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Application of DNA methyltransferase inhibitor to construction of neural tube defect mouse model

A technology for methyltransferase and neural tube defects, applied in the field of applied basic medicine, can solve problems such as no relevant reports, and achieve the effects of simple and easy operation, stable effect, and high rate of neural tube defects.

Inactive Publication Date: 2019-05-21
首都儿科研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DNA methyltransferase inhibitor described in the present invention is used for preparing and constructing the mouse model of neural tube defects, and no relevant reports have been seen so far

Method used

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  • Application of DNA methyltransferase inhibitor to construction of neural tube defect mouse model
  • Application of DNA methyltransferase inhibitor to construction of neural tube defect mouse model
  • Application of DNA methyltransferase inhibitor to construction of neural tube defect mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The establishment of embodiment 1 experimental animal model

[0036] The use of experimental animals follows the Order of the Ministry of Health of the People's Republic of China (No. 55) - Implementation Rules for the Management of Medical Experimental Animals, and the regulations of the Medical Experimental Animal Management Committee of the Capital Institute of Pediatrics. 7-8 weeks, SPF-grade C57BL / 6J mice weighing 19-21g were fed with breeding feed and drank water regularly. At 6:00 p.m., the male and female rats were caged together at a ratio of 1:1, and those who found the vaginal tie in the next morning were recorded as 0.5 days pregnant. The pregnant rats were randomly divided into experimental groups and control groups. At 7.5-8.5 days pregnant, the experimental The pregnant mice in the control group were given intraperitoneal injection of 5-azacitidine solution in the dose range of 0.5mg / kg-3mg / kg; the pregnant mice in the normal control group were given the ...

Embodiment 2

[0038] Embodiment 2 Pathomorphological examination

[0039] 2.1 Tissue block embedding and sectioning

[0040] 1) Take out the fixed embryo tissue and wash it with running water overnight.

[0041] 2) Alcohol gradient dehydration: the tissue blocks were placed in 70%, 80%, 90%, 95%, 100%, and 100% alcohol for 1.5 hours each.

[0042] 3) Transparency in xylene: place the dehydrated tissue block in xylene I for 30 minutes, and xylene II for 30 minutes until the specimen is completely transparent.

[0043] 4) Wax immersion of the tissue block: immerse the transparent embryonic tissue in melted paraffin wax I for 2 hours, and paraffin wax II for 2 hours;

[0044] 5) Tissue block embedding: put the wax-soaked embryonic tissue block vertically into the embedding box with the cut side down, add melted 100% paraffin, quickly adjust the position of the tissue block with tweezers, and place it flat on ice to make the paraffin wax Solidifies quickly.

[0045] 6) Slicing: Place the ti...

Embodiment 3

[0059] Example 3 Detection of DNA methyltransferase activity The standard product in the kit was diluted according to the experimental requirements, and blank wells, standard wells, and sample wells to be tested were respectively set. Accurately add 50 μl of the standard substance on the enzyme-labeled plate, add 40 μl of the sample diluent to the well of the sample to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the microplate well, try not to touch the well wall, shake gently to mix. Seal the plate with a sealing film and incubate at 37°C for 30 min. Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry. Add 50 μl of enzyme-labeled reagent to each well, except for blank wells. Incubate, wash. First add 50 μl of chromogenic reagent A to each well, then ad...

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Abstract

The invention relates to a construction method for a mouse neural tube defect model. When the neural tube of a pregnant mouse fetus grows, a DNA methyltransferase inhibitor is injected into a pregnantmouse, then, the fetus of the mouse has methylated metabolism abnormity, and accordingly the neural tube defect mouse mode is established; the mouse model can be used for biomedical research on neural tube defects, is beneficial to research on the nosetiology and pathogenesis of the neural tube defects, and is especially provided as a good animal model for research on all molecules and cells related to the neural tube defects. The method is easy to operate, high in feasibility, stable in effect and high in teratogenesis rate.

Description

Technical field: [0001] The invention belongs to the field of applied basic medical research, and relates to a method for preparing a neural tube defect mouse model, in particular to the application of a DNA methyltransferase inhibitor, and a method for using the inhibitor to construct a neural tube defect mouse model . Background technique: [0002] Neural tube defects are one of the most common and serious birth defects in humans. It is a group of defects caused by incomplete closure of the neural tube during embryonic development, including anencephaly, encephalocele and spina bifida. , Although a large number of factors related to the occurrence of neural tube defects have been discovered, most of their mechanisms of action are not very clear, which brings great difficulties to the targeted prevention and treatment of neural tube defects. Pathogenesis-based Research on prevention and treatment has also been severely limited. Constructing a suitable animal model of neur...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/706A01K67/027
Inventor 王秀伟官臻王建华牛勃张霆吴建新朱智强郭金王芳
Owner 首都儿科研究所
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