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Mesenchymal stem cell preparation used for glioma immunotherapy and meanwhile capable of realizing magnetic resonance tracing

A technology of mesenchymal stem cells and immunotherapy, applied in the fields of genetic engineering and biomedical engineering, can solve the problems of less than 10% survival rate and unsatisfactory prognosis of malignant glioma, and achieve the effect of broad application prospects

Inactive Publication Date: 2019-12-03
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the precision surgical treatment and radiotherapy and chemotherapy techniques for glioma have made some progress in recent years, the prognosis of malignant glioma is still very unsatisfactory, the 5-year survival rate is less than 10%, and the median survival time is only 12-15 months. moon
[0005] At present, there is no report on the simultaneous integration of IFNβ gene and Ferritin H gene into the genome of mesenchymal stem cells for the treatment of intracranial glioma

Method used

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  • Mesenchymal stem cell preparation used for glioma immunotherapy and meanwhile capable of realizing magnetic resonance tracing
  • Mesenchymal stem cell preparation used for glioma immunotherapy and meanwhile capable of realizing magnetic resonance tracing
  • Mesenchymal stem cell preparation used for glioma immunotherapy and meanwhile capable of realizing magnetic resonance tracing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Construction and Identification of FerritinH-T2A-IFNβ Overexpression Lentiviral Vector

[0056] 1. Acquisition of the target gene

[0057] According to the rat FerritinH (NM_012848) gene and IFNβ (NM_019127) gene sequences in GenBank, the FerritinH-T2A-IFNβ sequence was synthesized by whole gene chemical synthesis. Design specific primers as follows:

[0058] FerritinH-T2A-IFNβ upstream primer (SEQ ID NO:2): fusion gene(21000-2)-P1

[0059] 5'-GAGGATCCCCGGGTACCGGTCGCCACCATGACCACCGCGTCTCCCTCGCAAG-3'

[0060] FerritinH-T2A-IFNβ downstream primer (SEQ ID NO:3): fusion gene(21000-2)-P2

[0061] 5'-CACACATTCCACAGGCTAGTCAGTTCTGGAAGTTTCTATTAAG-3'.

[0062] Primers were used to amplify the FerritinH-T2A-IFNβ sequence by PCR. After the PCR product was electrophoresed on the agarose gel, the target band with a molecular weight of about 1210 bp was excised for later use.

[0063] 2. Construction and sequencing of recombinant plasmids

[0064] The recovered PCR pro...

Embodiment 2

[0072] Example 2 Transfection of MSCs with FerritinH-T2A-IFNβ overexpression lentivirus and determination of transfection efficiency

[0073] 1. Culture and passage of Wistar rat MSCs

[0074] After recovery of Wistar rat MSCs, use DMEM / F12 complete medium (containing 10% FBS + 1% penicillin-streptomycin mixed solution + 1% L-glutamine) to culture in a cell constant temperature incubator, every 2~ 3d liquid change. When the cell confluency reaches about 70%, digest and subculture at a ratio of 1:3.

[0075] 2. Transfect MSCs with FerritinH-T2A-IFNβ overexpression lentivirus

[0076] Take MSCs within 5 passages in the logarithmic growth phase, and use 1×10 5 Cells / well were seeded in a 12-well culture plate, and 1 mL of complete medium was added to each well. When the cell confluency was about 50%, the culture medium was removed, washed with PBS and then transfected. In order to determine the optimal MOI and transfection time, set the MOI (MOI=Volume of virus solution×Viru...

Embodiment 3

[0102] Example 3 In vitro effectiveness detection of FerritinH-T2A-IFNβ overexpression lentivirus transfected MSCs

[0103] In order to verify the effect of FerritinH overexpression and extracellular iron supplementation on intracellular iron content, the qualitative and quantitative detection of intracellular iron content was performed by Prussian blue staining, transmission electron microscopy, in vitro MR scanning and atomic absorption spectroscopy.

[0104] 1. Prussian blue dyeing

[0105] Press 2×10 5 Cells / well FerritinH-IFNβ-MSCs and WT-MSCs were inoculated in a six-well plate, each group of cells had 2 wells, and after adhering to the wall, one well was added with 350 μM ferric citrate (FC) The complete medium was incubated for 60 hours, and the other well was incubated with only complete medium for the same time. Wash once with PBS, fix with 4% paraformaldehyde for 20 min, and wash three times with PBS, 5 min each time. Add 2 mL of Perl's reaction solution (10% pot...

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Abstract

The invention belongs to the technical field of genetic engineering and biomedical engineering, and particularly discloses a mesenchymal stem cell preparation used for glioma immunotherapy and meanwhile capable of realizing magnetic resonance tracing. The mesenchymal stem cell preparation comprises mesenchymal stem cells capable of simultaneously secreting interferon-beta (IFN beta) and realizingmagnetic resonance (MR) imaging. The mesenchymal stem cells are jointly modified with IFN beta and FerritinH genes, can adjust the tumor immune microenvironment by secreting an immunomodulatory factorIFN beta, and exerts an inhibition effect on glioma growth; and meanwhile, through FerritinH reporter gene magnetic resonance (MR) imaging, the stem cells after transplanting are monitored dynamically, non-invasively and systematically in real time at the living body level, the experimental basis is provided for promoting stem cell therapy clinical transformation, a new means is provided for deeply illuminating the stem cell action mechanism, and the important scientific significance and clinical application prospects are achieved in aspects of stem cell therapy and transgenic therapy of glioma.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and biomedical engineering, in particular to a mesenchymal stem cell preparation that can be used for glioma immunotherapy and can be traced by magnetic resonance. Background technique [0002] Glioma is the most common tumor of the central nervous system and a major refractory disease that seriously endangers human health. The morbidity, morbidity and mortality of glioma in my country are high, which brings a heavy burden to individuals, families and society. Although the precision surgical treatment and radiotherapy and chemotherapy techniques for glioma have made some progress in recent years, the prognosis of malignant glioma is still very unsatisfactory, the 5-year survival rate is less than 10%, and the median survival time is only 12-15 months. moon. Immunotherapy inhibits or kills tumor cells by mobilizing the body's own immune system. It is currently a research hotspot in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61K49/06A61P35/00C12N15/867C12N5/10
CPCA61K35/28A61K49/06A61P35/00C12N5/0663C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 沈君毛家骥曹明慧
Owner SUN YAT SEN UNIV
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