KR20160036889A - Novel marker of nerve cell and uses thereof - Google Patents

Abstract

The present invention relates to: a differentiation method of neural cells comprising a step of overexpressing dual-specificity phosphatase 4 (DUSP4) which is a novel neuronal marker in stem cells; a composition for promoting neural cell differentiation including the DUSP4 gene; and a pharmaceutical composition for treating cranial nerve diseases including stem cells in which the DUSP4 gene is overexpressed. The differentiation into neuronal cells can be promoted, when overexpressing the DUSP4 provided from the present invention in the stem cells. Therefore, the present invention can be widely used in the effective treatment of various cerebral nerve diseases such as Alzheimer′s disease by using the stem cells in which the DUSP4 gene is overexpressed.

Description

Novel marker of nerve cell and uses thereof < RTI ID = 0.0 >

The present invention relates to a novel neuron cell marker and its use, and more specifically, to a neuron cell differentiation method comprising the step of overexpressing a novel neuron cell marker DUSP4 (dual-specificity phosphatase 4) A composition for promoting differentiation of neurons including the DUSP4 gene, and a stem cell overexpressing the DUSP4 gene.

In mammals, nerve cells may be damaged if the neurons are directly damaged due to extrinsic damage, wounds, or the like, or if a neurological disease accompanied by degenerative damage of nerve cells is caused by a genetic cause, When cells are damaged, they are no longer regenerated. Even if neuronal damage is caused by extrinsic damage, neurodegenerative diseases, such as stroke, Parkinson's disease or Alzheimer's disease, The development of therapies that can treat the underlying diseases has been actively studied all over the world for a long time, but no good therapies have yet been developed.

In recent years, studies have been actively conducted to develop a method of treating damaged neurons by differentiating stem cells, which are pluripotent cells capable of differentiating into various cells, into neurons. Since stem cells can be differentiated into various cells, it can be a fundamental method for treating diseases causing tissue damage, but it is not easy to obtain stem cells, and it is easy to differentiate stem cells into desired cells And the stem cell itself should not be rejected by the patient's immune system, so it has not yet been universally used. However, in the case of brain nervous system diseases accompanied by damage of nerve cells, it is considered to be most suitable for the treatment using stem cells than any other diseases, because the brain nervous system tissue has almost no immune rejection reaction unlike other tissues, When cells are transplanted from the outside, long-term survival of the transplanted cells can be expected.

Accordingly, studies for developing a method for applying stem cells to treat diseases such as stroke, Alzheimer's disease, Parkinson's disease, demyelinating disease, and spinal cord injury have been actively conducted. For example, in WO 2005/003320, stem cells are cultured by sequentially adding basic fibroblast growth factor, fibroblast growth factor 8, SHH (Sonic Hedgehog) and brain-derived neurotrophic factor, Discloses a method for inducing the differentiation of stem cells into neurons including co-culturing with astrocytes. In Korean Patent No. 495532, mesenchymal stem cells are treated with epidermal growth factor and hepatocyte growth factor (hepatocyte growth factor) to differentiate and proliferate into neuronal cells.

However, the nerve cells differentiated from the outside of the living body can not confirm whether or not the neurons in the brain can be matched with the conditions of the patient suffering from the neurological diseases, and even when the neural cells are implanted, There is a problem that it may be deteriorated. As a method to solve such a problem, a method of transplanting stem cells scheduled to be differentiated into neural cells into a site where nerve injury has occurred has been studied. However, the success rate of neural differentiation is still low or disadvantage that neural differentiation is not performed normally Is not solved.

Under these circumstances, the present inventors have made intensive researches to develop a method for promoting differentiation into neurons. As a result, it has been found that stem cells overexpressing the novel neuron cell marker DUSP4 (dual-specificity phosphatase 4) And the present invention has been completed.

One object of the present invention is to provide a method of differentiating neural cells comprising over-expressing DUSP4 (dual-specificity phosphatase 4) in stem cells.

It is another object of the present invention to provide a composition for promoting neuronal differentiation promotion comprising a DUSP4 gene.

It is still another object of the present invention to provide a pharmaceutical composition for treating cerebral neurological diseases, which comprises stem cells overexpressing the DUSP4 gene.

In order to develop a method for promoting differentiation into neural cells, the present inventors screened neuronal differentiation factors for proteins whose expression levels were increased during differentiation of neurons, and found that DUSP4 (dual-specificity phosphatase 4 ). The present inventors analyzed the effect of DUSP4 on differentiation of neurons. As a result, DUSP4 was expressed in a small amount from embryonic stem cells and embryoid-like cell mass, and the expression level was greatly increased by neuronal differentiation, At the time of completion of differentiation, the expression level thereof was confirmed to be the highest level. In addition, the DUSP4 increases the level in the embryonic cerebral cortex as the number of days of embryonic pregnancy increases, and does not increase further after birth. Therefore, the DUSP4 is a neuronal cell marker whose expression level increases with the development of the cranial nervous system . However, the function of the DUSP4 as a neuron marker has not been reported so far, and was first identified by the inventors of the present invention.

As a result of inhibiting the expression of DUSP4, the level of differentiation of neurons decreased and the expression of various neuron-specific markers was inhibited. On the other hand, when the expression of DUSP4 was restored, And the expression level of the neuron - specific marker was also restored to the normal level. As a result of analyzing the mechanism of action of DUSP4 on neuronal differentiation, when an external signal that induces neuronal differentiation is transferred into undifferentiated stem cells, DUSP4 inhibits phosphorylation of ERK, And the expressed Cav1.2 channel protein migrates to the cell membrane and calcium is introduced to phosphorylate CaMKK. As a result, CaMKI and MARK2 are phosphorylated and ultimately, stem cells are differentiated into neuronal cells .

Thus, it was found that overexpression of the DUSP4 promotes differentiation into neurons.

In one embodiment, the present invention provides a method of differentiating neural cells comprising overexpressing DUSP4 (dual-specificity phosphatase 4) in stem cells.

The term "DUSP4 (dual-specificity phosphatase 4) " of the present invention refers to a protein capable of dephosphorylating serine / threonine and tyrosine residues, also called MAPK (mitogen-activated protein kinase) MAPK phosphatases (MKPs) Tyrosine phosphatase is a type of human, the gene encoding DUSP4 is known to exist on chromosome 8. DUSP4 is involved in the regulation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 depending on the cell type and is also involved in the progression of breast cancer, liver cancer, ovarian cancer and acute myelogenous leukemia . In particular, it is known that DUSP4 gene can cause breast cancer if it is deleted, and DUSP4 is known to play a role as a cancer suppressor gene. In addition, DUSP4 plays an important role in the determination of endoderm in the process of zebrafish development, is involved in the development of the heart in embryonic stem cells, and can increase the expression level of DUSP4 in aged cells, It is known that apoptosis caused by oxidative stress can be used as a transcription target for p53.

Sequence information of the DUSP4 can be obtained from known databases (e.g., NM_001394, NM_176933, NP_001385, NP_795907, etc.) such as National Center for Biotechnology Information (NCBI). For example, in the present invention, the amino acid sequence of SEQ ID NO: 1 was used as the DUSP4 protein and the polynucleotide of SEQ ID NO: 2 was used as the DUSP4 gene.

In the present invention, as long as DUSP4 can promote the differentiation from stem cells to neurons, it includes all peptides to which various amino acid sequences are added at the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 1 . In addition, it may further comprise a targeting sequence, a tag, an amino acid sequence designed for specific purposes to increase the stability of the labeled residue, half-life or peptide. In addition, if it is possible to promote the differentiation of stem cells into neurons, mutant proteins in which some amino acids of the amino acid sequence of SEQ ID NO: 1 are mutated by addition, substitution, deletion or the like are also included in the category of DUSP4 .

For example, the DUSP4 provided in the present invention may comprise a polypeptide having a sequence that differs from the amino acid sequence of SEQ ID NO: 1 by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

In the present invention, the overexpression can be carried out by introducing a DUSP4 gene into a stem cell. The DUSP4 gene is introduced into a stem cell in the form of an expression vector containing the DUSP4 gene, and then the overexpressed . (PUC18, pBAD, pIDTSAMRT-AMP, etc.), Escherichia coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids pTTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), lambda-phage (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP), lentivirus, retrovirus, adenovirus, Vaccinia virus, baculovirus and the like can be used.

The nucleotide sequence constituting the DUSP4 gene may be a base sequence capable of encoding the amino acid sequence of SEQ ID NO: 1 or a variety of amino acid sequences capable of being added to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: The nucleotide sequence may be added to the 5'-end or the 3'-end of the nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 1, preferably the nucleotide sequence may be a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or the various amino acid sequences which can be added to the N-terminal or the C-terminal of the amino acid sequence of SEQ ID NO: 1 is the 5'-terminal or the 5'-terminal of the nucleotide sequence of SEQ ID NO: 2 Terminal < / RTI >

In addition, as long as it is capable of encoding a protein capable of promoting differentiation from stem cells to neurons, a polynucleotide including a nucleotide sequence homologous with the above nucleotide sequence is also included in the category of the polynucleotide provided in the present invention . The polynucleotide may be a polynucleotide including a nucleotide sequence preferably showing a homology of 80% or more, more preferably a polynucleotide including a nucleotide sequence having 90% or more homology, The polynucleotide may be a polynucleotide comprising a nucleotide sequence having 95% or more homology.

On the other hand, the polynucleotide may be mutated by substitution, deletion, insertion, or a combination of one or more bases. When the nucleotide sequence is prepared by chemically synthesizing, it is possible to use a method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988 , Triesters, phosphites, phosphoramidites and H-phosphate methods, PCR and other auto primer methods, and oligonucleotide synthesis on solid supports.

The term "stem cell " as used herein means a cell capable of self-replicating and capable of differentiating into two or more new cells. According to the ability to differentiate, a stem cell, And can be classified into pluripotent stem cells and multipotent stem cells, and can be classified into mesenchymal stem cells, embryonic stem cells, and degenerated stem cells depending on the stem cells.

