Human umbilical cordmesenchymal stem cell-derived exosome and application thereof

A technology of mesenchymal stem cells and membranous vesicles, applied in drug research and development, can solve the problems of low survival rate of MSC transplantation, limitations, hidden dangers of tumorigenic clinical application, etc., and achieve the effect of simple preparation technical scheme, convenient storage, and easy control of use time

Active Publication Date: 2010-11-24
江苏澳洋生物医药研究院有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] hucMSC transplantation for the treatment of liver, kidney and skin damage has attracted more and more attention. However, due to the low survival rate of MSC transplantation and the potential risk of tumorigenesis in long-term clinical application, its clinical application is limited. Therefore, it is necessary to find an operation Simple, safe and effective MSC-based cell-free therapy is very important

Method used

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  • Human umbilical cordmesenchymal stem cell-derived exosome and application thereof
  • Human umbilical cordmesenchymal stem cell-derived exosome and application thereof
  • Human umbilical cordmesenchymal stem cell-derived exosome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of membranous vesicles derived from umbilical cord mesenchymal stem cells (hucMSC-exosome)

[0027] 1. Isolation and culture of human umbilical cord mesenchymal stem cells

[0028] Human umbilical cord MSCs were cultured and expanded in vitro according to the method previously established by our research group for isolation, culture and identification of human umbilical cord MSCs (Chun Qiao, et al. Cell Biology International, 2008, 32: 8-15). Specifically: take the fresh umbilical cord of a sterile newborn, wash it repeatedly with phosphate buffer saline (PBS), and cut it into tissue pieces with a diameter of about 1-2 mm; L-DMEM nutrient solution containing 10% fetal bovine serum, 5% CO , Culture in saturated humidity at 37°C; remove non-adherent cells, and digest 0.25% trypsin after 80% confluence of adherent cells for subculture.

[0029] 2. Preparation of Human Umbilical Cord Mesenchymal Stem Cell Conditioned Medium (MSC-CM)

[0030] Select ...

Embodiment 2

[0034] Example 2: Observation of curative effect of hucMSC-exosome on mouse 20% deep second-degree SD rat scald model

[0035] (1) Experimental animals were divided into groups and 20% deep second-degree scald model to prepare clean-grade SD rats, weighing about 120-130g, with a total of 6 rats, half male and half male, divided into control group, umbilical cord mesenchymal stem cell treatment group, and umbilical cord mesenchymal stem cell treatment group. Mesenchymal stem cell exosome treatment group, 2 rats in each group, one male and one male. The anesthetized rat was placed on the operating table, and the 20% body surface area of ​​the rat was calculated according to the Rubner formula. Cut off the back hair, remove the hair with sodium thiosulfide, put it in 82°C water, take it out quickly after 8 seconds, dry it with gauze (to avoid further aggravating the wound damage), add 6ml of normal saline, and wrap it with sterile gauze .

[0036] (2) Treatment method 3 hours a...

Embodiment 3

[0038] Example 3: Repairing effect of hucMSC-exosome on rat renal tubular epithelial cells (NRK) hypoxia model

[0039] Establishment of NRK cell hypoxia model: NRK cells were anoxic for 4 hours under the condition of 5% CO2, 2% O2, 37°C and saturated humidity, and after adding exosome for 48 hours, various indicators were detected.

[0040] Dosage:

[0041] Low dose group (0.5mg / 5×10 5 cells)

[0042] Middle dose group (1mg / 5×10 5 cells)

[0043] High dose group (2mg / 5×10 5 cells)

[0044] Using flow cytometry, TUNEL, and immunohistochemistry to evaluate the repair effect of exosome secreted by hucMSCs on NRK cell hypoxia model in vitro. Quantitative PCR, Western blot and other techniques were used to preliminarily explore the main mechanism of damage repair. After hypoxia / reoxygenation or cisplatin treatment in NRK cells, the nuclei shrunk, TUNEL was deeply stained, and the cell shape was large and irregular. After adding exosome, the cell morphology was better, and ...

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Abstract

The invention relates to extraction of an active ingredient exosome from a human umbilical cordmesenchymal stem cell and application thereof in preparation of medicaments for treating liver and kidney injury and skin diseases. The human umbilical cordmesenchymal stem cell-derived exosome is prepared by the following steps of: (1) culturing a human umbilical cordmesenchymal stem cell; (2) preparing a cordmesenchymal stem cell conditioned medium; and (3) separating and purifying the exosome. The experiments for infusing the human umbilical cordmesenchymal stem cell exosome serving as a medicament into a mice acute liver injury model, a rat kidney injury model and a rat skin scald model show that the medicament can improve the liver function, the kidney function and the skin condition, promote the pathological recovery of the liver, the kidney and the skin, reduce the death rate, fulfill the aims of blocking and reversing the development of the liver and kidney injury and open up a new approach for clinical treating of the liver and kidney injury.

Description

technical field [0001] The invention belongs to the fields of drug research and development, cell biology and molecular biology. It involves the extraction of the active ingredient membranous vesicle (exosome) in umbilical cord mesenchymal stem cells (human umbilical cordmesenchymal stem cell, hucMSC) and its application in the preparation of drugs for the treatment of liver and kidney damage and skin diseases. Background technique [0002] hucMSC transplantation for the treatment of liver, kidney and skin damage has attracted more and more attention. However, due to the low survival rate of MSC transplantation and the potential risk of tumorigenesis in long-term clinical application, its clinical application is limited. Therefore, it is necessary to find an operation Simple, safe and effective MSC-based non-cell therapy is very important. Studies have shown that MSCs mainly improve myocardial function through paracrine effects, and the membranous vesicles secreted by MSCs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/44C12N5/0775A61P1/16A61P13/12A61P17/00A61K35/28
Inventor 许文荣钱晖朱伟严永敏毛飞黄玲沈俐
Owner 江苏澳洋生物医药研究院有限责任公司
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