KR101186218B1 - Use of EGF-like Domain Peptide of Heregulin Beta 1 - Google Patents

Abstract

The present invention relates to the use of the EGF-like domain peptide of hiregulin beta1 to improve memory or to prevent or treat memory impairment diseases.

The EGF-domain domain peptide of hiregulin beta 1 according to the present invention can enhance memory by inducing proliferation of neuronal progenitor cells and differentiation into neuronal cells that produce neurotransmitters and can be used for the prevention or treatment of memory impairment diseases. Can be.

Hiregulin, HRG, EGF, Nerve, Hyperplasia, Memory

Description

Use of EGF-like Domain Peptide of Heregulin Beta 1}

The present invention relates to the use of the EGF-like domain peptide of hiregulin beta1 to improve memory or to prevent or treat memory impairment diseases.

Mammalian brain has a complex function by securing a number of cells by proliferation of neural precursor cells and generating a systematic neural network through a series of processes such as differentiation, survival, death and synapse formation. Will be able to perform Recently, neural progenitor cells have been identified in adults, and the process of stem cell proliferation and differentiation (neurogenesis) in the adult brain can be called regeneration (Identification of a neural stem cell in the adult mammalian). central nervous system (1999), Cell 96, 25-34). Neural progenitor cells are found mainly in the subventricular zone of the striatum, which is in contact with the lateral ventricle, and also in the subventricular zone of the hippocampus. These cells can differentiate and function (Functional neurogenesis in the adult hippocampus (2002) Nature 415, 1031-1034). Even during adulthood, neuronal progenitor cells proliferate in some parts of the brain, such as the hippocampus, to replace dead cells. Neuronal cell proliferation occurs in areas affected by degenerative brain disease or brain damage, which promotes neurogenesis. Should be. The adult brain produces many substances necessary for nerve growth, grows axons and dendrites, and continuously remodels synaptic connections and neural networks with new learning and memory. Continues. The proliferation and differentiation of neural progenitor cells occurs mainly in the hippocampus in the adult brain, and the hippocampus forms spatial perception and memory, and dementia patients initially have entorhinal coetex and cholinergic neurons and glutamate neurons in the hippocampus. (Glutamatergic neurons), GABAergic neurons (regulating their activity) are killed by the loss of spatial memory. In addition, nerve cell death caused by cramps and ischemia in the hippocampus causes memory loss in epilepsy and stroke.

In the process of cell differentiation and synapse formation, nerve cells die when they do not receive a target-derived survival factor, such as nerve growth factors. Cell death by stress and cytotoxic agents is degenerative. It is a major cause of brain disease. Degenerative brain diseases in which death of neuronal cells is a major cause include various diseases such as dementia, Parkinson's disease, and Alzheimer's disease. Most degenerative brain diseases are accompanied by memory impairment (memory loss), and recent memory deterioration phenomenon has occurred in the general public due to deteriorating living environment such as stress and pollution caused by complex social activities.

The most likely candidate for neuroregeneration is the neurotrophic factor. Therefore, it is possible to promote neuronal regeneration by securing the number of cells by promoting the proliferation of neural progenitor cells using nerve growth factors and promoting their differentiation.Based on this, the survival and death of neurons, migration, synapse formation, etc. Modulation may help to restore memory and improve memory.

Nerve growth factors present in the nervous system generally induce neuronal responses by activating cell membrane receptors to regulate signaling. Among the growth factors, neuroregulin is a structurally related gene family and is expressed in over 30 different forms by alternative splicing (Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system (1993) Nature 362, 312-318; Neuregulins: functions, forms, and signaling strategies (2003) Exp . Cell Res . 284, 14-30). Neuregulin is divided into three types, Type I, Type II, and Type III, among which Type I Neuregulin is called NDF (Neu differentiation factor), HRG (Heregulin), ARIA (Acetylcholin receptor inducing activity), etc. Type I nuregulin is collectively called hiregulin). Type II nuregulin is called GGF (glial growth factor) and is mainly expressed in late nervous system development, and type III nuregulin is called sensory and motor neuron derived facto (SMDF) and cytyne rich domain (CRD) nuregulin.

Hiregulin (HRG) reacts with receptor tyrosine kinase (RTK) ErbB3 and ErbB4 to regulate signaling and induce neuronal responses by regulating phosphorylation of important intracellular signaling proteins. Another receptor, ErbB2, reacts after hyregulin binds to ErbB3 and ErbB4 (Expression of neuregulins and their putative receptors, ErbB2 and ErbB3, is induced during wallerian regeneration (1997) J. Neurosci . 17, 1642-1659). All nuregulins have an epidermal growth factor (EGF) -like domain, which only binds to the ErbB protein for cellular responses. Hiregulin is divided into hiregulin α or β by the α or β form domain next to the EGF-sexual domain, where α is found in non-neuronal cells and β is in nerve cells. Hiregulin induces the death and proliferation of Schwann cells and glial cells in the central and peripheral nervous system, survival and migration and differentiation of neurons, and axon growth (Role of neuregulins in glial cell development (2000) Glia 29, 104- 111; GDNF family ligands activate multiple events during axonal growth in mature sensory neurons (2004) Mol . Cell . Neurosci . 25, 453-459).

As a method of treating nerve growth factor for neurological disease, there is a method of directly implanting stem cells, but it is difficult to separate and maintain neural precursor cells. Another method of cell therapy using a virus as a gene transfer vector is have. Regenerative medicine based on stem cell theraphy will be the ultimate treatment for the disease.

Since the first clinical trials in humans began in 1990, about 600 clinical trials have been in progress in 3500 patients. However, 75% of the approved gene therapies are aimed at the treatment of monogenetic diseases such as cancer and cystic fibrosis, and the development of gene therapies for neurological diseases and nerve regeneration is not active. (2002); Gene therapy clinical trials (2002) J Gene Med . www. wiley.co.uk/genmed).

