Application of osteocalcin to regulation of exosomes of endothelial progenitor cells

Abstract

The invention relates to application of osteocalcin to the regulation of exosomes of endothelial progenitor cells. The regulation relates to number and diameter distribution of the exosomes. The regulated exosomes can obviously promote multiplication and migration of endothelial cells, and ensure that the endothelial cells rapidly produce vessels. The discovery of a regulation mechanism provides novel thought for the treatment of ischemic diseases.

Description

Technical field

[0001] The invention relates to the field of biomedicine, in particular to the application of osteocalcin (OCN) in regulating endothelial progenitor cell exosomes. Background technique

[0002] Osteocalcin (OCN), also known as bone Gla protein, or bone γ-Carboxyglutamic Acid Containing Protein (BGLAP or BGP), or bone-dependent vitamin K protein (BoneVitamin K Dependent Protein), It is a structural protein specifically synthesized and secreted by osteoblasts, odontoblasts and hypertrophic chondrocytes. It is one of the components that constitute the bone matrix. Glutamate residues in osteocalcin molecules are converted into gamma-carboxyglutamate (Gla) residues through vitamin K-dependent post-translational modification. The γ-carboxylated osteocalcin molecule contains 3 Gla residues. Active vitamin D promotes the biosynthesis of γ-carboxylated osteocalcin in osteoblasts. γ-carboxylated osteocalcin is called active osteocalcin, which can bind to calcium through ...

Examples

Embodiment 1

[0036] Example 1. Construction of osteocalcin (OCN) overexpressing cells

[0037] Experimental material: Osteocalcin (OCN), its amino acid sequence and its coding sequence can be referred to / selected from the prior art (such as mouse osteocalcin RAT-OCN: GenBank: AAA41761; GenBank: AAA53280; etc.), OCN coding Gene synthesis can be commissioned by commercial companies. Both pYr eukaryotic expression vector and endothelial progenitor cells (EPC) can be purchased through commercial companies.

[0038] Restricted medium: FBS was filtered through a 0.22μm filter in advance and centrifuged at 100000g for 12h to remove exosomes in FBS. Then 10% of the FBS was added to EBM-2 medium (containing EGM-2MVSingleQuots). Use FBS for exosomes.

[0039] (1) Construction of OCN overexpression vector

[0040] Using genetic engineering techniques, the OCN encoding gene was introduced into the pYr eukaryotic expression vector to construct the OCN overexpression vector pYr-RAT-OCN.

[0041] The main step...

Embodiment 2

[0057] Example 2: The effect of osteocalcin (OCN) on the secretion of exosomes

[0058] (1) Culture of transfected EPC cells and extraction of exosomes

[0059] 1. Cultivation of transfected cells

[0060] Experimental grouping (2 groups):

[0061] Group A: EPC cells transfected with OCN overexpression vector (pYr-RAT-OCN group)

[0062] Group B: EPC cells transfected with empty plasmid (control group, NC group)

[0063] The above-mentioned cells in the logarithmic growth phase cultured in a conventional medium were replaced with the same volume of restricted medium at the same cell concentration, and after 24 hours of culture, the exosomes were extracted.

[0064] 2. Extraction of exosomes (2 groups)

[0065] The exosomes in the cell culture were extracted using the exosomes extraction kit. The main steps include: the cell culture is centrifuged at 2000g for 30 minutes to remove the cells / cell debris. Take the supernatant, filter with a 1μm filter membrane, and take the filtrate. The f...

Embodiment 3

[0076] Example 3. Research on the mechanism of osteocalcin (OCN) regulating endothelial progenitor cell function through exosomes

Technical field

[0001] The invention relates to the field of biomedicine, in particular to the application of osteocalcin (OCN) in regulating endothelial progenitor cell exosomes. Background technique

[0002] Osteocalcin (OCN), also known as bone Gla protein, or bone γ-Carboxyglutamic Acid Containing Protein (BGLAP or BGP), or bone-dependent vitamin K protein (BoneVitamin K Dependent Protein), It is a structural protein specifically synthesized and secreted by osteoblasts, odontoblasts and hypertrophic chondrocytes. It is one of the components that constitute the bone matrix. Glutamate residues in osteocalcin molecules are converted into gamma-carboxyglutamate (Gla) residues through vitamin K-dependent post-translational modification. The γ-carboxylated osteocalcin molecule contains 3 Gla residues. Active vitamin D promotes the biosynthesis of γ-carboxylated osteocalcin in osteoblasts. γ-carboxylated osteocalcin is called active osteocalcin, which can bind to calcium through ...

Examples

Embodiment 1

[0036] Example 1. Construction of osteocalcin (OCN) overexpressing cells

[0037] Experimental material: Osteocalcin (OCN), its amino acid sequence and its coding sequence can be referred to / selected from the prior art (such as mouse osteocalcin RAT-OCN: GenBank: AAA41761; GenBank: AAA53280; etc.), OCN coding Gene synthesis can be commissioned by commercial companies. Both pYr eukaryotic expression vector and endothelial progenitor cells (EPC) can be purchased through commercial companies.

[0038] Restricted medium: FBS was filtered through a 0.22μm filter in advance and centrifuged at 100000g for 12h to remove exosomes in FBS. Then 10% of the FBS was added to EBM-2 medium (containing EGM-2MVSingleQuots). Use FBS for exosomes.

[0039] (1) Construction of OCN overexpression vector

[0040] Using genetic engineering techniques, the OCN encoding gene was introduced into the pYr eukaryotic expression vector to construct the OCN overexpression vector pYr-RAT-OCN.

[0041] The main step...

Embodiment 2

[0057] Example 2: The effect of osteocalcin (OCN) on the secretion of exosomes

[0058] (1) Culture of transfected EPC cells and extraction of exosomes

[0059] 1. Cultivation of transfected cells

[0060] Experimental grouping (2 groups):

[0061] Group A: EPC cells transfected with OCN overexpression vector (pYr-RAT-OCN group)

[0062] Group B: EPC cells transfected with empty plasmid (control group, NC group)

[0063] The above-mentioned cells in the logarithmic growth phase cultured in a conventional medium were replaced with the same volume of restricted medium at the same cell concentration, and after 24 hours of culture, the exosomes were extracted.

[0064] 2. Extraction of exosomes (2 groups)

[0065] The exosomes in the cell culture were extracted using the exosomes extraction kit. The main steps include: the cell culture is centrifuged at 2000g for 30 minutes to remove the cells / cell debris. Take the supernatant, filter with a 1μm filter membrane, and take the filtrate. The f...

Embodiment 3

[0076] Example 3. Research on the mechanism of osteocalcin (OCN) regulating endothelial progenitor cell function through exosomes

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