KR20200004208A - Pharmaceutical composition for preventing or treating the dementia comprising human neural crest derived stem cells and screening method thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising human neural crest derived stem cells as active ingredients, and a screening method thereof. According to the present invention, the human neural crest derived stem cells have effects of reducing expression of amyloid-β peptide (Aβ), inflammatory microglias, BDNF pro-domain, and p75NTR in the brain of an Alzheimer′s disease mouse model, increasing expression of NEP which is β amyloid-degradation enzymes and expression of LC3B, RAB7, etc. which are autophagy signaling proteins, and protecting nerve cells and being differentiated partially into nerve cells. Therefore, according to the present invention, the human neural crest derived stem cells can be effectively utilized as a pharmaceutical composition for preventing or treating dementia.

Description

Pharmaceutical composition for preventing or treating the dementia comprising human neural crest derived stem cells and screening method according to the present invention.

The present invention relates to a pharmaceutical composition for preventing or treating dementia comprising human neural stem cells derived from nasal stem cells as an active ingredient, and to a method for screening the same, wherein the BDNF pro-domain is reduced by using human nerve cell derived nasal stem cells. It relates to a pharmaceutical composition for preventing or treating dementia and a screening method thereof.

Dementia is a complex condition in which a mature brain is damaged or destroyed by an acquired trauma or disease, and the overall cognitive and higher mental functions such as intelligence, learning, and language are generally deteriorated. The rapid increase in the number of dementia-related dementia has become a serious social problem. Although dementia has a high incidence, there are currently no clear treatments or preventive agents that cause huge socioeconomic losses.

Alzheimer's disease, the most common form of dementia, is a degenerative brain disease that causes severe brain and cognitive impairment in the course of aging, causing severe disorders in memory and cognitive function. It is necessary to identify and study important factors affecting the rapid reduction of cognitive ability in Alzheimer's disease in order to develop new mechanisms and new treatments.

Many studies have been conducted on the mechanism of Alzheimer's disease so far, but it is not yet clearly identified. According to research reports on Alzheimer's disease, amyloid-β peptide (Aβ) is considered to be an important cause of disease progression. It is formed from amyloid precursor protein (APP). The β-pleated sheet form is insoluble and accumulates to form amyloid fibril aggregates, which are known to be neurotoxic by themselves and cause inflammatory reactions to cause brain cell damage.

Neurotrophic factors, on the other hand, regulate the survival and differentiation of neurons during development, maintain neuronal structure and ion channel activity, neurotransmitter release, and axon path-finding throughout the life of an individual. ) And other functions. Examples of such neurotrophic factors are neuronal growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (Neurotrophin-3, NT-3) and neurotropin -4/5 (Neurotrophin-4 / 5, NT-4 / 5) is present.

Among them, brain-derived neurotrophic factor (BDNF) is most present in the central nervous system, and is expressed in the hippocampus, the cortex and the base of the brain, which are responsible for learning, memory, and higher thinking ability in the central nervous system. BDNF is known to play an important role in the development, survival and differentiation of neurons. Precursor BDNF is divided into BDNF (mBDNF) and BDNF in pro-peptide (pro-domain) form of mature form through cleavage process. .

Previous studies have reported that the onset and progression of Alzheimer's disease is closely associated with decreased expression of BDNF (Nature Reviews 10: 209-219, 2011; Nat. Med. 15: 331-337, 2009; Gene Ther. 15: 966-977, 2008; J. Neurosci. 15: 7810-7820,1995), mature form BDNF (mBDNF) inhibits neuronal cell death and prevents neuronal cell death in animal models of intractable neurological diseases such as stroke and spinal cord injury and Alzheimer's disease. Increasing survival, studies have shown that mBDNF is effective in regulating inflammatory and immune responses in epileptic animals, and clinical trials have been conducted in patients with Lou Gehrig's disease, a degenerative neurological disorder, using recombinant BDNF.

However, the expression of BDNF pro-domain in brain tissues of Alzheimer's disease patients was found to be increased compared to normal subjects. Thus, the present inventors have expressed the expression of BDNF pro-domain and the treatment of dementia, which reduces the expression of BDNF pro-domain in dementia models. The aim of this study was to develop a method for screening therapeutic agents for dementia by measuring the amount.