In the disease of the nervous system, the overall research of neuroscience about the study of brain function is still in a poor state, and the development of therapeutic drugs for various chronic nervous system diseases is also in trouble. In particular, the use of neural progenitor cells is a revolutionary new treatment of the medical community that treats diseases in a completely different perspective, and can promote differentiation into neuronal cell types suitable for transplantation. In addition, direct injection of gene therapy that promotes the proliferation and differentiation of neuronal progenitor cells could make this treatment a reality and popularization.

It is an object of the present invention to provide a memory enhancing or preventive or therapeutic use of EGF-like domain peptides of hiregulin beta1.

In order to solve the above problems, the present invention provides a composition for improving memory or preventing or treating memory impairment disorders comprising a nucleic acid encoding the EGF-like domain peptide of hyregulin beta1.

In another aspect, the present invention provides a composition for improving memory or preventing or treating memory impairment disorders comprising the EGF-like domain peptide of hiregulin beta1.

The present invention also provides a DNA sequence encoding the EGF- sex domain peptide of hyregulin beta1; And a recombinant adenovirus comprising a DNA sequence encoding a secretory indication peptide, the composition for improving memory or preventing or treating a memory damage disease.

The present invention also provides a DNA sequence encoding the EGF- sex domain peptide of hyregulin beta1; And a mesenchymal stem cell incorporating a recombinant adenovirus containing a DNA sequence encoding a secreted indication peptide, and a composition for preventing or treating memory impairment.

EGF-domain domain peptide of the hiregulin beta 1 according to the present invention can improve memory by inducing differentiation into neurons necessary for the proliferation and memory formation of neural progenitor cells and can be used for the prevention or treatment of memory damage diseases. .

The present invention provides a composition for improving memory or preventing or treating memory impairment diseases, including a nucleic acid encoding an EGF-like domain peptide of hyregulin beta1 (hereinafter referred to as HRGβE).

The present inventors have revealed in previous studies that HRGβE has an effect on the migration, differentiation, neurite outgrowth and regeneration of peripheral nervous system neurons, and thus it is effective in the treatment of peripheral nervous system neuropathy diseases. 0765496). However, after further studies on HRGβE, it was surprisingly found that HRGβE has the effect of inducing the proliferation, survival, and differentiation of neuronal progenitor cells of the central nervous system, and more surprisingly, HRGβE has the effect of improving memory. Thus, the present invention has been completed. As described above, mammalian brains perform complex functions by obtaining a number of nerve cells and generating systematic neural networks through a series of processes, such as movement, differentiation, survival and death, and synapse formation, to appropriate locations within organs. do. Therefore, simply promoting the proliferation or regeneration of neuronal progenitor cells does not immediately perform a function of improving memory or repairing memory damage. Therefore, the finding that HRGβE has a therapeutic effect of memory enhancement and memory impairment disease is the discovery of surprising effects that could not be predicted at all from previous studies.

The present inventors transplanted mesenchymal stem cells into which a recombinant adenovirus containing a DNA sequence encoding HRGβE (hereinafter referred to as HRGβE-Ad) into the brain of rats through the following examples, thereby proliferating neural progenitor cells, It was confirmed that there was an effect of promoting differentiation and improving memory. That is, as a result of HRGβE-Ad infection of human bone marrow mesenchymal stem cells, HRGβE-Ad synthesized HRGβE and secreted it extracellularly, and co-cultured HRGβE-Ad-infected mesenchymal stem cells with neuronal progenitor cells. It was observed that HRGβE induces proliferation of neural progenitor cells. In addition, transplantation of the HRGβE-Ad-infected mesenchymal stem cells into the brain of a rat model of brain disease resulted in proliferation of endogenous neural progenitor cells, survival of proliferating cells, and differentiation into neurons. Differentiation into glutamate, Gaba and cholinergic cells required for neuronal cell types. In addition, the memory test results of the brain injury model rats completed the present invention by obtaining a result that the memory is improved.

In one embodiment of the invention, the nucleic acid encoding the HRGβE may be a nucleic acid having a nucleotide sequence of SEQ ID NO: 1, but not limited to. In addition, the nucleotide sequence of SEQ ID NO: 1 or more than 90% homology, and includes a nucleic acid encoding a variant peptide having an activity of inducing the proliferation of neural precursor cells, for example, GGF (glial growth factor), NDF ( Nucleic acids encoding other types of rat or human hyregulin alpha / beta EGF-like domain peptides such as Neu differentiation factor (ARIA), Acetylcholin receptor inducing activity (ARIA), or sensory and motor neuron derived factor (SMDF). have.

The present invention also provides a composition for improving memory or preventing or treating memory impairment diseases, including HRGβE.

In one embodiment of the invention, the HRGβE may be a peptide having an amino acid sequence of SEQ ID NO: 4, but not limited to. In addition to the amino acid sequence of SEQ ID NO: 4 or more than 90%, and includes a variant peptide having an activity that induces the proliferation of neural precursor cells, for example, GGF (glial growth factor), NDF (Neu differentiation factor) , EGF-like domain peptides of other types of rat or human hyregulin alpha / beta such as Acetylcholin receptor inducing activity (ARIA), and sensory and motor neuron derived factor (SMDF).

The present invention also provides a composition for improving memory or preventing or treating memory impairment disorders comprising a recombinant adenovirus (HRGβE-Ad) comprising a DNA sequence encoding HRGβE.

In one embodiment of the invention, the recombinant adenovirus may further comprise a DNA sequence encoding a secretion signal peptide (signal peptide).

In one embodiment of the present invention, the secretion indicator peptide may be a peptide having an amino acid sequence of SEQ ID NO: 5, the DNA sequence encoding the secretion indicator peptide may have a sequence of SEQ ID NO: 2, but is not limited thereto It is not intended to be construed as including variants or fragments thereof having substantially the same activity.

