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Method for promoting hippocampus neural stem cells to differentiate into cholinergic neurons

A technology for cholinergic neurons and neural stem cells, which is applied in the field of cell culture and can solve the problems of unclear mechanism of action of Lhx8

Pending Publication Date: 2020-10-30
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above studies have shown that Lhx8 plays an important role in the differentiation of cholinergic neurons, but the specific mechanism of Lhx8 in this process is still unclear

Method used

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  • Method for promoting hippocampus neural stem cells to differentiate into cholinergic neurons
  • Method for promoting hippocampus neural stem cells to differentiate into cholinergic neurons
  • Method for promoting hippocampus neural stem cells to differentiate into cholinergic neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: The hippocampal neural stem cells can be prepared by the following method:

[0026] 1 primary culture

[0027] (1) After intraperitoneal anesthesia of pregnant SD mice, disinfect the abdomen with alcohol cotton balls, expose the abdominal cavity, and take out the embryos;

[0028] (2) Isolating embryonic mouse hippocampal primordium in a sterile culture dish;

[0029] (3) Transfer the isolated hippocampus tissue to a 15ml centrifuge tube, add basal culture medium and gently blow and centrifuge (1200rpm, 5min);

[0030] (4) Discard the supernatant, add basal culture medium and centrifuge (1200rpm, 5min) after gently pipetting, repeat this step once;

[0031] (5) Discard the supernatant, add NSCs culture medium and pipette gently;

[0032] (6) Cell filter (40 μm) filter the cell suspension into a 50ml centrifuge tube;

[0033] (7) Inoculate into T25 culture flask after forming cell suspension again;

[0034] (8) After 5-6 days, larger and more neurospheres...

Embodiment 2

[0050] Example 2: The method of inducing hippocampal NSCs to differentiate into neurons is as follows (the method of infecting NSCs with GAS5 overexpression lentivirus):

[0051] (1) GAS5 overexpression lentivirus infection of hippocampal NSCs

[0052] 1 Determination of the best MOI value

[0053] (1) Set five concentration gradients with MOI values ​​of 5, 10, 20, 50, and 100;

[0054] (2) with 5×10 4 Density of cells / well Inoculate hippocampal NSCs in 24-well culture plate, culture with NSCs culture medium, set 3 duplicate wells for each gradient, add negative control virus (CON238) into the wells at the concentration calculated according to the corresponding MOI value, 16h Afterwards, the virus was removed, and fresh NSCs culture medium was replaced to continue culturing for 3 days; the GAS5 overexpression lentivirus was designed and synthesized by Shanghai Jikai Company (product number 41264-2), and the control negative virus was CON238

[0055] (3) The EVOSFLAuto auto...

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Abstract

The invention discloses a method for promoting differentiation of hippocampal neural stem cells to cholinergic neurons, comprising the following steps of: separating hippocampus NSCs, inoculating, culturing with an NSCs culture solution, adding viruses, removing the NSCs culture solution after a certain period of time, and replacing with a fresh NSCs culture solution to continue culturing; whereinthe virus is a GAS5 overexpression lentivirus; and the MOI value of the added virus is 5-100. According to the invention, the expression quantity of GAS5 can be remarkably increased, and the proportion of hippocampus neural stem cells differentiating to cholinergic neurons can be remarkably increased.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for promoting the differentiation of hippocampal neural stem cells into cholinergic neurons. Background technique [0002] The learning and memory functions of mammals are closely related to the cholinergic nervous system, including Alzheimer's disease (AD), Parkinson's disease (Parkinson's disease, PD), vascular dementia and traumatic brain injury (Traumatic brain injury). Injury (TBI) and other central nervous system (CNS) lesions are accompanied by varying degrees of cholinergic nervous system dysfunction, and patients mainly suffer from cognitive dysfunction mainly due to learning and memory impairment, which poses a serious threat to the family and society. caused a serious burden. Therefore, it is of great significance to study the key regulatory factors in the development and differentiation of cholinergic neurons and actively promote the regener...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/113C12N5/0797
CPCC12N15/86C12N15/113C12N5/0623C12N2740/15043C12N2800/106C12N2310/11C12N2330/51C12N2509/00
Inventor 赵荷艳张蕾
Owner NANTONG UNIVERSITY
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