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Application of 3,4-dihydroxybenzoic acid methyl ester in the preparation of drugs for inducing neural stem cell/neural precursor cell directed differentiation

A technology of methyl dihydroxybenzoate and neural precursor cells, which is applied in the field of medicine, can solve problems such as no reports of directional differentiation of neural stem cells/neural precursor cells, and achieve avoidance of false positive results, good cell purity and activity , Guarantee the effect of purity and activity

Active Publication Date: 2020-06-16
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, it is reported that it has antioxidant activity and promotes the growth of neurons, but there is no report that it induces the directed differentiation of neural stem cells / neural precursor cells

Method used

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  • Application of 3,4-dihydroxybenzoic acid methyl ester in the preparation of drugs for inducing neural stem cell/neural precursor cell directed differentiation
  • Application of 3,4-dihydroxybenzoic acid methyl ester in the preparation of drugs for inducing neural stem cell/neural precursor cell directed differentiation
  • Application of 3,4-dihydroxybenzoic acid methyl ester in the preparation of drugs for inducing neural stem cell/neural precursor cell directed differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Morphological differentiation effect of MDHB on neural stem / precursor cells

[0031] 1. Culture of rat hippocampal neural stem / precursor cells:

[0032]Get the required number of newborn SD rats P0-2 (Guangdong Provincial Animal Center), soak them in 75% (v / v) ethanol solution for 30s, cut off the head skin and skull, take out the rat brain, put the brain in In the HBSS buffer solution, take out the rat hippocampus, remove the meninges on the hippocampus, move the hippocampus into a penicillin bottle containing 3ml HBSS solution, cut it to 1mm with ophthalmic scissors, add 2ml 0.25% (w / v) to the bottle Trypsin + EDTA, pipetting gently until mixed, put into cell culture incubator (37°C, 5% carbon dioxide, constant humidity) for digestion for 15min. Stop the digestion with 10% (v / v) FBS+DMEM / F12, pipette gently several times, then add 900U DNaseI (deoxyribonuclease I) to remove DNA, pipette gently several times, so that the solution is fully mixed with DNaseI (...

Embodiment 2

[0037] Example 2 MDHB improves the differentiation of neural stem / precursor cells into neuron-specific antibody Tuj1 (axon-specific protein), and reduces the expression of glial cell-specific antibody GFAP (glial fibrillary acidic protein)

[0038] The night before, plate 12mm cell slides (neural stem / precursor cell slides) in a 24-well plate, add 500 microliters of 0.0125g / ml PLL to plate overnight, suck out the PLL the next day, and wash once with 1×PBS for 2 minutes , and then add 100 microliters of 10 μg / ml lamnin (laminin) to each slide for 2 hours, inoculate the digested neural stem / precursor cells on the slide, inoculate 30,000 cells per well, and use DMEM / F12+ Suspend the cells in 10% (v / v) FBS, then put them into the cell culture incubator and incubate for 60min, the cells adhere to the wall, discard the complete medium, add the differentiation medium, and add in proportion (0μM, 8μM, 16μM and 32μM) For MDHB, replace the medium every other day and a half, suck out the...

Embodiment 3

[0039] Example 3 MDHB enhances immunofluorescent staining of neuron-specific antibody MAP2 (microtubule-associated protein 2) during neural stem / precursor cell differentiation.

[0040] The night before, 12mm cell slides were plated in a 24-well plate, and 500 microliters of 0.0125g / ml PLL was added to plate overnight. The next morning, the PLL was aspirated, washed twice with 1×PBS for 2 minutes each time, and 100 μl of PLL was added to the slides. Incubate 10 μg / ml lamnin in the incubator for 2 hours, then inoculate the digested neural stem / precursor cells on the slide, inoculate 30,000 cells per well, suspend the cells with DMEM / F12+10% FBS and put them into the cells Incubate in the incubator for 60 min, the cells adhere to the wall, discard the complete medium, add the differentiation medium, and add MDHB in proportion (0 μM, 8 μM, 16 μM and 32 μM), change the medium every other day, suck out the medium after 7 days of action, add Wash with pre-cooled 1×PBS twice, 3 min e...

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Abstract

The invention discloses application of 3,4-methyl dihydroxybenzoate (MDHB) in preparation of drugs for inducing directional differentiation of neural stem cells or neural precursor cells. The invention discovers that MDHB has the effect of directionally inducing differentiation of neural stem cells or neural precursor cells to cholinergic neurons, can be used for developing drugs for curing related diseases (SCI, AD and the like) of cholinergic neuron damage and loss, can be used for cell transplantation therapy of related diseases (SCI, AD and the like) of cholinergic neuron damage and loss, and can be used for developing directional inducers and the like of neural stem cells or neural precursor cells. The new pharmacological activity of MDHB is determined, the illumination of a pesticide effect mechanism provides reference for developing new drugs with proprietary intellectual property rights, MDHB is applied to basic experiments in the field of neurosciences, and a novel method is provided for searching models of cholinergic neurons.

Description

technical field [0001] The invention belongs to the field of medicine, and particularly relates to the application of methyl 3,4-dihydroxybenzoate in the preparation of drugs for inducing neural stem cell / neural precursor cell directed differentiation. Background technique [0002] Cholinergic neurons are widely distributed in the central nervous system, such as motor neurons in the anterior horn of the spinal cord, including the collateral branches of their axons that emanate from the intercalated cells, and the specific sensory projection neurons in the ventral posterior part of the thalamus are all choline cholinergic neurons, each link of the ascending activating system of the brainstem reticular structure, the striatum, the basal ganglia of the forebrain, the piriform area of ​​the limbic system, the amygdala, and the hippocampus all contain cholinergic neurons. The damage and death of cholinergic neurons in different parts lead to many important neurological diseases, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793A61K35/30A61P25/00A61P25/28
CPCA61K35/30C12N5/0619C12N2501/999
Inventor 罗焕敏潘君平信怡榕胡阳米象男王嘉惠高勤
Owner JINAN UNIVERSITY
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