The present inventors have studied to develop a therapeutic agent for dementia and a method for screening the same, and as a result, self renewal ability to continuously proliferate itself, differentiation to specific tissue, and lesions Human neural crest derived nasal stem cells, one of the stem cells that have been effectively used as a therapeutic agent for various diseases because of their tropism, have been implanted into the brains of Alzheimer's disease mice. When the expression of the BDNF pro-domain was reduced.

Accordingly, an object of the present invention is a pharmaceutical composition for the prevention or treatment of dementia comprising human neural stem cells derived from nasal stem cells as an active ingredient, the pharmaceutical composition and screening thereof, characterized by reducing the expression of BDNF pro-domain To provide a way.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention is a pharmaceutical composition for the prevention or treatment of dementia comprising human neural crest derived nasal stem cells as an active ingredient,

The composition provides a pharmaceutical composition, characterized in that the expression of a brain-derived neurotrophic factor (BDNF) pro-domain is reduced.

In addition, the present invention comprises the steps of: a) treating the candidate substance to the brain of a dementia mouse model;

b) identifying the increase or decrease in the expression of brain-derived neurotrophic factor (BDNF) pro-domains in the brain of the demented mouse model after treatment with the candidate substance; And

c) selecting candidates that reduce the expression of BDNF pro-domain as compared to the control that did not treat the candidates;

It provides a screening method of treating dementia, comprising a.

In one embodiment of the present invention, the dementia may be selected from the group consisting of Alzheimer's disease, cerebrovascular dementia, senile dementia, Parkinson's disease, and dementia due to head injury.

In another embodiment of the present invention, the dementia may be Alzheimer's disease.

In another embodiment of the present invention, the step a) may be to process the candidate material in the hippocampus portion of the dementia mouse model.

As another embodiment of the present invention, step b) may further include increasing or decreasing the expression of the p75 neurotrophin receptor (p75NTR).

As another embodiment, the step c) may further comprise the step of selecting a candidate to reduce the expression of p75NTR compared to the control group not treated with the candidate.

The present invention also provides a method for preventing or treating dementia, comprising administering the pharmaceutical composition to a subject.

The present invention also provides a use of the pharmaceutical composition for preventing or treating dementia.

Human neural stem cells derived from human neural stem cells according to the present invention reduce the expression of amyloid-β peptide (Aβ), inflammatory microglia, BDNF pro-domain, and p75NTR in the brain of the Alzheimer's disease mouse model, and beta amyloid-degradation enzyme. In addition to the effect of increasing the expression of NEP and the expression of autophagy signals proteins LC3B, RAB7, etc., it was confirmed to protect neurons and some of them differentiate into neurons. Therefore, human neural stem cells derived from the neural stem cells according to the present invention are expected to be usefully used as a pharmaceutical composition for the prevention or treatment of dementia.

1A shows immunohistofluorescence staining for Aβ plaque deposition (green color) when human neural stem-derived nasal stem cells (hNCNSCs) were implanted into the brain hippocampus of an Alzheimer's disease mouse model according to an embodiment of the present invention. Results are shown, Figure 1b is a diagram showing the results of measuring the expression level of Aβ42 by ELISA.
Figure 2a shows the immunomodulatory effect on inflammatory microglial activity following transplantation of hNCNSCs by immunohistochemical staining using Iba-1 antibody (green color) in the brain of an Alzheimer's disease mouse model according to an embodiment of the present invention. Figure 2b is an enlarged view showing the results of implantation of PBS and hNCNSCs (white box portion) of the immunostaining results using the Iba-1 antibody.
Figure 3a is a result of confirming the neuronal protective effect of the transplantation of hNCNSCs by immunohistofluorescence staining method using NeuN antibody (green color) in the brain of the Alzheimer's disease mouse model according to an embodiment of the present invention, Figure 3b is NeuN Among the results of immunostaining using antibodies, the results of transplantation of hNCNSCs and bone marrow-derived mesenchymal stem cells (hBMSCs) (white box area) are enlarged.
Figure 4a is a transplant cell according to the transplantation of hNCNSCs and hBMSCs by immunohistochemical staining method using human nuclei specific antibody (red color) and NeuN antibody (green color) in the brain of Alzheimer's disease mouse model according to an embodiment of the present invention Is a result of confirming the survival and differentiation into neurons, Figure 4b is an enlarged view of a portion (white box portion) of Figure 4a.
5 is a diagram showing the expression of BDNF pro-domain, p75NTR, BDNF mature form, and sortilin according to the transplantation of hNCNSCs in the brain of the Alzheimer's disease mouse model according to an embodiment of the present invention through western blotting.
FIG. 6 is a diagram illustrating the expression of NEP and autophagy signals related proteins according to the transplantation of hNCNSCs in the brain of an Alzheimer's disease mouse model according to an embodiment of the present invention through western blotting.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, the present invention will be described in detail.