In the present invention, the stem cell can be interpreted as a stem cell capable of being differentiated into a neuron, and the stem cell can be differentiated into a cholinergic neuron through an embryo-like cell mass, a neuron cell, and a neuronal progenitor cell. Therefore, in the present invention, if it is possible to differentiate into the neurons, all kinds of stem cells such as mesenchymal stem cells, embryonic stem cells, and degenerative stem cells can be used. Preferably, embryonic stem cells, degenerated stem cells, May be mesenchymal stem cells derived from bone marrow, adipose tissue, teeth, dental tissue, blood, umbilical cord blood, liver, skin, gastrointestinal tract, placenta, uterus, fetus.

The term "embryoid body " of the present invention, also referred to as a soma, refers to a cell mass in which stem cells are formed in a ball shape at the initial stage of cell division. The embryoid-like cell mass exhibits a level of differentiation potential similar to that of ordinary embryonic stem cells, and can be differentiated into a variety of living tissues such as bone cells, muscle cells, nerve cells, epithelial cells and fibroblasts.

In the present invention, the embryoid-like cell mass may be used as an intermediate medium for differentiating stem cells into neural progenitor cells, which may be prepared by culturing stem cells with various components (for example, serum, NEAA, antibiotics, LDN193189, SB431542, (For example, DMEM / F12 medium, KO-DMEM / F12 medium, etc.) containing the above-mentioned medium.

The term "neural progenitor cell " of the present invention, also referred to as a" neuron precursor cell ", refers to all cells that can differentiate into neurons in a wide range or are present during the differentiation process . The neural progenitor cells divide neuron stem cells or other stem cells to form neuroblasts, and the formed neuroblasts move to a site where neural tube or nerve cell is formed. Then, And finally form neurons. All the cells from the stem cells to just before the completion of differentiation correspond to a wide range of neuronal progenitor cells, and the narrower range includes division This terminated neuroblastoma corresponds to neural progenitor cells.

The term "nerve cell" of the present invention is also referred to as a neuron, and is a unit cell constituting the cranial nervous system of an animal. It is composed of a cell body portion excluding a neuron, nucleus, and cytoplasm surrounding them. It means cells larger than other somatic cells. The nerve cells can be divided into apical neurons, unipotential neurons, bipolar neurons, gastric unipotential neurons, and multipolar neurons according to the number of neurons contained therein.

DUSP4 provided by the present invention was found to be a type of nerve cell marker whose expression level is increased in proportion to the level of differentiation of neurons during the developmental process of the embryo and its expression level is no longer increased when the differentiation of neurons is completed , The function of DUSP4 on such neuronal differentiation has not been known at all in the past and has been first identified by the present inventors.

According to one embodiment of the present invention, DUSP4 is expressed in a small amount from the embryonic stem cell and embryoid-like cell mass, and the amount of expression is greatly increased by the differentiation of neurons. However, when the differentiation into neurons is completed, (Fig. 2A). DUSP4 protein was detected in small amounts from embryonic stem cells and embryoid-like cell mass, and the level of detection of DUSP4 protein was greatly increased when neuronal differentiation was performed (Fig. 2B) (Fig. 2c). As the number of days of embryo gestation increases, the protein level of DUSP4 contained in the embryonic brain cortex increases, and the protein level of DUSP4 is no longer increased after birth (FIG. 2d), it was found that the DUSP4 is a type of neuronal marker that increases in expression level with development of the cranial nervous system.

When the expression of DUSP4 was inhibited (FIG. 3A), the length of the neurite significantly decreased (FIG. 3B) and the expression level of neuronal markers (MAP2 and β-tubulin III) , And the expression level of DUSP4 was inhibited, indicating that the level of neuronal differentiation was generally decreased.

In addition, when wild-type DUSP4 or wild-type DUSP4 or 284-amino acid cysteine serine-transformed DUSP4 was introduced after the suppression of the expression of DUSP4, when wild-type DUPS4 was expressed in nerve cells inhibiting DUSP4 expression by treating shDUPS4, It was confirmed that the length of the protrusions was extended but the length of the neurite was not changed when the control and the mutated DUSP4 were expressed (Fig. 4b). The nestin and neuronal markers (MAP2, β-tubulin III and synaptophysin ) Expression level of all markers was restored when wild-type DUSP4 was introduced into shDUSP4-introduced neurons, but when the mutant DUSP4 was introduced into shDUSP4-introduced neurons, expression of the marker (Fig. 4C), it was found that restoration of DUSP4 expression improved the level of neuronal differentiation as a whole.

On the other hand, when DUSP4 was expressed normally, the phosphorylated ERK was maintained at a certain level. However, when the expression of DUSP4 was suppressed, the phosphorylated ERK was continuously increased with the elapse of the differentiation time 5a). It was confirmed that the non-phosphorylated ERK was present in both the cytoplasm and the nucleus, but the phosphorylated ERK increased in inverse proportion to the expression level of DUSP4 as the DUSP4 expression level was lower (Fig. 5c) (FIG. 5d), phosphorylated CaMKK (FIG. 5g) involved in the phosphorylation of CaMKI (FIG. 5g) and phosphorylated phosphorylated MARK2 (FIG. 5h) in the neuronal differentiation factor receiving the phosphorylated CaMKI (Fig. 6A), which affects the activity of the intracellular calcium ion, and Cav1.2 channel protein (Fig. 6B), which affects the level of the intracellular calcium ion, Both were confirmed reduced.

Thus, when an external signal that induces neuronal differentiation is delivered into the cell, DUSP4 inhibits ERK phosphorylation and induces expression of Cav1.2 channel protein, and the expressed Cav1.2 channel protein migrates into the cell membrane to enter calcium , CaMKK was phosphorylated and CaMKI and MARK2 were phosphorylated and differentiated into neuronal cells (Fig. 7).

Thus, when the DUSP4 was overexpressed in the stem cells, it was found that the differentiation into neurons could be accelerated by the above-mentioned action mechanism.

As another embodiment for achieving the above-mentioned object, the present invention provides a composition for promoting neuronal differentiation promotion comprising a DUSP4 gene.

As described above, since overexpression of DUSP4 in stem cells can promote the differentiation of neural cells from stem cells, a composition capable of introducing the DUSP4 gene into stem cells can be used so as to overexpress DUSP4 in stem cells .

The composition for promoting differentiation of neurons including the DUSP4 gene is not particularly limited, but preferably includes an expression vector containing a DUSP4 gene as an active ingredient.

The term "expression vector" of the present invention means a gene construct comprising a gene insert that is capable of expressing a desired protein in an appropriate host cell, and an essential regulatory element operably linked to the expression of said gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

The term "operably linked" of the present invention means a state in which a nucleic acid sequence encoding a desired protein or RNA is functionally linked to a nucleic acid expression control sequence so as to perform a general function . For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In addition, the expression vector may further include a protein tag which can be optionally removed using endopeptidase to easily detect the expression of DUSP4 in stem cells or neurons differentiated from the stem cells have.

The term "tag " of the present invention means a molecule exhibiting quantifiable activity or property and includes a polypeptide fluorescent substance such as a fluorophore such as fluorescein, a fluorescent protein (GFP) Or may be a fluorescent molecule including; Myc tag, a Flag tag, a histidine tag, a Lucin tag, an IgG tag, or a strap tagidine tag. Particularly, in the case of using an epitope tag, a peptide tag composed of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues can be used.

In the present invention, the expression vector is not particularly limited as long as it is capable of producing the DUSP4 provided by the present invention by expressing the polynucleotide. The expression vector may be a mammalian cell (for example, a human, a monkey, a rabbit, a rat, a hamster, The vector may be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells including plant cells, yeast cells, insect cells or bacterial cells (for example, Escherichia coli, etc.) Is operably linked to a suitable promoter so that the polynucleotide can be expressed in the host cell and can be a vector comprising at least one selectable marker and more preferably an expression vector capable of being expressed in mammalian stem cells And most preferably is expressed in human stem cells to induce differentiation into neurons Can be expressed in the vector can be.

Since the amount of expression of the protein and the expression of the expression vector are different depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose. In the present invention, a lentiviral vector was used as the expression vector.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for the treatment of cerebral neurological diseases comprising stem cells overexpressing the DUSP4 gene or cells differentiated from the stem cells, and a pharmaceutical composition The present invention provides a method for preventing or treating a cerebral neurological disease, comprising the step of administering to an individual having or potentially causing a cerebral neurological disease in an effective amount.

The term "differentiation-induced cell" as used herein means a cell in which differentiation from a stem cell is under way or has been completed.

In the present invention, the differentiation-inducing cell may be any cell that is formed in the intermediate process in which the DUSP4 gene is differentiated from stem cells into neurons. For example, when a stem cell is differentiated into a neuron, it may be a first embryonic-like cell mass produced, a neuronal precursor cell differentiated from the embryoid-like cell mass, a neural cell differentiated from the neural progenitor cell, and the like .

As described above, it has been confirmed that when the DUSP4 gene is overexpressed in a cell showing a low level of differentiation into neurons, neuronal differentiation is restored to a normal level. Therefore, Therefore, when the DUSP4 gene is introduced into a patient in which nerve injury has been induced, the stem cells overexpressing the DUSP4 gene can produce new neurons in the injured area, and thus may be effective in treating the nerve injury.

The term "a stem cell overexpressing the DUSP4 gene" of the present invention refers to a stem cell capable of differentiating into a neural cell, into which a composition for promoting differentiation of a neural cell including the DUSP4 gene is introduced, Cells. At this time, stem cells, DUSP4 gene, compositions for promoting differentiation of neurons and the like are the same as described above.

The term "cranial nervous system disease" as used herein encompasses diseases in which neurons are directly damaged due to extrinsic damage, wounds, or the like, or degenerative damage of nerve cells due to a genetic cause. Exemplary neurodegenerative diseases include exogenous nerve damage, degenerative neurological diseases, and degenerative neurological diseases may include Alzheimer's disease, Parkinson's disease, Huntington's disease, tauopathy, and the like.

The term "individual" of the present invention includes, without limitation, mammals including rats, livestock, humans, and the like in which the cranial nerve system disease is likely to be or will be developed.

In addition, the pharmaceutical composition of the present invention may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions, in addition to stem cells overexpressing the DUSP4 gene used as an active ingredient. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Preferably, the pharmaceutical composition of the present invention can be formulated in the form of an injectable preparation by mixing a buffer, a preservative, an anhydrous agent, a solubilizing agent, an isotonic agent, a stabilizer, and the like in the nature of containing stem cells as an active ingredient , Preferably in unit dosage ampoules or in multiple dosage forms.

