M2 type bone marrow macrophage exosome, application thereof and spinal cord injury treatment preparation

A macrophage and exosome technology, applied in spinal cord injury treatment preparations, M2-type bone marrow macrophage exosomes and their application fields, can solve the problems of lack, limited regeneration ability, and difficulty in long-term sustained effect. , to achieve the effect of fast absorption, high action efficiency, local tissue survival and nerve regeneration

Active Publication Date: 2021-06-29
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The exosomes for the treatment of spinal cord injury are mainly derived from different types of stem cells. The nerve regeneration effect of stem cell exosomes can improve the functional recovery after spinal cord injury to a certain extent, but the regeneration of nerves is closely related to the local microenvironment. Under the condition of ischemia and hypoxia, its regenerative ability is often limited. At present, there is no exosome product mainly for the treatment of angiogenesis after spinal cord injury.
[0004] In addition, systemically injected exosomes tend to reach the liver and kidney with the blood and be metabolized, and only a small part reaches the target site to play a therapeutic role. Local exosome administration can only maintain a high effect concentration in the early stage, and it is difficult to maintain a long-term effect. Sustained effect
[0005] In summary, there is still a lack of exosomes that promote angiogenesis after spinal cord injury, and the use of such exosomes for continuous drug treatment of spinal cord injury

Method used

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  • M2 type bone marrow macrophage exosome, application thereof and spinal cord injury treatment preparation
  • M2 type bone marrow macrophage exosome, application thereof and spinal cord injury treatment preparation
  • M2 type bone marrow macrophage exosome, application thereof and spinal cord injury treatment preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1, Isolation and cultivation of M2 type bone marrow-derived macrophages:

[0078] C57BL / 6 mice aged 4-6 weeks were selected and sacrificed by decapitation. Separate the femur and tibia, wash the cells in the bone marrow with IMDM medium, and gently blow the cells repeatedly to blow away the larger tissue pieces in the bone marrow cavity, filter the cell suspension with a 70-mesh cell sieve, and collect the filtrate in a centrifuge tube , centrifuge at 1200 rpm for 5 minutes. Discard the supernatant, add 2mL erythrocyte lysate to resuspend, room temperature for 5min, add an equal volume of medium to stop the lysis of erythrocytes. Centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend the bone marrow cells in cell culture medium, and seed them evenly in a 100mm culture dish. After 12 hours, collect the supernatant, centrifuge at 1200 for 5 minutes, collect non-adherent cells, discard the supernatant, and supplement complete medium (IMDM, 10ng / ...

Embodiment 2

[0079] Example 2, acquisition of hiOTULIN-M2-Exos:

[0080] M2 macrophages were washed twice with PBS, and incubated with complete medium containing exosome-free serum (DMEM basal medium + 10% exosome-free serum + 1% penicillin and streptomycin) for 48 hours. The culture medium was centrifuged at 800×g for 10 minutes and 3000×g for 30 minutes; after centrifuging at 10000×g at 4°C for 1 hour, the supernatant was filtered with a 0.22 μm filter to remove cells and debris and collect exosomes. The exosomes were centrifuged at 140,000×g for 3 hours, resuspended in PBS, and then centrifuged at 140,000×g for 3 hours to obtain high-purity M2 bone marrow macrophage exosomes.

[0081] Characterization of the obtained exosomes, transmission electron microscopy and NTA measurements showed that the isolated exosomes exhibited a double-layer membrane spherical morphology with a diameter range of about 100 nm (see figure 2 A-B). Western blot verified the expression of exosome-specific mar...

Embodiment 3

[0082] Example 3, detection of components of hiOTULIN-M2-Exos:

[0083] The protein expression profile in hiOTULIN-M2-Exos was detected by ITRAQ technology. The PRM experiment found that the OTULIN protein was most significantly upregulated (the enrichment degree of this protein in exosomes was 7.22±0.06 times that in M2 bone marrow macrophages) (see image 3 A), Western blot further confirmed that OTULIN was significantly enriched in M2-Exos (M2 type bone marrow macrophage exosomes) (see image 3 B).

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Abstract

The invention discloses an M2 type bone marrow macrophage exosome, application thereof and a spinal cord injury treatment preparation, and belongs to the technical field of spinal cord injury treatment. The M2 type bone marrow macrophage exosome highly expressing the OTULIN protein can play roles in improving a local ischemia and hypoxia environment after spinal cord injury and promoting revascularization, and is more beneficial to nerve regeneration and survival of local tissues. An exosome sustained-release system is formed by combining with light-cured hydrogel and is applied to local spinal cord injury, so that the treatment efficiency of the exosome is improved, liver and kidney metabolism is avoided, long-time action can be realized by continuous sustained release, and functional recovery of mice after spinal cord injury is remarkably improved.

Description

technical field [0001] The invention belongs to the technical field of spinal cord injury treatment, and in particular relates to an M2 type bone marrow macrophage exosome, application thereof, and a treatment preparation for spinal cord injury. Background technique [0002] Spinal cord injury is a serious traumatic disease of the central nervous system. The global incidence rate is 15-40 cases per million people per year. There are more than 2 million patients with traumatic spinal cord injury in China, with an annual increase of 100,000-140,000, which is higher than global incidence. The spinal cord is the main bridge for information communication between the brain and the peripheral nervous system. Damage to the spinal cord will lead to loss of motor, sensory and autonomic functions, often causing severe disability and even death. A heavy economic burden on families and society. There is currently no effective treatment for patients with severe neurological impairment. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786A61K35/15A61L26/00A61P17/02A61P25/00
CPCC12N5/0645A61K35/15A61P25/00A61P17/02A61L26/008A61L26/0066A61L26/0057A61L2300/30A61L2300/41
Inventor 罗子翔彭伟曹勇胡建中段春岳吴天定许琰谢勇刘煜东
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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