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Application of M2 type macrophage exosome in preparation of medicine for treating osteoporosis

A technology for osteoporosis and macrophages, applied in the biological field, can solve problems such as application limitations, and achieve the effect of inhibiting bone loss.

Pending Publication Date: 2022-03-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the immunogenicity of allogeneic cells and other reasons, its application is limited.

Method used

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  • Application of M2 type macrophage exosome in preparation of medicine for treating osteoporosis
  • Application of M2 type macrophage exosome in preparation of medicine for treating osteoporosis
  • Application of M2 type macrophage exosome in preparation of medicine for treating osteoporosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Isolation and identification of M2 macrophage exosomes

[0020] Induction of S1.M2 macrophages

[0021] RAW264.7 (mouse mononuclear macrophage leukemia cells) was cultured in a 10cm dish containing 10% fetal calf serum in DMEM high-glucose medium, and when the cell proliferation was close to 70-80% confluent, it was passed on to a 10cm dish, The culture medium is DMEM high-glucose medium without fetal bovine serum, stimulated with 10ng / ml IL-4 every other day, and induced for 24 hours. figure 1 Display: After IL-4 stimulation, the cell morphology is "spindle-shaped", and RT-qPCR detection shows that the cells can express the classic markers of M2 macrophages: arginine (Arg-1) and mannitol receptor (Mannose receptor) . After confirming that the induced cells were M2 macrophages, the supernatant of the cells was collected for subsequent experiments.

[0022] S2. M2-EVs isolation

[0023] The supernatant collected above was subjected to exosome extraction. ...

Embodiment 2

[0029] Example 2, the inhibitory effect of M2-EVs on osteoclasts

[0030] S1. Isolation of mouse bone marrow-derived macrophages (BMM)

[0031] Mouse bone marrow-derived macrophages (BMM) were isolated according to conventional methods. After isolation, the BMMs were cultured in a 10 cm dish containing 10% fetal calf serum, 30ng / ml MCSF, and alpha MEM high-glucose medium containing double antibodies to penicillin and streptomycin , the medium was changed every 2 days, and on the 5th day of induction, the cells could proliferate to 70% confluence, and the cells were collected for subsequent experiments.

[0032] S2. Induction of osteoclasts

[0033] The above-mentioned cells were digested and seeded in a 96-well plate, and 100,00 BMMs were plated in each well. The medium was the same as above, and RANKL was added every other day to a final concentration of 100ng / ml, and the medium was changed every two days. image 3 It was shown that after induction, the cells began to fuse ...

Embodiment 3

[0037] Example 3, the rescue effect of M2-EVs on osteoporotic mice

[0038] Ovariectomized (OVX) mice were obtained by conventional ovariectomy in female mice of 8 weeks, and 1 μg / ul M2-EVs 100 μl (solvent was 10% PBS buffer) was injected through the tail vein after 1 week, and injected weekly 2 times. Eight weeks after injection, micro CT was used to analyze the femoral bone formation of OVX mice injected with M2-EVs regularly and the vehicle-injected control group (phosphate-buffered saline solution). Figure 5 The results showed that the addition of M2-EVs could effectively improve the bone loss in OVX mice.

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Abstract

The invention provides an application of an M2 type macrophage exosome in preparation of a medicine for treating osteoporosis. The preparation method comprises the following steps: preparing an exosome derived from M2 macrophages, and proving the inhibition effect of M2-EVs on osteoclasts through RT-qPCR, western blotting, immunofluorescence and other experiments at the cellular level; meanwhile, the effect of M2-EVs on promoting osteogenesis of ovarian extirpation mice is determined through in-vivo experiments. According to the invention, osteoclast formation and bone resorption capability can be inhibited, and bone loss capability of OVX mice can be inhibited in vivo; the pharmaceutical composition can be used for treating related diseases such as osteoporosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a new application of exosomes derived from macrophages, and specifically relates to an application of exosomes derived from M2 macrophages in osteoporosis. Background technique [0002] Osteoporosis (OP) is a systemic skeletal disease characterized by low bone mass and microarchitectural disruption of bone tissue, resulting in increased bone fragility and susceptibility to fracture. As the aging population grows, osteoporosis has become one of the most common bone diseases. Dysregulation of osteoblasts and osteoclasts is the main cause of osteoporosis. Studies have shown that changes in macrophage polarization can reduce bone resorption factors and promote osteogenesis and bone mineralization. [0003] Macrophages are highly heterogeneous and plastic cells that can differentiate into different types in vivo according to different stimuli and microenvironments. Among them, unactivate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/15A61P19/10
CPCA61K35/15A61P19/10
Inventor 谢志坚黄小园蓝燕华沈嘉慧
Owner ZHEJIANG UNIV
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