The present invention is a pharmaceutical composition for preventing or treating dementia comprising human neural crest derived nasal stem cells (hNCNSCs) as an active ingredient,

The composition provides a pharmaceutical composition, characterized in that the expression of a brain-derived neurotrophic factor (BDNF) pro-domain is reduced.

As used herein, the term "neural crest" is a group of ectoderm cells that appear temporarily immediately after embryonic development during vertebrate development, and corresponds to the last cutoff with the outer epidermis when the neural tube is formed. It is a part. Neural crest stem cells migrate from the rear of the neural fold during the migratory stage of the developmental stage. Neural crest stem cells migrate to a wide range of tissues and organs in the body. It is known to differentiate into neural cells, glial cells of the nervous system, melanocytes of the skin, endocrine cells, and various mesenchymal cells and distribute them in adult non-nervous tissues and organs.

The term "stem cell" used in the present invention is a cell that is the basis of a cell or tissue constituting an individual, and its feature is capable of repeated division and self-renewal, and a specific function according to the environment. It means a cell having a multipotent ability to differentiate into cells having a. It occurs in all tissues during the development of the fetus, and is found in some tissues, such as bone marrow and epithelial tissue, which are actively replaced even in adulthood. Stem cells are totipotent stem cells that form when the fertilized egg begins its first division, depending on the type of cells that can differentiate, and pluripotent stem cells in the endoplasmic linings where these cells continue to divide. It is classified as multipotent stem cells in mature tissues and organs. At this time, pluripotent stem cells are cells that can only differentiate into cells specific to the tissues and organs that contain the cells, and the homeostasis of adult tissues as well as the growth and development of individual tissues and organs in the prenatal, neonatal and adult periods. It is involved in the maintenance of regeneration in maintenance and tissue damage. These tissue specific pluripotent cells are collectively referred to as adult stem cells.

Mesenchymal stem cells, which are classified as adult stem cells, are well known as repair cells of various connective tissues, and these cells can differentiate into various types of cells of the mesenchymal system. In addition, due to the advantages of easy acquisition and clinical application, it is possible to supplement the shortcomings of neural stem cells, which are located in the deep part of the brain, are difficult to obtain a sufficient amount, and there is a risk of brain damage. It is attracting attention as a material and can be extracted from tissues such as bone marrow, umbilical cord blood, adipose tissue, and umbilical cord. It has the ability to differentiate into. In the present invention, mesenchymal stem cells isolated from human nasal nasal turbinate tissue were used.

The "harmonic palatal tissue" is an independent small bone representing the shell shape on the left and right sides of the nasal cavity, attached to the maxilla and palate, and there is a large distribution of reticular venous sinus and many mesenchymal stromal cells around them. As a submucosal tissue that is present, one obtained in the course of the paraplegic partial resection performed in the otorhinolaryngology area for the purpose of improving nasal congestion may be used.

In addition, it is possible to use a tissue biopsy easily within a short time without pain of the patient in the otolaryngology outpatient rather than the operating room.

Accordingly, the present invention can provide a material for self-donation and transplantation using tissue that is removed and discarded during many surgical procedures performed to improve the anatomical structure of the human body for the purpose of alleviating the symptoms. There is a characteristic that can contribute to the recycling and the establishment of customized tissue and cell bank of the patient using the same.

The term "differentiation" used in the present invention refers to a process in which the stem cells in the early stages have characteristics as individual tissues, and in the present invention, a part of human neural stem cells derived from human neural stem cells transplanted into the brain of Alzheimer's disease mouse Differentiation into neurons was confirmed.

As used herein, the term "dementia" refers to a significant decline in mental capacity, such as intelligence, will, memory, etc. due to cerebral diseases, the dementia in the present invention Alzheimer's disease, cerebrovascular dementia, senile dementia, Parkinson's disease And, and may be selected from the group consisting of dementia due to head injury, according to a specific embodiment or experimental example of the present invention may be Alzheimer's disease, but is not limited thereto.

