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
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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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