In another embodiment of the present invention, the recombinant adenovirus of the present invention may have a nucleotide sequence in which the DNA sequence encoding the HRGβE and the DNA sequence encoding the secretion indicating peptide. More specifically, but not limited thereto, the nucleotide sequence of the DNA sequence encoding the HRGβE and the DNA sequence encoding the secretion indicator peptide may be the nucleotide sequence of SEQ ID NO: 3, HRGβE and secretion indicator peptide is fused The peptide may have an amino acid sequence of SEQ ID NO: 6, but is not limited thereto, and may be interpreted to include variants or fragments thereof having substantially the same activity.

In another embodiment of the invention, the recombinant adenovirus of the invention may further comprise a viral promoter capable of expressing a DNA sequence, wherein the viral promoter may be a CMV promoter.

In another embodiment of the present invention, the recombinant adenovirus of the present invention may be deficient in parts of the genome required for replication in a target cell, wherein the deficient part is E1, E3, E4, and L1. It may be selected from the -L5 gene, preferably may be lacking the E1 gene.

The recombinant adenovirus used in the embodiment of the present invention is a replication defective viral vector in which the E1B region has been deleted, and has recently been studied to obtain a therapeutic effect on a disease site by injecting a therapeutic gene into a neurological disease ( Viral vectors for modulating expression in neurons (1996) Curr . Opin . Neurobiol . 6, 576-583; Adenovirus vectors for gene delivery (1999) Curr . Opin . Biotech .. 10, 440-447). Advantages of the adenovirus is that finally even possible to introduce different types of cells differentiated as nerve or muscle cells, unlike retroviruses to be introduced only in dividing cells (Gene delivery into the brain using virus vector (1993) Nature genetics 3 , 187-189; Gene therapy for cerebral vascular disease (1997), Stroke 27, 534-539). In addition, the cytotoxicity is low compared to other viral mediators, it is safe and can be purified at a high concentration, it has a relatively large capacity of 8kb and is easy to produce. In addition, unlike the retroviruses, the recombinant adenovirus used in the present invention is 100% introduced into mesenchymal stem cells and disappears after 3 months because it does not intervene in the chromosome, and the low risk of insertional mutagenesis is also a great advantage. .

Existing papers have confirmed that adenovirus is expressed to a high degree when inducing expression of E. coli β-galactosidase gene in epithelial cells, skin, and muscle of lung (Adenovirus-mediated in vivo gene transfer and expression). in normal rat liver (1992), Nature genetics 1, 372-378; Diversity of airway epitherial cell target for in vivo recombinant adenovirus-mediated gene transfer (1993) J. Clin Invest 91, 225-234;.. Intraperioneal in vivo gene transfer in the skin using replication-deficient receombinant adenovirus vector (1994) J. Invest Dermatol 102, 415-421;.... Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle (1996) Hum Mol 5 1703-1712). In addition, the present inventors reported that recombinant LacZ adenovirus is well introduced into HiB5 neuroprogenitor cells, is well introduced into the nervous system of rats, and is expressed over 6 weeks (Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy. of peripheral nerve injury (2000) Mol . cells . 10, 540-545). Therefore, the recombinant adenovirus HRGβE-Adv used in the present invention can be introduced and transplanted into mesenchymal stem cells as described below, or directly used for regeneration of degenerative brain diseases involving memory disorders.

The present invention also provides a composition for improving memory or preventing or treating memory impairment diseases, including mesenchymal stem cells incorporating a nucleic acid encoding HRGβE.

In one embodiment of the present invention, the introduction of the nucleic acid may be performed using a microinjection method, an electrointroduction method, a direct injection method, a particle injection method, a liposome method or an expression vector. Microinjection (microinjection) is not limited to this, for example, can be used stereotek microinjection, there is an advantage that it is well inserted into the genome and delivered to the offspring, regardless of the size of the DNA (50-200kb). The introduction of nucleic acid by electroporation is a method commonly used in ES cells, etc. The principle is that foreign DNA enters the cell suspension due to a high voltage applied to the cell suspension for a short time and its permeability changes. . Particle bombardment is a method of injecting DNA into metal cells together with metal particles by applying a desired DNA to the surface of microparticles (about 0.5-10 μm in diameter) such as gold and tungsten and spraying them into cells or tissues at high speed. . The liposome method is a method in which DNA is fused to a cell membrane using liposomes. In addition, there is a method of inserting a desired nucleic acid in a variety of expression vectors and introducing it into the host cell. Expression vectors may be, but are not limited to, plasmids or viruses, preferably recombinant adenoviruses.

In one embodiment of the invention, the recombinant adenovirus (HRGβE-Ad) is as described above.

Mesenchymal stem cells, commonly known as hematopoietic stem cells in the bone marrow, have the ability to differentiate into a variety of mesodermal cells, including bone, cartilage, fat, and muscle cells. The maintenance costs and effort are relatively low compared to other undifferentiated cells. As human bone marrow mesenchymal stem cells are known to have the potential to differentiate into glial cells in the brain (Kofen et al. 1999; Azizi et al. 1998), treatment of central nervous system diseases using mesenchymal stem cells as cell therapy It is presented as possible. Therefore, the method of delivering HRGβE using mesenchymal stem cells is not only HRGβE but also the effect of neuronal regeneration by the growth factor secreted by mesenchymal stem cells and the effect of replacing mesenchymal stem cells by neural cells. It is advantageous to have.

