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Apoptotic vesicle as well as preparation method and application thereof

A technology of microvesicles and apoptosis, applied in biochemical equipment and methods, cell dissociation methods, microorganisms, etc., can solve problems such as limiting therapeutic applications and side effects, and achieve simple extraction methods, improved adipogenic ability, and yield high effect

Pending Publication Date: 2022-01-18
PEKING UNIV SCHOOL OF STOMATOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the many side effects and deficiencies in the existing soft tissue defect repair and depression repair treatment methods, the present invention provides a safe and efficient extracellular vesicle that promotes fat regeneration, aiming to solve the problems in the existing soft tissue defect repair and depression repair treatment. Issues that limit its therapeutic use due to side effects

Method used

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  • Apoptotic vesicle as well as preparation method and application thereof
  • Apoptotic vesicle as well as preparation method and application thereof
  • Apoptotic vesicle as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Efficient extraction of apoVs derived from RAW264.7

[0033] RAW264.7 was cultured in vitro, and when the cell confluence reached 90-100%, 500nM STS was added to induce apoptosis, and the apoVs derived from RAW264.7 were obtained by gradient centrifugation, and its concentration was detected by nanoparticle tracking analysis, and its protein was detected by BCA method Quantity, obtain the optimal extraction conditions, and establish a standard extraction process. details as follows:

[0034] a) Centrifuge the cell culture supernatant at 800 g for 10 min at 4° C. to remove cell debris in the culture supernatant, and take the supernatant to obtain the first centrifugation supernatant;

[0035] b) Centrifuge the first centrifugation supernatant at 4° C. and 2000 g for 10 min to remove impurities such as apoptotic bodies in the first centrifugation supernatant, and take the supernatant to obtain the second centrifugation supernatant ;

[0036] c) centrifuging t...

Embodiment 2

[0038] Example 2 Characteristic analysis of apoVs derived from RAW264.7

[0039] The morphology, particle size, and concentration of apoVs derived from RAW264.7 were detected by cryo-TEM and nanoparticle tracking analysis.

[0040] Cryo-TEM:

[0041] (1) Pipette 5 μl of apoptotic microvesicle suspension onto the copper grid, and let it stand at room temperature for 1 min;

[0042] (2) Use filter paper to absorb excess liquid along the outside of the copper grid, absorb 5 μl of 2% uranyl acetate and drop it onto the copper grid, and let stand at room temperature for 30 seconds;

[0043] (3) Absorb the excess liquid along the outside of the copper mesh with filter paper, and let it dry at room temperature;

[0044] (4) Images were taken under a transmission electron microscope, and the voltage was set to 120kV.

[0045] Nanoparticle size tracking analysis detection:

[0046] (1) Use a nanoparticle tracking analyzer to record the trajectory of exosomes under Brownian motion; ...

Embodiment 3

[0049] Example 3 In vitro experiments to detect the effect of apoVs derived from RAW264.7 on the adipogenic differentiation of human adipose-derived mesenchymal stem cells in vitro

[0050]After 14 days of adipogenic induction, the effect of cell adipogenic differentiation was examined by Oil Red O staining.

[0051] Oil red O staining:

[0052] To prepare the dyeing solution, weigh 0.5 g of Oil Red O dry powder, dissolve it in 100 ml of 100% isopropanol, and after fully dissolved, it becomes Oil Red O stock solution, and store it in the dark at 4°C. Before staining, take Oil Red O stock solution and dilute it according to the ratio of stock solution: distilled water = 3:2. After filtering with filter paper, it becomes Oil Red O working solution, which is used for staining.

[0053] Aspirate the medium, rinse with PBS three times, and fix with 10% neutral formalin for 1 hour. Discard the formalin, rinse with PBS three times, and rinse with 60% isopropanol. After drying, add...

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Abstract

The invention provides a preparation method of apoptotic vesicle. The preparation method comprises the following steps: 1) culturing a mouse mononuclear macrophage leukemia cell line in vitro; (2) when the confluence degree of the cells reaches 90%-100%, adding STS (staurosporin) to induce cell apoptosis; and (3) collecting the supernatant of the culture solution, and then separating the supernatant by virtue of a gradient centrifugation method, so as to obtain the apoptotic vesicle (apoVs). The invention also provides the apoptotic vesicle and application of the apoptotic vesicle in preparation of a preparation for promoting adipogenic differentiation of mesenchymal stem cells. The apoptotic vesicle provided by the invention can promote adipogenic differentiation of mesenchymal stem cells, can be used for repairing soft tissue defects or recesses, and have no obvious side effects.

Description

technical field [0001] The invention relates to the technical field of biological tissue engineering, in particular to an apoptotic microvesicle which can be used to promote the adipogenic differentiation of mesenchymal stem cells, a preparation method and application thereof. Background technique [0002] Soft tissue defects or depressions caused by trauma, infection, surgery or congenital malformations are very common in clinical practice. There are two types of maxillofacial soft tissue defects: congenital factors and acquired factors. The most common congenital factors are cleft lip and palate, and acquired factors are mainly caused by trauma, such as traffic accidents, burns, infectious diseases and tumors. Bits can also cause soft tissue defects. In addition, postoperative breast deformation and absence of benign and malignant breast lesions are also common types of soft tissue defects. Tissue reconstruction is an important part of maxillofacial and breast postoperat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786A61L27/38A61L27/50
CPCC12N5/0645A61L27/3804A61L27/3895A61L27/3834A61L27/50C12N2509/10A61L2430/34
Inventor 周永胜张晓刘云松朱原
Owner PEKING UNIV SCHOOL OF STOMATOLOGY
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