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Application of naloxone in drug for regulating and controlling activity of DNA demethylase of Tet1 protein

A technology of methylase activity and naloxone, applied in the field of neurogenesis, can solve problems such as insignificant improvement, low input-output ratio, and impairment of spatial learning and memory abilities

Inactive Publication Date: 2018-11-16
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the input-output ratio of this method is not high, and the improvement obtained by spending a lot of time and energy is not obvious
In addition, this method has little effect on the impairment of spatial learning and memory ability caused by aging, drugs and diseases
2) Moderate stress (stress) can also have a positive impact on adult neurogenesis and spatial learning and memory ability, but too high pressure will have a negative impact on adult neurogenesis and spatial learning and memory ability
4) Some drugs have a certain neuroprotective effect, which can delay the decline of adult neurogenesis and spatial learning and memory ability affected by aging, drugs and diseases, but it is difficult to improve adult neurogenesis and spatial learning and memory ability under normal conditions
In addition, naloxone can also promote adult neurogenesis and improve the ability of spatial learning and memory, but its mechanism of action is still unknown

Method used

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  • Application of naloxone in drug for regulating and controlling activity of DNA demethylase of Tet1 protein
  • Application of naloxone in drug for regulating and controlling activity of DNA demethylase of Tet1 protein
  • Application of naloxone in drug for regulating and controlling activity of DNA demethylase of Tet1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Naloxone inhibits the DNA demethylation ability of Tet1CD

[0068] Two groups of controls were set up in this experiment: cells not transfected with the plasmid and cells transfected with the Flag plasmid of the control group. When the 293FT cells covered about 60% of the entire culture dish, transfect the pMX-Tet1CD plasmid and the Flag plasmid of the control group, change the medium after 12 hours, and continue to culture the cells for 3 days. Collect the cells that were not transfected with the plasmid, the cells transfected with the control plasmid, and the cells transfected with pMX-Tet1CD, extract the nuclear protein from them according to Experimental Technique 6, measure the protein concentration and prepare the reaction system. There are 6 groups of reactions in this experiment: a. Untransfected nuclear protein, b. Untransfected nuclear protein + naloxone, c. Transfected control plasmid nuclear protein, d. Transfected control plasmid nuclear protein ...

Embodiment 2

[0070] Example 2: Naloxone cannot affect the proliferation of Tet1 knockout neural stem cells

[0071] Experimental technique 8 was used to induce the differentiation of wild-type and Tet1 knockout embryonic stem cells into neural stem cells, and the selected neural stem cells were expanded for culture. Neural stem cells were planted in a 6cm cell culture dish at a density of 300,000 / dish, cultured with normal cell culture medium and medium containing 10 μM naloxone, collected cells after 3 days, and counted cells with a hemocytometer after digestion.

[0072] Such as Figure 4 As shown, the proliferation rate of Tet1-knockout neural stem cells was significantly slower than that of wild-type neural stem cells; when naloxone was added to the wild-type neural stem cell culture process, the number of cells was about 40% more than that of the control group; in Tet1 knockout neural stem cells After adding naloxone, the cell number did not change substantially. It shows that the p...

Embodiment 3

[0073] Example 3: Naloxone cannot affect the differentiation of Tet1 knockout neural stem cells

[0074] Spread the slides into a 24-well plate, add Matrigel and place in a 37°C incubator for 30 minutes. During the cell digestion and counting. After aspirating and discarding Matrigel, wild-type and Tet1 knockout neural stem cells were planted in 24-well cell culture plates at a density of 60,000 / well, and normal differentiation medium (control group) and differentiation medium containing 10 μM naloxone ( (experimental group) to induce neural stem cell differentiation, and after 2 weeks, the cells were fixed and stained. Nestin-positive cells represent neural stem cells, GFAP-positive cells represent glial cells, Tuj-positive cells represent neurons, O4-positive cells represent oligodendrocytes, and the total number of cells is represented by the number of DAPI.

[0075] Such as Figure 5 As shown, compared with the normal group, the proportion of neurons obtained after diff...

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Abstract

The invention relates to application of naloxone in a drug for regulating and controlling the activity of DNA demethylase of Tet1 protein. The invention further provides application of the inhibitionaction of the naloxone for the activity of the DNA demethylase of the Tet1 protein in preparation of a drug for improving the spatial learning memory ability. The invention further provides application of the Tet1 in mediation of the naloxone to promote nerves to take actions. The naloxone can effectively enhance the multiplication of neural stem cells and promote the neural stem cells to be differentiated towards neurons by inhibiting the activity of the DNA demethylase of the Tet1.

Description

technical field [0001] The present invention relates to the field of neurogenesis, specifically, the present invention relates to the application of naloxone in the medicine for regulating the activity of Tet1 protein DNA demethylase; using naloxone to change DNA methylation in neural stem cells, and thereby regulate Methods of Proliferation and Differentiation of Neural Stem Cells. Background technique [0002] The original theory was that new nerve cells do not grow in adult animals. However, with the advancement and deepening of scientific research, researchers have discovered that neural stem cells exist in adult animals. Neural stem cells can differentiate into different types of nerve cells under appropriate conditions, thereby supplementing lost nerve cells in the course of life or providing support for a variety of advanced neural activities. The process of differentiation of neural stem cells into different types of nerve cells in adult animals is generally called...

Claims

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Application Information

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IPC IPC(8): A61K31/485A61P25/28
CPCA61K31/485A61P25/28
Inventor 郑辉梁丽宁陈金龙
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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