Meanwhile, the pharmaceutical composition of the present invention can be transplanted or injected into a nerve-damaged region of a patient suffering from a cerebral neurological disease through parenteral administration. According to the anatomical characteristics of the nerve-damaged region, Or may be administered in a conventional manner.

In addition, the dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, dry state, diet, administration time, administration method and severity of the disease, Can be easily determined.

Since overexpression of DUSP4 provided by the present invention in stem cells can promote differentiation into neural cells, when the above-mentioned DUSP4 overexpressing stem cells are used, they can be widely used for effective treatment of various brain diseases such as Alzheimer's disease There will be.

FIG. 1A is a photomicrograph showing the appearance of rat embryonic stem cells derived from 129s4 / Jae, embryonic-like cell mass formed from the embryonic stem cells and differentiated nerve cells from the embryoid-like cell mass.
FIG. 1B shows the changes in the expression levels of neural stem cell markers (nestin) and neural cell markers (MAP2, β-tubulin III and synaptophysin) in embryonic stem cells, embryonic-like cell masses, And FIG.
FIG. 1C is a photograph showing immunostaining of neural cell markers (MAP2 and β-tubulin III) on cells in the differentiation process of embryonic stem cells and neurons.
2A is a graph showing the results of comparing changes in the expression level of DUSP4 in embryonic stem cells, embryonic-like cell masses, and cells in the differentiation process of neuronal cells.
FIG. 2B is a photograph and a graph showing the result of quantitative analysis of expression level of DUSP4 by performing Western blot on embryonic stem cells, embryonic-like cell masses, and cells in the differentiation process of nerve cells.
FIG. 2c is a photograph showing immunostaining for DUSP4 in embryonic stem cells and cells differentiated for 5 days. FIG.
FIG. 2d is a photograph showing Western blot analysis of DUSP4 in mouse embryo and neonatal brain cortex tissues. FIG.
FIG. 3A is a graph showing the results of comparison of mRNA levels of DUSP4 expressed in each embryonic stem cell (J1 ES) into which scrambled shRNA or shDUSP4 is introduced, and a graph (top) showing the results of the comparison between the respective embryonic stem cells (J1 ES) Western blot analysis showing the results of comparing levels of DUSP4 protein in differentiated neurons.
FIG. 3B is a graph comparing the lengths of the neurites and the phase-contrast microscope photographs showing the results of photographing the morphology of differentiated neurons in each embryonic stem cell into which scrambled shRNA or shDUSP4 was introduced.
Fig. 3c is a graph showing the distribution of the neural stem cell marker (nestin) and neuronal cell marker (MAP2, beta-tubulin III) in the embryonic stem cells, embryonic cell clusters and neuronal differentiation cells into which scrambled shRNA or shDUSP4 was introduced ≪ / RTI > and synaptophysin).
FIG. 3D is a photograph showing the result of performing immunostaining using neuron markers (MAP2 and? -Tubulin III) on scaffold shRNA or shDUSP4-introduced neuron cells.
FIG. 3E is a graph showing changes in the protein levels of neuronal markers (MAP2 and β-tubulin III) in cells in the differentiation process of embryonic stem cells, embryonic-like cell clusters and neurons into which scrambled shRNA or shDUSP4 was introduced And FIG.
FIG. 4A is a graph showing the results of a comparison between the shRNA-injected embryonic stem cell (J1 ES-shRNA) and the neuron differentiated from the embryonic stem cell (Neuron Day3-shRNA) as a vector, wild type DUSP4 gene (DUSP4) DUSP4 CS). The upper part shows the result of photographing the expression of GFP introduced into each transfected cell by fluorescence microscope, and the lower part shows the result of anti-FLAG antibody and anti-DUSP4 -GFP antibody using Western blot analysis.
FIG. 4B is a photograph and a graph showing the change in neurite length of the neuron transformed to express wild-type or mutated DUSP4, wherein the top is a photograph of the transformed neuron, and the bottom is a photograph of each transformed neuron A graph showing the length of nerve processes.
FIG. 4C shows the results of the addition of shDUSP4 or shDUSP4 (shDUSP4), addition of wild type DUSP4 after introduction of shDUSP4 (shDUSP4 + DUSP4) or addition of mutated DUSP4 after introduction of shDUSP4 (shDUSP4 + DUSP4 CS) Each embryonic stem cell, embryonic-like cell mass differentiated from each of these embryonic stem cells, and each differentiated neuron were subjected to nestin and nerve cell markers (MAP2, β-tubulin III < / RTI > and synaptophysin).
FIG. 4D is a photograph showing immunostaining of neuronal cell markers (MAP2 and β-tubulin III) on transformed neurons (vectors, DUSP4 and DUSP4 CS).
FIG. 4E is a photograph showing the results of Western blot analysis of changes in protein levels of neuron markers (MAP2 and β-tubulin III) in transformed neurons (vector, DUSP4 and DUSP4 CS).
5A is a graph showing the results of comparing the amounts of activated (phosphorylated) ERK and non-activated ERK in each embryonic stem cell into which scrambled shRNA or shDUSP4 was introduced and each neuron differentiated from the embryonic stem cell. It is a blot analysis photograph and graph.
FIG. 5B is a photograph showing immunostaining using antibodies against phosphorylated ERK in each neuron differentiated from embryonic stem cells into which scrambled shRNA or shDUSP4 was introduced.
FIG. 5c is a photograph showing the results of Western blot analysis of the content of DUSP4 and ERK in nucleus and cytoplasm of each neuron differentiated from embryonic stem cells into which scrambled shRNA or shDUSP4 was introduced.
FIG. 5D shows an example of a vector that induces over-expression of DN ERK (dominant-negative mutant ERK) after the introduction of a scrambled shRNA or shDUSP4, a vector inducing overexpression of ERK after shDUSP4 is introduced, or shDUSP4 introduced Western blot analysis of changes in protein levels of phosphorylated CaMKI, non-phosphorylated CaMKI and neuronal cell markers (MAP2 and β-tubulin III) on each embryonic stem cell and each neuron differentiated from each of the embryonic stem cells The results are shown in Fig.
FIG. 5E is a graph showing the results of a comparison between the neurons (Neuron Day 3) to which shRNA was not introduced, the cells (shDUSP4 + Vector) into which the empty vector was introduced into the differentiated nerve cells into which shDUSP4 was introduced, and the normal CaMKI gene (ShDUSP4 + CaMKI T117A) transfected with shDUSP4 and CaMKI gene (CaMKI T117A) transfected into differentiated neurons, and CaMKI gene (shDUSP4 + CaMKI T117A) transfected with shDUSP4 CaMKI T117A) (shDUSP4 + CaMKI T117D). The results are shown in Fig.
FIG. 5f shows a cell in which shDUSP4 is introduced and an empty vector is introduced into a differentiated neural cell (shDUSP4 + Vector), a cell in which a normal CaMKI gene has been introduced into neurons differentiated with shDUSP4 (shDUSP4 + CaMKI), and shDUSP4 (ShDUSP4 + CaMKI T117A) transfected with the CaMKI gene (ShDUSP4 + CaMKI T117A) mutated to the differentiated neuron and the CaMKI gene (CaMKI T117A) transfected into the differentiated neuron by the introduction of shDUSP4 (MAP2 and β-tubulin III), Western blot analysis of the protein levels of the neuronal markers (MAP2 and β-tubulin III).
FIG. 5G is a photograph showing immunological analysis results of confirming the phosphorylation of CaMKK in each embryonic stem cell into which scrambled shRNA or shDUSP4 has been introduced and each embryonic stem cell.
FIG. 5H is a photograph showing immunological analysis results of confirming the phosphorylation of MARK2 in each embryonic stem cell into which scrambled shRNA or shDUSP4 has been introduced and each embryonic stem cell.
FIG. 6A is a graph showing the results of the expression of the scFvs (shDUSP4) or shDUSP4 (shDUSP4), shDUSP4, shDUSP4 + vector, or shDUSP4 after induction of DN ERK overexpression shDUSP4 + DN ERK) is a graph showing the results of comparing the intracellular calcium levels of the respective neurons with the photographs and graphs and comparing the lengths of the neurites measured in each neuron.
FIG. 6B shows the results of the expression of the gene encoding the scFv-1 gene, in which a vector inducing overexpression of DN ERK was introduced (shDUSP4 + Vector) or shDUSP4 was introduced after shDUSP4, shDUSP4, shDUSP4, shDUSP4 + DN ERK) Western blot analysis showing the level of Cav1.2 channel protein in total lysate or cell membrane fraction (NeutrAvidin) of each embryonic stem cell and each differentiated neuron And graphs.
FIG. 6C shows a vector containing a scrambled shRNA (Scrambled) or shDUSP4 (shDUSP4), a vector inducing overexpression of DN ERK after introduction of shDUSP4 (shDUSP4 + Vector) or shDUSP4 shDUSP4 + DN ERK) photographs and graphs showing the results of immunological staining of each neuron differentiated from embryonic stem cells using an antibody against Cav1.2 channel protein.
7 is a schematic diagram showing a signal transduction process involving DUSP4 involved in neural differentiation.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Production of differentiated neurons from embryonic stem cells

Example 1-1: induction of neuronal differentiation

Rat embryonic stem cells J1 ES derived from 129s4 / Jae were cultured in ESC medium (15% ES FBS, 1% penicillin-streptomycin, 2 mM L-glutamine, 1% non essential amino acid (NEAA), 0.1 mM beta-mercapto (DMEM medium containing ethanol and 1 x 10 ³ U / ml of leukemia inhibitory factor (LIF)) at 5% CO ₂ and 37 ° C wet conditions on γ-irradiated mouse embryonic fibroblasts (MEFs).

The cultured embryonic stem cells were inoculated into a 90 mm culture dish at a density of 3 × 10 ⁶ cells / cm 2 and cultured for 8 days to obtain embryoid-like cell masses (EBs). At this time, the medium was changed every two days, and at the fourth day, 10 μM of retinoic acid was treated. After completion of the incubation, the embryoid-like cell masses (EBs) were unicellularized by adding trypsin, inoculated into a poly-D-lysine-laminin coated culture container, and cultured in N2 medium containing 20 ng / ml bFGF ) Was added and cultured for 5 days to differentiate the embryonic stem cells into neurons (Fig. 1A).

