / mL are bone marrow mesenchymal stem cell exosomes.

[0019] Furthermore, the present invention also provides a method for preparing a stem cell exosome preparation for preventing and treating pet bone and joint injuries, the preparation method comprising mixing the above raw materials and dissolving them in physiological saline, adjusting the pH to 7.0, and filtering and sterilizing to obtain the preparation.

[0020] Preferably, the stem cell exosome preparation can be prepared into a sterile injectable solution or suspension; for example, a solution suitable for administration by intra-articular injection, intravenous injection, intramuscular injection, subcutaneous injection or intraperitoneal injection.

[0021] Furthermore, the present invention also provides the use of RANKL monoclonal antibody 5F16 and bone marrow mesenchymal stem cell exosomes in combination in the preparation of a drug for preventing and treating pet bone and joint injuries.

[0022] Preferably, the dosage form of the drug is not limited to injection.

[0023] Further preferably, the drug is a sterile injectable solution or suspension;

[0024] The injection solution is a solution administered by intraarticular injection, intravenous injection, intramuscular injection, subcutaneous injection or intraperitoneal injection.

[0025] The advantages of the present invention are as follows: The present invention provides, for the first time, stem cell exosomes and their application in the preparation of drugs for the prevention and treatment of pet bone and joint diseases. The present invention combines, for the first time, RANKL monoclonal antibody 5F16 and bone marrow mesenchymal stem cell exosomes for the repair of bone and joint injuries. Experimental analysis shows that the preparation can effectively reduce the levels of IL-1β, TNF-ɑ and IL-6 in pet serum, inhibit the secretion of IL-1β, TNF-ɑ and IL-6, repair bone and joint injuries, improve and promote cartilage tissue repair, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The secretion changes of IL-1β, TNF-ɑ and IL-6 in the serum of tabby cats treated with different preparations. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0028] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0029] The present invention first provides a stem cell exosome preparation for preventing and treating pet bone and joint injuries. Each 1000 mL contains 10-40 mL of bone marrow mesenchymal stem cell exosomes, 20-50 μg of RANKL monoclonal antibody 5F16, 5-20 mg of chondroitin sulfate, and the balance is normal saline.

[0030] Preferably, the stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 10 mL of bone marrow mesenchymal stem cell exosomes, 20 μg of RANKL monoclonal antibody 5F16, 5 mg of chondroitin sulfate per 1000 mL, and the balance is normal saline.

[0031] Preferably, the stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 20 mL of bone marrow mesenchymal stem cell exosomes, 30 μg of RANKL monoclonal antibody 5F16, 10 mg of chondroitin sulfate per 1000 mL, and the balance is normal saline.

[0032] Preferably, the stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 40 mL of bone marrow mesenchymal stem cell exosomes, 50 μg of RANKL monoclonal antibody 5F16, 20 mg of chondroitin sulfate per 1000 mL, with the remainder being normal saline.

[0033] Preferably, the heavy chain variable region sequence of the RANKL monoclonal antibody 5F16 is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO:2.

[0034] Preferably, the heavy chain variable region of the RANKL monoclonal antibody 5F16 includes CDR-H1, CDR-H2, and CDR-H3, and the sequences of CDR-H1-3 are shown in SEQ ID NO.3-5.

[0035] Preferably, the light chain variable region of the RANKL monoclonal antibody 5F16 includes CDR-L1, CDR-L2, and CDR-L3, and the sequences of CDR-L1-3 are shown in SEQ ID NOs. 6-8.

[0036] Preferably, the method for extracting exosomes from bone marrow mesenchymal stem cells comprises the following steps:

[0037] 1) Bone marrow tissue was obtained and digested with trypsin to prepare single cell suspension, and CD29 was isolated by flow cytometry. + CD90+ CD105 + CD14 - CD34 - CD45 - bone marrow mesenchymal stem cell exosomes;

[0038] 2) The bone marrow mesenchymal stem cell exosomes obtained in step 1) were placed in DMEM / F12 medium for continuous subculture;

[0039] 3) After the cells have stabilized at the passage level, collect the cell supernatant by centrifugation;

[0040] 4) Filtering the supernatant from step 3) through a membrane filter, performing ultrahigh-speed centrifugation, and collecting the precipitate;

[0041] 5) Resuspend the pellet from step 4) in 1× PBS and perform ultracentrifugation again;

[0042] 6) Resuspend the precipitate from step 5) in 1× PBS, collect the filtrate through a filter membrane, and adjust the density to 1×10 4 -1×10 6 Exosomes / mL are bone marrow mesenchymal stem cell exosomes.

