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NTDs (neural tube defects) rat embryo animal model and constructing method thereof

A technology of animal models and construction methods, applied in the direction of animal cells, vertebrate cells, embryonic cells, etc.

Inactive Publication Date: 2017-11-21
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies in recent years have found that 30-50% of NTDs cannot be prevented by folic acid

Method used

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  • NTDs (neural tube defects) rat embryo animal model and constructing method thereof
  • NTDs (neural tube defects) rat embryo animal model and constructing method thereof
  • NTDs (neural tube defects) rat embryo animal model and constructing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Retinoic acid intervention to construct a C57BL / 6 mouse embryo NTDs model.

[0040] At 18:00 p.m., the C57BL / 6 mice were mated in a cage with a male-to-female ratio of 2:1, and the vaginal plug was taken out at 8:00 the next morning to check the vaginal plug. The day when the vaginal plug was found at 12:00 noon was defined as 0.5 days pregnant (E0.5d ).

[0041] The obtained pregnant mice were randomly divided into a control group (normal group) and an experimental group (NTDs group). On E7.5d, the pregnant mice in the experimental group were given 19-23 mg / kg of retinoic acid solution prepared with sesame oil by intragastric administration. model; control group of pregnant mice given the same volume of sesame oil.

[0042] On E10.5d, the pregnant mice were sacrificed by cervical dislocation, the abdomen was wiped with 75% ethanol, a small piece of skin was pinched, cut along the midline of the abdomen to the level of the nipple line, and then cut along bot...

Embodiment 2

[0047] Example 2: Culture of NTDs mouse embryonic NSCs.

[0048] The brain vesicle tissues of E10.5d mouse embryos in the normal group and the NTDs group were separated and transferred to 15ml centrifuge tubes. Add a small amount of DMEM / F12 medium, blow and suck repeatedly with a 1ml pipette until no tissue pieces can be seen with the naked eye, filter with a 200-mesh filter, centrifuge at 1000rpm for 5min, and discard the supernatant. Add NSCs proliferation medium (DMEM / F12 medium supplemented with 2% B27, 20ng / ml EGF and 20ng / ml bFGF), mix well by pipetting, and adjust the cell concentration to 1×10 6 / ml, inoculated in culture flasks, placed at 37℃ / 5%CO 2 Incubator suspension culture.

[0049] figure 2 It was shown that after culture, the proliferation ability of NSCs in normal group and NTDs group was significantly different. The NSCs in the normal group could form cell colonies on the 4th day of primary culture, and the colonies increased significantly on the 6th da...

Embodiment 3

[0050] Example 3: Identification of NTDs mouse embryonic NSCs.

[0051] Adjust the concentration of NSCs in normal group and NTDs group to 1×10 5 / ml, inoculated in a 24-well plate coated with polylysine, 1ml per well, placed at 37°C / 5% CO 2 Proliferate for 24 hours in the incubator. Using conventional immunofluorescence operation method, Nestin antibody was selected for identification of NSCs (anti-Nestin: 1:500).

[0052] From image 3 It can be seen from the results that, similar to NSCs of normal mouse embryos, the cells isolated and cultured from brain vesicles of NTDs mouse embryos also express Nestin protein, which proves that they are NSCs.

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Abstract

The invention discloses an NTDs (neural tube defects) rat embryo animal model and a constructing method thereof. Normal pregnant rats which have been pregnant for 6 to 10 days are injected with 19-23 mg / kg of retinoic acid at one time for intragastric administration intervention, then construction of the stable and efficient NTDs rat embryo animal mode can be induced rapidly after pregnant 10.5 days, and further, rat embryo brain vesicle tissue is separated out for cell culture while an NTDs cell model can be built. The animal model and the cell model constructed by the method can provide good platforms for research on NTDs molecular mechanism and cellular mechanism in vivo and vitro.

Description

technical field [0001] The invention relates to a method for constructing an animal model, in particular to a method for constructing an NTDs mouse embryo animal model, and the NTDs mouse embryo animal model and cell model constructed by the method. Background technique [0002] Neural tube defects (NTDs) are the most common birth defects in humans, and the incidence rate ranks second. [0003] NTDs are a type of congenital malformation caused by neural tube insufficiency during embryonic development. The main manifestations are abnormalities of the brain and spinal cord. Clinically, congenital anencephaly and spina bifida are common, but its pathogenesis is still unclear. [0004] There are 300,000 newborns suffering from it every year in the world, and the incidence rate is about 0.5-2 per thousand. The incidence rate is higher in developing countries, as high as 13.9‰ in some areas of Shanxi Province, which seriously affects the quality of the population, but also brings...

Claims

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Application Information

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IPC IPC(8): A01K67/02C12N5/073
CPCA01K67/02A01K2207/20A01K2227/105A01K2267/0356C12N5/0603
Inventor 解军于娟李仁科张霆徐钧刘志贞杨丽红刘丹靳宁
Owner SHANXI MEDICAL UNIV
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