Application of KDM6 as target in preparation of medicine for improving early-stage neuroectoderm differentiation efficiency

A neuroectodermal and drug technology, applied in the field of biomedicine, can solve the problems of neuronal apoptosis and decay that cannot be rooted out, and achieve the effect of improving neuronal apoptosis and neuronal metabolism

Active Publication Date: 2021-11-05
SHENZHEN BEIKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This drug that improves brain cell metabolism by dilating blood v

Method used

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  • Application of KDM6 as target in preparation of medicine for improving early-stage neuroectoderm differentiation efficiency
  • Application of KDM6 as target in preparation of medicine for improving early-stage neuroectoderm differentiation efficiency
  • Application of KDM6 as target in preparation of medicine for improving early-stage neuroectoderm differentiation efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1, knocking out KDM6A / KDM6B in human embryonic stem cells (HUES8)

[0040] 1. Obtain KDM6A / KDM6B knockout human embryonic stem cells

[0041] The cell lines DKO1 and DKO2 in which both KDM6A / B genes are knocked out are obtained by Crispr / Cas9 technology, and the method steps are as follows:

[0042] (1) Design and synthesis of sgRNA and genotype primers

[0043] sgRNA design: Analyze the target gene sequence, screen for suitable target sites, and design one sgRNA for each target site. In general, targeting Cas9 to exons encoding functional protein domains by sgRNAs is more likely to abrogate gene function than purely targeting the 5′ exon.

[0044] In the KDM6A knockout, the nucleotide sequence of the sgRNA targeting the target gene includes:

[0045] sgRNA1-KDM6A: 5'-CCTGGGAGATAAAGCCACCA-3' (as shown in SED ID NO.1);

[0046] sgRNA2-KDM6A: 5'-ATCCTAATTCTGGCCAGTCC-3' (as shown in SED ID NO.2);

[0047] In the KDM6B knockout, the nucleotide sequence of the s...

Embodiment 2

[0083] Example 2, adding KDM6 inhibitor-GSKJ1 during early neural differentiation

[0084] 1. Materials

[0085] Control group: human embryonic stem cells (HUES8) differentiated to early neuroectoderm.

[0086] Positive group: human embryonic stem cells (HUES8) added GSK J1 and GSK126 during early neuroectodermal differentiation.

[0087] 2. Method

[0088] The steps of cell culture and differentiation were the same as in Example 1, wherein the control group was exactly the same, and the positive group was added with GSK J1 and GSK 126 from the 0th day to the seventh day of differentiation.

[0089] 3. Total RNA extraction and qRT-PCR detection

[0090] The specific operation steps are the same as in Example 1, and the qRT-PCR experimental results are as follows Figure 6 shown;

[0091] Depend on Figure 6 The qRT-PCR experiment results showed that the immunofluorescence results showed that the KDM6 inhibitor GSKJ1 increased the expression of SOX1, NESTIN and PAX6 in th...

Embodiment 3

[0095] Example 3, adding KDM6 inhibitor-GSKJ1 during early neural differentiation

[0096] 1. Materials

[0097] Control group: human induced pluripotent stem cells (WTC) differentiated to early neuroectoderm.

[0098] Positive group: human induced pluripotent stem cells (WTC) added GSK J1 and GSK 126 during early neuroectodermal differentiation.

[0099] 2. Method

[0100] The steps of cell culture and differentiation were the same as in Example 1, wherein the control group was exactly the same, and the positive group was added with GSK J1 and GSK 126 from the 0th day to the seventh day of differentiation.

[0101] 3. Total RNA extraction and qRT-PCR detection

[0102] The specific operation steps are the same as in Example 1, and the qRT-PCR experimental results are as follows Figure 8 shown;

[0103] Depend on Figure 8 The results of qRT-PCR experiments showed that the immunofluorescence results showed that the KDM6 inhibitor GSKJ1 increased the expression of SOX1, NE...

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Abstract

The invention provides application of KDM6 as a target in preparation of a medicine for improving early-stage neuroectoderm differentiation efficiency. According to the application, the deletion of demethylase KDM6 of H3K27me3 can promote the improvement of the differentiation efficiency of human embryonic stem cells to the early neuroectoderm; and meanwhile, the research finds that the differentiation efficiency can also be improved by using a KDM6 small-molecule inhibitor (GSK-J1) in the early neuroectoderm differentiation process. Therefore, a KDM6A knockout reagent, a KDM6B knockout reagent and a KDM6 inhibitor can promote early generation of neurons by improving early neural differentiation efficiency and improve neuron apoptosis (not improving existing neuron metabolism), and are potential novel drugs for neurodegenerative diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application of KDM6 as a target in the preparation of drugs for improving the differentiation efficiency of early neuroectoderm. Background technique [0002] Degenerative neurological disease (Neurodegenerative disease) seriously affects people's daily life, and it affects millions of people around the world. These include epilepsy, Alzheimer's disease (AD), Parkinson's disease (Parkinson's disease), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), etc. All are common degenerative neurological diseases that seriously affect people's quality of life. Degenerative neurological diseases have varying degrees of damage to neurons and myelin sheaths. At present, the main role of therapeutic drugs for degenerative diseases is to delay the progress of the disease by improving brain metabolism. For example, Vinpocetine is a cerebral vasodilator , mainly by inhibi...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/7088A61K31/55A61P25/08A61P25/28A61P25/16A61P25/14A61P25/00
CPCA61K45/00A61K31/7088A61K31/55A61P25/08A61P25/28A61P25/16A61P25/14A61P25/00
Inventor 蒋卫孟雅婧
Owner SHENZHEN BEIKE BIOTECH
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