In addition, the present invention comprises the steps of: a) treating the candidate substance to the brain of a dementia mouse model;

b) identifying the increase or decrease in the expression of brain-derived neurotrophic factor (BDNF) pro-domains in the brain of the demented mouse model after treatment with the candidate substance; And

c) selecting candidates that reduce the expression of BDNF pro-domain as compared to the control that did not treat the candidates;

It provides a screening method of treating dementia, comprising a.

At this time, the step b) may further comprise the step of confirming the increase or decrease in the expression of the p75 neurotrophin receptor (p75 neurotrophin receptor, p75NTR).

In addition, step c) may further comprise the step of selecting a candidate to reduce the expression of p75NTR compared to the control group did not process the candidate.

As used herein, the term "neutropin receptor" is also referred to as a neurotrophic factor receptor, and is divided into Trk (tropomyosin-related kinase) and p75NTR. In the present invention, the p75NTR may be a receptor to which the BDNF pro-domain binds.

The term "expression" used in the present invention means to express the information possessed by the gene in the form of a gene product having a function of RNA or protein according to the program, and the transcription, post-transcription modification, translation until the gene is expressed This is done by means of control at individual stages called post-translational equations.

The candidate substance of the present invention means an unknown substance used in screening for measuring the increase and decrease of the expression of the BDNF pro-domain, stem cells, stem cell culture, compounds, microbial culture or extracts, natural extracts, nucleic acids, and peptides It may be selected from the group consisting of, wherein the nucleic acid is microRNA, siRNA, shRNA, antisense RNA, aptamer (aptamer), LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino It may be selected from the group consisting of, but is not limited thereto.

The method for measuring the increase and decrease of the expression of the BDNF pro-domain is a conventional method known in the art Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA) ), Immunoprecipitation, flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, or protein chip Can be measured by one or more methods selected from, and according to a specific embodiment or experimental example of the present invention may be Western blotting, enzyme immunoassay (ELISA), or immunofluorescence (immunofluorescence) This is not restrictive.

In the present invention, the step a) may be to process the candidate material in the hippocampus portion of the dementia mouse model, but is not limited thereto.

The term "hippocampus" used in the present invention is located inside the cerebral lobe of the cerebrum and occupies a part of the arc in the middle of the circumferential system, involved in learning, memory, and recognition of new things, emotional behavior and part Adjust your exercise.

The present invention also provides a method for preventing or treating dementia, comprising administering the pharmaceutical composition to a subject.

The present invention also provides a use of the pharmaceutical composition for preventing or treating dementia.

As used herein, the term "prevention" means any action that inhibits a disease or delays the onset by administration of a pharmaceutical composition according to the invention.

As used herein, the term "treatment" refers to any action by which administration of the pharmaceutical composition according to the present invention improves or advantageously alters the condition for the disease.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

As used herein, the term "individual" refers to a subject in need of treatment of a disease, and more specifically, human or non-human primates, mice, dogs, cats, horses, cattle, and the like. Mean mammal.

As used herein, the term "pharmaceutical composition" means prepared for the purpose of preventing or treating a disease, and may be used by formulating in various forms according to conventional methods, respectively. For example, they may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and the like, creams, gels, patches, sprays, ointments, warnings, lotions, linings, pastas or It may be used in the form of external preparations such as cataplasma, suppositories, and sterile injectable solutions.

The pharmaceutical compositions according to the invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions. At this time, carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug, It may be determined according to the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art. Administration may be administered once a day or may be divided several times.

In one experimental example of the present invention, when the human neural crest-derived nasal stem cells were transplanted into the brain of Alzheimer's disease mice, it was confirmed that Aβ plaque deposition and Aβ42 expression were reduced compared to the control group (see Experimental Example 1).

In another experimental example of the present invention, when the human neural stem cell-derived nasal stem cells were transplanted into the brain of Alzheimer's disease mice, it was confirmed that the expression of inflammatory microglia was reduced compared to the control group (see Experimental Example 2).

In another experimental example of the present invention, it was confirmed that when the human neural stem cell-derived nasal stem cells were transplanted into the brain of Alzheimer's disease mice, neuronal protective effects were shown (see Experimental Example 3).

In another experimental example of the present invention, when the nasal stem cells derived from human neural crest were implanted into the brain of Alzheimer's disease mice, the effect of differentiation into neurons was confirmed (see Experimental Example 4).

