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Application of thymidylate synthase inhibitor in preparation and construction of mouse model with NTDs (neural tube defects)

A thymidylate synthase and neural tube deformity technology, which is applied in the field of applied basic medical research to achieve the effects of stable effect, low fetal absorption lethality and high neural tube deformity rate

Active Publication Date: 2015-07-29
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 present invention uses thymidylate synthase inhibitors to intervene in the metabolism of thymidine deoxynucleoside (dTMP) in pregnant mice, and induces the construction of NTDs mouse models. So far, there have been no relevant reports at home and abroad.

Method used

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  • Application of thymidylate synthase inhibitor in preparation and construction of mouse model with NTDs (neural tube defects)
  • Application of thymidylate synthase inhibitor in preparation and construction of mouse model with NTDs (neural tube defects)
  • Application of thymidylate synthase inhibitor in preparation and construction of mouse model with NTDs (neural tube defects)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] "Example 1" Thymidylate Synthase Inhibitor Preparation and Construction of Neural Tube Defect Mouse Model

[0032] 1. Experimental animals and materials

[0033] SPF grade adult C57BL / 6J mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., China, license number: SCXK (Beijing) 2006-0009), 7-8 weeks old, weighing 19-20g, fed for more than a week after purchase Adapt to the environment of SPF level animal room. The temperature of the animal room is controlled at 18-29°C; relative humidity: 50%-80%; number of air changes: 10-20 times / h; air velocity <0.18m / s; air cleanliness: equivalent to 10,000 grades. Feed feeding (purchased from Beijing Huafukang Biotechnology Co., Ltd.), regular drinking water. 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...

Embodiment 2

[0045] "Example 2" Determination of Thymidylate Synthase Activity and Biochemical Index Detection of Folic Acid Metabolism

[0046] 2.1 Extraction of total tissue protein and determination of protein concentration

[0047] Refer to the kit (CelLytic MTTM Cell Lysis Reagent) operating instructions to extract the protein in the brain tissue of the fetal rats in the normal group and the experimental intervention group. Weigh an appropriate amount of fetal mouse brain tissue sample, add an appropriate amount of CelLytic MT reagent, transfer it to a pre-cooled grinder and grind it, transfer it to an EP tube, centrifuge at low temperature for 10 minutes, and take the supernatant to determine its protein by Bradford method concentration.

[0048] Dissolve protein standards to a final concentration of 0.5 mg / ml. Add 0, 1, 2, 4, 8, 12, 16, 20 ul of the standard to the standard wells of the 96-well plate, and add standard diluent to make up to 20 ul. Add an appropriate volume of samp...

Embodiment 3

[0054] 《Example 3》Pathological Morphology Inspection

[0055] 3.1 Paraffin embedding and sectioning

[0056] 1. Dehydration and transparency

[0057] Take out the fetal mice in the fixative solution, dehydrate and clear, and dehydrate step by step through low to high concentration ethanol: 50% alcohol → 70% alcohol → 85% alcohol → 95% alcohol → 95% alcohol → 100% alcohol.

[0058] Transparent: xylene I, xylene II (pure xylene), observe the condition of the specimen in the experiment until the specimen is completely transparent.

[0059] 2. Wax penetration and embedding

[0060] Use paraffin wax with a melting point of 52-60°C, and put the transparent tissue block in 1:1 xylene and paraffin to infiltrate overnight at 37°C, then adjust the temperature to 58°C and change to pure paraffin to continue infiltration, usually at intervals of 3 hours Pure paraffin once, until there is no smell of xylene, the subsequent embedding can be carried out, the treated fetal mouse is put int...

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Abstract

The invention relates to an application of a thymidylate synthase inhibitor in preparation and construction of a mouse model with NTDs (neural tube defects). Specifically, the thymidylate synthase inhibitor is used for intervening a pregnant mouse to cause dysbolism of thymidine monophosphate (dTMP) and dysplasia of a mouse embryo, so that the mouse model with the NTDs is constructed. The application is high in feasibility, easy to operate, good in repeatability, stable in effect and high in teratogenicity rate, the mouse model close to the pathogenesis of human NTDs is simulated through inhibition of metabolism of dTMP, and good animal model is provided for further research of birth defects, especially for research of the mechanism of all molecules and cells associated with the NTDs.

Description

Technical field: [0001] The invention belongs to the field of applied basic medical research, and relates to the application of a thymidylate synthase inhibitor to prepare and construct a neural tube defect mouse model. Background technique: [0002] Neural Tube Defects (NTDs) refer to the abnormalities of the brain and spine caused by incomplete closing of the neural tube in the early stages of embryonic development. Cerebral or meningocele, spina bifida, etc., the mechanism of its occurrence is still unclear. Constructing an animal model with human NTDs disease-simulating manifestations will help to understand the occurrence and development of human NTDs more conveniently and effectively, and understand its etiology, pathogenesis, and pathological characteristics, which is of great significance for research on prevention and control measures. Folic acid deficiency in early pregnancy is an important risk factor for the occurrence of NTDs, supplementing 0.4-4.0mg / d of folic...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/513
Inventor 官臻牛勃张霆王秀伟王建华朱智强王芳
Owner 首都儿科研究所
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