According to this embodiment, HRGβE-induced mesenchymal stem cells were more differentiated into neurons after brain transplantation, and GABAergic neurons, glutamatergic neurons, which are the types of neurons necessary for the transplantation site. , Cholinergic neurons (cholinergic neuron) and can be seen that differentiation. In particular, neurons expressing glutamic acid dehydrogenase (GAD), an enzyme that produces GABA, a neurotransmitter secreted early in neuronal differentiation, differentiated more. As such, the mesenchymal stem cells can be effectively prevented or treated for memory decay due to neuronal death by differentiating into glutamate, gabb, and cholinergic neurons, which are important for memory formation in the hippocampus.

In one embodiment of the present invention, the mesenchymal stem cells may be, but is not limited to, bone marrow mesenchymal stem cells or umbilical cord blood mesenchymal stem cells, preferably bone marrow mesenchymal stem cells, more preferably Mesenchymal stem cells extracted from the bone marrow of the subject to which the composition is to be administered. The subject includes, but is not limited to, humans, orangutans, chimpanzees, mice, rats, dogs, cattle, chickens, pigs, goats, sheep, and the like. Mesenchymal stem cells extracted from the bone marrow of the subject can be proliferated through cell division, and thus, a large amount of cells can be easily obtained without the help of an oncogene.

The composition for improving memory or preventing or treating memory impairment diseases of the present invention can be used as a pharmaceutical composition. Accordingly, the present invention provides the use of mesenchymal stem cells infected with nucleic acids, HRGβE peptides, HRGβE-Ad or HRGβE-Ad encoding HRGβE for the manufacture of a medicament for the prevention or treatment of amelioration or memory impairment disease; Pharmaceutical compositions for preventing or treating memory enhancement or memory impairment diseases, including mesenchymal stem cells infected with nucleic acids encoding HRGβE, HRGβE peptides, HRGβE-Ad or HRGβE-Ad; And mesenchymal stem cells infected with a therapeutically effective amount of a nucleic acid encoding HRGβE, HRGβE peptide, HRGβE-Ad or HRGβE-Ad to a subject.

In one embodiment of the invention, the memory impairment disease may be a disease such as, but not limited to, dementia, Parkinson's disease, Alzheimer's disease, epilepsy, stroke, schizophrenia or traumatic brain injury. However, in addition to the memory impairment diseases exemplified herein, diseases exhibiting memory impairment due to impaired function of neurons are well known in the art. In the present invention, as long as the HRGβE peptide promotes the proliferation of neuronal progenitor cells and differentiation into neurons and consequently improves or restores memory, those skilled in the art will recognize nucleic acids encoding the HRGβE for the prevention or treatment of diseases related to memory damage. Mesenchymal stem cells infected with HRGβE peptide, HRGβE-Ad or HRGβE-Ad may be used.

An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to achieve the effect of improving memory or preventing or treating memory impairment disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like. For example, when administered once or several times a day for adults, 0.1ng / kg to 10g / kg for HRGβE peptide, 0.01 for mesenchymal stem cells infected with nucleic acid encoding HRGβE, HRGβE-Ad or HRGβE-Ad. It can be administered at a dose of ng / kg ~ 10g / kg.

In the present invention, the 'object' includes, but is not limited to, a human, an orangutan, a chimpanzee, a mouse, a rat, a dog, a cow, a chicken, a pig,

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants. Or solubilizers such as flavoring agents can be used.

The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

Pharmaceutical formulation forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. Can be.

The pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

HRG βE- Name Manufacturing

HRGβE-Ad used in the examples of the present invention was prepared according to the method described in the examples of Korean Patent No. 10-0765496. The sequence of SEQ ID NO: 1 was used for the DNA sequence encoding HRGβE, and the sequence of SEQ ID NO: 2 was used for the DNA sequence encoding the secretion indicating peptide.

Experimental animal

The rats used for the experiment were 6 to 7 week old (190-240 g) Sprague-Dawley (CrjBgi: CD (SD) IGS, Orient, Korea), and were used in the experimental animal breeding room (20-23 ° C, day / night (12h / 12h) 1 to 2 weeks after the cycle was used in the brain transplant experiment.

Intermediate lobe  Stem cell Brain transplant  Pretreatment

hMSCs were labeled with fluorescent 1,1-dioctadecyl-3,3,3,3-tetra-methyllindocarbocyanine perchlorate (DiI-C18- (3)) for 2 hours at room temperature in the dark. Fluorescently labeled hMSCs were cultured in 37 ° C., 10% FBS α-MEM medium and treated for 2 hours to differentiate in 0.5% FBS α-MEM, a serum deficient condition. Thereafter, the cells were attached so that the number of viable cells in saline-containing 0.7% penicillin / streptomycin, 20 mM Hepes (pH = 7.2) and 0.5% glucose was 6 × 10 4 cells / μl, followed by brain transplantation by 2 μl.

Brain transplant

For stereotaxic surgery, firstly, rats were subjected to general anesthesia by intraperitoneal injection of 50 mg / ml equithensin. To prepare a memory impairment model, 1.5 μl of a neurotoxin, ibothenic acid (1 mg / ml), was injected into the olfactory cortex of the lateral brain, and 6 × 10 ⁴ cells / μl on the hippocampal white plate in the hippocampus. HMSCs were injected in a stereotactic manner using a 2.0 µl automicroscopy. After the recovery phase, the memory test was performed, and after 10 weeks, the brain was removed by perfusion. Proliferating cells were labeled by intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine, Sigma) at 50 mg / kg for 3 days after transplantation of mesenchymal stem cells. Twenty four hours after the last intraperitoneal injection, brain tissue was obtained through perfusion.

Fixation and Extraction of Rat Brain

Before performing the perfusion method, the rats that were killed by injecting carbon dioxide for about 5 minutes were opened, and about 400 ml of 4% PFA (paraformaldehyde, Sigma, USA) PBS at pH 7.25 was injected into the right ventricle. An injection needle with a diameter of 1 mm connected with a peristaltic pump (manhatan company, South Korea) was used to inject the needle into the lower ventricle to allow it to pass through the artery. It burst the right atrium, preventing blood from entering the heart at the same time, while removing the blood flowing through the perfusion.

