KR20170030216A - Composition for the prevention or treatment of cerebral infarction and stroke comprising immortalized neural stem cells - Google Patents

Abstract

The present invention relates to immortalized neural stem cells, a use of a culture medium thereof or an extract thereof for treating cerebral infarction (stroke). The immortalized neural stem cells of the present invention, in particular, transgenic and immortalized neural stem cells in which choline acetyltransferase gene were introduced, effectively reduce the volume of the cerebral infarction when applied to a cerebral infarction animal model; inhibit an inflammation-inducing substance such as a lipid peroxide and a carbon monoxide in brain tissue; inhibit the expression of inflammatory cytokines in the brain tissue; and are excellent in protecting the brain. Ultimately, the present invention can be usefully used as the pharmaceutical composition capable of preventing and treating cerebral infarction or stroke.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing and treating cerebral infarction or cerebral apoplexy including immortalized neural stem cells as an active ingredient,

The present invention relates to the use of immortalized neural stem cells, cultures thereof or extracts thereof for cerebral infarction (stroke) treatment.

Stroke is a neurological symptom that occurs when a blood vessel supplying blood to the brain is blocked or burst and the brain of the part is damaged. There are two types of stroke: ischemic stroke and hemorrhagic stroke. Ischemic stroke occurs when cerebral tissue becomes ischemic due to decrease or interruption of blood supply to brain tissue. Hemorrhagic stroke occurs when blood vessels burst into brain tissue and ischemic stroke occurs in approximately 80% of all stroke patients Respectively. When ischemic stroke occurs, the flow of blood is temporarily cut off and the cells can not get oxygen and nutrients. However, the procedure that resumes vascular flow by using surgery or thrombolytic agent is called reperfusion, which is called reoxygenation ) Process. Although reperfusion is essential for the recovery process in that it supplies oxygen and nutrients to the cells, excessive oxygenation, or reactive oxygen species (ROS), is produced in this process and infarction is accompanied by inflammatory reaction. It is known that the area where this occurs is widened.

Stem cells, on the other hand, are cells that are capable of differentiating into all kinds of cells that constitute the body, such as nerves, blood, and cartilage, if necessary, while maintaining their differentiation into specific cells. Due to their diverse functions, these stem cells have recently become commercially valuable as medicines and cosmetics.

Therefore, the present inventors have conducted various experiments on stem cells. As a result, the present inventors have found that immortalized neural stem cells (NSCs), especially immortalized neural stem cells (ChAT) transfected with human choline acetyltransferase immortalized NSCs) were significantly superior to the treatment of cerebral infarction.

Korean Patent Publication No. 10-2007-0028640 Korean Patent Publication No. 10-2006-0085915

Accordingly, the present invention relates to a pharmaceutical composition for preventing and treating cerebral infarction or stroke using immortalized neural stem cells.

The present invention provides a pharmaceutical composition for preventing or treating cerebral infarction or stroke, which comprises an immortalized neural stem cell, a culture solution thereof or an extract thereof as an active ingredient.

In one embodiment of the present invention, the immortalized neural stem cells may be transgenic immortalized neural stem cells into which a choline acetyltransferase gene has been introduced.

In one embodiment of the present invention, the choline acetyltransferase may have the amino acid sequence of SEQ ID NO: 1 (NP_065574).

In one embodiment of the present invention, the choline acetyltransferase gene may be a polynucleotide sequence of SEQ ID NO: 2 (NM_020549.4).

In one embodiment of the present invention, the transformed immortalized neural stem cells into which the choline acetyltransferase gene has been introduced may be the accession number KCTC 12885BP.

In one embodiment of the present invention, the neural stem cells may be contained in the composition at 1 × 10 ³ to 1 × 10 ⁹ cells.

In one embodiment of the present invention, the cerebral infarction may be caused by focal cerebral ischemia.

