CN-121975726-A - Method for separating myocardial cell-derived small extracellular vesicles from plasma and application thereof

Abstract

The invention relates to a method for separating myocardial cell-derived small extracellular vesicles from in vitro plasma and application thereof. The method comprises the steps of ⑴ providing a ligand capable of specifically binding to RYR2 of a Raney receptor 2 protein, ⑵ separating total small extracellular vesicles from the isolated plasma sample, ⑶ incubating the ligand with the total small extracellular vesicles to enable the ligand to specifically bind to the small extracellular vesicles of myocardial cell origin with RYR2 expressed on the surface to form a ligand-vesicle complex, ⑷ binding the ligand-vesicle complex to a solid support, thereby separating the small extracellular vesicles of myocardial cell origin with RYR2 expressed on the surface from the mixture, and ⑸ washing and/or eluting the ligand-vesicle complex bound on the solid support to obtain enriched small extracellular vesicles of myocardial cell origin.

Inventors

• QIN GANGJIAN
• HUANG JIANRONG

Assignees

• 南方科技大学

Dates

Publication Date

20260505

Application Date

20260326

Claims (10)

1. 1. A method for isolating cardiomyocyte-derived small extracellular vesicles from an ex vivo plasma sample, the method being an in vitro procedure for non-diagnostic purposes, comprising the steps of: ⑴ Providing a ligand capable of specifically binding to RYR2 of the ranitidine receptor 2 protein; ⑵ Isolating total small extracellular vesicles from the ex vivo plasma sample; ⑶ Incubating the ligand with total small extracellular vesicles such that the ligand specifically binds to cardiomyocyte-derived small extracellular vesicles with surface expressed RYR2 to form ligand-vesicle complexes; ⑷ Binding the ligand-vesicle complex to a solid support, thereby separating small extracellular vesicles from the mixture that are derived from cardiomyocytes that express RYR2 on their surface; ⑸ Washing and/or eluting the ligand-vesicle complex bound on the solid support to obtain enriched cardiomyocyte-derived small extracellular vesicles.
2. 2. The method of isolating cardiomyocyte-derived small extracellular vesicles from an isolated plasma sample according to claim 1 wherein the ligand is a biotin-labeled RYR2 antibody; the solid phase carrier is a microsphere coated with streptavidin or neutravidin.
3. 3. The method of claim 1, wherein the RYR2 antigen target is obtained by screening for small extracellular vesicles derived from cardiomyocytes from an isolated plasma sample by: (1.1) constructing a transgenic animal expressing a mutant of methionyl-tRNA synthetase L274G specifically in cardiomyocytes; (1.2) administering L-azido norleucine to the transgenic animal, labeling cardiomyocyte neogenesis proteins; (1.3) isolating heart tissue and heart-derived small extracellular vesicles from said transgenic animal, identifying proteins in which ANL is labeled by mass spectrometry; (1.4) Cross-analysis of ANL-labeled proteins in heart tissue and heart-derived small extracellular vesicles, screening for overlapping and highly expressed membrane protein RYR2.
4. 4. The method of claim 1, wherein the separation of the total small extracellular vesicles from the isolated plasma sample is by ultracentrifugation combined with density gradient centrifugation.
5. 5. A cardiomyocyte-derived small extracellular vesicle obtained by the method of any one of claims 1-4.
6. 6. Use of a ligand capable of specifically binding to RYR2, a lanine receptor 2 protein, for the preparation of a product for the in vitro diagnosis of cardiovascular diseases.
7. 7. The use according to claim 6, wherein the product is an in vitro test kit, the biological sample tested by the kit is an ex vivo biological sample comprising total small extracellular vesicles, the ex vivo biological sample is an ex vivo plasma sample, and the cardiovascular disease is acute myocardial infarction or heart failure.
8. 8. An in vitro diagnostic kit for cardiovascular diseases, characterized in that it comprises a ligand capable of specifically binding to the RYR2 protein of the ranitidine receptor 2.
9. 9. The in vitro diagnostic kit according to claim 8, wherein the ligand is a biotin-labeled anti-RYR 2 antibody, a streptavidin or neutravidin coated solid support, a reagent for isolating total small extracellular vesicles from an ex vivo plasma sample, a detection reagent for in vitro assisted detection of the expression level of RYR2 protein, and a wash buffer.
10. 10. An application of RYR2, a lanine receptor 2 protein, in the preparation of products for in vitro diagnosis of cardiovascular diseases.