When each tissue was fixed, the brain was extracted and placed in 4 ° C., 4% PFA solution for 4 hours, and then placed in a fixed solution (30% sucrose 1 × PBS) for 10 hours to be fixed and dehydrated. After freezing at -40 ℃, fixed to freeze cutting with OCT compound (Sakura co. Japan), and then coronally sectioned to a thickness of 35 ㎛ using Cryostat (Microm). At this time, the storage buffer was stored in 24well and stored at 4 ° C. containing the beginning to the end of the hippocampus. Since DiI, a fluorescent dye, diffuses from the cell membrane from the moment of death of the living body, all subsequent experiments were performed within 48 hours from the time of the perfusion method.

BrdU  process of dyeing

Frozen sections were stored in acryl 24well, washed three times with PBS and then reacted with a 0.5% Triton X-100 in PBS solution in a stirrer for 25 minutes. After transferring the tissue to 2N HCl solution of 37 ℃ and reacted for 30 minutes at 37 ℃. The tissues were then washed three times with PBS, followed by a 1 hour blocking step for non-specific immune responses in 10% normal serum in 3% Bovine Serum Albumin (BSA) with 0.1% Triton X-100 in PBS solution. The primary antibody was then attached at 4 ° C. for 16 hours. The antibody was used diluted with 1% BSA PBS. The antibody used at this time is BrdU (BD bioscience, 1: 500), Nestin (1: 1000), Tuj1 (Sigma, 1: 1500), GFAP (Chemicon, 1: 700), ChAT (Chemicon, 1: 500), GAD67 (Chemicon, 1: 2000), VGluT1 (Chemicon, 1: 500), and TH (Chemicon, 1: 700). The secondary antibody complementary to the primary antibody was then attached for 45 minutes at room temperature. Secondary antibodies used at this time are fluorescently labeled Cy3 (Jackson, 1: 500), FITC (Chemicon, 1: 500), and Alexa488 (Molecular probe, 1: 700).

The number of BrdU cells was measured by counting the cells in the nucleus of the counterstained signal, and BrdU was round in the shape of a nucleus and did not count nonspecific signals at the edges or tears of tissues. Data analysis was performed using one-way batch analysis (Anova).

< Example  1> HRG βE- Name  Infected Intermediate lobe  From stem cells HRG βE Secretory  Measure

After introducing sHRGβE adenovirus into human bone marrow mesenchymal stem cells, culture medium was taken 2 days after the introduction and analyzed using standard ELISA method to analyze the secretion amount of sHRGβE. Specifically, human bone marrow mesenchymal stem cells were planted in a 35 mm culture vessel at 3 x 10 ^5, and then introduced with LacZ-Ad or HRGβE-Ad for 2 hours in a medium containing 2% FBS for one hour, followed by 10% FBS medium. After 12 hours of exchange, it was finally exchanged again with 1 mL of alpha-MEM. After 24 hours, the medium was obtained and stored at −70 ° C. until used with the protease inhibitor. An ELISA kit (R & D Systems, Germany) was used to measure the amount of sHRGβE in the medium.

As shown in Figure 1, human mesenchymal cells introduced with HRGβE adenovirus produced 83.5 ng of HRGβE per 10 ^7-8 virus particles and naturally produced 30-40 ng of HRGβ protein in the control group in which LacZ-Ad was introduced. It became. That is, HRGβE (6.5kDa) was introduced at 100moi per 3 × 10 ⁵ cells in HRG ELISA, and about 36.6 ng / mL was produced compared to the control group. However, since the HRGβE peptide (6.5kDa) is a very small molecular weight compared to the molecular weight of the total HRGβ protein (45kDa), it can be seen that the total protein was produced at 5.6 nM, which is about 5 times when produced with peptides compared to 1.17nM.

As a result, it can be seen that HRGβE is secreted out of cells effectively by HRGβE-Ad.

< Example  2> sHRG β E of  Neuroprogenitor cell proliferation promoting effect in vitro )

To determine whether secreted sHRGβE affects the proliferation of neural progenitor cells, neuronal progenitor cells obtained from mesenchymal stem cells and embryonic day 14 were co-cultured. Human bone marrow mesenchymal stem cells were planted in a cover glass and introduced with LacZ-Ad or sHRGβE-Ad for 2 hours in a medium containing 2% FBS the next day and then exchanged with 10% FBS medium. After 12 hours, cortical neural stem cells isolated from embryonic day 14 of rats were planted in mesenchymal stem cells and incubated for 12 hours in N2 medium containing FGF, followed by N2 medium. After 12 hours of exchange, BrdU was treated with 10 uM. One day after the treatment with BrdU, the cells were fixed with 4% paraformaldehyde and immunostained with BrdU antibody and HuNu (human nuclear protein) antibody.

Neural progenitor cells require substrates such as polyornithine and polylysine and extracellular matrix proteins such as fibronectin and laminin on the surface when cultured. However, in the present invention, when the co-culture of neural progenitor cells on HRGβE-secreting mesenchymal stem cells, it was observed that the neural progenitor cells grew by seating the mesenchymal stem cells as a support without the stroma and extracellular matrix protein, The effect of proliferating progenitor cells was observed. According to (a) and (b) of FIG. 2, the neural progenitor cells grown on the mesenchymal stem cells into which LacZ-Ad was introduced as a control group were labeled with BrdU of 22.4% of all neural progenitor cells and proliferated. It was confirmed that the 22.4%, the neuronal progenitor cells grown on the mesenchymal stem cells introduced HRGβE-Ad was confirmed that 39.9% of all neuronal progenitor cells proliferated. Based on the control group of LacZ mesenchymal stem cells, the proliferation of neuronal progenitor cells co-cultured with HRGβE mesenchymal stem cells was increased by about 78%, indicating that HRGβE affected the proliferation of neural progenitor cells. . In addition, the proliferative effect of HRGβE peptide secreted from mesenchymal stem cells is suppressed by treating HRGβE neutralizing antibody at a concentration of about 6 times (33.3nM, 5ug / mL) of peptide (5.6nM) produced in mesenchymal stem cells. It was confirmed.