[0043] Furthermore, the present invention also provides a method for preparing a stem cell exosome preparation for preventing and treating pet bone and joint injuries, the preparation method comprising mixing the above raw materials and dissolving them in physiological saline, adjusting the pH to 7.0, and filtering and sterilizing to obtain the preparation.

[0044] Preferably, the stem cell exosome preparation can be prepared into a sterile injectable solution or suspension; for example, a solution suitable for administration by intra-articular injection, intravenous injection, intramuscular injection, subcutaneous injection or intraperitoneal injection.

[0045] Furthermore, the present invention also provides the use of RANKL monoclonal antibody 5F16 and bone marrow mesenchymal stem cell exosomes in combination in the preparation of a drug for preventing and treating pet bone and joint injuries.

[0046] Preferably, the dosage form of the drug is not limited to injection.

[0047] Further preferably, the drug is a sterile injectable solution or suspension;

[0048] The injection solution is a solution administered by intraarticular injection, intravenous injection, intramuscular injection, subcutaneous injection or intraperitoneal injection.

[0049] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0050] Example 1

[0051] A method for extracting exosomes from bone marrow mesenchymal stem cells, comprising the following steps:

[0052] 1) Bone marrow tissue of tabby cats was digested with trypsin to prepare single cell suspension, and CD29 was isolated by flow cytometry. + CD90 + CD105 + CD14 - CD34 - CD45 - bone marrow mesenchymal stem cell exosomes;

[0053] 2) The bone marrow mesenchymal stem cell exosomes obtained in step 1) were placed in DMEM / F12 medium for continuous subculture;

[0054] 3) After the cells have stabilized through passage, collect the cell supernatant at 4°C, 2000×g;

[0055] 4) Filter the supernatant from step 3) through a membrane filter and then perform ultracentrifugation at 4°C, 100,000 × g, for 60 min. After completion of the centrifugation, collect the precipitate.

[0056] 5) Resuspend the pellet from step 4) in 1× PBS and ultracentrifuge again at 4°C, 100,000×g, 60 min.

[0057] 6) Resuspend the precipitate from step 5) in 1× PBS, collect the filtrate through a filter membrane, and adjust the density to 1×10 4 Exosomes / mL are bone marrow mesenchymal stem cell exosomes.

[0058] Example 2

[0059] Preparation of RANKL monoclonal antibody: BALB / c mice were immunized with RANKL protein as antigen using hybridoma technology, and then mouse spleen cells were isolated and fused with SP2 / 0 myeloma cells. After screening, a high-affinity RANKL monoclonal antibody 5F16 was obtained.

[0060] Sequence analysis revealed that the heavy chain variable region sequence of RANKL monoclonal antibody 5F16 is shown in SEQ ID NO: 1, and the light chain variable region sequence is shown in SEQ ID NO: 2. The heavy chain variable region of RANKL monoclonal antibody 5F16 includes CDR-H1, CDR-H2, and CDR-H3, with the sequences of CDR-H1-3 shown in SEQ ID NOs. 3-5. The light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, with the sequences of CDR-L1-3 shown in SEQ ID NOs. 6-8.

[0061] Affinity analysis of RANKL monoclonal antibody 5F16: The affinity kinetic constants of RANKL monoclonal antibody 5F16 were measured using GE Biacore T200. The specific experimental procedures were as described in the instrument manual. Experimental results: Based on the test results, the antibody affinity data are shown in Table 1 below:

[0062] Table 1. Antibody affinity kinetic analysis

[0063] Antibody antigen Ka(1 / Ms) Kd(1 / s) KD(M) 5F16 RANKL <![CDATA[1.126×10 6 ]]> <![CDATA[6.254×10 -5 ]]> <![CDATA[5.554×10 -11 ]]>

[0064] Experimental conclusion: The RANKL monoclonal antibody 5F16 obtained in the present invention has a high affinity with the RANKL (human) recombinant protein, and the equilibrium dissociation constant is only 5.554×10 -11 .

[0065] Example 3

[0066] A stem cell exosome preparation for preventing and treating pet bone and joint injuries. Each 1000 mL contains 10 mL of bone marrow mesenchymal stem cell exosomes, 20 μg of RANKL monoclonal antibody 5F16, 5 mg of chondroitin sulfate, and the balance is normal saline.

[0067] The preparation method of the stem cell preparation comprises mixing the above raw materials and dissolving them in physiological saline, adjusting the pH to 7.0, and filtering and sterilizing to obtain the product.

