Method for genetically modifying and inducing intracerebral transplantation mesenchymal stem cells to be differentitated into neuron
A mesenchymal stem cell and gene modification technology, applied in the field of BDNF gene-modified mesenchymal stem cell construction, can solve problems such as short half-life
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Embodiment 1
[0010] Embodiment 1, the preparation of the retrovirus of recombinant hBDNF gene
[0011] Centrifuge to collect 2~3×10 6 Total RNA was extracted from neuroblastoma (SK-N-SH) cells, and a cDNA library was constructed. Using SK-N-SH cDNA as a template, PCR primers: 5′-ATGACCATCCTTTTCCTTAC-3′, 5′-CTATCTTCCCCTTTTAATGG-3′, cDNA of hBDNF was cloned and ligated to T-vector for sequencing. The obtained CDS nucleic acid sequence of hBDNF is shown in SEQ.1 in the sequence listing.
[0012] Cut the CDS of hBDNF from the pGEM-T vector with restriction endonucleases EcoR I and Xho I, and then connect it to pMSCV-neo with T4 ligase, that is, construct the retroviral plasmid pMSCV-hBDNF of the recombinant hBDNF gene .
[0013] PT67 cells grown in several phases were transfected with pMSCV-hBDNF plasmid and liposome (LipofectAMINE 2000), and then drug-resistant cells were screened with 600 μg / ml G418. After the formation of resistant clones, culture them in culture medium without G418 for...
Embodiment 2
[0014] Embodiment 2, transducing BDNF gene by retrovirus infection
[0015] Will P 1 Generation of MSCs by 1 x 10 4 cells / cm 2 Density inoculation in culture dishes, and then according to the ratio of cells and virus particles 1:10 after 8 hours of infection, cultured in virus-free medium for 24 hours, and then cultured with 300μg / ml G418 selection. After the formation of resistant clones, replace the medium without G418. The results of ELISA assay showed that the BDNF level in the culture medium of BDNF-MSCs was significantly higher than that in the culture medium of MSCs without BDNF gene modification.
Embodiment 3
[0016] Example 3. Directional induction of BDNF-MSCs into neurons by intrabrain transplantation in rats
[0017] Wistar rats were irradiated with 2.4cGy γ-rays for immunodepletion. 24 hours later, using a rat brain stereotaxic instrument, according to the instructions of "Rat Brain Stereotaxic Atlas", inject BDNF-MSCs stained with DAPI nucleus in the anterior part of rat striatum. The coordinates are: 1.0mm anterior to the anterior chimney, 2.5mm lateral, and 4.5mm subdural. The amount of injected cell suspension was 20ml (containing 2×10 6 BDNF-MSCs), the injection speed was 1 μl / min, the needle was retained for 10 minutes, and the needle was withdrawn slowly at 1 mm / min. 1 day, 3 days, 6 days, 15 days, 30 days after cell transplantation, the rats were sacrificed by heart shearing, and the brain tissue was fixed by aortic perfusion with 4% paraformaldehyde / 0.1MPBS fixative solution, and frozen section. NeuN, GFAP, Nestin immunohistochemical antibody binding, fluorescent s...
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