Preparation method and application of personalized induced neural stem cell exosome

A neural stem cell and inducible technology, which is applied in the field of the preparation of individualized induced neural stem cell exosomes, can solve the problems that have not existed, and achieve the effect of promoting growth, restoring tissue integrity and high safety.

Pending Publication Date: 2020-11-10
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The microenvironmental change based on neural stem cells is a new strategy for the treatment of aging and UV damage. The application of this strategy will repair damaged skin fibroblasts and has great clinical value, but there is no relevant report

Method used

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  • Preparation method and application of personalized induced neural stem cell exosome
  • Preparation method and application of personalized induced neural stem cell exosome
  • Preparation method and application of personalized induced neural stem cell exosome

Examples

Experimental program
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Embodiment 1

[0031] This embodiment provides a preparation method and identification process of personalized induced neural stem cells, including the following steps:

[0032] I. Acquisition of Skin Fibroblasts

[0033] The acquisition of skin fibroblasts has been disclosed in the prior art [9] . Skin tissue was obtained surgically under local anesthesia. The cell culture medium includes: DMEM (Dulbecco's Modified Eagle Medium), 10% FBS, 5% non-essential amino acids, 100 U / ml penicillin, 100 g / ml streptomycin. Skin fibroblasts were replaced by passaged 2nd to 6th passages for subsequent operations.

[0034] 2. Retrovirus preparation and packaging

[0035] Human SOX2, FOXG1, and Brn2 gene fragments were cloned and transferred into pMXs retroviral vector (CellBiolabs). Virus packaging was transfected into Plat-E cells by retroviral vector plasmids (purchased from Shanghai Gemma Company and transfected according to the company's recommended transfection method). The transfected Plat-E sup...

Embodiment 2

[0041] This example provides a method for extracting and identifying exosomes from personalized induced neural stem cells. The specific steps and results are as follows:

[0042] 1. Extraction of exosomes from individualized induced neural stem cells

[0043] The individualized induced neural stem cell exosomes will be extracted using gradient ultracentrifugation: first, the supernatant of the induced neural stem cells obtained in Example 1 is centrifuged at a speed of 300g to remove free cells; then, the supernatant of the cells is removed. The supernatant will be centrifuged at 3,000g to remove cell debris; finally, the supernatant to remove free cells and debris will be centrifuged at 100,000g to collect exosomes. After centrifugation, the exosomes were washed with filtered PBS and centrifuged again at 100,000 g to obtain exosomes derived from personalized induced neural stem cells. The exosomes can be stored in a -80°C refrigerator for a long time and still maintain biolo...

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Abstract

The invention discloses a preparation method and application of a personalized induced neural stem cell exosome. The preparation method comprises the following steps: taking skin fibroblasts, carryingout subculture on the skin fibroblasts by using a liquid culture medium, and inducing the skin fibroblasts to become personalized induced neural stem cells by using a somatic cell reprogramming technology; and collecting a large number of exosomes secreted by the induced neural stem cells. The prepared personalized induced neural stem cell exosome has good skin penetrability and can directly permeate into the corium layer to improve the local regeneration microenvironment. Therefore, a skin anti-aging product prepared from the skin anti-aging composition has the effects of naturally keeping skin moist, whitening, resisting wrinkles and resisting pigmentation on normal skin, has the effects of promoting growth and recovering tissue integrity on aged and ultraviolet-damaged skin, has the effect of resisting scar formation on traumatic skin, does not irritate the skin, and is high in safety.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of personalized induced neural stem cell exosomes. Background technique [0002] Skin aging is often caused by endogenous cellular aging and exogenous factors, such as excessive sun exposure and oxidative stress [1] , its clinical manifestations include increased wrinkles, surface roughness, and loss of elasticity. Research shows that the pathological basis of skin aging is the change of subcutaneous connective tissue extracellular matrix [2～3] . Skin fibroblasts (fibroblasts) are the most common cells in connective tissue, which are differentiated from embryonic mesenchymal cells. In connective tissue, the main function of fibroblasts is to secrete a variety of proteins that make up the extracellular matrix, including matrix proteins, collagen, and fibroelastin. These proteins are critical to maintaining the structural integrity of connective...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0797C12N15/867C12N7/01A61K35/30A61P17/18
CPCC12N5/0623C12N15/86C12N7/00A61K35/30A61P17/18C12N2506/09C12N2501/602C12N2501/60C12N2501/603C12N2500/50C12N2740/10021C12N2740/10043
Inventor 郑加麟夏骁寰竺映波张汉
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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