阅读说明：本技术 Kdm6作为靶标在制备用于提高早期神经外胚层分化效率的药物中的应用 (Application of KDM6 as target in preparation of medicine for improving differentiation efficiency of early neuroectoderm ) 是由 蒋卫 孟雅婧 于 2021-08-03 设计创作，主要内容包括：本发明提供了KDM6作为靶标在制备用于提高早期神经外胚层分化效率的药物中的应用。本申请发现,H3K27me3的去甲基化酶KDM6的缺失可以促进人胚胎干细胞向早期神经外胚层分化效率的提高；同时,研究发现在早期神经外胚层分化过程中使用KDM6小分子抑制剂(GSK-J1)也可以提高其分化效率。因此,KDM6A的敲除试剂、KDM6B的敲除试剂和KDM6抑制剂可以通过提高早期神经分化效率从而促进神经元的早期生成,改善神经元凋亡(而不是改善已有神经元代谢),是一种潜在的神经退行性疾病新型药。(The invention provides application of KDM6 as a target in preparation of a medicament for improving differentiation efficiency of early neuroectoderm. The application discovers that the deletion of demethylase KDM6 of H3K27me3 can promote the efficiency of differentiation of human embryonic stem cells to early neuroectoderm to be improved; meanwhile, research finds that the KDM6 small-molecule inhibitor (GSK-J1) can be used for improving the differentiation efficiency in the early neuroectodermal differentiation process. Therefore, the KDM6A knockout reagent, the KDM6B knockout reagent and the KDM6 inhibitor can promote the early generation of neurons by improving the early neural differentiation efficiency, improve the neuron apoptosis (rather than improve the existing neuron metabolism), and are potential novel medicines for neurodegenerative diseases.)

Use of KDM6 as a target in the manufacture of a medicament for increasing the efficiency of differentiation of early neuroectoderm.

2. The agent according to claim 1, wherein the agent for increasing the differentiation efficiency of early neuroectoderm is an agent for treating a degenerative disease.

3. The use according to claim 1, wherein the agent for increasing the efficiency of differentiation of early neuroectoderm is an agent that promotes the efficiency of differentiation of human embryonic stem cells or human induced pluripotent stem cells into early neuroectoderm.

4. The use according to any one of claims 1 to 3, wherein the medicament for increasing the efficiency of early neuroectodermal differentiation comprises at least one of the following components:

a knock-out agent of KDM 6A;

a knock-out agent of KDM 6B;

inhibitors of KDM 6.

5. The use of claim 4, wherein the KDM6A knockout agent comprises: the sgRNA of the target gene is targeted, and the nucleotide sequence of the sgRNA is shown in SED ID NO.1 or/and SED ID NO. 2.

6. The use of claim 4, wherein the KDM6A knockout agent comprises: the sgRNA of the target gene is targeted, and the nucleotide sequence of the sgRNA is shown in SED ID NO.3 or/and SED ID NO. 4.

7. The use of claim 4, wherein the inhibitor of KDM6 comprises: GSK-J1, or grnas and/or shrnas that inhibit KDM6 expression.

Use of a knockout reagent of KDM6A/KDM6B or/and an inhibitor of KDM6 in the preparation of a medicament for increasing the efficiency of differentiation of early neuroectoderm.

9. A medicament for increasing the efficiency of early neuroectodermal differentiation, comprising at least one of the following:

a knock-out agent of KDM 6A;

a knock-out agent of KDM 6B;

inhibitors of KDM 6.

Technical Field

The invention relates to the technical field of biomedicine, in particular to application of KDM6 as a target in preparation of a medicine for improving differentiation efficiency of early neuroectoderm.

