A kind of extracellular vesicle derived from human amniotic mesenchymal stem cells and its application

A technology of stem cells and human amniotic membrane, applied in the field of biomedicine to achieve the effect of inhibiting the hypertrophy of cardiomyocytes

Active Publication Date: 2022-03-11
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The relevant prior art is as follows: the Chinese invention patent with the publication number CN106635976A discloses a method and a kit for obtaining amniotic mesenchymal stem cells; the Chinese invention patent with the publication number CN104488850A discloses a method for preparing human amniotic mesenchymal stem cells However, there is no report on extracellular vesicles derived from hAMSCs and its application in the preparation of drugs for the prevention and treatment of pathological cardiomyocyte hypertrophy.

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  • A kind of extracellular vesicle derived from human amniotic mesenchymal stem cells and its application
  • A kind of extracellular vesicle derived from human amniotic mesenchymal stem cells and its application
  • A kind of extracellular vesicle derived from human amniotic mesenchymal stem cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of Hamscs-EVS of Human Ferrous Metachate Stem Cell Source

[0046] Separation of Hamscs:

[0047] (1) Rinse the amniotic tissue repeatedly, then cut the amniotic tissue, then cut the amniotic membrane, according to the plasma of the amniotic membrane: (1-2), the volume ratio of the digestion, 37 ° C Incubate 7-10 min, obtained after digestion; the digestion is a PBS containing 2.4 U / mL protease DISPase;

[0048] (2) Add a digestion of amniotic tissue to α-MEM complete medium containing 10% fetal bovine serum, after standing for 5-10 min, according to amniotic tissue: Digestion = 1: (2-3) volume Digestion was digested at 37 ° C to give 0.75 mg / mL collagenase COLLAGEN D and 5% fetal bovine serum α-MEM complete medium;

[0049] (3) Cut the tissue liquid after digestion (1500 rpm is centrifuged for 10min), go to the supernatant, leave the precipitate, and repeatedly rinsed, centrifuge, precipitate until it is clear, abandon the supernatant, with 15% fe...

Embodiment 2

[0063] Example 2Hamscs-EVS is transmitted into a hypertrophy of intraocular intraocular cells via cyclone

[0064] Hamscs-EVS, DII, using fluorescently active dyes DII (1'-Dioctadecyl-3, 3, 3 ', 3'-Tetramethylindocarbocyanine Perchlorate), DII is one of the most commonly used cell membrane fluorescent probes, presenting orange-red fluorescent, entering cell membrane After late diffusion, the cell membrane of the entire cell can be gradually dyed, and the extracted Hamscs-EVS extracted in Example 1 was resuspended with PBS, and 1 μM DII dye was 37 ° C, including 5% CO. 2In the cell incubator, it was incubated for 5 min and then incubated into centrifuge muscle, centrifuged at 4 ° C, 100000 g from 1 h, abandoned out excess of dye, washed with PBS 3 times, and the PBS was resuspended. Dii marked Hamscs-EVS co-cultured with myocardial hypertrophy of cells, PBS was rinsed 3 times, and the cell membrane of myocardial fertilizer (WGA) was labeled with WGA staining with WGA, and laser Foc...

Embodiment 3

[0066] Example 3Hamscs-EVS prevention and pathological myocardial cell hypertrophy

[0067] Construct a cardiac hypertrophy: In order to clearly construct a highly treated concentration concentration of cardiac III, use 5 × 10 -7 M, 5 × 10 -6 M and 1 × 10 -5 M Vastrial Angiothen II (Ang II) The three gradient concentrations induce human myocardial cell AC16 hypertrophy, and the compass of myocardial fertilizer major cell model was determined by Real-Time PCR. Figure 8 After Hamscs-EVS, the cardiocyte membrane was labeled by wheat nodule (WGA), and the laser confocal microscope was observed in the cell surface area, and the results showed that 5 × 10 -6 Mang II Treats AC16 Heartocytes, with 5 × 10 -7 M and 1 × 10 -5 Compared to cell hypertrophic phenotype, the cell surface area has increased significantly ( Figure 8 . Figure 9 Cardiomyopathic peptide (ANP), brain sodium urine peptide (BNP), β-myocardin heavy chain (BNP), β-myocardium (β-MHC) and myocardium β-MHC MRNA and myocardial...

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Abstract

The invention relates to an extracellular vesicle derived from human amniotic mesenchymal stem cells. The preparation method is as follows: after obtaining human amniotic mesenchymal stem cells through separation and culture, culturing and carrying out subculture amplification, and culturing the 3-5th generation human amniotic mesenchymal stem cells. When the density reaches 70%-80%, discard the medium, wash with PBS and replace with α-MEM (no phenol red) medium containing 5% extracellular vesicle-free fetal bovine serum, and continue to culture for 48h Afterwards, the cell culture supernatant was collected, and extracellular vesicles were finally prepared by gradient and high-speed centrifugation. The extracellular vesicles can directly target the 3'-UTR region of PIK3R1 mRNA in hypertrophic cardiomyocytes through the carried miRNAs21‑5p / 128‑3p / 221‑3p, and inhibit the PI3K / Akt pathway, so they can be used for the preparation of Drugs for the prevention and treatment of pathological cardiomyocyte hypertrophy.

Description

Technical field [0001] The present invention belongs to the field of biopharmaceutical technology, and more particularly to cellular vesicles and its application thereof in a human amniotic mesenchymal stem cell (HAMSCS). Background technique [0002] Pathological cardiomytes hypertrophy, pch) is mainly caused by aging and neuronal fluid loss (such as angiotensin II elevation), is a variety of cardiovascular diseases (such as hypertension, myocardial infarction, ischemic heart The common process of sick, etc.) is considered to be an important independent risk factor that causing heart failure and death, and the prevention and treatment of PCH has important significance to treat multiple cardiovascular disease. Traditional drugs and interventions can only be partially improving clinical symptoms, explore new clinical treatment methods and strategies to inhibit and even gradually become an important direction of research in cardiovascular. [0003] Humanamniotic Mesenchymal Stem Ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/073A61K35/50A61P9/00
CPCC12N5/0668C12N5/0605A61K35/50A61P9/00C12N2509/00C12N2509/10
Inventor 靳建亮谢春凤朱剑云苗登顺王嵘于珍珍孙海建陈海云周佳雯
Owner NANJING MEDICAL UNIV
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