Description

Method for separating myocardial cell-derived small extracellular vesicles from plasma and application thereof Technical Field The invention belongs to the technical field of biotechnology and medical diagnosis, and particularly relates to a method for specifically separating small extracellular vesicles from blood plasma, the small extracellular vesicles from myocardial cells, and a method and a kit for diagnosing heart diseases based on a vesicle surface marker RYR 2. Background Heart disease, particularly acute myocardial infarction and heart failure, is a leading cause of death worldwide. Its early diagnosis and accurate disease assessment are critical to improving patient prognosis. The development of novel biomarkers capable of reflecting myocardial cell injury and stress state dynamically and sensitively in real time has important clinical significance. Small extracellular vesicles (sEV) serve as important media for intercellular communication, carry molecular fingerprints of their parent cells, and are considered to be extremely potential liquid biopsy targets. Cardiomyocytes secrete sEV into the blood circulation under normal physiological or pathological stress conditions. Theoretically, by analyzing sEV derived from these cardiomyocytes, real-time molecular information of the cardiomyocytes can be obtained noninvasively. However, the source of sEV in plasma is extremely complex, intermixing vesicles from blood cells, endothelial cells and other tissue cells, making direct analysis of total sEV impossible to obtain specific information for cardiomyocytes. Therefore, realizing the specific separation of myocardial cell source sEV is a precondition for developing a detection kit for in vitro diagnosis of heart diseases. The reference 1("Circulating small extracellular vesicles as blood-based biomarkers of muscle health in aging nonhuman primates", Mishra S et al, Geroscience. 47(3):3709-3723,2025 Jun) discloses a method for separating skeletal muscle derived small extracellular vesicles from serum comprising incubating serum sEV with biotin-labeled SGCA (alpha-actin) or MuSK antibodies, followed by further incubation with streptavidin-conjugated magnetic beads, and preparing sEVSKM by elution and magnetic separation (see methods section "sEV isolation"). The method uses alpha-actin SGCA as a skeletal muscle specific sEV surface marker, incubating serum total sEV with biotin-labeled anti-SGCA antibodies, followed by binding with streptavidin-conjugated magnetic beads, and preparing skeletal muscle source sEV by elution and magnetic separation. Comparative document 1 verifies the purity and specificity of the isolated sEV and uses it for biomarker studies of muscle aging. Distinguishing technical characteristics Comparing the technical scheme of the invention right 1 with the technical scheme of the comparison document 1, the two are different in that: ⑴ The target protein is completely different, namely the invention uses the Raney receptor 2 protein RYR2 as a specific marker, and the comparison document 1 uses alpha-muscin SGCA as a marker. RYR2 is quite different from SGCA in molecular structure, biological function and cellular localization. RYR2 is a calcium release channel receptor positioned in sarcoplasmic reticulum membrane and participates in myocardial cell excitation-contraction coupling, SGCA is a component of a myoglycan complex, is positioned in sarcolemma and participates in maintaining the stability of the sarcolemma. ⑵ The tissue of origin of the target vesicles was different, the invention was aimed at isolating sEV of cardiomyocyte origin and comparative document 1 was aimed at isolating sEV of skeletal muscle origin. ⑶ The screening method of the markers is different, the RYR2 is obtained through the screening of an innovative in vivo marking method, the method comprises the steps of constructing a transgenic mouse of which the myocardial cells specifically express MetRS x L274G mutant, giving ANL marked myocardial cell neogenesis protein, carrying out mass spectrometry and cross comparison on the ANL marked protein in heart tissue and heart sEV, and finally screening out the RYR2 as the specific marker of myocardial cell source sEV. The comparison document 1 directly selects the known muscle marker SGCA, and does not disclose any systematic screening method. Based on the distinguishing characteristics, the invention aims to provide a novel separation method of myocardial cell sources sEV and establish a quantitative diagnosis technology of heart diseases based on the separation method. Non-obvious analysis of the present invention relative to comparative document 1: The technical problems are provided with the following creativity: There is a long felt need in the art to isolate tissue-specific sEV from plasma, but isolation of cardiomyocyte source sEV has not been achieved. Although the separation of skeletal muscle sEV is achieved in comparative document 1, there are substantia