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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

Inactive Publication Date: 2013-04-17
北京清美联创干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the half-life of BDNF protein in the brain is very short, so the mixed transplantation of BDNF and MSCs cannot make BDNF fully exert its biological function, so it is necessary to find a method that can make BDNF act on the implanted MSCs for a long time

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a method for promoting bone mesenchymal stem cells transplanted in the rat brain to be directionally differentiated into neurons. The method specifically comprises the steps of: separating mesenchymal stem cells from medulla ossiums and carrying out amplification and purification; cloning into a cDNA (complementary deoxyribonucleic acid ) sequence coded by a human brain derived neurotrophic facto (hBNDF) protein through PCR, constructing an expression vector carrying a hBNDF coding gene, and finally transplanting the mesenchymal stem cells expressing a hBNDF coding gene into the rat brain through a brain stereotaxic method. Under the induction of the hBNDF, the quantity of the mesenchymal stem cells directionally differentiated into the neurons is remarkably increased. According to the method provided by the invention, the quantity of the mesenchymal stem cells which are transplanted in the brain and directionally differentiated into the neurons can be greatly increased, a novel method is developed for the stem cell treatment of the nervous system disease, and a wide clinical application prospect is achieved.

Description

technical field [0001] The invention relates to a method for in vitro transduction of neurotrophic factor encoding genes by virus vectors to promote efficient and directional differentiation of bone marrow mesenchymal stem cells transplanted into the brain into neurons, and at the same time paracrine neurotrophic factors nourish and support peripheral nerve cells. Background technique [0002] Mesenchymal stem cells (MSCs) originate from the mesoderm and are the precursor cells of various stromal cells such as fibroblasts, osteoblasts, and adipocytes, and have strong proliferation ability and multi-differentiation potential. MSCs can achieve higher purity through adherent culture. MSCs can be induced to differentiate into neurons expressing NeuN and glial cells expressing GFAP in vitro. Transplanted MSCs into brain tissue can survive for a long time and differentiate into nerve cells. However, experiments have shown that most of the MSCs implanted into the brain differenti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/12A61K35/28A61K48/00A61P25/00A61P25/16A61P25/28
Inventor 田杰
Owner 北京清美联创干细胞科技有限公司
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