MiRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome

A technology of mesenchymal stem cells and exosomes, which is applied in the application field of antagonizing scar treatment in joints, to achieve the effect of promoting wound healing and preventing excessive hyperplasia

Active Publication Date: 2016-04-13
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no literature report on miR-145-5p modified exosomes; there is no related report on miR-145-5p modified exosomes secreted by umbilical cord mesenchymal stem cells for wound treatment

Method used

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  • MiRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome
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  • MiRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Acquisition of umbilical cord mesenchymal stem cells stably and highly expressing MicroRNA145-5p

[0050] 1. Obtaining umbilical cord mesenchymal stem cells

[0051] The umbilical cord came from the Obstetrics and Gynecology Department of Changhai Hospital. Primary cultured mesenchymal stem cells. After harvesting the umbilical cord, put it in a sterile Petri dish or bottle containing sterile PBS or DMEM medium, and operate as soon as possible. After repeated washing with phosphate buffer, soak in L-DMEM containing penicillin and streptomycin (concentration of penicillin and streptomycin is 100IU / ml) for about 5 minutes, and cut into tissue pieces with a diameter of about 1.5mm; containing 10% Fetal bovine serum L-DMEM nutrient solution, 5% CO 2 , Culture in saturated humidity at 37°C; after 7-10 days, the mesenchymal stem cells can be seen crawling out of the tissue, and after removing the tissue block, digest with 0.25% trypsin for subculture. Subsequen...

Embodiment 2

[0070] Example 2: Extraction and identification of exosomes of microRNA145-5p-UMSC

[0071] 1. Preparation of exosome-free medium

[0072] Fetal bovine serum (life) was put into an ultracentrifuge (Beckman Company) in batches for centrifugation at 150,000 g at 4°C for 12 hours to remove exosomes in fetal bovine serum. Serum without exosomes was formulated into stem cell medium in proportion. This purpose is to obtain more pure stem cell exosomes later.

[0073] 2. Extraction of microRNA145-5p-UMSC exosomes

[0074] Replace the previously prepared exosome-free FBS medium in the mesenchymal stem cell culture system with high expression of microRNA145-5p, collect the culture supernatant after 48h-72h, and temporarily freeze it in - Store at 80°C. After a certain amount (about >150ml) of the conditioned medium is collected, centrifuge at 300×g at 4°C for 10 minutes to remove cell debris; centrifuge at 2000×g at 4°C for 10 minutes to remove impurities such as dead cells; filter...

Embodiment 3

[0083] Example 3: Cytological study on the ability of exosomes secreted by umbilical cord mesenchymal stem cells modified with miRNA145-5p to regulate the proliferation and differentiation of fibroblasts

[0084] 1. The effect on the cell proliferation ability of fibroblasts was detected by flow cytometry

[0085] 1) Spread fibroblasts in a six-well plate, and when the cell density is 70%, add TGFβ cytokines to one well and add exosomes (100ug / ml) from umbilical cord mesenchymal stem cells overexpressing Mir-145-5p ( Experimental group), one well was added with TGFβ cytokine and overexpressed NC exosomes at the same time, one well was only added with TGFβ cytokine (positive control group), and one well was without any intervention.

[0086] 2) After 48 hours, cells were digested with 0.25% trypsin, and flow cytometry experiments were performed. The results can be seen: the proportion of fibroblasts added with overexpressed Mir-145-5p umbilical cord mesenchymal stem cell exoso...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of the miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome. The invention provides a preparation method of the highly-miRNA145-5p-expressed umsc-exosome (umbilical cord mesenchymal stem cell exosome) and application of various biological preparations for accelerating full-thickness skin defect healing and antagonizing scar contracture. The preparation method of the exosome includes (1), adopting fresh umbilical cords to culture human umbilical cord mesenchymal stem cells; (2) preparing mesenchymal stem cells highly expressed in miRNA145-5p; (3), storing conditional media; (4) performing exosome extraction and purification. The highly-miRNA145-5p-expressed umbilical cord mesenchymal stem cell exosome is transferred into a full-thickness wound model of rat backs by serving as a biological preparation, so that skin granulation tissue proliferation can be promoted effectively, wound healing is accelerated and scar contracture can be antagonized; novel approaches and novel methods are provided for wound treatment.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for extracting and preparing microRNA145-5p and modified umbilical cord mesenchymal stem cells (humanumbilicalcordmesenchymalstemcell, hucMSC) exosomes (exosome) and its role in promoting wound healing and inhibiting scar hyperplasia, especially Application in the treatment of joint site antagonistic scars. Background technique [0002] Myofibroblasts (myofibroblasts) are highly differentiated cells from fibroblasts expressing smooth muscle actin (α-SMA), which participate in both fiber formation and extracellular matrix remodeling in the fibrosis cascade. stages (VanDeWaterL, VarneyS, TomasekJJ, Mechanoregulation of the MyofibroblastinWoundContraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention. 2013May;2(4):122-141). Its main function is to generate force and change tissue tension, and it is the main cell in wound granulation contractio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N15/86A61K35/28A61P17/02
Inventor 方硕刘厚奇王越邢新薛春雨杨超章云童毕宏达戴海英
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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