Use of human-derived mitochondria in preparation of drug for treating achilles tendon injury

Abstract

Use of human-derived mitochondria in the preparation of a drug for treating Achilles tendon injury and in the preparation of a drug used in conjunction with Achilles tendon suture surgery for treating Achilles tendon rupture. By means of constructing a monkey Achilles tendon injury model, it has been verified that human-derived mitochondria, especially mitochondria derived from human platelets, can make the Achilles tendon tissue denser and improve the rehabilitation effect, providing a simple, efficient and safe method for repairing Achilles tendon injury for Achilles tendon injury patients.

Description

Application of human mitochondria in the preparation of drugs for treating Achilles tendon injury Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of human mitochondria in the preparation of medicines for treating Achilles tendon injuries. Background Art

[0002] Achilles tendon injury refers to an open or closed injury to the Achilles tendon due to external force. It is a common disease. The main cause is related to violent factors. The main clinical symptoms include pain, swelling, and movement disorders. It can lead to complications of Achilles tendon rupture, seriously affecting the patient's work and life.

[0003] Achilles tendon injuries are characterized by concentrated stress and a lack of blood supply. Healing takes a long time, and the wound typically heals as a scar with poor mechanical properties. Currently, treatments are primarily through medication and surgery, but the prognosis is generally poor. In addition to surgical repair, platelet-rich plasma (PRP) is often used in combination with other treatments for Achilles tendon injuries. However, there are many types of PRP with complex components, and the platelet content and quality of different patients have a significant impact on the quality and therapeutic effect of PRP. Therefore, the therapeutic effect on Achilles tendon injuries is not stable.

[0004] Mitochondria have been considered a therapeutic target in medicine for the past three decades. In recent years, the use of mitochondrial transplantation for tissue repair has garnered significant interest. Platelets are rich in mitochondria, and their use in treating Achilles tendon injuries holds great promise, but no relevant reports have been reported.

[0005] Summary of the Invention

[0006] In order to overcome the defects in the prior art, the present invention provides the use of human mitochondria in the preparation of a drug for treating Achilles tendon injury, providing a simpler, more efficient and convenient method for treating Achilles tendon injury.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention is to provide the use of human mitochondria in the preparation of a drug for treating Achilles tendon injury.

[0009] Furthermore, the human mitochondria are mitochondria derived from human platelets.

[0010] Furthermore, the human platelet-derived mitochondria are prepared by the following method:

[0011] Step 1: Take human anticoagulated whole blood, centrifuge it, take the supernatant, centrifuge it again, and precipitate platelets;

[0012] Step 2: After removing part of the supernatant, the platelets are resuspended in the remaining supernatant to prepare platelet-rich plasma;

[0013] Step three: extract mitochondria from platelet-rich plasma.

[0014] Furthermore, in step 1, the centrifugation conditions are 150-450 g for 7-15 min, and the centrifugation conditions are again 600-1200 g for 7-15 min.

[0015] Furthermore, in step 2, the volume of the remaining supernatant is 600-1000 μl.

[0016] Furthermore, in step three, the steps of extracting mitochondria are as follows:

[0017] (1) Add platelet-rich plasma to extract solution A, vortex and place on ice;

[0018] (2) Add extract B and place on ice, vortexing periodically.

[0019] (3) Add extract C, mix well, centrifuge, take the supernatant, centrifuge again, discard the supernatant, and precipitate mitochondria;

[0020] Wherein, based on 100 ml of 0.6% sodium chloride solution, the extract solution A is prepared by the following method: 60-90 mg EDTA and 200-450 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride solution, and filtered to prepare solution A; preferably, 87.6 mg EDTA and 310 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride solution, and filtered to prepare solution A;

[0021] The extract B is a heparin solution with a mass volume ratio of 1 to 10%, preferably 4%;

[0022] The extract solution C is prepared by dissolving 2-6 g mannitol, 60-90 mg EDTA, and 200-450 mg sodium lactate in 100 ml of 0.9% sodium chloride solution and filtering to prepare solution C. Preferably, 4.55 g mannitol, 87.6 mg EDTA, and 310 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution and filtered to prepare solution C.

[0023] Furthermore, the above medicine is an injection.

[0024] The second aspect of the present invention is to provide the use of human mitochondria in the preparation of a drug for treating Achilles tendon rupture in conjunction with Achilles tendon suture surgery.

[0025] Furthermore, the human mitochondria are mitochondria derived from human platelets.

[0026] Furthermore, the human platelet-derived mitochondria are prepared by the following method:

[0027] Step 1: Take human anticoagulated whole blood, centrifuge it, take the supernatant, centrifuge it again, and precipitate platelets;

[0028] Step 2: After removing part of the supernatant, the platelets are resuspended in the remaining supernatant to prepare platelet-rich plasma;

[0029] Step three: extract mitochondria from platelet-rich plasma.

