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Method for extracting exosome derived from human hair follicle dermal papilla cells

A technique for dermal papilla cells and an extraction method, which is applied in the field of exosome extraction derived from human hair follicle dermal papilla cells, can solve the problems that the regulation effect has not been studied, and achieves the effects of enhancing proliferation and migration and delaying degenerative period.

Pending Publication Date: 2019-06-07
ZHEJIANG UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0006] In summary, the problem with the prior art is that whether DPC-secreted exosomes have a regulatory role in HF growth and development has not been investigated

Method used

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  • Method for extracting exosome derived from human hair follicle dermal papilla cells
  • Method for extracting exosome derived from human hair follicle dermal papilla cells
  • Method for extracting exosome derived from human hair follicle dermal papilla cells

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Embodiment

[0090] 1. Materials and methods

[0091] 1.1. Isolation and culture of DPC and ORSC from healthy human scalp;

[0092] Informedly obtained healthy human scalp specimens from patients without systemic disease undergoing plastic surgery; ORSC and DPC were isolated from scalp HF by a two-step enzymatic digestion method; the scalp was cut along the dermal-epidermal junction and soaked In dispase, overnight at 4°C, pull out the HF and trypsinize for 5-10 minutes; wash the ORSC suspension with pre-cooled PBS after sifting through a filter and centrifuge at 800×g for 5 minutes, then resuspend in complete keratinocytes In cell culture medium (CKSFM); the dermis was digested with collagenase at 37°C for 3-6 hours, washed with PBS through filter sieved DPC and resuspended in DMEM / F-12 (1:1) containing 10% FBS (Hyclone, Logan, UT, USA); ORSC and DPC were transferred to a 25ml culture flask and incubated at 37°C, 5% CO 2 The study protocol was approved by the Institutional Review Board ...

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Abstract

The invention belongs to the technical field of cell secretion extraction, and discloses a method for extracting exosome derived from human hair follicle dermal papilla cells (DPC-Exos). The method isas below: identifying DPC-Exos by controlled resistance pulse sensing (TRPS) analysis, electron microscopy and Western Blot; and adding the DPC-Exos to a culture medium of human hair follicle outer root sheath cells (ORSCs) cultured in vitro. The DPC-Exos are identified to be about 105 nm in diameter and expressing tumor susceptibility gene (TSG) 101, differentiation cluster (CD) 9 and CD63; DPC-Exos promote proliferation and migration of human ORSCs, and mRNA and protein levels of beta-catenin and Sonichedgehog (Shh) in ORSCs are detected to be up-regulated after treatment; transdermal injection of DPC-Exos accelerates an HF growth phase of mice and delays the occurrence of regression. Immunohistochemical analysis shows that the expression levels of beta-catenin and Shh are up-regulatedin the hair follicles of an experimental group. DPC-Exos help regulate the growth and growth cycle of the HF and provide a new potential for the treatment of clinical hair loss.

Description

technical field [0001] The invention belongs to the technical field of cell secretion extraction, and in particular relates to a method for extracting exosomes derived from human hair follicle dermal papilla cells. Background technique [0002] Dermal papilla cells (DPCs) originate from the hair follicle (HF) mesenchyme and play a key role in HF epithelial-mesenchymal communication. DPC is considered to regulate the growth and development of HF through a paracrine mechanism, in which exosomes may play an important role. In androgenetic alopecia, the hair manifests as HF becomes thinner and softer, the hair bulb becomes smaller, and the anagen period becomes shorter, leading to hair loss. Hair loss greatly affects the external image, self-confidence and quality of life, but currently there are two main drugs approved by the FDA, finasteride for oral administration and minoxidil for external use. With the cessation of medication, the hair follicles gradually recover and conti...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/6883G01N33/68
Inventor 吕中法周丽娟王晗余丽娟吴贤杰景璟陈钰虹
Owner ZHEJIANG UNIV
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