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Micromolecule compound combination for inducing skin fibroblast to realize direct transdifferentiation into nerve cell and application

A technology of small molecule compounds and fibroblasts, applied in the field of biotechnology and neurodevelopment, can solve problems such as no clinical treatment methods, achieve significant curative effect and improve exercise ability

Active Publication Date: 2020-02-18
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite being extensively studied, there is still no effective clinical treatment for SCI

Method used

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  • Micromolecule compound combination for inducing skin fibroblast to realize direct transdifferentiation into nerve cell and application
  • Micromolecule compound combination for inducing skin fibroblast to realize direct transdifferentiation into nerve cell and application
  • Micromolecule compound combination for inducing skin fibroblast to realize direct transdifferentiation into nerve cell and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Combination screening of small molecules for efficiently inducing transdifferentiation of skin fibroblasts into neurons

[0029] 1 cell culture

[0030] 1.1 Preparation and inoculation of fibroblasts

[0031]Animals were used in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Soochow University Animal Care and Use Committee. Experiments involving human subjects were performed in accordance with the World Medical Association Code of Ethics (Declaration of Helsinki). According to literature reports, skin fibroblasts were extracted and prepared from rat, mouse or human body surface. Briefly, a piece of skin was dissected from the back of a neonatal rat or mouse, minced and digested, and cultured adherently to obtain RDFs (rat dermal fibroblasts) and MDFs (mouse dermal fibroblasts). Human adult dermal fibroblasts (HDFs) were obtained from neonatal foreskins of healthy post-circumcision individu...

Embodiment 2

[0052] Identification of chemically induced neurons (CiNs)

[0053] 2.1 Immunofluorescence staining

[0054] Cells seeded in 2D-PDL or 3D-SF were fixed in cold 4% paraformaldehyde (PFA) for 30 min, washed 3 times with PBS (10 min each), and incubated for 1.5 h at room temperature or overnight at 4 °C. anti. Diluent for primary antibody (PBS / 0.02%NaN 3 / 3% bovine serum albumin (BSA) / 0.2% Triton X-100) diluted and used at the following working concentrations: mouse monoclonal antibody (mAb) anti-tubulin β3 (Tubb3) (1:500 dilution; BioLegend), rabbit mAb anti-synapsin 1 (1:200 dilution; Cell Signaling Technologies) and rabbit mAb anti-NeuN (1:500 dilution; Cell Signaling Technologies). Thereafter, cells were washed 3 times (10 min) with PBS. Secondary antibody Alexa FluorTM 594 goat anti-mouse or rabbit IgG (H + L) (Invitrogen) in PBS / 0.02%NaN 3 1:200 dilution in 3% BSA and incubate the cells for 1 hour at room temperature in the dark. Cells were subsequently washed 3 times...

Embodiment 3

[0062] Transplantation of chemically induced neurons (CiNs) for spinal cord injury

[0063] 3.1 Construction of 3D-SF porous scaffold of silk protein micro-nanofibers

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PUM

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Abstract

The invention provides a micromolecule compound combination for inducing skin fibroblast to realize direct transdifferentiation into a nerve cell. The micromolecule compound combination comprisesCHIR99021, Forskolin, LDN193189, SB431542, SP600125, VPA and Y27632, is used for inducing the transdifferentiation of the skin fibroblast to the nerve cell, and is use for repairing long-distance peripheral nerve defects and central nervous damage. According to a transdifferentiation technique, the nerve cell can be efficiently obtained, and can be transplanted to an animal model with spinal cord injury to obtain notable treatment effects. Through improvement of athletic ability, cortex evoked potential restoration and other indexes are obtained. The invention opens up a new way for treatment of correlation nervous system diseases including spinal cord injury.

Description

technical field [0001] The invention relates to the fields of biotechnology and neurodevelopment, in particular to a small molecular compound combination for inducing direct transdifferentiation of fibroblasts to neurons and its application. Background technique [0002] CNS damage is irreversible because terminally differentiated neurons cannot regenerate. Although stem cells have the potential to differentiate into a variety of nerve cells including neurons, stem cell therapy has also been considered a promising solution for central nervous system injury repair, but studies have shown that neural stem cells transplanted into damaged tissues More readily differentiate into astrocytes or oligodendrocytes, less differentiate into functional neurons. Furthermore, the application of stem cell transplantation still faces several major obstacles, including tumor formation, graft rejection, and ethical issues. Therefore, finding more appropriate treatments for nerve injuries and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793A61K31/506A61K31/352A61K31/519A61K31/4439A61K31/416A61K31/19A61K31/4409A61P43/00
CPCA61K31/19A61K31/352A61K31/416A61K31/4409A61K31/4439A61K31/506A61K31/519A61P43/00C12N5/0619C12N2501/15C12N2501/155C12N2501/35C12N2501/70C12N2501/73C12N2506/1307A61K2300/00
Inventor 胡雅楠张焕相张锋杨敏何其圣
Owner SUZHOU UNIV