Method for inducing and acclimating epidermal stem cells into nerve cells

A technology of epidermal stem cells and nerve cells, applied in the field of directional induction and domestication of epidermal stem cells into nerve cells, which can solve the problems of limited application range of epidermal stem cells

Inactive Publication Date: 2010-06-30
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The scope of application of epidermal stem cells is also limited by the influence of unipotent cells

Method used

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  • Method for inducing and acclimating epidermal stem cells into nerve cells
  • Method for inducing and acclimating epidermal stem cells into nerve cells
  • Method for inducing and acclimating epidermal stem cells into nerve cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of Epidermal Stem Cells Before Induction and Domestication

[0036] 1. Preparation of epidermal stem cells:

[0037] Direct source and original source: With the consent of the patient, it was collected from healthy circumcision patients in the Surgical Outpatient Department of 301 Hospital.

[0038] (1) Take fresh adult foreskin (in vitro time is about 1-4 hours, stored at 4°C);

[0039] (2) soak the foreskin in dilute iodophor solution (containing iodophor 20%) for 1 min;

[0040] (3) Rinse with PBS / normal saline for 3 times to remove blood clots (to prevent affecting the digestion of enzymes);

[0041] (4) Trim the foreskin, remove the fat tissue, rinse with PBS / saline several times, and finally cut into 1cm 2 left and right organizational blocks;

[0042] (5) Place the tissue block in a 6cm dish containing neutral protease (3mg / ml; D-hanks as solvent) solution and treat it at 4°C for 14-16h. If the treatment is not enough, it can also be place...

Embodiment 2

[0118] Example 2 Directed induction and domestication of epidermal stem cells into nerve cells

[0119] 1. Culture medium composition

[0120] (1) The components and dosage of nerve induction medium are:

[0121] DMEM / F12 (Invitrogen, cat.no.11330-32); 2% FBS (Gibco, cat.no.10437010) 45ng / ml b-FGF (Invitrogen, cat.no.13256-029); 20ng / ml EGF ( invitrogen, cat.no.); 0.5mM retinoic acid (RA) (sigmaR2625); 0.5mM valproic acid (Sigma, cat.no.T8552); 2000U / ml leukemia inhibitory factor (Chemicon, cat.lif1010); 100mM sodium pyruvate ( sigma, cat.no.P2256); 0.5mM β-mercaptoethanol (sigma cat.no.m7522);

[0122] (2) The two components and dosage of nerve induction medium are:

[0123] DMEM / F12 (Invitrogen, cat.no.11330-32) 2% FBS (Gibco, cat.no.10437010); 20ng / ml b-FGF (Invitrogen, cat.no.13256-029); invitrogen, cat. no.); 0.5 uM RA (sigma R2625); 1×B27 (invitrogen Cat. No. 17504); 100 ug / ml butylated hydroxyanisole (sigma, cat. no. B1253).

[0124] (3) The composition and dosage ...

Embodiment 3

[0129] Identification and result analysis of the target cell of embodiment 3

[0130] 1. Immunofluorescence identification of induced differentiation results

[0131] 1. Identification method: In the experimental verification of induction of nerve cells, the identification method of the nerve cell marker (a-SMA) detected by immunofluorescence is as follows:

[0132] (1) Cells three weeks after induction and epidermal stem cells were taken as controls, and cells were washed twice with PBS.

[0133] (2) Add 1ml of 4°C pre-cooled Tribule Lysis Solution to each six-well plate, and see that the cells are all lysed, about 5min

[0134] (3) Use a nuclease-free gun tip to suck the cell lysate into a 1.5ml centrifuge tube

[0135] (4) Add 200ul chloroform, shake vigorously up and down four to five times to make it fully mixed

[0136] (5) 12000rpm, 5min 4°C. The liquid is divided into three layers, and the upper transparent liquid layer is sucked into a new 1.5ml centrifuge tube

...

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Abstract

The invention discloses a method for inducing and acclimating epidermal stem cells into nerve cells, comprising the following steps of: mixing an eplife culture medium and an induction culture medium according to different proportions, and ensuring that the epidermal stem cells have enough time fit for the continuous reduction of the eplife culture medium and the gradual increase of the induction culture medium, which is beneficial to reducing the differentiation to the epidermis and increasing the transformational probability to targeted cells. The method is simple and very effective to directively induce and acclimate the epidermal stem cells into the nerve cells, overcomes the prejudice from technical personnel in the filed, subverts a judgment that the epidermal stem cells are unipotent stem cells for the first time, and proves that the epidermal stem cells have the multi-directional differentiation potential.

Description

technical field [0001] The invention relates to a method for inducing cells, in particular to a method for directional induction and domestication of epidermal stem cells into nerve cells. Background technique [0002] Humans have been studying epidermal stem cells for decades, but due to their difficulty in inducing differentiation, they have always been considered as unipotent stem cells, that is, they can only differentiate into related components of the epidermis. Regenerative medicine is hot in the field of medicine, and the research on stem cells has occupied an important position in the field of regenerative medicine. Stem cells, due to their multi-differentiation potential, can differentiate into various tissues and organs under specific conditions have attracted the attention of scientists. . Embryonic stem cells are totipotent stem cells that can differentiate into all tissues and organs except the placenta. However, because research on embryonic stem cells invol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08
Inventor 付小兵韩为东陈美霞
Owner GENERAL HOSPITAL OF PLA
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