[0068] Example 4

[0069] A stem cell exosome preparation for preventing and treating pet bone and joint injuries. Each 1000 mL contains 20 mL of bone marrow mesenchymal stem cell exosomes, 30 μg of RANKL monoclonal antibody 5F16, 10 mg of chondroitin sulfate, and the balance is normal saline.

[0070] The preparation method of the stem cell preparation comprises mixing the above raw materials and dissolving them in physiological saline, adjusting the pH to 7.0, and filtering and sterilizing to obtain the product.

[0071] Example 5

[0072] A stem cell exosome preparation for preventing and treating pet bone and joint injuries. Each 1000 mL contains 40 mL of bone marrow mesenchymal stem cell exosomes, 50 μg of RANKL monoclonal antibody 5F16, 20 mg of chondroitin sulfate, and the balance is normal saline.

[0073] The preparation method of the stem cell preparation comprises mixing the above raw materials and dissolving them in physiological saline, adjusting the pH to 7.0, and filtering and sterilizing to obtain the product.

[0074] Comparative Example 1

[0075] The preparation method and steps of Comparative Example 1 are the same as those of Example 3, except that the stem cell exosome preparation does not contain RANKL monoclonal antibody 5F16, but is supplemented with an equal amount of physiological saline. The preparation method is the same as that of Example 3.

[0076] Comparative Example 2

[0077] The preparation method and steps of Comparative Example 2 are the same as those of Example 3, except that the preparation does not contain bone marrow mesenchymal stem cell exosomes, but instead uses an equal amount of physiological saline as a supplement. The preparation method is the same as that of Example 3.

[0078] Test example

[0079] The preparation method of the bone and joint injury model of the tabby cat includes the following steps: after weighing, 50 mg / kg 1% sodium pentobarbital was intraperitoneally injected into the tabby cats of each group for anesthesia; when the tabby cats showed anesthesia symptoms, they were fixed on the tabby cat table and kept in a supine position. Before the operation, the hair of the right hind limb bone joint of the tabby cat was trimmed, and the bone joint was disinfected with iodine tincture; under a microscope, the skin of the medial side of the right bone joint was longitudinally cut with ophthalmic scissors 2 cm proximal to the right bone joint, avoiding the medial transverse and longitudinal arteries, cutting the medial collateral band, opening the joint cavity, and cutting the joint capsule along the upper edge of the medial collateral ligament; the joint capsule was cut to expose the patella, which was fixed by eversion, and the joint capsule was continued to be cut until the kneecap was completely exposed; the right bone joint of the tabby cat was bent to expose the anterior cruciate ligament and medial meniscus of the tibia, and the anterior cruciate ligament was cut with a surgical blade and the meniscus was removed; after removing the meniscus, the tabby cat was treated for hemostasis and the joint cavity was cleaned, then the patella was placed in its original position and the layers of tissue were sutured in turn, and the affected area was bandaged under sterile conditions; after the operation, the tabby cat was injected with ampicillin (4.0 mg / kg) intramuscularly for 5 consecutive days to prevent incision infection, and then the tabby cat was allowed to eat freely.

[0080] The efficacy evaluation of the stem cell exosome preparation includes the following steps: dividing the civet cat bone and joint injury model into 6 groups, each with 5 animals, wherein the blank group is a normal civet cat, which is treated with only physiological saline throughout the process; the control model group is a civet cat model treated with only physiological saline throughout the process; control group 1 is a civet cat model treated with the preparation of Example 1; control group 2 is a civet cat model treated with the preparation of Comparative Example 2; treatment groups 1-3 are treated with the stem cell exosome therapeutic agent described in Examples 3-5 of the present invention, respectively. The treatment process is to inject 200 μL of sample into the bone and joint cavity of the civet cat, each course of treatment is 1 week, and after 2 consecutive weeks of injection, the serum of the civet cat samples is collected and the secretion of IL-1β, TNF-ɑ and IL-6 in the serum is analyzed by ELISA kit.

[0081] The results are as follows Figure 1 As shown: the IL-1β, TNF-ɑ and IL-6 in the serum of the tabby cat in the control model group were significantly higher than those in the blank group, confirming that the pet bone and joint injury model of the present invention was successfully prepared. The comparative example shows that neither the stem cell exosome preparation nor the monoclonal antibody preparation alone can effectively reduce the levels of IL-1β, TNF-ɑ and IL-6 in the serum. However, the stem cell exosome preparation prepared by any one of Examples 3-5 of the present invention has good inhibitory effects on the secretion of IL-1β, TNF-ɑ and IL-6 in the serum, and effectively alleviates joint damage caused by cytokines. In the present invention, it was also found that the combined use of bone marrow mesenchymal stem cell exosomes and RANKL monoclonal antibody 5F16 also has a synergistic effect, which can inhibit the secretion of IL-1β, TNF-ɑ and IL-6, repair bone and joint injuries, and improve and promote cartilage tissue repair.