As shown in FIG. 1A, it was confirmed that embryoid-like cell clumps obtained from embryonic stem cells can be cultured for 5 days to differentiate into neurons.

Example 1-2: Verification of mRNA level of neuron marker

The embryo-like cell mass was cultured for 5 days by the method of Example 1-1, and the specimens were collected on days 2, 3 and 5 while neurons were differentiated, and mRNA was analyzed from each collected sample. Specifically, Trizol RNA reagent (Qiagen, Hilden, Germany) was added to each sample to disrupt the cells of each sample and centrifuged (10,770 x g, 10 minutes, 4 ° C) to obtain a supernatant. 0.2 ml of chloroform was added to the supernatant obtained above and mixed, and the mixture was centrifuged (10,770 x g, 2 minutes, 4 캜) to obtain a supernatant. The same volume of isopropanol was added to the supernatant obtained above, and the reaction was allowed to proceed at room temperature for 10 minutes. Then, the reaction product was centrifuged (10,770 x g, 15 minutes, 4 캜) to obtain precipitated RNA. The obtained RNA was washed with 0.5 ml of cold 70% ethanol, centrifuged again (10,770 x g, 1 min, 4 캜), and the precipitated total RNA was obtained.

CDNA was obtained by performing a reverse transcription reaction using 2 μg of the obtained total RNA, 500 ng of random primer, 200 units of M-MLV reverse transcriptase and 25.2 units of Rnasin RNase inhibitor.

PCR was performed using the above-obtained cDNA as a template using the CFX96TM Real-Time system (Bio-Rad Laboratories, Hercules, Calif.) To obtain nestin, MAP2, tubulin III) and synaptophysin (Fig. 1B). At this time, embryonic stem cells (J1 ES) and embryoid-like cell mass (EB) were used as a control group.

nestin F: 5'-GAGGTGACCCTTGGGTTAGA-3 '(SEQ ID NO: 3)

nestin R: 5'-TAGCCCTACCACTTCCTGCT-3 '(SEQ ID NO: 4)

β-tubulin III F: 5'-AGCCCTCTACGACATCTGCT-3 '(SEQ ID NO: 5)

β-tubulin III R: 5'-ATTGAGCTGACCAGGGAATC-3 '(SEQ ID NO: 6)

MAP2 F: 5'-CACTGGACATCAGCCTCACT-3 '(SEQ ID NO: 7)

MAP2 R: 5'-CACCCTGGTCCTTCATCTCT-3 '(SEQ ID NO: 8)

synaptophysin F: 5'-GAGAACAACAAAGGGCCAAT-3 '(SEQ ID NO: 9)

synaptophysin R: 5'-GCACATAGGCATCTCCTTGA-3 '(SEQ ID NO: 10)

As shown in FIG. 1B, the neural stem cell marker (nestin) was expressed at a certain level from the embryo-like cell mass, and the expression level was greatly increased when neuron differentiation was performed. However, when the neural stem cell differentiation was completed, Respectively. In contrast, neuronal markers (MAP2, β-tubulin III, and synaptophysin) are not expressed in embryonic-like cell masses, but their expression levels are greatly increased as neuronal differentiation is performed, and they are expressed at the highest level at the completion of differentiation Respectively.

Example 1-3: Immunostaining using neuron markers

Each of the samples thus obtained was subjected to immunostaining using an antibody against neuronal markers (MAP2 and β-tubulin III).

Specifically, each sample thus obtained was treated and fixed with 4% paraformaldehyde, treated with PBS containing 0.1% Triton X-100 to impart permeability to the sample, and then washed with 5% chlorine Serum (GS) was treated to prevent nonspecific binding. Immunoreactivity was then performed by treatment with PBS containing anti-MAP2 antibody (Abcam, Cambridge, UK) or anti-beta-tubulin III antibody (Cell signaling, Danvers, MA) and 0.2% BSA. After completion of the immune reaction, each sample was washed with PBS (PBS-T) containing 0.1% Tween-20 and washed with Alexa-488 secondary antibody (Invitrogen) or 594 secondary antibody (Invitrogen) And reacted at room temperature for 1 hour. After completion of the reaction, each sample was treated with PBS-T, washed and photographed with a Leica microscope (Leica Microsystems) or an Axiovert 200 microscope (Carl Zeiss, Oberkochen, Germany) (FIG. At this time, embryonic stem cells (J1 ES) were used as a control group.

As shown in FIG. 1C, it was confirmed that each nerve cell marker was not expressed in embryonic stem cells but only in cells in the differentiation process of neurons.

From the results of FIGS. 1B and 1C, it can be seen that the differentiated cells were nerve cells.

Example 2: Detection of novel neuronal differentiation marker DUSP4 (dual-specificity phosphatase 4)

In order to identify new candidates for protein tyrosine phosphatases (PTPs) associated with neurogenic differentiation, differences in gene frequencies between embryonic stem cells and differentiated neurons were analyzed by qPCR sequencing. As a result, DUSP4 (dual-specificity phosphatase 4 Respectively.

Example 2-1: Verification of mRNA level of DUSP4

We examined the changes of mRNA level of DUSP4 during neuronal differentiation.

The embryoid-like cell mass was cultured for 5 days by the method of Example 1-2, and the specimens were collected on days 1 to 5 while neurons were differentiated. The mRNA level of DUSP4 was confirmed by performing the method of Example 1-2 except that each of the samples thus obtained and the following primers were used (Fig. 2A).

DUSP4 F: 5'-CTGGCCTACCTGATGATGAA-3 '(SEQ ID NO: 11)

DUSP4 R: 5'-TGAGACTCAAACTGCAGCAA-3 '(SEQ ID NO: 12)

As shown in FIG. 2A, DUSP4 was expressed in a small amount from the embryonic stem cell and embryoid-like cell mass, and the expression level was greatly increased when neuron differentiation was performed. However, when the differentiation into neuron was completed, the expression level of DUSP4 was the highest Respectively. The level of expression of DUSP4 was slightly different from that of neural stem cell markers (nestin) and neuronal markers (MAP2, β-tubulin III and synaptophysin) previously identified, but the level of expression was not clearly decreased at the time of completion of differentiation Is distinguished from neural stem cell markers and distinguished from neural cell markers in that they are expressed in small amounts from embryonic stem cells and embryonic-like cell masses. However, DUSP4 was found to be a type of neuronal marker in that it showed the highest level at the end of neuronal differentiation.

Example 2-2: Western blot analysis using DUSP4

From the results of Example 2-1, it was determined that DUSP4 is a kind of neuron marker, so Western blot analysis using it was performed.

Specifically, embryonic stem cells (J1 ES), embryo-like cell masses (EB), and cells undergoing differentiation of neurons were washed with PBS and incubated with protease inhibitor and phosphatase inhibitor cocktail (Roche, Penzberg, Germany) (1% NP-40, 0.5% sodium-deoxycholate, 0.1% SDS, 150 mM sodium chloride, 2 mM EDTA, 50 mM sodium fluoride, 50 mM Tris-HCl, pH 7.4) The supernatant thus obtained was subjected to SDS-PAGE to obtain a gel containing the developed protein bands, and the developed protein bands were transferred to a PVDF membrane (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) was added to the reaction solution, followed by the reaction with an anti-DUSP4 antibody and an HRP-conjugated secondary antibody (Santa Cruz, Santa Cruz, CA) DUSP4 was detected using Image J software (Fig. 2B). At this time, actin was used as an internal control group.

As shown in FIG. 2b, the DUSP4 protein was detected in a small amount from embryonic stem cells and embryoid-like cell clusters, and the detection level of DUSP4 protein was significantly increased by the differentiation of neurons.

Example 2-3: Immunostaining using DUSP4

Immunostaining analysis using anti-DUSP4 antibody was performed to confirm the position of DUSP4 in the cells. Approximately, the method of Example 1-3 was carried out except that the embryonic stem cells and the cells differentiated for 5 days were used and the anti-DUSP4 antibody was used to confirm the position of the intracellular DUSP4 (Fig. 2C).

As shown in FIG. 2C, DUSP4 is found mainly in the nucleus but also in the cell matrix.

Example 2-4: Change in expression level of DUSP4 in the development of the nervous system

We examined how the expression level of DUSP4 changes in the neural developmental process.

Specifically, the embryos at the 10th, 12th, 15th, and 18th gestations of pregnant C57BL / 6J mice (KOATECH, Pyeongtaek, Korea) and the brains of mice 1 and 4 days after birth were dissected, Lt; / RTI > Crushing the excised tissue in a buffer _{(1 mM MgCl 2, 2 mM} EGTA, 1 mM DTT, 1% Triton X-100, 1% protease mixture, phosphatase inhibitors and 20 mM HEPES), centrifuged the lysate ( 4 ° C, 15, 10,770 xg) to give a supernatant.

Western blot analysis was carried out in the same manner as in Example 2-2, except that the obtained supernatant was used as a sample (Fig. 2D). At this time, GAPDH was used as an internal control group.

As shown in FIG. 2d, it was confirmed that the protein level of DUSP4 contained in the embryonic cerebral cortex increased as the number of days of embryo gestation increased, and that the protein level of DUSP4 did not increase any more after birth.

Therefore, it was found that DUSP4 is a protein whose expression amount increases with the development of the cranial nervous system.

Example 3: Functional analysis by inhibiting expression of DUSP4

Example 3-1: Obtaining Embryonic Stem Cells Suppressing DUSP4 Expression and Differentiated Neurons

From the results of Example 2-4, it was found that DUSP4 is a protein whose expression level increases with development of the cranial nervous system. Therefore, in order to confirm whether DUSP4 is essential for neuronal differentiation, , The expression of DUSP4 was inhibited and its effect was analyzed.

