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Schwarm progenitor cell derived from marrow neural crest cell and application of Schwarm progenitor cell to promotion of nerve regeneration

A nerve regeneration and neural crest technology, applied in the field of tissue engineering, can solve problems such as limited application

Active Publication Date: 2016-09-07
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the source and purity of SC still limit its application in cell therapy

Method used

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  • Schwarm progenitor cell derived from marrow neural crest cell and application of Schwarm progenitor cell to promotion of nerve regeneration
  • Schwarm progenitor cell derived from marrow neural crest cell and application of Schwarm progenitor cell to promotion of nerve regeneration
  • Schwarm progenitor cell derived from marrow neural crest cell and application of Schwarm progenitor cell to promotion of nerve regeneration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Culture and identification of bone marrow neural crest cells (BM-NCC)

[0045] For the cultivation of BM-NCC, the following repeated two-step conditional culture procedure was applied to obtain BM-NCC:

[0046] In the first step, after adult Wistar rats (8 weeks old) were sacrificed, the bone marrow was washed out from the tibia and femur immediately, and the bone marrow cells were inoculated and cultured in IMDM medium containing 10% FBS, and BMSCs were grown on plastic culture dishes. Characteristics, repeated medium changes to remove floating hematopoietic stem cells, and primary rat BMSCs were isolated and cultured;

[0047] The second step, with 20ng / ml EGF, 20ng / ml FGF2, 1% N2 (v / v), 2% B27 (v / v)), 2mM glutamine, 100U / ml penicillin, 0.1mg / ml chain Mycin DMEM / F12 (v / v, 1:1) ball culture medium, the primary BMSCs were cultured into balls, and the seeding cell density was 2~3×10 5 cells / ml to form primary cell spheres (Sphere). The sphere culture medium...

Embodiment 2

[0054] Example 2.Isolation and proliferation of BM-NCC clones and detection of cell markers

[0055] BM-NCC clones were obtained by limited dilution method [Glejzer A, Laudet E, Leprince P, Hennuy B, Poulet C, Shakhova O, et al. Wnt1 and BMP2: two factors recruiting multipotent neural crest progenitors isolated from adult bone marrow. Cell Mol LifeSci .2011;68:2101-14.]. After the cell spheres were subcultured to the fourth generation or above, the cell spheres were digested into a single cell suspension with accutase enzyme, and the cells were inoculated into 96-well cell culture plates at a density of 0.7 cells / well for single-cell sphere culture (cells per well The number is random as 0, 1 or 2, etc.), and half the amount of medium is changed twice a week. Observe the cells in each well with a microscope. When the number of cells in a single cell sphere formed by the proliferation of a single cell reaches more than 100, the cell sphere is digested into a single-cell suspen...

Embodiment 3

[0060] Embodiment 3.BM-NCC directed differentiation to Schwann cell (SC)

[0061] BM-NCC monoclonal cells (N1, N3, N4 and N13) were induced to differentiate into SC by using specific differentiation medium.

[0062] The isolated cell clones were separated into single cells at passages P5-P7, and inoculated on small round glass slides coated with PLL / laminin, treated with 5% FBS, 35ng / ml tRA, 5μM forskolin, 200ng / ml HRG -1β, 10ng / ml FGF2, 10ng / ml PDGF-AA and 1% N2 in DMEM / F12 (v / v, 1:1) culture solution (i.e. SC directional induction differentiation medium) for incubation, and change the medium every 3 days .

[0063] After N1 cells were induced to differentiate for 12 h, the adherent cells began to elongate, and the cell shape changed from round to spindle ( Figure 5 A); 48h later, the spindle-shaped cells were arranged side by side ( Figure 5 B); After 60 h, the cells gradually aged and died. The cells induced for 48 hours were selected for ICC staining of S100β to conf...

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Abstract

The invention relates to the technical field of tissue engineering, in particular to a Schwarm progenitor cell derived from a marrow neural crest cell and application of the Schwarm progenitor cell to promotion of nerve regeneration. The rat marrow-derived neural crest cell N1, namely, BM-NCC clone N1, is provided, the preservation number is CCTCC NO:C201647, and the rat marrow-derived neural crest cell N1 has the performance of the Schwarm progenitor cell (SCP). The invention further provides a subculture method of the BM-NCC clone N1 and a method for induced directional differentiation to the Schwarm cell (SC). The BM-NCC clone N1 is expected to be applied to nerve regeneration promotion therapy based on cells.

Description

technical field [0001] The invention relates to the technical field of tissue engineering, in particular to a Schwann progenitor cell derived from bone marrow neural crest cells and its application in promoting nerve regeneration. Background technique [0002] In the nervous system, Schwann cells (Schwann cells, SC) are the glial cells of the peripheral nervous system, which are involved in the myelination of axons, the organization of peripheral neurons, the maintenance of normal function of neurons, the formation of synapses and It plays a key role in the response to nerve injury and neuralgia. In the Wallerian degeneration stage after peripheral nerve injury, SC dedifferentiates into immature SC or precursor-like SC, participates in the removal of myelin and axon debris, produces a variety of growth factors and cytokines, proliferates and migrates to form axon regeneration. Growing Bungner ribbons, which eventually redifferentiate into mature myelinating SCs, support axo...

Claims

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Application Information

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IPC IPC(8): C12N5/079A61K35/30A61L27/38A61P25/00
CPCA61K35/30A61L27/383A61L27/3878A61L2400/06A61L2430/32C12N5/0622C12N2500/38C12N2501/01C12N2501/115C12N2501/135C12N2533/32C12N2533/52
Inventor 施海燕丁斐强亮余英奇孙晓婷胡静静孙晓欢何欢
Owner NANTONG UNIVERSITY
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