[0082] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A stem cell exosome preparation for preventing and treating pet bone and joint injuries, characterized in that: Each 1000 mL contains 10-40 mL of bone marrow mesenchymal stem cell exosomes, 20-50 μg of RANKL monoclonal antibody 5F16, 5-20 mg of chondroitin sulfate, and the balance is normal saline; the heavy chain variable region sequence of the RANKL monoclonal antibody 5F16 is as shown in SEQ ID NO.1, and the light chain variable region sequence is as shown in SEQ ID NO:2, wherein the pet is a tabby cat, and the bone marrow mesenchymal stem cell exosomes are derived from the bone marrow tissue of the tabby cat.

2. The stem cell exosome preparation according to claim 1, wherein The stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 10 mL of bone marrow mesenchymal stem cell exosomes, 20 μg of RANKL monoclonal antibody 5F16, 5 mg of chondroitin sulfate per 1000 mL, and the remainder is normal saline.

3. The stem cell exosome preparation according to claim 1, wherein The stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 20 mL of bone marrow mesenchymal stem cell exosomes, 30 μg of RANKL monoclonal antibody 5F16, 10 mg of chondroitin sulfate per 1000 mL, and the remainder is normal saline.

4. The stem cell exosome preparation according to claim 1, wherein The stem cell exosome preparation for preventing and treating pet bone and joint injuries contains 40 mL of bone marrow mesenchymal stem cell exosomes, 50 μg of RANKL monoclonal antibody 5F16, 20 mg of chondroitin sulfate per 1000 mL, and the remainder is normal saline.

5. The stem cell exosome preparation according to claim 1, wherein The heavy chain variable region of the RANKL monoclonal antibody 5F16 includes CDR-H1, CDR-H2, and CDR-H3, and the sequences of CDR-H1-3 are shown in SEQ ID NO.3-5.

6. The stem cell exosome preparation according to claim 1, wherein The light chain variable region of the RANKL monoclonal antibody 5F16 includes CDR-L1, CDR-L2, and CDR-L3, and the sequences of CDR-L1-3 are shown in SEQ ID NOs. 6-8.

7. The stem cell exosome preparation according to claim 1, wherein The method for extracting exosomes from bone marrow mesenchymal stem cells includes the following steps: 1) Bone marrow tissue was obtained and digested with trypsin to prepare single cell suspension, and CD29 was isolated by flow cytometry. + CD90 + CD105 + CD14 - CD34 - CD45 - bone marrow mesenchymal stem cell exosomes; 2) The bone marrow mesenchymal stem cell exosomes obtained in step 1) were placed in DMEM / F12 medium for continuous subculture; 3) After the cells have stabilized at the passage level, collect the cell supernatant by centrifugation; 4) Filtering the supernatant from step 3) through a membrane filter, performing ultrahigh-speed centrifugation, and collecting the precipitate after the centrifugation is complete; 5) Resuspend the pellet from step 4) in 1× PBS and perform ultracentrifugation again; 6) Resuspend the precipitate from step 5) in 1× PBS, collect the filtrate through a filter membrane, and adjust the density to 1×10 4 -1×10 6 Exosomes / mL are bone marrow mesenchymal stem cell exosomes.

8. The method for preparing the stem cell exosome preparation for preventing and treating pet bone and joint injuries according to any one of claims 1 to 7, characterized in that: The preparation method comprises mixing the above raw materials and dissolving them in physiological saline, adjusting the pH to 7.0, and filtering and sterilizing to obtain the product; wherein the pet is a tabby cat, and the bone marrow mesenchymal stem cell exosomes are derived from the bone marrow tissue of the tabby cat.

9. Use of the RANKL monoclonal antibody 5F16 and bone marrow mesenchymal stem cell exosomes in combination in the preparation of a medicament for preventing and treating bone and joint injuries in pets, wherein the heavy chain variable region sequence of the RANKL monoclonal antibody 5F16 is shown in SEQ ID NO. 1, and the light chain variable region sequence is shown in SEQ ID NO: 2, wherein the pet is a tabby cat, and the bone marrow mesenchymal stem cell exosomes are derived from the bone marrow tissue of the tabby cat.

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