< Example  3> HRG by βE Immanence  Neuroprogenitor Cell Proliferation and Differentiation of Neuroprogenitor Cells

HRG by βE Immanence  Neuroprogenitor Cell Proliferation

The human mesenchymal stem cells were divided into a control group in which LacZ-Ad was introduced and a test group in which HRGβE-Ad was introduced. BrdU was intraperitoneally injected for 3 days from 11 days after brain transplantation to label endogenous neural progenitor cells. At 2 weeks after transplantation, the brains of rats were extracted after perfusion and detected with BrdU-labeled proliferating cells by immunohistochemistry (FIG. 3).

Figure 4 shows a schematic diagram of the transplantation site of the mesenchymal stem cells and compare the seated sites of the mesenchymal stem cells transplanted by fluorescent labeling. The seating site and shape of the transplanted mesenchymal stem cells indicate that they are transplanted to the correct site.

 (See Figures 5A, 5B). As shown in FIG. 5 (a), BrdU-labeled proliferative cells are mainly present in the subgranular zone (SGZ) and hippocampal white plate region of the CA1 region of the hippocampus. According to FIG. 5 (b), the number of proliferating cells of neural progenitor cells transplanted with HRGβE-Ad infected mesenchymal stem cells increased more than twice in the dentate gyrus and CA1 region of the hippocampus.

HRG Differentiation of Neuroprogenitor Cells by βE

In order to observe early neuronal differentiation of endogenous neural progenitor cells with increased proliferation, double fluorescent staining was performed using the neuron-specific markers Nestin and Tuj-1 and GFAP, astrocyte-specific markers of glial cells ( 6a, 6b).

As shown in FIG. 6 (a), the expression of Nestin was increased and the expression of GFAT was decreased in BrdU labeled cells of the HRGβE-Ad group. Figure 6 (b) shows the quantitative expression of the differentiated cells, the number of cells expressing nestin, a neuronal marker in the HRGβE-Ad group is increased, while the number of cells expressing GFAP expressing astrocytes Was reduced compared to the control. Therefore, it can be seen that HRGβE promotes differentiation of neural progenitor cells into early neurons, while inhibiting differentiation into glial cells.

< Example  4> HRG by βE Immanence  Survival of neural progenitor cells and differentiation of neural progenitor cells

Four weeks after intraperitoneal injection of BrdU, the brains of control and experimental animals were removed after perfusion and evaluated for long-term survival of BrdU-labeled cells.

The BrdU-labeled cells shown in FIG. 7 (a) are cells that divide at 2 weeks and survive for 4 weeks, and in the HRGβE-Ad group, a large number of cells survive compared to the control so (stratum oriens) in the hippocampus CA1 region. , py (stratum pyramidale; pyramidal layer), sr (stratum radiatum), etc. are moved to exist, and the gcl (granule cell layer) in the dentate gyrus (DG) area is proliferated compared to the control group, Able to know.

7 (b) shows that after 4 weeks of fluorescence staining with endogenous neural progenitor cells differentiated into markers for each neuronal cell type, they differentiate into neuronal cells. VGluT1 (vesicular glutamate transporter 1) as a marker for glutamate neurons using glutamate, GAD67 (glutamic acid decarboxylase 67) as a marker for GABA neurons, and choline acetyl as a marker for cholinergic neurons using acetylcholine as a neurotransmitter Fluorescent tissue staining using TH (tyrosine hydroxylase) as markers of transferase) and dopaminergic neurons revealed that the cells stained with the markers of each neuron type in the group transplanted with HRGβE-Ad-infected mesenchymal stem cells. The number increased.

As a result, it can be seen that endogenous neural progenitor cells proliferated by HRGβE induce differentiation into glutamate neurons, GABA neurons, cholinergic neurons, and dopaminergic neurons, which are neurons that regulate memory formation in the hippocampus.

< Example  5> implanted Intermediate lobe  Stem Cell Differentiation

To investigate the differentiation pattern of HRGβE-Ad-infected mesenchymal stem cells 10 weeks after brain transplantation, double fluorescent tissue staining was performed with NeuN, a marker of neurons, GFAP, a marker of astrocytes among glial cells, and O4, a marker of oligodendrocytes. Was implemented. Brain transplanted mesenchymal stem cells were fluorescently labeled with DiI and used for double fluorescent tissue staining. Specifically, the frozen sections were stored in acryl 24well, washed twice with PBS, and then placed on ice in a chamber at 20 ° C. for 20 minutes with 0.5% Triton X-100 in PBS solution and shaken to prevent DiI diffusion. A blocking step for nonspecific immune responses in 15% normal serum in 3% Bovine Serum Albumin (BSA) with 0.1% Triton X-100 in PBS solution was then performed on a shaker for 2 hours and then the primary antibody was attached for 4 hours. (3 hours 4 ° C., 1 hour room temperature). At this time, the antibody was diluted with 1% BSA in PBS. The antibody used was ChAT (Chemicon, 1: 500), GAD65 / 67 (Chemicon, 1: 500), GAD67 (Chemicon, 1: 2000), VGluT1 (Chemicon, 1: 500), NeuN (Chemicon, 1: 700). ), GFAP (Chemicon, 1: 700), O4 (Chemicon, 1: 700). The secondary antibody complementary to the primary antibody was then attached for 45 minutes at room temperature. The secondary antibodies used were fluorescently labeled Cy2 (Jackson, 1: 500) and FITC (Chemicon, 1: 500) to set different wavelength bands from Rhodamine of DiI. After the whole process was mounted and observed and photographed by confocal laser scanning microscope (LSM510, Carl Zeiss, Germany).

