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Intermittent hypoxia treated stem cell derived exosome and application in cardiac muscle tissue

An exosome and stem cell technology, applied in animal cells, tissue culture, vertebrate cells, etc., can solve problems such as major biological safety hazards, affecting the function of exosomes, and adverse clinical transformation, and achieve significant cell and tissue repair. , good clinical translation potential, high cardioprotective effect

Active Publication Date: 2019-12-17
SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some results have been achieved, some other obstacles have been set up for the clinical application of exosomes: on the one hand, the biological effects of exosomes are the "combined force" produced by their contents, and the single process Expressing or knocking out a single molecule may not change the biological function of exosomes to a large extent; Infection or electroporation to introduce nucleic acid for exosome modification will affect the survival status of mesenchymal stem cells, which may affect the function of exosome synthesis
Third, from the perspective of clinical transformation, biological modification methods such as virus infection currently have great biological safety hazards, and the cost is too high, which is not conducive to clinical transformation

Method used

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  • Intermittent hypoxia treated stem cell derived exosome and application in cardiac muscle tissue
  • Intermittent hypoxia treated stem cell derived exosome and application in cardiac muscle tissue
  • Intermittent hypoxia treated stem cell derived exosome and application in cardiac muscle tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Isolation and preparation of adipose-derived mesenchymal stem cells, intermittent hypoxia treatment and identification

[0061] Take SD rats aged 4-6 weeks; kill them (killed by anesthesia) without shearing; soak in 75% alcohol for 10 minutes, not too long, so as not to affect cell activity.

[0062] Configure digestion solution (DMEM-F12 complete medium containing double antibody and 10% FBS, collagenase): add collagenase (tested, collagenase I, II and IV types can be used in this step) with 0.075% to 0.1% The ratio is added to the F-12 complete medium. Generally, the number of rats in one experiment needs 5ml of digestive solution, and so on. After mixing, filter with a 0.22um filter, and then use it for later use.

[0063]Take the rat out of the alcohol solution in the fume hood, put it in a prone position in a sterile petri dish, cut the back skin along the coronal axis of the back from 1em above the tail of the rat, and cut it to the level of the chest ca...

Embodiment 2

[0069] Example 2 Extraction and identification of exosomes from adipose-derived mesenchymal stem cells

[0070] Extraction of exosomes: Extract the cell supernatant, transfer the supernatant to a new tube and centrifuge (200g, 20 minutes, 4°C); carefully transfer the supernatant to a new tube and centrifuge (10,000g , 30 min, 4°C) to remove larger vesicles; samples at this stage can be filtered through a 0.22 μm syringe filter (Millipore) and centrifuged at 110,000 g (Sorvall WXULTRA SERIES, rotor F65L) for 2 hours at 4°C with cold After resuspension in PBS, ultracentrifuge again (110,000g, 1 hour, 4°C), dry exosomes carefully and resuspend in cold PBS, exosomes should be used immediately or refrigerated at -80°C.

[0071] Electron microscope identification of exosomes: Mix the exosome solution and 4% paraformaldehyde at a ratio of 1:1 (total volume 10-20 μl is sufficient), drop on a clean plastic film to form droplets, and then place the front surface of the electron microsco...

Embodiment 3I

[0078] Embodiment 3 INTEXO and EXO in vitro cardiomyocyte protective effect research

[0079] The experiment was divided into four groups, namely: normal cell group (NC group), hypoxia / reoxygenation group (IR), hypoxia / reoxygenation + exosomes group (IR+EXO) and hypoxia / reoxygenation + intermittent Hypoxic preconditioning of ADMSC-derived exosomes (IR+INTEXO)

[0080] Changes in the expression of pyroptosis and apoptosis pathway-related proteins: add 30 μg of exosomes secreted by ADMSCs and ADMSCs pretreated by the present invention into the corresponding culture medium of rat cardiomyocytes that have been subjected to hypoxia treatment in a 6-well plate After incubation for 12 hours, the cells were collected; the preparation of protein lysate: take a clean centrifuge tube, add 10ml of RIPA strong lysate, and then add 1 tablet of protease inhibitor and 1 tablet of phosphatase inhibitor to it, fully After dissolving and mixing, divide into several EP tubes and store in -20°C r...

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Abstract

The invention relates to an exosome derived from a mesenchymal stem cell. The exosome is prepared by intermittent hypoxic stimulation of the mesenchymal stem cell. The exosome prepared from the invention has stronger tissue and organ ischemia-reperfusion injury protective effect, and the biological effect of unit exosomes is improved, and treatment of diseases such as ischemic heart disease is more facilitated.

Description

technical field [0001] The present invention relates to an induced extracellular membrane vesicle, in particular to an induced exosome, which has a specific composition and content and is suitable for tissue repair, such as myocardial tissue. Background technique [0002] As an important mediator of paracrine, exosomes have attracted more and more attention due to their special biological functions. Exosomes are membrane vesicles secreted by living cells. They have the functions of transporting proteins and nucleic acids, specifically targeting recipient cells, exchanging proteins and lipids or triggering downstream signaling events, and participating in intercellular communication. Pay attention to. Because exosomes can exist in the extracellular environment for a long time without being degraded or diluted, and can be transported to distant target cells through interstitial fluid or blood, and exosomes are almost non-immunogenic, and no rejection will occur. Therefore, e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P9/10
CPCA61K35/28A61P9/10C12N5/0662C12N5/0667C12N2500/02
Inventor 王长谦毛承誉
Owner SHANGHAI NINTH PEOPLES HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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