Immortalized human neural stem cell line and preparation method thereof, and recombinant virus vector and application thereof

A neural stem cell, recombinant lentivirus technology, applied in the field of cell biology and neuroscience, can solve the problem of increasing the complexity of coding and tumorigenic risk, carcinogenesis and other issues

Active Publication Date: 2019-09-13
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current methods of immortalization of these cells are not stable enough, or are accompanied by potential cancer risks, such as hTERT gene introduction alone may not be sufficient for immortalization, and the combination of SV40 antigen increases the complexity of coding and potential tumor risk

Method used

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  • Immortalized human neural stem cell line and preparation method thereof, and recombinant virus vector and application thereof
  • Immortalized human neural stem cell line and preparation method thereof, and recombinant virus vector and application thereof
  • Immortalized human neural stem cell line and preparation method thereof, and recombinant virus vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Human source neural stem cells (Neural Progenitor Cell Origin ATCCBXS0117Normal; Human ( ACS5003 TM )) Cultivation and Identification:

[0044] Culture method: After culturing cells with complete medium (DMEM / F12+2% B27, 20ng / mL basic FGF, 20ng / mL EGF, 2nM L-glutamine, 2μg / ml Heparin sodium, 1%penicillin–streptom--ycin) , the cells were subcultured and expanded. Morphologically, it can be seen that the human neural stem cells grow in suspension and form typical neurosphere signs ( figure 1 ).

[0045] Pre-coat 24-well plate slides with polylysine (PDL), coat at 37°C for 2 hours or overnight, wash with PBS 3 times and let it dry for at least 2 hours. Take out the cultured cells, digest the neural stem cell spheres with Accutase enzyme to form single cells, and press 5×10 4 Cells were plated in each well, and a sufficient amount of complete medium was added after the cells adhered to the wall; the other 96-well plates did not need to be coated, and 1×10...

Embodiment 2

[0049] Establishment of Immortalized Human Neural Stem Cell Line

[0050] 1. Construction of recombinant lentiviral vector pLenti-EF1a-EGFP-P2A-Puro-CMV-MYCL-3Flag containing L-myc gene

[0051] Query the sequence of the gene L-myc, GenBank ID is NM_001033081; select the special empty vector pLenti-EF1a-EGFP-P2A-Puro-CMV-MCS-3Flag( Figure 4 ), the L-myc gene inserted at the EcoRI site ( Figure 5 ), the effective fragment size is 1095bp; the recombinant plasmid vector is sequenced and identified ( Figure 6 )

[0052] (1) Obtaining the target gene fragment

[0053] The target gene and upstream and downstream sequences were queried from GenBank, and primers were designed with VectorNTI software.

[0054] PCR amplification of the target gene: use the high-fidelity PrimeSTAR enzyme to amplify the target gene, then perform agarose gel electrophoresis on the PCR product to detect the amplification effect, and cut the target gene band from the gel after agarose gel electrophore...

Embodiment 3

[0085] Example 3: Biological characteristics and toxicological evaluation of immortalized human neural stem cell lines

[0086] 1. Amplify and subculture the immortalized cell lines, and detect the proliferation ability and three-lineage differentiation of the cells;

[0087] Each cell proliferation, differentiation experiment and detection method were basically the same as before, and the biological phenotype experiment was carried out after the immortalized human neural stem cell line was passed down to 25 generations, and the 24-well plate was coated with polylysine (PDL, Sigma), Accutase enzyme digested the cell ball into a single cell and pressed 5-10×10 4 cells were plated. In the proliferation identification experiment, the immunofluorescence experiment was performed after culturing for 2-3 days with complete medium; for the differentiation identification experiment, the immunofluorescence experiment was performed after culturing for 7-10 days with the differentiation ...

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Abstract

The invention discloses an immortalized human neural stem cell line and a preparation method thereof, and a recombinant virus vector and an application thereof. The preparation method includes the following steps: (1) constructing the recombinant lentiviral vector pLenti-EF1a-EGFP-P2A-Puro-CMV-MYCL-3Flag of an L-myc gene; (2) packaging and producing lentivirus, and collecting supernatant; and (3)adopting the lentivirus supernatant to perform transfection, screening and construction of the immortalized human neural stem cell line on human neural stem cells. The preparation method is a novel, stable, safe and effective immortalization coding strategy; the prepared immortalized human neural stem cell line has all the biological characteristics of primary neural stem cells and also has normalcharacteristics of three-line differentiation, so that the cell line can successfully differentiate neurons, astrocytes and oligodendrocytes; and the growth speed and the ability to become neurospheres of the cell line are stronger than primary cells, so that the cell line can still be rapidly propagated after the successful passing of 25 generations and has no tumorigenicity.

Description

technical field [0001] The invention belongs to the technical field of cell biology and neuroscience, and specifically relates to an immortalized human neural stem cell line, a preparation method, a recombinant virus vector and application. Background technique [0002] Neural stem cells (Neural stem cells, NSCs) were initially thought to exist only before birth and shortly after birth, but recent evidence has shown that they also exist in the adult lateral ventricle and the dentate gyrus of the hippocampus, with the ability to differentiate into neurons, astrocytes, and Multidirectional potential of stromal and oligodendrocytes, self-renewal, low immunogenicity and good histocompatibility. Since NSCs can provide a variety of nerve cells to maintain and repair damaged brain tissue, they are considered to be good tools and the most promising natural resources for the treatment of neurological diseases. Numerous preclinical studies have confirmed that transplanting exogenous ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0623C12N15/86C12N2500/32C12N2501/11C12N2501/115C12N2501/91C12N2533/52C12N2740/15043
Inventor 陈陆馗张桂龙
Owner SOUTHEAST UNIV
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