In another experimental example of the present invention, the expression of BDNF pro-domain, p75NTR, BDNF mature form, and sortilin was confirmed when human neural stem cells were transplanted into the brain of Alzheimer's disease mice. It was confirmed that the expression of p75NTR was significantly reduced compared to the control (see Experimental Example 5).

In another experimental example of the present invention, when the human neural stem cell-derived nasal stem cells were transplanted into the brain of Alzheimer's disease mice, it was confirmed that the expression of NEP and the expression of autophagy signals proteins LC3B and RAB7 were higher than those of the control group (Experimental Example 6 Reference).

Hereinafter, preferred embodiments and experimental examples of the present invention will be described in detail with reference to the accompanying drawings. However, these Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these Examples and Experimental Examples.

Example 1 . Brain Tissue Preparation in Alzheimer's Disease Mice

Stereotaxic injection of APP / PS1 transgenic Alzheimer's disease mouse animal model (5xFAD) 1 x 10 ⁵ human neural crest-derived nasal stem cells (hNCNSCs, 2 x 10 ⁵ stem cells) in each hippocampus region of the brain the implant, and mouse models both hippocampus regions of the brain of the other group, the bone marrow-derived MSCs (hBMSCs) were each transplanted by 1 x 10 ^5. Another group was intraperitoneally injected once daily for three days with no cell transplantation, a signal inhibitor of p75NTR (TAT-PEP5, TAT5), a receptor for BDNF pro-domain or BDNF precursor.

Experimental Example  1. a person Nervous power  Transplantation of Derived Nasal Stem Cells Aβ  Confirmed plaque deposition and reduced Aβ42 expression

1-1. Immunological Tissue fluorescence  Dyeing Aβ  Check for reduced plaque deposition

Six weeks after the cell transplantation by the method of Example 1, brain tissues were extracted by sacrificing Alzheimer's disease mice by PFA perfusion, the tissues were embedded in the OCT compound to form blocks, and sections were sectioned at a thickness of 14-16 μm to Aβ plaque deposition ( immunohistofluorescence staining for green color).

As a result, as shown in FIG. 1A, the cortex and hippocampus of the mouse animal model of the brain when transplanted with human neural stem-derived nasal stem cells than when injected with PBS as a control for two stem cells or TAT5 Hippocampus) showed a significant decrease in Aβ plaque deposition, and the brains of the TAT5-injected model showed a decrease in Aβ, but no significant difference was observed.

1-2. Using ELISA Of Aβ42  Reduction of expression

After 6 weeks after the cell transplantation by the method of Example 1, brain tissues of Alzheimer's disease mice were extracted without perfusion process, soluble proteins were isolated from tissues using RIPA buffer, and the expression level of Aβ42 was measured by ELISA.

As a result, as shown in FIG. 1B, it was confirmed that the expression of Aβ42 protein was significantly increased in the brain of the Alzheimer's disease animal model injected with PBS as compared to normal animals, and the amount of Aβ42 expression in the group transplanted with human neural stem cells derived from nasal stem cells. It was confirmed that this significantly decreased.

Experimental Example  2. The person Nervous power  Reduction of Inflammatory Microglial Expression Following Transplantation of Derived Nasal Stem Cells

After 6 weeks after the cell transplantation, the brain tissues were sacrificed at the expense of Alzheimer's disease mice by PFA perfusion, and the tissues were embedded in the OCT compound to form blocks and sections of 14-16 μm thick to inflammatory microglia (inflammatory We confirmed the immune modulatory effect of TAT5 or two stem cell transplantation by immunohistochemical staining using Iba-1 antibody (green color).

As a result, as shown in Figures 2a and 2b, the expression of inflammatory microglia is reduced in the cortex and hippocampus of the mouse animal model when transplanted human neural stem cells derived from nasal stem cells than when injected with PBS However, bone marrow-derived mesenchymal stem cell transplantation model was confirmed that the expression does not decrease significantly. In addition, it was confirmed that the microglia expression is reduced in the brain of the model injected with TAT5.

Experimental Example  3. The person Nervous power  Confirmation of Neuronal Cell Protective Effects of Transplanted Nasal Stem Cells

After 6 weeks after the cell transplantation by the method of Example 1, brain tissues were extracted by sacrificing Alzheimer's disease mice by PFA perfusion, and the tissues were embedded in the OCT compound to make blocks and sectioned at a thickness of 14-16 μm to be neuronal markers. Immunological tissue fluorescence staining using NeuN antibody (green color) confirmed neuroprotective effects of TAT5 or two stem cell transplants.

