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Immortalized human neural stem cell line and preparation method thereof

A neural stem cell and immortalization technology, applied in the fields of cell biology and neuroscience, can solve the problems of increased coding complexity, tumorigenic risk, carcinogenicity, etc., and achieve the effect of strong growth speed and ability to form neurospheres

Inactive Publication Date: 2018-11-23
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
  • Immortalized human neural stem cell line and preparation method thereof
  • Immortalized human neural stem cell line and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Extraction, culture and identification of primary neural stem cells from human hippocampus

[0055] 1. Extraction of primary human hippocampus-derived neural stem cells

[0056] Under the review and approval of the Clinical Research Ethics Committee of Zhongda Hospital Affiliated to Southeast University (approval number: 2017ZDSYLL048-P01), and the informed consent of pregnant women with abortion was obtained, the fetal brain tissue of the terminated pregnancy was collected in about 3 months, strictly in accordance with the clinical neurosurgical procedures General procedures and standards, the material is collected through minimally invasive surgery (the hippocampus tissue in the fetal brain tissue), and the integrity of the fetal fetal brain and the dignity of the fetus are guaranteed as much as possible after surgery. Reasonable and safe destruction.

[0057] The obtained cranial nerve hippocampus tissue was aseptically lysed by trypsin and centrifuged at 800 rpm fo...

Embodiment 2

[0065] Establishment of Immortalized Human Neural Stem Cell Lines

[0066] 1. Construction of recombinant lentiviral vector pLenti-EF1a-EGFP-P2A-Puro-CMV-MYCL-3Flag containing L-myc gene

[0067] The sequence of the query gene L-myc, the GenBank ID is NM_001033081; the empty vector pLenti-EF1a-EGFP-P2A-Puro-CMV-MCS-3Flag ( Figure 5 ), the L-myc gene inserted at the EcoRI site ( Image 6 ), the effective fragment size was 1095bp; the recombinant plasmid vector was sequenced and identified.

[0068] (1) Obtaining the target gene fragment

[0069] Query the target gene and upstream and downstream sequences from GenBank, and use VectorNTI software to design primers.

[0070] PCR amplification of the target gene: use the high-fidelity PrimeSTAR enzyme to amplify the target gene, then the PCR product is subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band is cut from the gel after agarose gel electrophoresis. Use TaKaRa MiniBEST ...

Embodiment 3

[0100] Biological Characteristics and Toxicological Evaluation of Immortalized Human Neural Stem Cell Lines

[0101] 1. Expand and subculture the immortalized cell line to detect the proliferation ability and tri-lineage differentiation of the cells;

[0102] Cell proliferation, differentiation experiments and detection methods are basically the same as before, pre-coated 24-well plates with polylysine (PDL, Sigam), digested with Accutase enzyme to form single cells, press 5-10 × 10 4 cells were seeded. For the proliferation identification experiment, the immunofluorescence experiment was performed after culturing in complete medium for 2-3 days; for the differentiation identification experiment, the immunofluorescence experiment was performed after culturing in the differentiation medium (DMEM / F12+2%FBS) for 7-10 days. For immunofluorescence experiments, cells were washed 2-3 times with PBS (pH 7.4), fixed with 4% paraformaldehyde for 15-30 minutes, permeabilized with 0.3% T...

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Abstract

The invention discloses an immortalized human neural stem cell line and a preparation method thereof. The method comprises the following steps: (1) isolating and culturing primary human hippocampus-derived neural stem cells; (2) constructing a recombinant lentiviral vector containing L-myc gene; (3) carrying out packaging production of the above recombinant lentiviral and collecting supernatant; (4) selecting virus supernatant with appropriate concentration for transfection and screening of stem cells and constructing the immortalized human neural stem cell line. The preparation method of immortalized cell line provided by the invention is a novel stable and safe and effective immortalized coding strategy; the prepared immortalized human neural stem cell line has all the biological characteristics of primary neural stem cells, and also has the feature of normal three-line differentiation at the same time, can be successfully differentiated into neurons, astrocytes and oligodendrocytes,and the growth rate of such cell line and ability to form neurospheres are stronger than the growth rate of primary cells, and the cell line can still proliferate rapidly after 20 generations withouttumorigenicity.

Description

technical field [0001] The invention belongs to the technical field of cell biology and neuroscience, and particularly relates to an immortalized human neural stem cell line and a preparation method thereof. Background technique [0002] Neural stem cells (NSCs) were initially thought to exist only before birth and shortly after birth, but recent evidence shows that they also exist in the subventricular layer of the lateral ventricle and the dentate gyrus of the hippocampus in adults. pluripotent, self-renewal, low immunogenicity and good histocompatibility. Because NSCs can provide various nerve cells to maintain and repair damaged brain tissue, they are considered as a good tool and the most promising natural resource for the treatment of neurological diseases. Numerous preclinical studies have confirmed that transplantation of exogenous neural stem cells can have a certain therapeutic effect in diseases such as ischemic stroke. However, before the clinical transformatio...

Claims

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Application Information

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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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