Specifically, lentivirus was prepared using MISSION Lentiviral Packaging Mix (Sigma-Aldrich). Lipofectamine 2000 (Invitrogen) is a vector or targetless shRNA control vector (scrambled-shRNA, SHC-002 (Invitrogen) containing shRNA sequences targeting Lenti X 293T cells with DUSP4 (TRCN0000054348, TRCN0000054349, TRCN0000054350, TRCN0000054335, TRCN0000054351, and TRCN0000054352) , Sigma-Aldrich). Embryonic stem cells that stably inhibited the expression of DUSP4 were produced by lentiviral transduction of shRNA (shDUSP4) inhibiting DUSP4 or scrambled shRNA at a condition treated with 8 μg / ml polybrene (Sigma-Aldrich). The selection process was carried out in medium containing 2 ug / ml of puromycin (Invitrogen) for 10 days. MRNA levels of expressed DUSP4 expressed in embryonic stem cells into which the scrambled shRNA or shDUSP4 was produced were confirmed by the method of Example 1-2 (Fig. 3A).

The thus prepared scrambled shRNA or shDUSP4-introduced embryonic stem cells (J1 ES) were subjected to the method of Example 1-1 to obtain respective neurons differentiated for 3 days from the embryoid-like cell mass, The level of DUSP4 protein contained in each nerve cell was confirmed by Western blot analysis of Example 2-2 (Fig. 3a).

As shown in Fig. 3A, the level of mRNA of DUSP4 was significantly decreased in shDUSP4-introduced embryonic stem cells compared with embryonic stem cells into which shRNA was introduced (control group), and the shDUSP4-introduced embryonic stem cells And the level of DUSP4 protein was significantly lower than that of the control group.

Example 3-2: Analysis of neuronal differentiation efficiency

In order to compare the differentiation efficiency of each neuron differentiated from each embryonic stem cell into which the scramble shRNA or shDUSP4 obtained in Example 3-1 was introduced, the neurite length of the neuron was compared.

Specifically, the neurites differentiated for 3 days from embryonic-like cell masses derived from the respective embryonic stem cells were measured using a phase contrast microscope (FIG. 3B). At this time, the length of the neurites was defined as being longer than or equal to one cell body, and the average neurite length was measured from each cell using MetaMorph 7.1 (Universal Imaging, Media, PA).

As shown in FIG. 3B, it was confirmed that the length of neurites differentiated from embryonic stem cells into which shDUSP4 was introduced significantly decreased in comparison with the control group.

Therefore, when the expression of DUSP4 is inhibited, the differentiation efficiency of neurons decreases.

Example 3-3: Analysis of expression levels of neural stem cells and neuronal marker

Except that each embryonic stem cell into which the scrambled shRNA or shDUSP4 obtained in Example 3-1 was introduced, embryoid-like cell clusters differentiated from each of these embryonic stem cells, and each neuron differentiated therefrom was used, The method of Example 1-2 was performed to compare the changes in the expression levels of neural stem cell markers (nestin) and neuronal markers (MAP2, β-tubulin III and synaptophysin) (FIG. 3c).

As shown in Fig. 3C, the expression level of all markers did not show any difference by scrambled shRNA or shDUSP4 in embryonic stem cells and embryo-like cell clusters, but shDUSP4 was observed in the cells in the differentiation process of neurons rather than the scrambled shRNA- It was confirmed that their expression level was significantly reduced in the introduced neuron.

Example 3-4: Immunostaining assay

The method of Example 1-3 was carried out except that each neuron differentiated from each embryonic stem cell into which scrambled shRNA or shDUSP4 obtained in Example 3-1 was introduced was used to prepare a neural cell marker (MAP2 and < RTI ID = 0.0 > P-tubulin III) < / RTI >

As shown in FIG. 3D, it was confirmed that the content of neuronal markers (MAP2 and β-tubulin III) in shDUSP4-introduced neurons was significantly lower than that of the control group in which scrambled shRNA was introduced.

Example 3-5: Western blot analysis

Each of the embryonic stem cells into which the scrambled shRNA or shDUSP4 obtained in Example 3-1 was introduced, the embryoid-like cell clusters differentiated from the embryonic stem cells, and the differentiated neurons were used as the primary antibody The method of Example 2-2 above was performed to compare the expression levels of neuronal markers (MAP2 and beta-tubulin III), except that antibodies against neuronal markers (MAP2 and beta-tubulin III) were used (Fig. 3E). At this time, actin was used as an internal control group.

As shown in FIG. 3E, it was confirmed that the protein levels of neuronal markers (MAP2 and β-tubulin III) in shDUSP4-transfected neurons were significantly lower than those in the control group in which scrambled shRNA was introduced.

The results of Examples 3-2 to 3-5 show that inhibition of DUSP4 expression in embryonic stem cells results in a decrease in neuronal differentiation level as a whole.

Example 4: Functional analysis through recovery of DUSP4

In Example 3, it was confirmed that when the expression of DUSP4 was inhibited, the efficiency of differentiation of neurons was decreased. Therefore, it was examined whether the efficiency of differentiation of neurons was increased when the expression of DUSP4 was restored. To this end, the shDUSP4-transfected embryonic stem cells prepared in Example 3 were infected with a lentivirus containing no gene, a test group infected with lentivirus containing the wild-type DUSP4 gene, and a test group containing the mutated DUSP4 gene Experimental groups infected with lentivirus were prepared and their differentiation efficiencies were compared after differentiation into neurons.

Example 4-1: Preparation of transformant

FLAG-encoding wild-type DUSP4 gene or the 2874 amino acid cysteine serine-transformed DUSP4 gene was introduced into a pLentiX-GFP-c1 vector (Clontech Laboratories, Mountain View, CA) And then transfected into Lenti X 293T cells using Lipofectamine 2000 (Invitrogen) and then cultured. 24. Collect culture supernatant while culturing for 48 and 72 hours, then centrifuge and concentrate to obtain lentivirus concentrate. The lentivirus concentrate was infected with the shRNA-injected embryonic stem cells prepared in Example 3 and the neurons differentiated from the embryonic stem cells under the condition that polybrene was treated at 8 / / ml. The infected cells were selected using a FACS Aria cell sorter (BD Biosciences, San Jose, Calif.) And further cultured in growth medium to obtain transformed embryonic stem cells. Further, the transformed embryonic stem cells obtained above were differentiated by the method of Example 1-1 to obtain differentiated neurons. Ectopic expression of FLAG-tagged DUSP4 was confirmed by Western blot analysis (Fig. 4A). At this time, as the control group, embryonic stem cells into which the pLentiX-GFP-c1 vector without the gene was introduced were used.

As shown in FIG. 4A, the wild-type DUSP4 gene or the 284-amino acid cysteine serine-transformed DUSP4 gene was transfected into embryonic stem cells into which shRNA was introduced and neurons differentiated from the embryonic stem cells Respectively.

Example 4-2: Analysis of neuronal differentiation efficiency

Except that each of the transformed neurons (vector, DUSP4 and DUSP4 CS) prepared in Example 4-1 was used and Neuron Day3-shRNA, which was not transformed into the control group, was used. The method of Example 3-2 was performed to compare changes in the length of the nerve processes of neurons upon restoration of DUSP4 expression (Fig. 4B).

As shown in FIG. 4B, when shDUPS4 was treated to express wild-type DUPS4 in neurons whose expression of DUSP4 was inhibited, the length of the neurite was elongated, but when the control and mutated DUSP4 were expressed, the length of the neurite was changed Respectively.

Example 4-3: Analysis of expression levels of neural stem cells and neuronal marker

The wild-type DUSP4 is introduced after the introduction of the shredded shRNA or shDUSP4 obtained in Example 3-1, the shDUSP4 obtained in the above-mentioned Example 4-1, or the mutated DUSP4 after the introduction of shDUSP4 is further added Using the method of Example 1-2 above, except that each introduced embryonic stem cell, embryonic-like cell mass differentiated from each of these embryonic stem cells, and each differentiated neuron were used, the neural stem cells The changes in the expression levels of the nestin and neuronal markers (MAP2, beta-tubulin III and synaptophysin) were compared (FIG. 4C).

As shown in FIG. 4C, the expression levels of all the markers did not differ in the embryonic stem cell and embryoid-like cell mass, but in the cells in the differentiation process of the neuron, the shDUSP4- Expression levels were significantly decreased, and when wild-type DUSP4 was introduced into shDUSP4-introduced neurons, expression levels of all markers were restored. However, when DUSP4 mutated in shDUSP4-introduced neurons was further introduced, Was not recovered.

Example 4-4: Immunostaining assay

Except that each of the transformed neurons (vector, DUSP4 and DUSP4 CS) prepared in Example 4-1 was used and Neuron Day3-shRNA, which was not transformed into the control group, was used. Immunostaining was performed using antibodies against neuronal markers (MAP2 and beta-tubulin III) by performing the methods of Examples 1-3 (Fig. 4d).

As shown in FIG. 4D, the expression level of neuronal markers (MAP2 and β-tubulin III) was increased at a lower level than that of the control group in the wild-type DUSP4 transduced cells (DUSP4), but the transformed cells transduced with mutated DUSP4 DUSP4 CS), the expression level of the neuronal cell marker was increased to a level lower than that of the wild-type DUSP4 transduced cell (DUSP4), and the neuronal marker was not expressed in the non-transformed cell (vector) .

Example 4-5: Western blot analysis

Neuron Day3-shRNA, which was not transformed into the control group, was used for each of the transformed neurons (vector, DUSP4 and DUSP4 CS) prepared in Example 4-1, The method of Example 2-2 above was performed to compare the expression levels of neuronal markers (MAP2 and beta-tubulin III), except that antibodies against neuronal markers (MAP2 and beta-tubulin III) were used (Fig. 4E). At this time, actin was used as an internal control group.

As shown in FIG. 4E, the expression of both neuronal markers was increased in wild-type DUSP4 transduced cells (DUSP4). However, in transformed cells (DUSP4 CS) transformed with mutant DUSP4, tubulin III) expression was increased.

The results of Examples 4-2 to 4-5 show that restoration of expression of DUSP4 improves the differentiation level of neurons as a whole.

Example 5: Analysis of action mechanism of DUSP4 associated with ERK activation

It is known that DUSP4 can deactivate the kinase of interest through the dephosphorylation of phospho-serine / threonine and phospho-tyrosine residues, in particular it is known to bind to inactive ERK, JNK and p38 have. Thus, we investigated the effect of the inhibition of DUSP4 on ERK activation.

