Application of human platelet apoptosis microvesicles

A technology of microvesicles and platelets, applied in blood/immune system cells, medical science, animal cells, etc., can solve problems such as unsatisfactory results, avoid ethical issues and immune issues, rapid detection, and broad clinical application prospects Effect

Active Publication Date: 2022-04-12
PEKING UNIV SCHOOL OF STOMATOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems and unsatisfactory effects of existing bone defect repair treatment methods, the present invention provides a safe and efficient apoptotic microvesicle that promotes bone regeneration, aiming to solve the existing bone defect repair treatment effects and promote its therapeutic application

Method used

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  • Application of human platelet apoptosis microvesicles
  • Application of human platelet apoptosis microvesicles
  • Application of human platelet apoptosis microvesicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Efficient extraction of apoVs derived from human platelets

[0058] Human platelets were isolated and purified in vitro, resuspended in culture medium, and 5000 nM STS was added to induce apoptosis. Platelet-derived apoVs were obtained by gradient centrifugation, and their concentration was detected by nanoparticle tracking analysis, and their protein content was detected by BCA method. Optimized extraction conditions and established a standard extraction process.

[0059] details as follows:

[0060] a) Centrifuge the supernatant of the cell culture liquid at 4° C. at 800 g for 10 min to remove cell debris in the supernatant of the culture liquid, and take the supernatant to obtain the first centrifugation supernatant;

[0061] b) centrifuging the first centrifugation supernatant at 4°C and 16000g for 30min, and taking the precipitate to obtain crude apoptotic microvesicles;

[0062] c) The crude apoptotic microvesicles were washed with sterile PBS, and the...

Embodiment 2

[0063] Example 2 Characteristic analysis of apoVs derived from human platelets

[0064] The morphology, particle size, and concentration of platelet-derived apoVs were detected by cryo-TEM and nanoparticle tracking analysis.

[0065] Cryo-TEM:

[0066] (1) Pipette 5 μl of apoptotic microvesicle suspension onto the copper grid, and let stand at room temperature for 1 min;

[0067] (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 on the copper grid, and let it stand at room temperature for 30 seconds;

[0068] (3) Use filter paper to absorb excess liquid along the outside of the copper mesh, and let it dry at room temperature;

[0069] (4) Images were taken under a transmission electron microscope, and the voltage was set to 120kV.

[0070] Nanoparticle size tracking analysis detection:

[0071] (1) Use a nanoparticle tracking analyzer to record the trajectory of apoptotic microvesicles under Brow...

Embodiment 3

[0075] Example 3 In vitro experiments to detect the effect of human platelet-derived apoVs on the differentiation of human bone marrow mesenchymal stem cells into osteogenesis in vitro

[0076] Human bone marrow mesenchymal stem cells were cultured under the following three culture conditions:

[0077] 1) Proliferation medium (PM): MEMα medium containing 10% FBS and 1% penicillin-streptomycin double antibody.

[0078] 2) Osteogenic induction (OM): Contains 10% FBS, 1% penicillin-streptomycin double antibody, 10mM β-sodium glycerophosphate (β-Sodium Glycerophosphate), 0.2mM L-ascorbic acid (Ascorbic Acid) and 100nM dexamethasone (dexamethasone) MEMα medium.

[0079] 3) Add 0.225 μg / mL human platelet-derived apoVs (OM+ 0.225 μg / mL PLT-apoVs) to the osteogenic induction medium.

[0080] After 10 days of osteogenic induction, the effect of osteogenic differentiation of cells was examined by alizarin red staining.

[0081] Alizarin red staining:

[0082] To prepare the dye solu...

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Abstract

The invention discloses application of human platelet apoptosis microvesicles in preparation of a preparation for promoting osteogenic differentiation of mesenchymal stem cells and provides a bone defect repair preparation which comprises a PLGA (poly (lactic-co-glycolic acid)) support material, and the human platelet apoptosis microvesicles are carried on the surface of the PLGA support material. The apoptosis microvesicles derived from human platelets provided by the invention can be extracted from blood of a patient, and ethical problems and immune problems are avoided. According to the bone defect repairing preparation constructed by the PLGA / pDA support carrying the human platelet apoptosis microvesicles, the defects that PLGA is hydrophobic and low in osteogenic activity are overcome, particularly, the human platelet apoptosis microvesicles are carried after a pDA film is loaded on the surface of the PLGA support, so that the human platelet apoptosis microvesicles can be slowly released in vivo, and the bone defect repairing preparation is used for repairing bone defects of the human platelet apoptosis microvesicles. The in-vivo osteogenesis capability of the mesenchymal stem cells is further improved.

Description

technical field [0001] The invention relates to the technical field of biological tissue engineering, in particular to the application of human platelet apoptosis microvesicles. Background technique [0002] Apoptosis is a highly regulated cell death process in which specific cells are sacrificed for the greater good of the organism. This is a normal physiological process in multicellular organisms. Apoptosis confers advantages on multicellular organisms to maintain homeostasis and fine-tune the life cycle in a coordinated manner. Cell death by apoptosis proceeds through several stages, starting with condensation of nuclear chromatin, followed by membrane vesicle rupture, and subsequent disintegration of cell contents into distinct membrane-encased vesicles called apoptotic bodies or apoptotic microsomes. Vesicles (apoptotic vesicles, apoVs). Apoptotic microvesicles are different from exosomes (also called exosomal microvesicles or exosomes), microvesicles (also called mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/19A61P19/08A61L27/34A61L27/18A61L27/54C12N5/078
CPCA61K35/19A61L27/18A61L27/34A61L27/54A61P19/08C12N5/06
Inventor 周永胜张晓江雨荷邵玉子杨坤坤
Owner PEKING UNIV SCHOOL OF STOMATOLOGY
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