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Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof

A technology of oligodendrocytes and secondary injury, applied to nervous system cells, genetically modified cells, botanical equipment and methods, etc., can solve problems such as hindering axon elongation and affecting nerve regeneration

Active Publication Date: 2016-10-12
江西美奥生物技术有限公司
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] In the early stage of nerve injury, astrocytes are in the early stage of maturation, which can secrete cytokines to maintain and promote the function of neurons, and play the role of nerve tissue scaffolding; but after they mature, a variety of beneficial functions they had in the early stage gradually disappear, and instead secrete Harmful factor, forms a chemical glial barrier, affects nerve regeneration, hinders axon elongation

Method used

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  • Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof
  • Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof
  • Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof

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Experimental program
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Effect test

Embodiment 1

[0067] Construction and packaging of embodiment 1 recombinant GAP-43 and GGF-2 lentiviral vector

[0068] Human GAP-43 and GGF-2 sequences were amplified from normal human peripheral blood genomic DNA by PCR, and the target fragments of GAP-43 and GGF-2 were respectively digested with XhoI / NdeI (Japan TAKARA Company) pET28a vector (U.S. Invitrogen Company) fragment, under the action of T4 DNA ligase (Japan TAKARA Company), prepare the clone connection solution at 4°C for 12 hours, transform Escherichia coli competent cell DH5a (U.S. Invitrogen Company) and carry out positive clone PCR identification and sequencing identification; the size of the identified GAP-43 and GGF-2 gene fragments is about 706bp and 1268bp, which conforms to the size and sequence of GAP-43 and GGF-2. Transfer the bacteria liquid with correct sequencing to 10ml LB liquid medium containing ampicillin antibiotic, culture overnight at 37°C, and use Beijing Tiangen Biological Endotoxin-Free Plasmid Small-sca...

Embodiment 2

[0072] Example 2 Preparation of human oligodendrocyte progenitor cells

[0073] Collect 20ml of bone marrow from donated volunteers, separate and purify mononuclear cells with lymphocyte separation medium, trypan blue count is 3×10 7 For each, serum-free oligodendrocyte culture medium (i.e. DMEM / F12 (1:1) (Gibco, USA) containing 1% N2, 2mM L-glutamine (Gibco, USA), 30ng / ml alkaline Fibroblast growth factor, 5ng / ml fibroblast growth factor 4, 10ng / ml stem cell growth factor and 5ng / ml FMS-like tyrosine kinase 3 ligand (PeproTech, USA)) were resuspended to 3×10 6 / ml, pipette 10ml and add to 50μg / ml polylysine-coated 75cm 2 In cell culture flasks, at 37°C, 5% CO 2 Incubate for 48 hours in a cell culture incubator.

[0074] After culturing for 48 hours, replace with fresh serum-free oligodendrocyte medium containing cytokines and continue culturing, and then replace with fresh serum-free oligodendrocyte medium containing cytokines at intervals of 2 days, until the cell growth ...

Embodiment 3

[0076] Example 3 Preparation of human oligodendrocyte progenitor cells transfected with recombinant GAP-43 and GGF-2 lentivirus

[0077] Add 10 μl 1E+7TU / ml of recombinant GAP-43 and GGF-2 lentiviruses to human oligodendrocyte progenitor cells in 50 μg / ml poly-lysine-coated six-well culture plate, the system is 2ml, mix Uniform, 37°C, 5% CO 2 After incubation in the incubator for 8-12 hours, replace the complete culture solution DMEM / F12 (1:1), which contains 1% N2, 2mM L-glutamine, 30ng / ml basic fibroblast growth factor, 5 ng / ml fibroblast growth factor 4, 10 ng / ml stem cell growth factor and 5 ng / ml FMS-like tyrosine kinase 3 ligand; when the cell growth reached 90% confluency, digest with 0.25% trypsin and transfer to 50 μg / ml polylysine coated 25cm 2 Grow in a culture bottle, 1 well corresponds to 1 culture bottle, at 25cm 2 Continue to digest and subculture when the fusion in the culture flask reaches 90%; growth transfection is divided into 3 groups: non-transfected ...

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Abstract

The invention provides a preparation method of a human oligodendrocyte precursor cell inhibiting nerve secondary. Specifically, the method includes: constructing a virus expression vector of two glial growth factors, conducting cotransfection of a human oligodendrocyte precursor cell to prepare the human oligodendrocyte precursor cell, which can achieve high expression of the two glial growth factors, i.e. a growth related factor 43 and a glial growth factor 2, give play to the combined action of the two to promote oligodendrocyte repair, and inhibit nerve secondary injury caused by proliferative response and glial scar formation after oligodendrocyte injury. Also, the proliferation and differentiation ability of oligodendrocyte precursor cell itself is also utilized to participate in nerve function repair, and no tumorigenesis exists. The invention also provides a kit for preparation of the human oligodendrocyte precursor cell inhibiting nerve secondary. The human oligodendrocyte precursor cell provided by the invention has very good therapeutic effect on nervous system diseases.

Description

technical field [0001] The present invention relates to a preparation method of human oligodendrocyte progenitor cells that inhibit secondary nerve injury and obtain human oligodendrocyte progenitor cells that inhibit secondary nerve injury through the method. In particular, the present invention relates to the construction of two A viral expression vector of a glial growth factor enables human oligodendrocyte progenitor cells to stably express the two glial growth factors, and jointly exert their biological functions; A kit for human oligodendrocyte progenitor cells. The human oligodendrocyte progenitor cells provided by the invention maintain good proliferative ability, participate in the repair of nerve function, and have no tumorigenicity, and are expected to be used in the clinical treatment of nervous system diseases to promote the repair of damaged nerve cells. Background technique [0002] Nervous system diseases, such as ischemia, hypoxia and injury, cause signific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/867C12N15/12C12N5/10A61K35/30A61P25/00
CPCA61K35/30C07K14/475C07K14/4756C12N5/0622C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 李一民李一汉陈宗明
Owner 江西美奥生物技术有限公司
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