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

Inactive Publication Date: 2008-08-06
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY +1
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  • 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 effects, so it is necessary to find a method that can make BDNF have a long-term effect on implanted MSCs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention is aimed at providing a method for utilizing external gene operation to induce the intracerebral transplanted mesenchymal stem cell to implement directed differentiation and make it into neurone. Its technical scheme includes the following steps: (1). using self-body biological characteristics to separate, amplify the purify mesenchymal stem cell; (2). cloning brain-derived neurotrophic factor cDNA sequence from human neuroblast tumor cDNA library, constructing retroviral vector of recombinant human brain-derived neurotrophic factor gene and introducing the brain-derived neutrotrophic factor gene into mesenchymal stem cell; and (3). utilizing cerebral stereo-orientation method to make the mesenchymal stem cell modified by brain-derived neurotrophic factor gene be transplanted into brain. Under the induction of neurotrophic factor the ratio of defferentiating mesenchymal stem cell into neurone can be obviously raised. Said invention also provides its application value.

Description

technical field [0001] The invention relates to a method for transducing neurotrophic factor gene in vitro by virus gene carrier to promote the differentiation of bone marrow mesenchymal stem cells transplanted in the brain into neurons, and at the same time, paracrine neurotrophic factor nourishes and supports 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, endothelial cells, 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 differentiate into...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08C12N5/10C12N15/12C12N15/867A61K48/00A61P25/00
Inventor 裴雪涛徐小虎赵连旭张杰王冬梅
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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