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Retroviral vector for expression of glial cell line-derived neurotrophic factor and use thereof

A neurotrophic factor and lentiviral vector technology, applied in the field of medical biology, can solve the problems of slow division and proliferation of neural stem cells and a decrease in the proportion of cells

Inactive Publication Date: 2009-04-01
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the division and proliferation of neural stem cells is relatively slow, especially after long-term subculture, the proportion of cells in the state of division and proliferation will gradually decrease

Method used

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  • Retroviral vector for expression of glial cell line-derived neurotrophic factor and use thereof
  • Retroviral vector for expression of glial cell line-derived neurotrophic factor and use thereof
  • Retroviral vector for expression of glial cell line-derived neurotrophic factor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Neural stem cell culture

[0021] 1. Methods: Primary neural stem cells were obtained from human embryos of spontaneously aborted fetuses (8-16w) (provided by the Obstetrics and Gynecology Department of Xuanwu Hospital, approved by the Ethics Committee). After collection, single-cell suspension was passed through a 400-mesh filter to remove tissue residues, counted with trypan blue and counted as 2.5×10 5 / ml density inoculation. The medium is DMEM-F12 (GIBICO, USA), N 2 Supplement (US GIBICO Company), 20ng / ml bFGF (US R&D Company), 20ng / ml EGF (US R&D Company), penicillin and streptomycin (US GIBICO Company). 5%CO 2 cultured in an incubator. Small cell colonies can be seen in about 3 days, and neurospheres can be seen in a week. The medium was changed every three and a half days, and when the neurospheres grew to about 50um, they were cut and passaged. After the number of neurospheres expanded to a certain amount, the neurospheres were broken up into a...

Embodiment 2

[0023] Example 2. Construction of lentiviral vector containing GDNF gene

[0024] 1. Material source: The plasmid PLNCX-2 containing human GDNF was donated by the Institute of Neuroscience, Capital Medical University. The full length of the GDNF gene sequence is 558bp, which is the sequence shown in SEQ ID No.3 in the sequence table. The lentiviral vector plasmid DUET101 was donated to Professor Linzhao Cheng from Johns Hopkins University.

[0025] 2. Method:

[0026] 1. PCR method to amplify the GDNF gene sequence containing the corresponding restriction site

[0027] According to the structure of the lentiviral vector plasmid, primers for amplifying GDNF containing two restriction sites, XbaI and BsrGI, were designed, and the upstream primer: 5'-T TCTAGA CCACCATGAAGTTATGGGATGTCGTG-3'(SEQ ID NO.1), the underlined part is the enzyme cutting site XbaI added, the protective base T is added in front of the enzyme cutting site, and the following ccacc is the Kozark sequence, fo...

Embodiment 3

[0060] Example 3. Conditions and optimization of transfected neural stem cells

[0061] 1. Method:

[0062] 1. Transfection: The packaging of lentiviral particles was carried out by liposome-mediated transient transfection method. The day before transfection, 293T cells (given from the School of Life Sciences, Peking University, see literature: Xu Xingang, Hu Jianhe, Zhang Yanming, Deng Hongkui; Retroviral vector-mediated expression of HCV envelope protein gene and animal immunity test; "Chinese Virology"; 200419(6):563-567.) Passage in 100mm culture dish, culture plate is used before inoculation Coat with 100ug / ml polylysine for 1 hour and wash twice with DPBS before use. The total number of seeded cells was 5×10 6 , 5% CO 2 Cultivate in the incubator for 24 hours, and transfect after the cells are 70%-80% confluent. The constructed transfer plasmid DUET101 encoding GDNF and GFP, and the plasmid CMV△8.91 encoding the lentiviral vector gag and pol structure (given to Johns...

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Abstract

The invention discloses a lentiviral vector for expressing the glial cell line-derived neurotrophic factor and applications thereof, which belong to the field of medical biotechnology. The preservation number of the lentiviral vector is CGMCC No.2086. The invention has the advantages: the lentiviral vector constructed by the invention has two promoter structures and can simultaneously express GDNF gene and hygromycin resistance gene, the lentiviral vector supernatant packaged by the transient transfeetion method can carry out high-efficiency transfection on the neural stem cells which divide relatively slowly, thus leading the GDNF gene to be integrated to the target cell genom and be stably expressed for a long time; the neural stem cells after transfection can remove the neural stem cells which are not successful for transfection by the screening of the hygromycin so as to obtain the uniform over-expression GDNF cell system; and the neural stem cells which carry out gene modification by the lentiviral vector can still keep the intrinsical biological characteristics.

Description

technical field [0001] The invention relates to a lentiviral vector expressing glial-derived neurotrophic factor and its application, belonging to the field of medical biotechnology. Background technique [0002] Glial cell derived neurotrophic factor (GDNF) is a neurotrophic factor with neurotrophic and neuroprotective effects. Research experiments in various clinical animal models have confirmed that GDNF can promote dopaminergic neurons, motor Survival of neurons and reconstruction of synapses after injury. However, since GDNF is a macromolecular protein, it is difficult to pass through the blood-brain barrier to local diseased brain tissue, which has become the main reason hindering its clinical application. [0003] Neural stem cells have the ability to self-proliferate and differentiate into neurons and glial cells, which brings hope for cell therapy of neurodegenerative diseases. Experimental studies on neural stem cell transplantation have confirmed that neural ste...

Claims

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Application Information

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IPC IPC(8): C12N15/86A61K48/00A61P25/00A61P25/16
Inventor 王淑艳关云谦任萍朱宛宛王旸徐艳玲张愚
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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