As the immortalized neural stem cells according to the present invention, the transgenic immortalized neural stem cells into which the choline acetyl transferase gene has been introduced effectively reduce the volume of cerebral infarction when applied to a cerebral infarction model; Inhibiting inflammatory agents such as lipid peroxides and carbon monoxide in brain tissue; Inhibit the expression of inflammatory cytokines in brain tissue; And thus can be usefully used as a pharmaceutical composition capable of ultimately preventing and treating cerebral infarction or stroke.

1 is a vector map of pLPCX used for establishing a transformant of an immortalized neural stem cell into which a choline acetyltransferase gene of the present invention has been introduced.
FIG. 2A shows the results of immunohistochemical staining for the expression of ChAT gene in immortalized stem cells (CBNU-NSC.ChAT) into which the nestin-positive choline transferase gene (ChAT) is introduced.
FIG. 2B shows the results of western blot analysis of growth / neurotrophic protein expression in immortalized neural stem cells (CBNU-NSC) and immortalized neural stem cells (CBNU-NSC.ChAT) into which the ChAT gene is introduced. ChAT, choline acetyltransferase; DAPI, 4,6-diamino-2-phenylindole; GDNF, glial cell-skinned neurotrophic factor; VEGF, vascular endothelial growth factor; CNTF, ciliary neurotrophic factor; NGF, nerve growth factor
FIG. 3A is a representative photograph of brain tissue stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) to measure cerebral infarct volume.
FIG. 3B is a graph showing quantitative results of total cerebral infarction volume, wherein '*' indicates a significant difference (P <0.05) compared to the normal group, '#' indicates middle cerebral artery occlusion (MCAO) (P <0.05) compared to those induced by Cerebral Infarction.
FIG. 4A is a graph showing the results of immunohistochemical staining for CBNU-NSC (CBNU-NSC), a normal group (Normal), a cerebral artery occlusion-induced cerebral infarction induction group (MCAO) . CHAT) The result of measurement of malondialdehyde (MDA) content in each rat brain homogenate.
FIG. 4B is a graph showing the results of immunohistochemical staining for CBNU-NSC (CBNU-NSC) in the normal group (Normal), middle cerebral artery occlusion and cerebral infarction induction group (MCAO) . CHAT) The concentration of nitric oxide (NO) in cerebrospinal fluid was measured in each rat.
FIG. 5 is a graph showing the results of immunohistochemical staining for CBNU-NSC (CBNU-NSC) and CBNU-NSC treated with a ChAT gene in a cerebral infarction model in a normal group, a cerebral artery occlusion-induced cerebral infarction induction group (MCAO) .ChAT) Morris water maze test was conducted for each rat and the time required to reach the platform was measured. '*' Showed significant difference (P <0.05) compared to the normal group and '#' showed significant difference (P <0.05) compared to the cerebral artery occlusion induced cerebral infarction group.
FIG. 6 is a graph showing the results of immunohistochemical staining for CBNU-NSC (CBNU-NSC), a normal group (Normal), a cerebral artery occlusion-induced cerebral infarction induction group (MCAO) .ChAT) As a result of evaluating the physical vitality of each rat, 6a shows the latency time and 6b shows the resting, slow-moving, and fast- And a fast-moving time ratio. '*' Showed significant difference (P <0.05) compared to the normal group and '#' showed significant difference (P <0.05) compared to the cerebral artery occlusion induced cerebral infarction group.
FIG. 7 shows the results of immunohistochemical staining for CBNU-NSC (CBNU-NSC) cells transfected with normal cells, normal cerebral artery occlusion induction group (MCAO), cerebral infarction animal model (CBNU-NSC) ChAT) As a result of analysis of inflammatory cytokine gene expression in brain tissue of each rat, 7a is the result of RT-PCR analysis of inflammatory cytokine, 7b is the quantitative analysis result of TNF-α gene expression, 7c is IL- 1 &lt; / RTI &gt; gene expression. '*' Showed significant difference (P <0.05) compared to the normal group and '#' showed significant difference (P <0.05) compared to the cerebral artery occlusion induced cerebral infarction group.
FIG. 8 shows the results of immunohistochemical staining for CBNU-NSC (CBNU-NSC) and CBNU-NSC treated with ChAT gene in normal model, normal cerebral artery occlusion induction group (MCAO) ChAT) 8a is a cytoskeletal protein (MAP-2), an astrocyte skeletal protein (GFAP) and a cell adhesion protein (ICAM-1) in the brain tissue of each rat. western blot analysis, 8b is MAP-2 quantitative analysis, 8c is GFAP quantitative analysis, and 8d is ICAM-1 quantitative analysis. '*' Showed significant difference (P <0.05) compared to the normal group and '#' showed significant difference (P <0.05) compared to the cerebral artery occlusion induced cerebral infarction group.

