Osteoclast vesicles triggered by pH regulation in closed region and application thereof

An osteoclast, closed area technology, applied in animal cells, vertebrate cells, cell dissociation methods, etc., can solve problems such as low biosafety, and achieve high biosafety, clear effects, and innovative guarantee mechanisms. Effect

Pending Publication Date: 2021-10-29
THE AFFILIATED SIR RUN RUN SHAW HOSPITAL OF SCHOOL OF MEDICINE ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patents such as these are limited to exosome materials derived from allogeneic cells, which have low biological safety

Method used

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  • Osteoclast vesicles triggered by pH regulation in closed region and application thereof
  • Osteoclast vesicles triggered by pH regulation in closed region and application thereof
  • Osteoclast vesicles triggered by pH regulation in closed region and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Bone-targeted sodium bicarbonate liposomes target the closed area of ​​osteoclasts, and obtain osteoclast vesicles by differential centrifugation + ultrafiltration

[0026] 1. Cultivate bone marrow-derived mononuclear macrophages and induce by adding 20ng / mL M-CSF.

[0027] 2. After 5 days, osteoclasts were induced with 20ng / mL M-CSF+50ng / mL RANKL.

[0028] 3. After 3 days, tetracycline-modified bone-targeted sodium bicarbonate liposomes were added, and the serum-free medium was changed, and the supernatant was collected every day.

[0029] 4. Gradient centrifugation, 200×g for 15 minutes, 2500×g for 15 minutes.

[0030] 5. Take the supernatant and filter the supernatant with a 0.22 μm filter to remove residual cells and debris.

[0031] 6. Transfer the solution using the ultracentrifuge filter unit. Centrifuge the solution at 4,000 x g until the volume in the upper compartment is concentrated to approximately 200 μL.

[0032] 7. Wash the ultrafiltration ...

Embodiment 2

[0034] Example 2. Bone-targeted disodium hydrogen phosphate disodium hydrogen phosphate liposome targets the closed area of ​​osteoclasts, and obtains osteoclast vesicles by density gradient method

[0035] 1. Cultivate bone marrow-derived mononuclear macrophages and induce by adding 20ng / mL M-CSF.

[0036] 2. After 5 days, osteoclasts were induced with 20ng / mL M-CSF+50ng / mL RANKL.

[0037] 3. After 3 days, tetracycline-modified bone-targeted disodium hydrogen phosphate liposomes were added, and the serum-free medium was changed, and the supernatant was collected every day.

[0038] 4. Use Percoll density gradient centrifugation medium to centrifuge at 1000×g for 20 minutes.

[0039] 5. Aspirate the exosome layer liquid at the interface of the upper layer, add 2 times the volume of PBS and mix well.

[0040] 6. Filter using a 0.22 μm filter to remove residual cells and debris, and transfer the solution using an ultracentrifugal filter device. Centrifuge the solution at 4,00...

Embodiment 3

[0043] Example 3. The pH change of the medium regulates the pH of the closed zone of osteoclasts, and the osteoclast vesicles are obtained by differential centrifugation

[0044] 1. Cultivate bone marrow-derived mononuclear macrophages and induce by adding 20ng / mL M-CSF.

[0045] 2. After 5 days, osteoclasts were induced with 20ng / mL M-CSF+50ng / mL RANKL.

[0046] 3. After 3 days, replace the serum-free medium, adjust the pH to 8.0, and collect the supernatant every day.

[0047] 4. Gradient centrifugation at 300×g for 10 minutes, and the supernatant was taken.

[0048] 5. Centrifuge at 2000×g for 10 minutes, and take the supernatant.

[0049] 6. Centrifuge at 10,000×g for 30 minutes, and take the supernatant.

[0050] 7. Continuously centrifuge at 4°C for 90 minutes at 100,000×g, remove the supernatant, resuspend the remaining pellet in PBS, and centrifuge again at 100,000×g for 90 minutes.

[0051] 8. Resuspend the pellet with 200 μL PBS

[0052] The concomitant expressi...

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PUM

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Abstract

The invention discloses an osteoclast vesicle triggered by pH regulation in a closed region and application thereof. The osteoclast can secrete a large number of extracellular vesicles by regulating the pH value of a closed region of osteoclast, and the vesicles are rich in RANK and can be used for antagonizing RANKL, so that the effect of inhibiting the osteoclast is achieved. The osteoclast vesicles are triggered and generated through pH regulation of the closed region, and the mechanism is innovative; the osteoclast-derived vesicles are adopted for osteoclast regulation, so that the method has autologous advantages and high biological safety; the effect is clear, and osteoclast can be effectively inhibited.

Description

technical field [0001] The invention belongs to the field of biological materials, and in particular relates to an osteoclast vesicle triggered by pH regulation in a closed area and an application thereof. Background technique [0002] Extracellular vesicles are a type of microparticles secreted by cells, usually carrying bioactive molecules such as proteins, mRNAs, miRNAs, and lipids, which can realize cell interaction and play an important role in cell signal transmission. Exosomes, in particular, have been widely studied in the field of tissue repair, and the functions and mechanisms of their bioactive molecules are clear. [0003] The closed area of ​​osteoclasts is a closed absorption area formed by mature osteoclasts, with an internal pH of about 4.5, rich in matrix degrading enzymes such as TRAP and CTSK, which play an important role in the function of osteoclasts, and at the same time serve as a microenvironment for osteoclasts Its formation mechanism and influencin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078A61K35/14A61P19/08
CPCC12N5/0643A61K35/14A61P19/08C12N2501/22C12N2500/90C12N2501/999C12N2509/00
Inventor 顾辰辉林贤丰范顺武
Owner THE AFFILIATED SIR RUN RUN SHAW HOSPITAL OF SCHOOL OF MEDICINE ZHEJIANG UNIV
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