Background

Degenerative neurological disease (neuro-degenerative disease) seriously affects people's daily lives, with the patients spreading over millions of people worldwide. These diseases include epilepsy (epilepsy), Alzheimer Disease (AD), Parkinson's disease, Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), and the like, which are common degenerative neurological diseases and seriously affect the quality of life of people. Degenerative neurological diseases have damage to neurons and myelin sheaths in different degrees, and treatment drugs for the current degenerative neurological diseases mainly have the effect of delaying the disease process by improving brain metabolism, for example, Vinpocetine (vinpocetin) is a cerebral vasodilator, and mainly increases vascular smooth muscle relaxation by enhancing c-GMP (phosphodiester kinase) through inhibiting phosphodiesterase activity, so that the cerebral blood flow is finally increased, the blood viscosity of a human body is reduced, the blood fluidity and microcirculation are improved, and the glucose uptake by brain tissues is promoted, so that the brain metabolism is improved. This drug that improves brain cell metabolism by dilating blood vessels cannot fundamentally solve neuronal apoptosis and decay, etc.

What is needed is a drug for improving differentiation efficiency of early neuroectoderm, which can promote early generation of neurons by improving differentiation efficiency of early neuroectoderm, improve apoptosis of neurons, and be used as a potential drug for treating degenerative diseases.

Disclosure of Invention

The invention aims to provide application of KDM6 as a target in preparation of a medicine for improving early neuroectodermal differentiation efficiency, and experiments show that targeting KDM6 (deletion) or KDM6 inhibitor in human embryonic stem cells or human induced pluripotent stem cells can improve the early neuroectodermal differentiation efficiency.

In a first aspect of the invention, there is provided the use of KDM6 as a target in the manufacture of a medicament for increasing the efficiency of differentiation of early neuroectoderm.

Further, the drug for improving differentiation efficiency of early neuroectoderm is a drug for treating a degenerative disease.

Further, the drug for improving the differentiation efficiency of early neuroectoderm is a drug for promoting the differentiation efficiency of human embryonic stem cells or human induced pluripotent stem cells into early neuroectoderm.

Further, the medicament for improving the early neuroectodermal differentiation efficiency includes at least one of the following components:

a knock-out agent of KDM 6A;

a knock-out agent of KDM 6B;

inhibitors of KDM 6.

Further, the KDM6A knock-out agent comprises: the nucleotide sequence of the sgRNA targeting the target gene is shown in SED ID NO.1 and SED ID NO. 2.

Further, the KDM6B knock-out agent comprises: the nucleotide sequence of the sgRNA targeting the target gene is shown in SED ID NO.3 and SED ID NO. 4.

Further, the KDM6 inhibitors include: GSK-J1, or grnas and/or shrnas that inhibit KDM6 expression.

In a second aspect of the invention, there is provided the use of a knockout agent for KDM6A/KDM6B or/and an inhibitor of KDM6 in the preparation of a medicament for increasing the efficiency of differentiation of early neuroectoderm.

In a third aspect of the present invention, there is provided a medicament for increasing the efficiency of differentiation of early neuroectoderm, the medicament comprising at least one of the following components:

a knock-out agent of KDM 6A;

a knock-out agent of KDM 6B;

inhibitors of KDM 6.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides application of KDM6 serving as a target in preparation of a medicine for improving differentiation efficiency of early neuroectoderm, and researches show that deletion of KDM6 can promote improvement of differentiation efficiency of human embryonic stem cells to the early neuroectoderm, and because KDM6 family is demethylase of H3K27me3, researches show that KDM6 inhibitor (GSK-J1) can be used in differentiation process of the early neuroectoderm to improve differentiation efficiency. Therefore, the KDM6A knockout reagent, the KDM6B knockout reagent and the KDM6 inhibitor can promote the early generation of neurons by improving the early neural differentiation efficiency, improve the neuronal apoptosis (rather than improve the existing neuronal metabolism), and are potential novel medicines for degenerative diseases.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a schematic diagram of an embodiment of the present invention;

FIG. 2 is a schematic diagram of a cultivation process according to an embodiment of the present invention;

FIG. 3 shows the result of immunofluorescence assay of KDM6 showing the increase in the expression levels of SOX1, NESTIN and PAX6 during early neural differentiation after being administered to KO;

FIG. 4 shows the qRT-PCR results of the rising expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process after KO of KDM 6;