Example 5-1: Western blot analysis

The amount of activated (phosphorylated) ERK and unactivated ERK in each embryonic stem cell into which the scrambled shRNA or shDUSP4 obtained in Example 3-1 was introduced and in each neuron differentiated from the embryonic stem cell was measured Western blot analysis (Fig. 5A). At this time, actin was used as an internal control group.

As shown in FIG. 5A, when DUSP4 was normally expressed (scrambled), the phosphorylated ERK remained constant, but when the expression of DUSP4 was suppressed, it was confirmed that the phosphorylated ERK was continuously increased with the lapse of the differentiation time .

Example 5-2: Immunostaining assay

Immunostaining was performed using antibodies against phosphorylated ERK in each neuron differentiated from embryonic stem cells into which the scrambled shRNA or shDUSP4 obtained in Example 3-1 was introduced (FIG. 5B).

As shown in FIG. 5B, it was confirmed that the content of phosphorylated ERK was increased in the neurons in which the expression of DUSP4 was inhibited.

Example 5-3: Cell division analysis

Each neuron differentiated from each embryonic stem cell into which the scrambled shRNA or shDUSP4 obtained in Example 3-1 was introduced was centrifuged (574 xg, 4 ° C, 20 minutes) and the precipitate was diluted with buffer (250 mM sucrose, (10 mM KCl, 1.5 mM MgCl ₂ , 1 mM EDTA, 1 mM DTT, 10 mM Tris-HCl, pH 7.4) and incubated for 10 minutes under ice conditions. 4 ° C, 5 minutes) to obtain a supernatant containing a nucleus-containing precipitate fraction and a cytoplasmic portion, respectively. The supernatant was centrifuged again (20,000 xg, 4 캜, 20 minutes) to obtain a precipitate containing cytoplasmic components. Western blot analysis for DUSP4 and ERK was performed on the resulting nuclear fraction and cytoplasmic components (Figure 5c). At this time, shRNA-free neurons were used as a control group, and α-tubulin, which is a cytoplasmic marker, and histone H1, a nuclear marker, were used as an internal control for Western blot analysis .

As shown in FIG. 5C, the non-phosphorylated ERK was present in both the cytoplasm and the nucleus, but phosphorylated ERK was inversely proportional to the expression level of DUSP4, and it was confirmed that the expression level of DUSP4 was increased as the level was lowered.

Example 5-4: CaMKI assay

In order to analyze the effect of phosphorylation of ERK on CaMKI (Ca ²⁺ / calmodulin-dependent kinase I), which is known to be involved in neurite outgrowth among signal molecules associated with ERK, the scramble shRNA Or shDUSP4, shDUSP4, or shDUSP4, respectively, or a vector that induces the overexpression of DN ERK (dominant-negative mutant ERK) is introduced into each embryonic stem cell And the changes in protein levels of phosphorylated CaMKI, non-phosphorylated CaMKI and neuronal cell markers (MAP2 and β-tubulin III) in each neuron differentiated from each embryonic stem cell were compared by Western blot analysis 5d). At this time, actin was used as an internal control group.

As shown in FIG. 5D, when normal ERK was overexpressed, the protein levels of phosphorylated CaMKI and neuronal markers (MAP2 and β-tubulin III) were markedly decreased. However, when abnormal ERK was overexpressed, phosphorylated CaMKI and nerve cells It was confirmed that the protein levels of the markers (MAP2 and β-tubulin III) were not significantly reduced.

Example 5-5: Analysis of differentiation efficiency using mutated CaMKI

First, a gene coding for mutated CaMKI (CaMKI T177D) mimicking a phosphorylated form was synthesized by performing PCR using the following primers.

T177D F: 5'-CAGTGTGCTCTCCGACGCCTGTGGGACTCC-3 '(SEQ ID NO: 13)

T177D R: 5'-GGAGTCCCACAGGCGTCGGAGAGCACACTG-3 '(SEQ ID NO: 14)

Next, a gene encoding mutated CaMKI (CaMKI T177A) which can not be phosphorylated was synthesized by performing PCR using the following primers.

T177A F: 5'-CAGTGTGCTCTCCGCAGCCTGTGGGACTCC-3 '(SEQ ID NO: 15)

T177A R: 5'-GGAGTCCCACAGGCTGCGGAGAGCACACTG-3 '(SEQ ID NO: 16)

Each of the thus-synthesized mutated CaMKI genes was introduced into neurons differentiated from embryonic stem cells into which shDUSP4 obtained in Example 3-1 was introduced, and the lengths of neurites were compared (Fig. 5E). At this time, as a control group, shRNA-free neurons, shDUSP4-introduced neuron-derived cells, and shDUSP4-introduced neuron cells were used to introduce normal CaMKI gene.

As shown in FIG. 5E, neurons expressing the DUSP4 (shDUSP4 + Vector) and the mutant CaMKI (CaMKI T177A), which can not be phosphorylated in the neurons inhibiting DUSP4 expression, On the other hand, in the neurons expressing mutant CaMKI (CaMKI T177D) mimicking normal CaMKI and phosphorylated forms in neurons whose DUSP4 expression was suppressed, the length of the neurites was similar to that of the control (Neuron Day 3) Level.

The fact that the length of the nerve procession was similar to that of the control group (Neuron Day 3) indicates that the neural differentiation was normally performed. Therefore, normal CaMKI and NK cells which can be phosphorylated on nerve cells in which the expression of DUSP4 is suppressed, The expression of mutated CaMKI (CaMKI T177D) mimicking the phosphorylated form increased the level of neurites to a level similar to that of normal, indicating that phosphorylation of CaMKI was carried out by expression of DUSP4, It is analyzed that it implies that differentiation is performed.

In order to confirm the above analysis, shDUSP4 + vector in which shDUSP4 was introduced and an empty vector was introduced into differentiated neurons, shDUSP4 + vector, shDUSP4 + (ShDUSP4 + CaMKI T117A) introduced with shDUSP4 and CaMKI gene (CaMKI T117A) mutated into differentiated neuron, and CaMKI gene (CaMKI T117A) mutated into differentiated neuron by introduction of shDUSP4 In one cell (shDUSP4 + CaMKI T117D), changes in protein levels of neuronal markers (MAP2 and beta-tubulin III) were compared via western blot analysis (Fig. 5f). At this time, normal embryonic stem cells were used as a control group, and actin was used as an internal control of western blot analysis.

As shown in FIG. 5F, one neuronal marker (β-tubulin III) was normally detected in all cells, while another neuronal marker (MAP2) was CaMKI (CaMKI T177D) mutated mimicking normal CaMKI and phosphorylated forms, Was expressed only in the neurons expressing. This indicates that the phosphorylation of CaMKI is carried out by the expression of DUSP4 and that the phosphorylation of CaMKI results in the differentiation of neurons.

Example 5-6: CaMKK assay

It was confirmed through the above Examples 5-4 and 5-5 that phosphorylation of CaMKI is involved in neuronal differentiation by DUSP4. Therefore, whether or not phosphorylation of CaMKK involved in phosphorylation of CaMKI is involved in neuronal differentiation by DUSP4 .

Each cell lysate was obtained from each embryonic stem cell into which the scramble shRNA or shDUSP4 obtained in Example 3-1 was introduced and each neuron differentiated from each embryonic stem cell, Anti-CaMKK antibody (Novus, Littleton, CO, USA) was added to the seafood and reacted overnight at 4 ° C. Protein A / G plus agarose beads (Calbiochem, Billerica, Mass.) Was added to the reaction and reacted at 4 ° C with stirring for 2 hours. After completion of the reaction, the reaction product was centrifuged at 3000 xg to recover the precipitate, and the recovered precipitate was washed three times with NP-40 buffer, and then this was incubated with phosphorylated serine / threonine antibody (ECM Biosciences, Versailles, KY) (Fig. 5G). At this time, Western blot analysis using an anti-CaMKK antibody was used as an internal control.

As shown in FIG. 5G, it was confirmed that the expression level of CaMKK was not changed but the level of phosphorylated CaMKK was decreased in cells in which DUSP4 expression was suppressed.

Therefore, phosphorylation of CaMKK was expected to be involved in neuronal differentiation by DUSP4.

Example 5-7: MARK2 assay

Since the phosphorylation of CaMKK was expected to be involved in the differentiation of neurons by DUSP4 through the above Examples 5-6, it was confirmed whether or not the involved phosphorylation of MARK2, which is affected by CaMKK phosphorylation, is involved in neuronal differentiation by DUSP4 .

Each cell lysate was obtained from each embryonic stem cell into which the scramble shRNA or shDUSP4 obtained in Example 3-1 was introduced and each neuron differentiated from each embryonic stem cell, The anti-MARK2 antibody (Abcam) was added to the seafood and reacted at 4 ° C overnight. Protein A / G plus agarose beads (Calbiochem, Billerica, Mass.) Was added to the reaction and reacted at 4 ° C with stirring for 2 hours. After completion of the reaction, the reaction product was centrifuged at 3000 xg to recover precipitates. The recovered precipitates were washed three times with NP-40 buffer, and subjected to western blot analysis using a phosphorylated serine / threonine antibody (Fig. 5H). At this time, Western blot analysis using an anti-MARK2 antibody was used as an internal control.

As shown in FIG. 5h, it was confirmed that the expression level of MARK2 was not changed but the level of phosphorylated MARK2 was decreased in cells in which DUSP4 expression was suppressed.

Thus, phosphorylation of MARK2 was expected to be involved in neuronal differentiation by DUSP4.

Example 6: Analysis of the action mechanism of DUSP4 associated with the regulation of intracellular calcium level

Example 6-1: Analysis of intracellular calcium level

It was confirmed through the above Examples 5-4 and 5-5 that phosphorylation of CaMKI is involved in neuronal differentiation by DUSP4, so that the change of intracellular calcium concentration directly affecting CaMKI activity was examined.

Each of the neurons into which the vector inducing overexpression of DN ERK was introduced after introduction of the scramble shRNA or shDUSP4 obtained in Example 3-1, shDUSP4 introduced, or an empty vector introduced or shDUSP4 introduced The cells were cultured in N2 medium (Invitrogen) containing Fluo-4 / AM dye (Invitrogen) for 30 min at 37 ° C, 5% CO ₂ and washed twice with N2 medium. Then, the level of intracellular calcium in the fluorescent stained cells was measured using a fluorescence microscope (Leica Microsystems), and the measured values were quantitatively analyzed by applying MetaMorph 7.1 (Universal Imaging) (Fig. 6A) .

