Novel method for promoting bone mesenchymal stem cells to be differentiated into neurons in rat brain
A technology of bone marrow mesenchymal and stromal stem cells, applied in the new field of promoting the differentiation of bone marrow mesenchymal stem cells into neurons in the rat brain, can solve problems such as short half-life
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Embodiment 1
[0009] Embodiment 1, the preparation of the retrovirus carrying hBDNF coding gene
[0010] Centrifuge to collect 2~3X10 6 Neuroblastoma (SK-N-SH) cells, extract total RNA, construct a cDNA library, use SK-N-SH cDNA as a template, and PCR primers are:
[0011] 5'-ATGACCATCCTTTTCCTTAC-3', 5'-CTATCTTCCCCTTTTAATGG-3'
[0012] The cDNA of hBDNF was cloned and ligated into T-vector for sequencing. The amino acid sequence of hBDNF obtained is as follows:
[0013] MTILFLTMVI SYFGCMKAAP MKEANIRGQG GLAYPGVRTH GTLESVNGPK AGSRGLTSLA DTFEHVIEEL
[0014] LDEDQKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSMRVRRHS DPARRGELSV
[0015] CDSISEWVTA ADKKTAVDMS GGTVTVLEKV PVSKGQLKQY FYETKCNPMG YTKEGCRGID KRHWNSQCRT
[0016] TQSYVRALTM DSKKRIGWRFIRIDTSCVCT
[0017] The constructed pMSCV-hBDNF retroviral plasmid uses pMSCV-neo as a vector and contains the complete hBDNF amino acid coding sequence.
[0018] The pMSCV-hBDNF plasmid and liposomes were added to PT67 cells grown in logarithm...
Embodiment 2
[0019] Example 2, transduction of BDNF coding gene by retrovirus infection
[0020] Divide P1 generation MSCs by 1X10 4 cells / cm 2 The density was inoculated in a culture dish, and then infected at a ratio of 1:10 of cells to virus particles for 8 hours, cultured in virus-free medium for 24 hours, and then cultured with 250ug / ml G418 for 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
[0021] Example 3. Directional induction of BDNF-MSCs transplanted into the rat brain into neurons
[0022] 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 is 20ul (including 5X10 6 BDNF-MSCs), the injection speed was 1ul / min, the needle was retained for 10min, and the needle was withdrawn slowly at 1mm / min. 1 day, 3 days, 6 days, 15 days, and 30 days after cell transplantation, the rats were sacrificed by heart shearing, and the brain tissue was fixed by aortic perfusion with 4% paramethanol / 0.1M PBS fixative solution, and frozen sections. NeuN, GFAP, Nestin immunohistochemical antibodies combined with fluorescent seconda...
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