FIG. 5 shows the immunofluorescence results of KDM6 inhibitor GSKJ1 in human embryonic stem cells for increasing the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process, and EZH2 inhibitor GSK126 for hardly influencing the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process;

FIG. 6 shows the qRT-PCR results of KDM6 inhibitor GSKJ1 in human embryonic stem cells for increasing the expression level of SOX1 and NESTIN in the early neural differentiation process, and EZH2 inhibitor GSK126 for not influencing the expression level of SOX1 and NESTIN in the early neural differentiation process;

FIG. 7 shows the immunofluorescence results of KDM6 inhibitor GSKJ1 in human induced pluripotent stem cells for increasing the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process, and EZH2 inhibitor GSK126 for substantially not influencing the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process;

FIG. 8 shows the qRT-PCR results of KDM6 inhibitor GSKJ1 in human induced pluripotent stem cells for increasing the expression level of SOX1 and NESTIN in the early neural differentiation process, and EZH2 inhibitor GSK126 for not influencing the expression level of SOX1 and NESTIN in the early neural differentiation process.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

the first part is that KDM6A/KDM6B is knocked out in human embryonic stem cells (HUES8), differentiation of a knocked-out cell line to early neuroectoderm is induced, and the knock-out of KDM6A/KDM6B can promote differentiation of the early neuroectoderm;

the second part is that KDM6 inhibitor-GSKJ 1 is added in the early nerve differentiation process of human embryonic stem cells (HUES8) so as to promote the early nerve differentiation;

the third part is that KDM6 inhibitor-GSKJ 1 is added in the early nerve differentiation process of human induced pluripotent stem cells (WTC), thereby promoting the early nerve differentiation.

The use of KDM6 of the present application as a target for the preparation of a medicament for increasing the efficiency of differentiation of early neuroectoderm will be described in detail below with reference to examples and experimental data.

Example 1 knock-out of KDM6A/KDM6B in human embryonic Stem cells (HUES8)

1. Obtaining KDM6A/KDM6B gene knockout human embryonic stem cells

Cell lines DKO1 and DKO2 with KDM6A/B both of which are knocked out are obtained by a Crispr/Cas9 technology, and the method comprises the following steps:

(1) synthesis of sgRNA and genotype primers

sgRNA design: and analyzing the target gene sequence, screening appropriate target sites, and designing 1 sgRNA in each target site. Generally, targeting Cas9 to sgrnas encoding exons of functional protein domains is more likely to eliminate gene function than to target the 5' exon alone.

In the KDM6A knock-out, the nucleotide sequence of sgRNA targeting the target gene comprises:

sgRNA1-KDM 6A: 5'-CCTGGGAGATAAAGCCACCA-3' (shown as SED ID NO. 1);

sgRNA2-KDM 6A: 5'-ATCCTAATTCTGGCCAGTCC-3' (shown as SED ID NO. 2);

in the KDM6B knock-out, the nucleotide sequence of sgRNA targeting the target gene comprises:

sgRNA1-KDM 6B: 5'-AGGCTGGATGCATCGGGCAG-3' (shown as SED ID NO. 3);

sgRNA1-KDM 6B: 5'-CCGCATTGGCCGACTGCAGC-3' (shown as SED ID NO. 4);

(2) vector construction

Oligo DNA was synthesized based on the designed sgRNA sequence. Carrying out enzyme digestion on an empty vector (with a screening gene (puromycin)) with a Cas9 gene to obtain a linearized plasmid, mixing Oligo DNA with the linearized empty vector, connecting with T4 ligase, adding buffer and ddH₂O, 16 ℃ overnight.

The ligation product was transformed into DH 5. alpha. E.coli and cultured overnight at 37 ℃. And selecting a single colony for amplification, extracting plasmids, and carrying out enzyme digestion identification. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, selecting the enzyme digestion product with the correct size for sequencing verification, and carrying out plasmid extraction and quality detection.

(3) The verified plasmid was transferred to a new DH5a competent E.coli and cultured overnight at 37 ℃. And selecting a single colony for amplification culture, and extracting the plasmid. Detecting endotoxin; carrying out absorbance detection (the ratio of 260nm to 280nm is 1.8-2.0); and the integrity of the plasmid was checked by agarose electrophoresis.