As shown in FIG. 6A, when the expression of DUSP4 is suppressed (shDUSP4 or shDUSP4 + Vector), the level of intracellular calcium is low and the length of neurite is decreased, while DUSP4 is normally expressed (Scrambled) When the expression was restored (shDUSP4 + DN ERK), the level of intracellular calcium was increased and the length of neurite was increased.

Therefore, it was found that the neuronal cell in which the expression of DUSP4 was inhibited was impaired in intracellular calcium release function, which is a key function for neuron differentiation.

Example 6-2: Analysis of expression level of Cav1.2 channel on cell surface

In Example 6-1, it was confirmed that the neuronal cell in which the expression of DUSP4 was inhibited was impaired in intracellular calcium release function, which is a key function for neuron differentiation. Therefore, Of the expression level of DUSP4.

Each embryonic stem cell into which a vector inducing overexpression of DN ERK was introduced after introducing the scrambled shRNA or shDUSP4 obtained in Example 3-1, the empty vector after shDUSP4 was introduced, or shDUSP4 was introduced, Each of the differentiated neurons was washed three times with cold PBS, allowed to stand at 4 ° C for 30 minutes, and PBS containing 0.5 mg / ml of sulfo-NHS-SS-biotin as a cell membrane-binding reagent was slowly added Shake it. Then, each of the cells was washed with PBS containing 10% BSA and reacted with the same solution except for biotin for 5 minutes to remove NHS-SS-biotin not bound to the cells. The cells were then washed three times with PBS and resuspended in the recovery buffer (137 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 10% glycerol, 0.5% Triton X-100, 50 mM sodium fluoride, 20 mM benzamide , Protease inhibitor cocktail and 20 mM Tris-HCl, pH 8.0), followed by sonication for 30 seconds to obtain a cell lysate. The obtained cell lysate was centrifuged (5,000 xg, 4 캜 for 10 minutes) , And a supernatant was obtained. Fixed NeutrAvidin resin (Thermo Fisher Scientific Inc.) was added to the supernatant obtained above, and reacted at 4 캜 for 12 hours while stirring slowly. After the reaction was completed, the reaction product was centrifuged (6,000 x g, 5 minutes) to obtain a precipitate containing the cell membrane fraction, and the obtained precipitate was washed three times with the recovery buffer. The washed precipitate and supernatant were subjected to Western blot analysis using anti-Cav 1.2 antibody (Thermo Fisher Scientific Inc.) (Fig. 6B). At this time, pan-cadhelin was used as a cell membrane marker, and actin was used as an internal control of western blot analysis.

As shown in FIG. 6B, it was confirmed that Cav1.2 channel protein was not expressed in embryonic stem cells and that expression level was increased as neuronal differentiation progressed. When suppression of DUSP4 expression (shDUSP4 or shDUSP4 + Vector ), The expression level of Cav1.2 channel protein was increased in the case that DUSP4 was normally expressed (scrambled) or its expression was restored (shDUSP4 + DN ERK). In addition, it was confirmed that the cell membrane fraction showed a larger variation in the expression level of Cav1.2 channel protein.

Therefore, when the expression of DUSP4 is suppressed, the expression level of Cav1.2 channel protein is decreased in neurons and the migration of already expressed Cav1.2 channel protein to cell membrane is inhibited.

Example 6-3: Immunochromatographic analysis of Cav1.2 channel

Each of the neurons into which a vector inducing overexpression of DN ERK was introduced after introduction of the scramble shRNA or shDUSP4 obtained in Example 3-1, shDUSP4 introduced, or an empty vector introduced or shDUSP4 introduced Immunostaining was performed using an antibody against Cav1.2 channel protein (Fig. 6C).

As shown in FIG. 6C, it was confirmed that the level of Cav1.2 channel protein was decreased in the neurons in which the expression of DUSP4 was inhibited.

The results of Examples 4 to 6 above show that DUSP4 provided by the present invention is involved in various intracellular signal transduction pathways involved in the differentiation of stem cells into neurons (Fig. 7).