The number of transplanted hMSCs labeled with DiI on the double-photographed slides was counted, and the number of FITC cells of each of the differentiation markers ChAT, GAD65 / 67, VGluT1, NeuN, GFAP, and O4 was counted. The number of differentiation markers in the hippocampus was percentaged by DiI number. At this time, the number of FITCs in the shape and position of the cells and the shape of the cells was counted.

8 is a schematic diagram illustrating a process of performing differentiation experiments and memory test of mesenchymal stem cells 10 weeks after transplantation of human mesenchymal stem cells into which HRGβE-Ad or LacZ-Ad is introduced into the hippocampus of a memory impaired animal model. to be.

Figure 9 (a) is a photograph showing the differentiation of the transplanted mesenchymal stem cells into neurons of each type as a result of performing fluorescent tissue staining with differentiation markers for each neuron type. These cells express markers of cells constituting the central nervous system such as ChAT, GAD65 / 67, TH, NeuN, GFAP, O4 in both the HRGβE-Adv group and the control group, indicating that they differentiate into neural cells.

Figure 9 (b) is a graph showing the quantitative analysis of the differentiation degree for each neuron type. In the HRGβE-Ad group, the differentiation of ChAT-labeled cells increased by 1.5-fold, the differentiation of GAD65 / 67-labeled cells by 1.8-fold and 1.3-fold in NeuN. However, the differentiation of GFAP-labeled cells decreased by 1.8-fold and 1.5-fold in the case of O4. In contrast, the expression of TH, a marker of dopaminergic neurons, was not significantly different between the experimental and control groups.

As a result, HRGβE-Ad-infected mesenchymal stem cells were observed to differentiate into cholinergic neurons and GABA neurons, which are neurons necessary for memory formation in the hippocampus when transplanted into the brain, and the number of cells differentiated into neurons (NeuN-expressing cells). The number) increases but the differentiation into astrocytic and oligodendrocytes is rather reduced.

< Example  6> HRG Memory improvement experiment by βE

Two behavioral tests were performed to investigate the ability of HRGβE to restore memory.

Y-shaped maze (Y maze ) Experiment

Y-shaped maze experiments were performed 6 weeks after brain transplantation. A Y-shaped maze is placed in a dark space where objects can be identified, and visual signs are installed around it. After that, the test rats were moved to the space, and then adjusted to the space for about 1 hour or more. Each branch passage was randomly labeled, and after 8 minutes, 10 minutes, and 12 minutes, the order and number of passages into each branch passage by the rats were recorded. These scores are given when entering a branch passage other than the branch passage that was selected in the previous stage. When another branch passage is selected three times in a row, the score is given and expressed as a percentage of the total selection. Rats remember the visual signs installed around them to search for a new basin. The higher this value is, the higher the probability of alteration can be.

As shown in FIG. 10 (a), in the experimental group in which the HRGβE-Ad-treated mesenchymal stem cells were transplanted with the brain, the memory behavior was measured for 8 minutes, 10 minutes, and 12 minutes compared with the mesenchymal stem cells transplanted without the pretreatment. Showed 1.2 times improvement in memory behavior.

Passive avoidance reaction passive avoidance ) Experiment

For passive avoidance reaction experiments were trained 4-5 days before the 10th week of brain transplantation. Training was an evasive reaction to light, blocking the passage of the two distinct chambers, turning on the lights in the chamber containing the rats, and opening the passage to record the time of movement to the opposite dark chamber. This training was conducted 4-6 times a day, and again 24 hours after the training, the experiment was conducted when the average travel time was about 20 seconds for 4-5 days. The experiment was conducted by light avoidance and moved to another chamber, giving an electric shock (1mA) for 3 seconds. After 24 hours of electric shock, they were placed in the chamber under the same conditions and the lamps were turned on, and the time when they moved to the opposite chamber was recorded. The higher this number, the more you can compare the degree of memory recovery for the stimulus.

According to Figure 10 (b) it can be seen that memory is significantly recovered for the passive avoidance experiment when the brain transplantation of HRGβE-Ad infected mesenchymal stem cells. Numerically, memory for stimulation was increased approximately four-fold in sHRGβE-Ad compared to the LacZ-Ad control.

Therefore, when brain-transplanted human mesenchymal stem cells infected with HRGβE-Ad to the hippocampus in the memory impairment model rats, HRGβE secreted from them induces proliferation of human mesenchymal stem cells and differentiation into functional neurons. It can be seen that it replaces apoptosis neurons and establishes neuronal connections and consequently contributes to memory recovery.

Figure 1 is a graph quantified by analyzing the amount of HRGβE released extracellularly after introduction of HRGβE-Ad to human bone marrow mesenchymal stem cells by ELISA.

2 shows the effect of HRGβ on the proliferation of neural progenitor cells. (a) is a micrograph showing cell proliferation after treatment of LacZ, sHRGβE, and HRGβE neutralizing antibodies using BrdU immunostaining, and (b) is a graph showing quantification.

Figure 3 is a schematic diagram showing the process of experimenting the proliferation and regeneration effect of the endogenous neuronal progenitor cells after transplanting adenovirus-infected mesenchymal stem cells in the animal model of memory impairment.

4 is a diagram showing the schematic diagram of the mesenchymal stem cell transplantation site and the site and shape of the human mesenchymal stem cell transplanted by fluorescent labeling.

Figure 5 shows the results of examining the proliferation of endogenous neural progenitor cells after transplanting the adenovirus-infected mesenchymal stem cells in the animal model of memory impairment. (a) is a micrograph showing cell proliferation by BrdU immunostaining, and (b) is a graph showing quantification.