(4) Cell transfection was performed: HUES8 cells that grew well were plated in 6-well plates 1 day before transfection and cultured overnight in a cell incubator to a cell density of 90% -95%. Reagents required for transfection were preheated in a 37 ℃ water bath (including Opti-MEM, plasmid).

The results are shown in table 1:

TABLE 1 KDM6 KO genotype

Cell line name	KDM6A deletion base	KDM6B deletion base
			DKO1	(del 10bp)	(del 753bp knock-out start codon)
DKO2	(del 53bp)	(del 728bp)

In other embodiments, other knockouts or knockouts may be used.

2. Cell culture and differentiation

Control group: human embryonic stem cells

Positive group: KDM6A/KDM6B gene knockout human embryonic stem cells obtained by the steps

After the control group and the positive group are subjected to cell culture cell passage, counting is carried out by a cell counting plate, and each hole of a 24-well plate is 2.0 multiplied by 10⁵And (4) carrying out cell culture, and placing the cells into a 37-degree incubator.

Early neuroectodermal differentiation (FIG. 2) N2B27 medium was prepared on days 0-4 and small molecules 2.5. mu.M SB431542(Selleck, # S1067) and 0.05. mu.M LDN193189(Selleck, # S7507) were added. The culture was performed with mTeSR1(Stemcell Technologies, #1000023391) on day-1.

N2B27 medium and small molecule 0.05. mu.M LDN193189(Selleck, # S7507) were used on days 5-7.

Early neuroectodermal cells were obtained on day 7.

The basal differentiation medium used from day 0 to day 7: KnockOut^TMDMEM/F12(Gibco, # 12660012); 0.5 XN 2 (Shanghai culture Biotechnology Co., Ltd., # M430721),0.5 XB-27 SUPPLEMENT W/O VIT A (Shanghai culture Biotechnology Co., Ltd., # B430805), 10% penillin-Streptomycin (Thermo, #15140163), 1% 2-Mercaptoethanol (Gibco, #2121115), 1% GlutaMAX^TMSupplement(Thermo,#35050061),1％MEM Non-Essential Amino Acids(Gibco,#11140-050)；

3. Immunofluorescence detection of early neural progenitor cell specific protein expression

Cells in 24-well dishes were rinsed 1 time with DPBS and then fixed with 4% paraformaldehyde at 200ul per well for 10 minutes at room temperature. The mixture is rinsed 3 times with DPBS for 5 minutes each time, and then sealed for 30 minutes at room temperature by adding a sealing solution (10% donkey serum with DPBS as a solvent and 3% Triton). Then, DPBS was rinsed 3 times for 5 minutes, and OCT3/4(C-10) (SANTA CRUZ, # sc-5279, 1: 200), SOX1(CST, #4194S, 1: 200), NESTIN (10C2) (SANTA CRUZ, # sc-23927, 1: 200), PAX6(Biolegend, #901301, 1: 400) was added overnight at 4 degrees. Following day, 3 washes with DPBS for 5 minutes each, followed by secondary antibody: TRITC-conjugated Donkey anti-Rabbit IgG (Jackson immune Research, # 711-. Then rinsed 3 times with DPBS, 5 minutes each, then applied with DAPI (1: 5000) at room temperature for 10 minutes, and finally rinsed 3 times with DPBS, 5 minutes each, and fluorescence can be tapped (microscope: olympus).

The immunofluorescence results are shown in FIG. 3, and it is found that the expression levels of SOX1, NESTIN and PAX6 are increased in the early neural differentiation process after KDM6 is expressed by KO.

4. qRT-PCR detection of early neural progenitor cell RNA expression

(1) Extraction of RNA from kit

The kit used was: HiPure Total RNA Mini Kit (magenta, # R4111-03)

(2) Reverse transcription into cDNA

Reagents used for reverse transcription: 5XABScript II RT Mix (ABClonal, # 9620520803).