[0030] Furthermore, in step 1, the centrifugation conditions are 150-450 g for 7-15 min, and the centrifugation conditions are again 600-1200 g for 7-15 min.

[0031] Furthermore, in step 2, the volume of the remaining supernatant is 600-1000 μl.

[0032] Furthermore, in step three, the steps of extracting mitochondria are as follows:

[0033] (1) Add platelet-rich plasma to extract solution A, vortex and place on ice;

[0034] (2) Add extract B and place on ice, vortexing periodically.

[0035] (3) Add extract C, mix well, centrifuge, take the supernatant, centrifuge again, discard the supernatant, and precipitate mitochondria;

[0036] Wherein, based on 100 ml of 0.6% sodium chloride solution, the extract solution A is prepared by the following method: 60-90 mg EDTA and 200-450 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride solution, and filtered to prepare solution A; preferably, 87.6 mg EDTA and 310 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride solution, and filtered to prepare solution A;

[0037] The extract B is a heparin solution with a mass volume ratio of 1 to 10%, preferably 4%;

[0038] The extract solution C is prepared by dissolving 2-6 g mannitol, 60-90 mg EDTA, and 200-450 mg sodium lactate in 100 ml of 0.9% sodium chloride solution and filtering to prepare solution C. Preferably, 4.55 g mannitol, 87.6 mg EDTA, and 310 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution and filtered to prepare solution C.

[0039] Furthermore, the above medicine is an injection.

[0040] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0041] The present invention verifies that human mitochondria, especially mitochondria derived from human platelets, can make Achilles tendon tissue denser and improve the rehabilitation effect by constructing a monkey Achilles tendon injury model, providing patients with Achilles tendon injury with a simple, efficient and safe method for repairing Achilles tendon injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG1 shows a precipitate of human platelet mitochondria extracted in one embodiment of the present invention;

[0043] FIG2 shows a schematic diagram of the surgical process for constructing a monkey Achilles tendon injury model and using mitochondria for treatment according to one embodiment of the present invention; wherein, I: monkey heel surgical area, II: free Achilles tendon, III: severed Achilles tendon, IV: suturing the severed Achilles tendon, V: local injection of platelet-derived mitochondria, VI: postoperative lower limb plaster fixation;

[0044] Figure 3 shows the histological section staining results of the macaque Achilles tendon injury model after treatment with human platelet-derived mitochondria; the left figure is the control group, in which the Achilles tendon was severed and sutured and then local injection of normal saline was performed; the right figure is the mitochondria treatment group, in which the Achilles tendon was severed and sutured and then mitochondria resuspended in an equal amount of normal saline were locally injected around the stump. DETAILED DESCRIPTION

[0045] The present invention provides the use of platelet-derived mitochondria in the preparation of a drug for treating Achilles tendon injury. Specifically, for patients with Achilles tendon rupture, the extracted mitochondria are locally injected at the suture site after Achilles tendon suture surgery; the wound is closed; and for patients with Achilles tendon injury, the Achilles tendon pain point is injected percutaneously after local skin disinfection.

[0046] In the present invention, platelet-derived mitochondria can be extracted using conventional methods. After mechanical or chemical membrane disruption, differential centrifugation can be performed to obtain mitochondrial fractions (referenced literature PMID: 18600228, PMID: 17406588). Currently available commercial kits can stably obtain mitochondrial fractions (such as Thermo Scientific TM In a preferred embodiment of the present invention, platelet-derived mitochondria are prepared by the following method:

[0047] (1) Take 20 ml of human anticoagulated whole blood, centrifuge at 300 g for 10 min, transfer the supernatant (about 8 ml) to a new centrifuge tube, and centrifuge at 800 g for 10 min to precipitate platelets.

[0048] (2) After removing most of the supernatant, the platelets were resuspended in the remaining serum (about 1 ml) to prepare platelet-rich plasma (PRP).

[0049] (3) Add 5 ml of mitochondrial extract solution A, shake and let it stand for 2 minutes; add 60 μl of mitochondrial extract solution B to lyse for 5 minutes, shaking for 5 seconds every 1 minute; add 5 ml of mitochondrial extract solution C, centrifuge at 700g for 10 minutes; transfer the supernatant to a new centrifuge tube, centrifuge at 12000g for 15 minutes to precipitate mitochondria; resuspend the mitochondria in 100 μl of physiological saline for later use.

[0050] In the above preparation method, solution A was prepared by dissolving 87.6 mg EDTA and 310 mg sodium lactate in 100 ml of 0.6% sodium chloride and filtering through a 0.22 μm filter to prepare solution A; solution B was prepared by dissolving 200 mg heparin in 5 ml of deionized water and filtering through a 0.22 μm filter to prepare solution B; and solution C was prepared by dissolving 4.55 g mannitol, 87.6 mg EDTA, and 310 mg sodium lactate in 100 ml of 0.9% sodium chloride and filtering through a 0.22 μm filter to prepare solution C.