As a result, as shown in FIGS. 3A and 3B, when the human neural stem-derived nasal stem cells were transplanted than when the PBS, TAT5, and bone marrow-derived stem cells were injected, the nerve cells were much higher in the cortex and hippocampus of the mouse animal model brain. Many things were confirmed.

Experimental Example  4. People Nervous power  Confirmation of Differentiation Effect of Derived Nasal Stem Cells into Neurons

6 weeks after the cell transplantation by the method of Example 1, the brain tissues were sacrificed at the expense of Alzheimer's disease mice by PFA perfusion, and the tissues were embedded in the OCT compound to make blocks, and the cells were transplanted into the mouse brain by sectioning 14-16 μm thick. Immunological tissue fluorescence staining using human nuclei specific antibody (red color) and neuron marker NeuN antibody (green color) against human cells for survival and differentiation of transplanted cells into neurons Confirmed.

As a result, as shown in Figs. 4a and 4b, when the human neural stem cells derived from nasal stem cells were transplanted than when the bone marrow-derived stem cells were transplanted, it was confirmed that the survival of the transplanted cells was much higher around the implanted site. Some of them were confirmed to differentiate into neurons.

Experimental Example  5. People Nervous power  Following Nasal Stem Cell Transplantation BDNF  pro-domain, p75NTR, BDNF  mature form, and sortilin  Expression level check

After 6 weeks after the cell transplantation, the brain tissues of Alzheimer's disease mice were extracted without perfusion process, and soluble proteins were isolated from the tissues using RIPA buffer and sortilin, BDNF pro-domain, which was co-receptor with p75NTR. Expression levels of BDNF mature form and the like were confirmed by western blotting.

As a result, as shown in FIG. 5, the expression of BDNF pro-domain and its receptor, p75NTR, was significantly reduced in the mouse animal model brain when transplanted with human neural stem-derived nasal stem cells as compared to when PBS was injected. It was.

Experimental Example  6. People Nervous power  Following Nasal Stem Cell Transplantation NEP  And autophagy  Expression level of signals related proteins

After 6 weeks after the cell transplantation, the brain tissues of Alzheimer's disease mice were extracted without perfusion, and soluble proteins were isolated from the tissues using RIPA buffer, and the beta amyloid-degradation enzyme NEP expression and autophagy signals were related. Expression of the proteins was confirmed by western blotting.

As a result, as shown in FIG. 6, when the human neural stem cells derived from nasal stem cells were transplanted than when injected with PBS, the expression of beta amyloid-degradation enzyme NEP and autophagy signals proteins LC3B and RAB7 in the mouse animal model brain. It was confirmed that the expression of is high.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

As a pharmaceutical composition for the prevention or treatment of dementia comprising human neural crest derived nasal stem cells as an active ingredient,
The composition is characterized in that to reduce the expression of brain-derived neurotrophic factor (BDNF) pro-domain (pro-domain), pharmaceutical composition.
The method of claim 1,
The dementia is selected from the group consisting of Alzheimer's disease, cerebrovascular dementia, senile dementia, Parkinson's disease, and head injury.
The method of claim 2,
The dementia is Alzheimer's disease, characterized in that the pharmaceutical composition.
a) treating candidates in the brain of a dementia mouse model;
b) identifying the increase or decrease in the expression of brain-derived neurotrophic factor (BDNF) pro-domains in the brain of the demented mouse model after treatment with the candidate substance; And
c) selecting candidates that reduce the expression of the BDNF pro-domain as compared to the control that did not treat the candidates;
It comprises a, screening method of the dementia treatment.
The method of claim 4, wherein
Step a) is a method for screening a dementia therapeutic agent, characterized in that the candidate material is treated in the hippocampus portion of the dementia mouse model.
The method of claim 4, wherein
The step b) further comprises the step of confirming the increase or decrease in the expression of the p75 neurotrophin receptor (p75 neurotrophin receptor, p75NTR), the screening method of the dementia treatment agent.
The method of claim 4, wherein
The step c) further comprises the step of selecting a candidate to reduce the expression of p75NTR compared to the control group not treated with the candidate, screening method for treating dementia.

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