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Telomerase immortalized human fetal forebrain neural precursor cell line and construction method thereof

A technology of precursor cells and brain nerves, which can be applied in the fields of biochemical equipment and methods, nervous system diseases, and botanical equipment and methods, and can solve problems such as tumorigenic danger.

Inactive Publication Date: 2009-01-28
首都医科大学北京神经科学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the expression of myc gene is not only related to cell proliferation and differentiation, but also closely related to cell canceration, transplantation to patients has a considerable risk of tumorigenesis.

Method used

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  • Telomerase immortalized human fetal forebrain neural precursor cell line and construction method thereof
  • Telomerase immortalized human fetal forebrain neural precursor cell line and construction method thereof
  • Telomerase immortalized human fetal forebrain neural precursor cell line and construction method thereof

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Experimental program
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Embodiment 1

[0085] Primary culture and characterization of human embryonic forebrain neural precursor cells

[0086] 1. Primary Isolation of Embryonic Brain Tissue

[0087] 1. Take the aborted embryo (12 weeks of pregnancy), take out the whole brain tissue under strict aseptic conditions, and store it in DMEM / F12 medium containing penicillin and streptomycin at 4°C. Cells should be isolated and cultured within 4 hours after the sample is taken Finish.

[0088] 2. Soak the isolated brain tissue in PBS solution containing penicillin and streptomycin twice.

[0089] 3. Place the soaked brain tissue in a plate containing NSA basal medium, carefully remove the meninges under a dissecting microscope, then take out the forebrain striatum with a scalpel and soak it in NSA basal medium for 3 times, Soak for 5 minutes each time.

[0090] 4. Put the soaked brain tissue into a centrifuge tube containing protease XXIII (3mg / ml) for digestion at 37°C for 6 minutes.

[0091] 5. Centrifuge at 1000 rp...

Embodiment 2

[0115] Establishment and Characterization of Telomerase Immortalized Human Embryonic (Fetal) Forebrain Neural Precursor Cells

[0116] 1. Establishment of telomerase-immortalized human embryonic forebrain neural precursor cells

[0117] 1. Establishment of packaging cells PG13 / JH1 / hTERT containing human telomerase catalytic subunit gene

[0118] (1) construct retroviral vector, its steps are as follows Figure 30 shown.

[0119] Target gene hTERT and reporter gene (human low-affinity human growth factor receptor (NGFR) membrane surface segment gene and enhanced green fluorescent protein (Enhanced green fluorescent protein, EGFP) gene fusion) They are connected by the internal ribozyme entry site (IRES) regulatory structure; IRES can control the expression of two genes from one mRNA, that is, the target gene hTERT and the reporter gene are expressed simultaneously, and the expression level of the reporter gene can be detected To determine whether the target gene is expressed...

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Abstract

The invention relates to a cell line of precursor of forebrain nerve of a telomerase immortalization human embryo and an establishing method and application thereof; the cell line is preserved in China culture collection and management committee general biology center in 21st February 2008, with the collection number of CGMCC No.2372. The cell line of the invention is named as hSN12W-TERT; the cell line expresses growth factor receptor of human nerves and green fluorescent protein, has high activity of telomerase, double core type than normal human, continuous passage culture in vitro of 36 months with 45th generation at the present and population doubling of 100 to 200 times and has the property of contact inhibition and density inhibition; the neuron which is formed by differentiation of the cell line has the property of electrophysiology; the cell line can be differentiated into Gamma-aminobutyric acid (GABA) cholinergic neuron under the induction of bone marphogenic protein 2(BMP2).

Description

technical field [0001] The invention relates to a cell line and its establishment method and application, in particular to a telomerase immortalized human embryo forebrain neural precursor cell line and its establishment method and application, which belongs to the field of basic research and clinical application of nervous system diseases . Background technique [0002] Central nervous system diseases, such as degenerative diseases Parkinson's disease (PD), Alzheimer's disease (AD) and acute injury diseases cerebral hemorrhage, cerebral infarction, etc., are generally difficult to cure, causing endless harm to individuals and families. This is because the neurons that make up the nervous system have completed division, proliferation and differentiation after birth, and cannot be replaced by new neurons after death. Once the brain tissue is damaged, it will cause permanent loss of function. [0003] The discovery of neural stem cells, an epoch-making development in the fiel...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/52C12N15/867A61K35/54A61P25/00
Inventor 张海燕徐群渊王一松赵咏梅赵春礼
Owner 首都医科大学北京神经科学研究所
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