Figure 6 shows the results of observation of early neuronal differentiation of endogenous neural progenitor cells proliferated after transplantation of adenovirus infected mesenchymal stem cells. (a) is a micrograph showing early neuronal differentiation, and (b) is a table showing the number of cells expressing each marker.

Figure 7 shows the results of observation of survival and differentiation of neural progenitor cells after 4 weeks of proliferating endogenous neural progenitor cells after transplantation of adenovirus infected mesenchymal stem cells. (a) shows that proliferated neuronal progenitor cells migrate to each hippocampal region and survive. (b) shows that neural progenitor cells proliferated by fluorescent tissue staining with markers for each neuron type are required for memory formation. The neurotransmitter glutamate or Gaba secretion is shown to differentiate into glutamate or Gaba neuronal cell types.

8 is a schematic diagram showing the process of performing differentiation and memory test of mesenchymal stem cells 10 weeks after transplanting human mesenchymal stem cells infected with adenovirus into a memory impaired animal model.

9 shows the results of observing the differentiation of mesenchymal stem cells 10 weeks after transplanting human mesenchymal stem cells infected with adenovirus into the animal model of memory impairment. (a) is a micrograph showing the results of staining with markers for each neuronal cell type, (b) is cholinergic to secrete acetylcholine, GABA, which are neurotransmitters necessary for memory formation of transplanted mesenchymal stem cells; Differentiation into Gabar neuronal cell types.

Figure 10 shows the results of the memory recovery ability after transplantation of adenovirus infected human mesenchymal stem cells. (a) is a graph showing the results of the Y-shaped labyrinth test to test the short-term spatial memory, (b) is a graph showing the passive avoidance response to test the long-term memory.

<110> Industry Academic Cooperation Foundation of KyungHee University <120> Use of EGF-like Domain Peptide of Heregulin Beta 1 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 189 <212> DNA <213> EGF-like domain peptide of Heregulin beta1 <400> 1 atgtccacga ctgggaccag ccatctcata aagtgtgcgg agaaggagaa aactttctgt 60 gtgaatgggg gcgagtgctt cacggtgaag gacctgtcaa acccgtcaag atacttgtgc 120 aagtgcccaa atgagtttac tggtgatcgt tgccaaaact acgtaatggc cagcttctac 180 aagcattaa 189 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> signal peptide <400> 2 atgtcgggac caggaactgc agca 24 <210> 3 <211> 219 <212> DNA <213> fusion of EGF-like domain peptide of Heregulin beta1 and signal peptide <400> 3 atgtcgggac caggaactgc agcaggtacc atgtccacga ctgggaccag ccatctcata 60 aagtgtgcgg agaaggagaa aactttctgt gtgaatgggg gcgagtgctt cacggtgaag 120 gacctgtcaa acccgtcaag atacttgtgc aagtgcccaa atgagtttac tggtgatcgt 180 tgccaaaact acgtaatggc cagcttctac aagcattaa 219 <210> 4 <211> 62 <212> PRT <213> EGF-like domain peptide of Heregulin beta1 <400> 4 Met Ser Thr Thr Gly Thr Ser His Leu Ile Lys Cys Ala Glu Lys Glu   1 5 10 15 Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Thr Val Lys Asp Leu              20 25 30 Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly          35 40 45 Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys His      50 55 60 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 5 Met Ser Gly Pro Gly Thr Ala Ala   1 5 <210> 6 <211> 72 <212> PRT <213> Fusion of EGF-like domain peptide of Heregulin beta1 and signal peptide <400> 6 Met Ser Gly Pro Gly Thr Ala Ala Cys Thr Met Ser Thr Thr Gly Thr   1 5 10 15 Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn              20 25 30 Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr          35 40 45 Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr      50 55 60 Val Met Ala Ser Phe Tyr Lys His  65 70  

Claims (11)

A composition for the treatment of schizophrenia, which is a memory-improving or memory-damaging disorder comprising a nucleic acid encoding an EGF-like domain peptide of hiregulin beta1. A composition for the treatment of schizophrenia, which is a memory-improving or memory-damaging disorder, comprising an EGF-sex domain peptide of hiregulin beta1. A composition for the treatment of schizophrenia, which is a memory-improving or memory-damaging disorder, comprising a recombinant adenovirus comprising a DNA sequence encoding the EGF-like domain peptide of hiregulin beta1. A composition for treating schizophrenia, which is a memory-improving or memory-damaging disorder, comprising mesenchymal stem cells incorporating a nucleic acid encoding an EGF-like domain peptide of hiregulin beta1. 5. The method of claim 4, The introduction of the nucleic acid is a micro-implantation method, electro-introduction method, direct injection method, particle injection method, liposome method or a composition for the treatment of schizophrenia, which is a memory impairment disorder, characterized in that the expression vector is used. The method of claim 5, The expression vector is a recombinant adenovirus for improving memory or a composition for treating schizophrenia that is a memory impairment disease. The method of claim 6, The recombinant adenovirus is a composition for treating schizophrenia, which is a memory-improving or memory-damaging disorder comprising a DNA sequence encoding a secretory indication peptide. The method of claim 6, The recombinant adenovirus is a composition for the treatment of schizophrenia, which is a memory-improving or memory-damaging disorder, further comprising a viral promoter capable of expressing a DNA sequence. 9. The method of claim 8, The viral promoter is a composition for improving memory, which is a CMV promoter, or for treating schizophrenia, a memory impairment disease. The method of claim 6, The recombinant adenovirus is characterized by a lack of a portion of the genome required for replication in the target cell, wherein the deficient portion is selected from the E1, E3, E4, and L1-L5 genes memory enhancement Composition for the treatment of schizophrenia which is a dragon or memory impairment disease. delete

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