As shown in FIG. 7, when an external signal that induces neuron differentiation is delivered into cells, DUSP4 inhibits ERK phosphorylation and induces expression of Cav1.2 channel protein, and the expressed Cav1.2 channel protein migrates to cell membrane CaMKI and MARK2 were phosphorylated and differentiated into neuronal cells.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel marker of nerve cell and uses thereof <130> KPA140717-KR <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> recombinant DUSP4 <400> 1 Met Val Thr Met Glu Glu Leu Arg Glu Met Asp Cys Ser Val Leu Lys   1 5 10 15 Arg Leu Met Asn Arg Asp Glu Asn Gly Gly Gly Ala Gly Gly Ser Gly              20 25 30 Ser His Gly Thr Leu Gly Leu Pro Ser Gly Gly Lys Cys Leu Leu Leu          35 40 45 Asp Cys Arg Pro Phe Leu Ala His Ser Ala Gly Tyr Ile Leu Gly Ser      50 55 60 Val Asn Val Arg Cys Asn Thr Ile Val Arg Arg Arg Ala Lys Gly Ser  65 70 75 80 Val Ser Leu Glu Gln Ile Leu Pro Ala Glu Glu Glu Val Arg Ala Arg                  85 90 95 Leu Arg Ser Gly Leu Tyr Ser Ala Val Ile Val Tyr Asp Glu Arg Ser             100 105 110 Pro Arg Ala Glu Ser Leu Arg Glu Asp Ser Thr Val Ser Leu Val Val         115 120 125 Gln Ala Leu Arg Arg Asn Ala Glu Arg Thr Asp Ile Cys Leu Leu Lys     130 135 140 Gly Gly Tyr Glu Arg Phe Ser Ser Glu Tyr Pro Glu Phe Cys Ser Lys 145 150 155 160 Thr Lys Ala Leu Ala Ala Pro Pro Pro Val Pro Pro Ser Ala Thr                 165 170 175 Glu Pro Leu Asp Leu Gly Cys Ser Ser Cys Gly Thr Pro Leu His Asp             180 185 190 Gln Gly Gly Pro Val Glu Ile Leu Pro Phe Leu Tyr Leu Gly Ser Ala         195 200 205 Tyr His Ala Ala Arg Arg Asp Met Leu Asp Ala Leu Gly Ile Thr Ala     210 215 220 Leu Leu Asn Val Ser Ser Asp Cys Pro Asn His Phe Glu Gly His Tyr 225 230 235 240 Gln Tyr Lys Cys Ile Pro Val Glu Asp Asn His Lys Ala Asp Ile Ser                 245 250 255 Ser Trp Phe Met Glu Ala Ile Glu Tyr Ile Asp Ala Val Lys Asp Cys             260 265 270 Arg Gly Arg Val Leu Val His Cys Gln Ala Gly Ile Ser Arg Ser Ala         275 280 285 Thr Ile Cys Leu Ala Tyr Leu Met Met Lys Lys Arg Val Val Leu Glu     290 295 300 Glu Ala Phe Glu Phe Val Lys Gln Arg Arg Ser Ser Ile Ser Pro Asn 305 310 315 320 Phe Ser Phe Met Gly Gln Leu Leu Gln Phe Glu Ser Gln Val Leu Ala                 325 330 335 Thr Ser Cys Ala Ala Glu Ala Ala Ser Pro Ser Gly Pro Leu Arg Glu             340 345 350 Arg Gly Lys Thr Pro Ala Thr Pro Thr Ser Gln Phe Val Phe Ser Phe         355 360 365 Pro Val Ser Val Gly Val His Ser Ala Pro Ser Ser Leu Pro Tyr Leu     370 375 380 His Ser Pro Ile Thr Ser Ser Ser Cys 385 390 <210> 2 <211> 5625 <212> DNA <213> Homo sapiens <400> 2 acctagcgcc ccctcccccg ggagcgcgga ggagcattaa taaacctcta agccgaggag 60 aaaactctgg ctggggcagt gcgctgagcg ccggaggagc gtaggcaggg cagcgctggc 120 gccagtggcg acaggagccg cgcgaccggc aaaaatacac gggaggccgt cgccgaaaag 180 agtccgcggt cctctctcgt aaacacactc tcctccaccg gcgcctcccc ctccgctctg 240 cgcgccgccc ggctgggcgc ccgaggccgc tccgactgct atgtgaccgc gaggctgcgg 300 gaggaagggg acagggaaga agaggctctc ccgcgggagc ccttgaggac caagtttgcg 360 gccacttctg caggcgtccc ttcttagctc tcgcccgccc ctttctgcag cctaggcggc 420 ccgggttctc ttctcttcct cgcgcgccca gccgcctcgg ttcccggcga ccatggtgac 480 gatggaggag ctgcgggaga tggactgcag tgtgctcaaa aggctgatga accgggacga 540 gaatggcggc ggcgcgggcg gcagcggcag ccacggcacc ctggggctgc cgagcggcgg 600 caagtgcctg ctgctggact gcagaccgtt cctggcgcac agcgcgggct acatcctagg 660 ttcggtcaac gtgcgctgta acaccatcgt gcggcggcgg gctaagggct ccgtgagcct 720 ggagcagatc ctgcccgccg aggaggaggt acgcgcccgc ttgcgctccg gcctctactc 780 ggcggtcatc gtctacgacg agcgcagccc gcgcgccgag agcctccgcg aggacagcac 840 cgtgtcgctg gtggtgcagg cgctgcgccg caacgccgag cgcaccgaca tctgcctgct 900 caaaggcggc tatgagaggt tttcctccga gtacccagaa ttctgttcta aaaccaaggc 960 cctggcagcc atcccacccc cggttccccc cagtgccaca gagcccttgg acctgggctg 1020 cagctcctgt gggaccccac tacacgacca ggggggtcct gtggagatcc ttcccttcct 1080 ctacctcggc agtgcctacc atgctgcccg gagagacatg ctggacgccc tgggcatcac 1140 ggctctgttg aatgtctcct cggactgccc aaaccacttt gaaggacact atcagtacaa 1200 gtgcatccca gtggaagata accacaaggc cgacatcagc tcctggttca tggaagccat 1260 agagtacatc gatgccgtga aggactgccg tgggcgcgtg ctggtgcact gccaggcggg 1320 catctcgcgg tcggccacca tctgcctggc ctacctgatg atgaagaaac gggtgaggct 1380 ggaggaggcc ttcgagttcg ttaagcagcg ccgcagcatc atctcgccca acttcagctt 1440 catggggcag ctgctgcagt tcgagtccca ggtgctggcc acgtcctgtg ctgcggaggc 1500 tgctagcccc tcgggacccc tgcgggagcg gggcaagacc cccgccaccc ccacctcgca 1560 gttcgtcttc agctttccgg tctccgtggg cgtgcactcg gcccccagca gcctgcccta 1620 cctgcacagc cccatcacca cctctcccag ctgttagagc cgccctgggg gccccagaac 1680 cagagctggc tcccagcaag ggtaggacgg gccgcatgcg ggcagaaagt tgggactgag 1740 cagctgggag caggcgaccg agctccttcc ccatcatttc tccttggcca acgacgaggc 1800 cagccagaat ggcaataagg actccgaata cataataaaa gcaaacagaa cactccaact 1860 tagagcaata acggctgccg cagcagccag ggaagacctt ggtttggttt atgtgtcagt 1920 ttcacttttc cgatagaaat ttcttacctc atttttttaa gcagtaaggc ttgaagtgat 1980 gaaacccaca gatcctagca aatgtgccca accagcttta ctaaaggggg aggaagggag 2040 ggcaaaggga tgagaagaca agtttcccag aagtgcctgg ttctgtgtac ttgtcccttt 2100 gttgtcgttg ttgtagttaa aggaatttca ttttttaaaa gaaatcttcg aaggtgtggt 2160 tttcatttct cagtcaccaa cagatgaata attatgctta ataataaagt atttattaag 2220 actttcttca gagtatgaaa gtacaaaaag tctagttaca gtggatttag aatatattta 2280 tgttgatgtc aaacagctga gcaccgtagc atgcagatgt caaggcagtt aggaagtaaa 2340 tggtgtcttg tagatatgtg caaggtagca tgatgagcaa cttgagtttg ttgccactga 2400 gaagcaggcg ggttgggtgg gaggaggaag aaagggaaga attaggtttg aattgctttt 2460 taaaaaaaaa agaaaagaaa aagacagcat ctcactatgt tgccaaggct catcttgaga 2520 agcaggcggg ttgggtggga ggaggaagaa agggaagaat taggtttgaa ttgctttttt 2580 aaaaaaaaaaaaaagaaaaa aaaagacagc atctcactat gttgccaagg ctcatctcaa 2640 gctcttgggc tcaagagatc ctcccacctc ggcctcctga gtagctggga ctgcaggtgt 2700 gtgtcatcat gaccaatgtg aattgctttt gaagctggtt catgggcatg taggccaccg 2760 aagcaatttt agaccacagt aagtcaagct tttttccctc cgatgatcac tgggtggttg 2820 cagcattttt tgcataaacc tgcctaagac ttgtctatcg tctgtgatca atatgccata 2880 ttacactaag gtgctcctgg aaaattgggt gcagttcaaa ttttcctaca gcaaatcatt 2940 tggcaaggcc agccattggg gaaaccagac aactagagat aaccctgaaa tgaatccttt 3000 tgtaaattga agcaccatct tttctttttt tgcataaatt ggaggtttta attttagggc 3060 agttacctga agtgaaatat accaacaatt tcttgtgttc tttaaattcc tagttaggtg 3120 aatatttttg aaggtcctct tttgaataaa gaggggaatg gacaccacat ttcaggtctt 3180 ctcgaagtgt ggaagggcaa gagagcatca gtgagctgat ggtggattgc ttacatcgga 3240 ttccattggt atgaatttcc caaactggaa atcaaagcgc cagggtgggg ttggggctga 3300 ctgctggtga gggggctggc cgctggctcc cgtgacgtgc gtcatgggca cgcaggcgcc 3360 attttgaatc tatcgtcggc acgtgggtgc cattttgaat ccttagttgg gcctttctaa 3420 atggagaatg gctttggagg gagacacgtt ttctgtgggg agggtttggg ggggagggag 3480 gagggaacaa gctacatgct attttgtttg tagtattgtg gaacagtctt gttatggagt 3540 gccagcttag aggttgttgc aaacttgtct agaagtgaga gcatggtttt ttttagccct 3600 ttgagagtct acatctaatg aacattcttg ctcacccata aataacgtca agcctcaatg 3660 tcaccgtcac gttgggatac tctttctcat ctggcatcct agacaggaca aggttggtta 3720 cctttccttc catgaaccat gaacctgtga cggcatcatt catcctgact tcaccaagct 3780 ccgcctgtgg gtgaggccag agctcccact ggcaattttt agaagagcca gaggctccct 3840 gcttcctcta gaaataacag ttcagggtga agcatggagg gtttcagttc ccagacaatg 3900 gaaccattta gagacaacac agttggacat ttccactttt tccttgattc ctggaagtcc 3960 agtgggttct gcagctgaaa aagccctggg tcccagcagc agagagacag gacagagggg 4020 atgcttgggc ggggagggac ggtaacctgc agaacagatt ccatttttat agaacgagta 4080 cacgtttgct aaaacagtcc tgctttccca gactggattc ccaccacagg gacagtcgga 4140 actcaggact agctccagcg acatctttcc tccgaattca agccttctat cacaatgtca 4200 aaacagctat ttataaagcc attttcattg tacttgataa cagcacgagt cccaaaactt 4260 ttagaaataa aataggacat tggcttgatt gaaaagaggg actttttaaa aattgttctt 4320 tcgtcagaag ccttttggat gacttacaat agctctgatg aagataccac cccagcgtca 4380 gtccaatagg tcagtgagtt tcaacaggca tccatccctc ccatgaaggg attctggtga 4440 tgggaagttt ctgtaatgac aggaaagcat tgaccctcat tgattgtcaa ctttggtatt 4500 agccatgaaa gacaggatgc tcattgggtg ttctgtagag tgaggaatgc tgcctattcc 4560 ctcccagaac gtctgaccca ggggtgtgtg ttgaggagcc ctgggggaaa tggaccaagt 4620 tttcccacag agcagtatta ggctgaagag caggtgactg gtaggcccca gctcccatca 4680 ttccctccca aagccatttt gttcagttgc tcatccacgc tggattccag agagttttcc 4740 aatttgggaa gccatgagaa aggtttttaa atcttgggaa gatggagaga gggacatagg 4800 atagttgact ccaacatgac aggaagaggc tggagattgg gaattggcca tcaaccaagc 4860 ctgtagtagt aaagccatgg tcccgcattg gaattacttg gggaacttat acagttctga 4920 tacccaggct ctcctagacc agttcaacca attctaggtg ggggactcag gcatcagtgt 4980 gtttcgtagc tccccgggtg ttttccctgt gcagccgagc ttgggaaact gccatgcttt 5040 ttggatgtca aggcgctgtt ggaggctggg tgtgacagca cagagccagg ttgtcttgtg 5100 gaaaccacag ccacgggttt gccactggct cagcatggcc tcactgccag tcccagcctg 5160 gctgagggac aagatggttt ctcttgggag ttcctgagtg gagcaccctt ccaggctttt 5220 tgaaagccag ctgatctgtg gagccttgtt aagggactca atacggtgtt tggatattga 5280 tgtttttcct tgagactgtc ttgtccatca ataaagatgg aggatgtctc ctctttgaac 5340 cccgcttccc caccagtact ctctctccct tagagtttat gagttattca aggaggagac 5400 ttcttaaaga cagcaacgca attcttgtaa cttgtgtaaa tagccccatc tttcagagtg 5460 ataccatttc tacatttgat aatgcctgta ttcctgtagg atgtatatag tttaggggat 5520 tttttttttg tttggttttg ttttttagaa gtcaatatgt ctggttttat ttattgcttg 5580 aaaaagatca tttgaaaaaa ataaatacat tttcaaccac taaaa 5625 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gaggtgaccc ttgggttaga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tagccctacc acttcctgct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agccctctac gacatctgct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 attgagctga ccagggaatc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cactggacat cagcctcact 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 caccctggtc cttcatctct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gagaacaaca aagggccaat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcacataggc atctccttga 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctggcctacc tgatgatgaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tgagactcaa actgcagcaa 20 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cagtgtgctc tccgacgcct gtgggactcc 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ggagtcccac aggcgtcgga gagcacactg 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cagtgtgctc tccgcagcct gtgggactcc 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggagtcccac aggctgcgga gagcacactg 30

Claims (13)

A method of differentiating neural cells comprising the step of overexpressing DUSP4 (dual-specificity phosphatase 4) in stem cells.
The method according to claim 1,
Wherein the overexpression is performed by introducing the DUSP4 gene into the stem cells.
3. The method of claim 2,
Wherein said DUSP4 gene is introduced into a stem cell in the form of an expression vector comprising said DUSP4 gene.
3. The method of claim 2,
Wherein the DUSP4 gene comprises the nucleotide sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein said stem cells are embryonic stem cells, degenerated stem cells or mesenchymal stem cells.
The method according to claim 1,
Wherein the stem cell overexpressing the DUSP4 gene is differentiated into a neural cell via an embryo-like cell mass, a neural progenitor cell, and a neural progenitor cell.
A composition for promoting differentiation of neurons comprising DUSP4 (dual-specificity phosphatase 4) gene.
8. The method of claim 7,
RTI ID = 0.0 &gt; DUSP4 &lt; / RTI &gt; gene.
9. The method of claim 8,
Wherein the DUSP4 gene comprises the nucleotide sequence of SEQ ID NO: 2.
A stem cell having dual-specificity phosphatase 4 (DUSP4) gene overexpression, or a cell differentiated therefrom.
11. The method of claim 10,
Wherein the differentiation-induced cell is an embryonic-like cell mass, a neural progenitor cell or a neuroblastoma.
11. The method of claim 10,
Wherein the cranial nerve system disease is an exogenous nerve injury or degenerative nerve disease.
13. The method of claim 12,
Wherein the degenerative neurological disease is a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, tauopathy, and combinations thereof.

Images