[0051] The components of the mitochondrial extract are clinically verified to be safe and reliable for clinical treatment.

[0052] The present invention will be described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention. However, the following embodiments do not limit the scope of the present invention.

[0053] In the examples, conventional methods were used unless otherwise specified, and reagents used were conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0054] Example 1

[0055] This example establishes a monkey Achilles tendon injury model to verify the effect of human platelet mitochondria in treating Achilles tendon injury. The specific experimental steps and results are as follows:

[0056] 1. Extraction of human platelet mitochondria

[0057] (1) Take 20 ml of human anticoagulated whole blood, centrifuge at 300 g for 10 min, transfer the supernatant (about 8 ml) to a new centrifuge tube, and centrifuge at 800 g for 10 min to precipitate platelets.

[0058] (2) After removing most of the supernatant, the platelets were resuspended in the remaining serum (about 1 ml) to prepare platelet-rich plasma (PRP).

[0059] (3) Add 5 ml of mitochondrial extract solution A, shake and let it stand for 2 minutes; add 60 μl of mitochondrial extract solution B to lyse for 5 minutes, shaking for 5 seconds every 1 minute; add 5 ml of mitochondrial extract solution C, centrifuge at 700g for 10 minutes; transfer the supernatant to a new centrifuge tube, centrifuge at 12000g for 15 minutes, and precipitate the mitochondria (see Figure 1); resuspend the mitochondria in 100 μl of physiological saline for later use.

[0060] 2. Establishing a monkey Achilles tendon injury model and using mitochondria for treatment (see Figure 2)

[0061] After conventional anesthesia, a 5-cm incision was made on the dorsal aspect of the ankle on the operative side to expose the Achilles tendon. The tendon was scalpel-severed and sutured using the Kessler suture technique. The extracted mitochondria were injected locally at the suture site, and the wound was closed.

[0062] Four weeks after surgery, the macaques were sacrificed and the Achilles tendon tissues were obtained for routine fixation, paraffin embedding, histological sectioning and HE staining.

[0063] The results are shown in Figure 3. The Achilles tendon tissue of the monkeys in the mitochondrial treatment group was denser, suggesting a better recovery effect.

[0064] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. It will be apparent to those skilled in the art that any equivalent modifications and substitutions to the present invention fall within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to fall within the scope of the present invention.

Claims

1. Application of human mitochondria in the preparation of drugs for the treatment of Achilles tendon injury.

2. The application of human mitochondria in the preparation of drugs for the treatment of Achilles tendon rupture in conjunction with Achilles tendon suture surgery.

3. The use according to claim 1 or 2, characterized in that: The human mitochondria are mitochondria derived from human platelets.

4. The use according to claim 3, characterized in that: The human platelet-derived mitochondria are prepared by the following method: Step 1, taking human anticoagulated whole blood, centrifuging, taking the supernatant, centrifuging again, and precipitating platelets; Step 2, after removing part of the supernatant, resuspending the platelets with the remaining supernatant to prepare platelet-rich plasma; Step three, extracting mitochondria from platelet-rich plasma.

5. The use according to claim 4, characterized in that: In step 1, the centrifugation condition is 150-450 g for 7-15 min, and the centrifugation condition is again 600-1200 g for 7-15 min.

6. The use according to claim 4, characterized in that: In step 2, the volume of the remaining supernatant is 600-1000 μl.

7. The use according to claim 4, characterized in that: In step 3, the steps for extracting mitochondria are as follows: (1) Add platelet-rich plasma to extraction solution A, vortex and place on ice; (2) Add Extraction Solution B and place on ice, vortexing periodically; (3) Add extract solution C, mix well, centrifuge, take the supernatant, centrifuge again, discard the supernatant, and precipitate mitochondria; Wherein, based on 100ml of 0.6% sodium chloride solution, the extract A solution is prepared by the following method: 60-90mg EDTA and 200-450mg sodium lactate are dissolved in 100ml of 0.6% sodium chloride solution, and filtered to prepare A solution; preferably 87.6mg EDTA and 310mg MgCl2 are dissolved in 100ml of 0.6% sodium chloride solution. The solution was filtered to prepare solution A; The extract B is a heparin solution, and its mass volume ratio is 1-10%, preferably 4%; Based on 100 ml of 0.9% sodium chloride solution, the extract C solution is prepared by the following method: 2-6 g mannitol, 60-90 mg EDTA, and 200-450 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution, and filtered to prepare C solution; preferably, 4.55 g mannitol, 87.6 mg EDTA, and 310 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution, and filtered to prepare C solution.

8. The use according to claim 1 or 2, characterized in that: The medicine is an injection.

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