20ul of the system used for reverse transcription, 1ug of RNA, the remainder was supplemented with RNase-free water.

The procedure used for reverse transcription: 5min at 25 ℃, 15min at 42 ℃, 30s at 85 ℃ and stop at 4 ℃. Then put into a refrigerator with 20 degrees below the zero to preserve.

(3) qRT-PCR System and procedure

Reagents used for qRT-PCR: 2 × SYBR Green qPCR Mix (Biomake, # B21202).

10ul system for qRT-PCR: 2 × SYBR Green qPCR Mix-5 μ l, 10ng cDNA, the remainder was made up with RNase-free water.

Procedure used for qRT-PCR: (95 ℃ for 5min, 95 ℃ for 15s, 60 ℃ for 30s) 39 cycles were repeated, 95 ℃ for 15s, and 0.5 ℃ to 95 ℃ for 15s increments.

The results are shown in FIG. 4, and the results of qRT-PCR show that the expression level of SOX1, NESTIN and PAX6 is increased in the early neural differentiation process after KDM6 is KO.

In summary, from the immunofluorescence results in FIG. 3 and the qRT-PCR results in FIG. 4, it can be seen that the expression levels of early neural marker molecules SOX1 and NESTIN are increased during the differentiation of KDM6A/6B-DKO cell lines DKO1 and DKO2 into early neural progenitor cells by using WT ESCs as a control group.

Example 2 addition of KDM6 inhibitor-GSKJ 1 during early neural differentiation

1. Material

Control group: human embryonic stem cells (HUES8) differentiated towards early neuroectoderm.

Positive group: human embryonic stem cells (HUES8) added GSK J1 and GSK126 during differentiation of early neuroectoderm.

2. Method of producing a composite material

The procedure of cell culture and differentiation in example 1 was the same as in the control group, and the positive group was supplemented with GSK J1 and GSK126 from day 0 to day seven of differentiation.

3. Total RNA extraction and qRT-PCR detection of cells

The specific operation steps are the same as example 1, and the qRT-PCR experiment result is shown in FIG. 6;

as can be seen from the qRT-PCR experimental results shown in FIG. 6, the immunofluorescence results show that the KDM6 inhibitor GSKJ1 improves the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process, and the EZH2 inhibitor GSK126 has no influence on the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process.

4. And (4) performing immunofluorescence.

The specific operation steps are the same as example 1, and the immunofluorescence results are shown in FIG. 5;

from the immunofluorescence results in fig. 5, it is known that KDM6 inhibitor GSK J1 can promote expression of early neuroectodermal important marker molecules, and EZH1/2 inhibitor GSK126 has no influence on expression of early neuroectodermal important marker.

Example 3 addition of KDM6 inhibitor-GSKJ 1 during early neural differentiation

1. Material

Control group: human induced pluripotent stem cells (WTCs) differentiate into early neuroectoderm.

Positive group: human induced pluripotent stem cells (WTCs) add GSK J1 and GSK126 during differentiation of early neuroectoderm.

2. Method of producing a composite material

The procedure of cell culture and differentiation in example 1 was the same as in the control group, and the positive group was supplemented with GSK J1 and GSK126 from day 0 to day seven of differentiation.

3. Total RNA extraction and qRT-PCR detection of cells

The specific operation steps are the same as example 1, and the qRT-PCR experiment result is shown in FIG. 8;

as can be seen from the qRT-PCR experimental results shown in FIG. 8, the immunofluorescence results show that the KDM6 inhibitor GSKJ1 increases the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process, and the EZH2 inhibitor GSK126 has no influence on the expression levels of SOX1, NESTIN and PAX6 in the early neural differentiation process.

4. And (4) performing immunofluorescence.

The specific operation steps are the same as example 1, and the immunofluorescence results are shown in FIG. 7;

from the immunofluorescence results in fig. 7, it is known that KDM6 inhibitor GSK J1 can promote expression of early neuroectodermal important marker molecules, and EZH1/2 inhibitor GSK126 has no influence on expression of early neuroectodermal important marker.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

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