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Preparation and application of cardiac cell exosome

A technology of heart cells and exosomes, applied in the field of biomedicine

Pending Publication Date: 2020-10-30
李刚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports at home and abroad about the local use of exosomes from human c-Kit+ heart cells to promote angiogenesis and repair myocardium. The preparation method of the present invention is simple and stable, which brings opportunities for the treatment of repairing damaged myocardium

Method used

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  • Preparation and application of cardiac cell exosome
  • Preparation and application of cardiac cell exosome
  • Preparation and application of cardiac cell exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Overall process of the present invention

[0034] Such as figure 1 Shown, be the detailed flow process of the present invention:

[0035] 1: Cardiac tissue processing: collect the myocardial tissue that will be discarded after the operation, cut it into tissue fragments with a size of 1mm3, put it into calcium-free cardioplegia, and then use the heart cell digestion and separation liquid to separate all heart cells in the heart tissue. Use a microporous filter to filter;

[0036] 2: Obtain a large number of heart cells: use a special medium for heart cells to expand in large quantities;

[0037] 3: Sorting c-Kit+ cells: Human cardiac c-Kit+ cells were screened by immunomagnetic bead sorting method, and all cells separated and amplified were labeled with CD117 (c-kit) antibody, and then labeled with a secondary antibody labeled with magnetic beads Cells are adsorbed by a magnet, and other cells are washed to obtain magnetically labeled heart CD117 (c-kit) positive cel...

Embodiment 2

[0042] Isolation of human cardiac c-kit+ cells

[0043] The use of heart tissue was approved by the Ethics Committee of the Cardiovascular Hospital Affiliated to Xiamen University. The source of the heart tissue was a child with congenital heart disease hospitalized in the Department of Cardiac Surgery of the Cardiovascular Hospital Affiliated to Xiamen University. There was no microbial infection, and the collection age was 0-5 years old. Right atrial appendage tissue surgically removed from children with chronic heart disease was placed in peroxygen-precooled calcium-free cardioplegia in time; the heart tissue was transferred to a small metal beaker with surgical forceps, and filtered and sterilized with calcium-free cardioplegia. The heart tissue is washed repeatedly with the beating fluid until the fat and blood clots attached to the tissue are removed. Transfer the rinsed heart tissue to a new small metal beaker, and then use ophthalmic scissors to cut the heart tissue in...

Embodiment 3

[0045] Identification of human cardiac c-kit+ cells.

[0046] Such as image 3 Above, c-Kit+ cardiac cells were detected and identified by immunofluorescence, showing that all isolated cells positively expressed c-Kit (CD117) protein red; image 3 Next, for the surface antigen expression of the cells detected by flow cytometry, it can be seen that the expression of CD117 (c-kit) exceeds 99%, thus indicating that the purity of the isolated and screened c-Kit+ heart cells exceeds 99%.

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Abstract

The invention discloses preparation and application of a cardiac cell exosome, and aims to provide an exosome for treating cardiovascular and limb ischemic diseases. The exosome has the advantages ofsimple preparation method, high efficiency and low toxicity, and can effectively prevent and treat myocardial infarction related diseases and limb ischemic diseases. The invention particularly relatesto a method for purifying c-Kit + cells from human heart tissue, a culture method and an exosome separation method, and the exosome separation method overcomes the defects of a traditional method andis high in yield, short in period and good in effect. Besides, no effective medicine for effectively treating heart failure, myocardial hypertrophy and ventricular wall tumor caused by myocardial infarction exists clinically. The exosome can promote angiogenesis of human umbilical vein endothelial cells in vitro, repair of limb ischemic tissues and blood vessels can be effectively improved through in-vivo local injection, the exosome can be applied to treatment of heart failure, myocardial hypertrophy, ventricular wall tumors, limb ischemia and the like caused by myocardial infarction, and the exosome is clear in pharmacological action, small in dosage and free of toxic and side effects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the preparation and application of cardiac cell exosomes. Background technique [0002] The cardiac cells of the present invention are cardiac c-Kit+ cells, which are a small group of immature and differentiated cardiac cells in cardiac tissue, which can be differentiated into precursor cells. The cardiac c-Kit+ cells have the functions of promoting cardiomyocyte proliferation and inhibiting apoptosis. It can increase myocardial blood flow, reduce inflammation, reduce scar area after myocardial infarction, prevent myocardial injury after ischemia / reperfusion, and improve heart function. Cardiac progenitor cells can be classified according to different characteristics and surface markers of cardiac cells: c-Kit+, side population stem cells, Sca-1+, myosphere cell clusters and myosphere-derived cell clusters, etc. Among them, c-Kit+ cardiac cells are the earliest d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61K35/34A61P9/10A61P35/00
CPCA61K35/34A61P9/10A61P35/00C12N5/0657C12N2500/32C12N2500/44C12N2500/84C12N2501/11C12N2501/115C12N2509/00C12N2509/10
Inventor 李刚
Owner 李刚
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