The present invention is characterized in that it provides the use of immortalized neural stem cells, cultures thereof or extracts thereof for cerebral infarction or stroke prevention and treatment.

In general, 'neural stem cells' are immature cells that are mainly in the nervous system. They are self-renewal which continues to proliferate in an undifferentiated state. Neurons and neurons (glia) It is defined as a cell showing the multipotency of differentiation. Neural stem cells are present in various anatomical regions throughout the fetal nervous system of mammals including humans. In recent years, neural stem cells have existed not only in the fetus but also in specific parts of the adult nervous system. Throughout the life of the neural stem cells, It is known to proliferate and produce new neurons.

Therefore, in the present invention, the neural stem cells have self-renewal ability (self-renewal ability), and the neurons or meuronal cells constituting the central nervous system, astrocytes, oligodendrocytes) that are capable of differentiating into differentiated cells.

On the other hand, non-immortalized stem cells have a problem in that their proliferative capacity is deteriorated in the primary culture or passage, and the content of the active ingredient in the culture liquid decreases. Therefore, there is a need to establish an immortalized cell line through the process of immortalization in order to continue to use the pre-distributed stem cells.

The inventors of the present invention have established immortalized cell lines by introducing v-myc oncogene into each of the above stem cells in order to overcome the above problems and maintain the characteristics of neural stem cells to stably obtain the same culture medium.

In addition, the present inventors have established a transformant into which choline acetyltransferase (hereinafter, abbreviated as 'ChAT') gene has been introduced to improve the function of immortalized neural stem cells.

The term &quot; choline acetyltransferase &quot; in the present invention is an enzyme having a function of binding acetyl-CoA to choline to produce acetylcholine which is a neurotransmitter. In one embodiment of the present invention, Sequence (NP_065574). It is synthesized in the body of neurons and moved to the nerve end through axoplasmic flow. In humans, the choline acetyltransferase enzyme is encoded by the ChAT gene (polynucleotide sequence of SEQ ID NO: 2 (NM_020549.4)).

In the following Experimental Example 2 of the present invention, the cerebral infarction volume reduction was confirmed by the administration of the immortalized stem cell (CBNU-NSC) or ChAT gene of the present invention to the cerebral infarction animal model by administration of the immortalized neural stem cell (CBNU-NSC.ChAT) As a result, the infarction volume of cerebral artery occlusion (MCAO) -induced group was 29.1% of the total brain tissue, whereas the infarcted neural stem cell (CBNU-NSC) group had a significant decrease in cerebral infarction volume by 5.9% In particular, in the group administered with the immortalized neural stem cells (CBNU-NSC.ChAT) into which the ChAT gene was introduced, the volume of cerebral infarction was 3.1%, which was significantly decreased compared to the induction group (see FIG. 3 ).

In Experimental Example 3 of the present invention, inhibition of lipid peroxidation and NO production in cerebral tissue following administration of immortalized stem cell (CBNU-NSC) or immortalized neural stem cell (CBNU-NSC.ChAT) (MDA), which is a lipid peroxide that increases with the induction of cerebral artery occlusion and reperfusion, can be effectively reduced by administration of immortalized neural stem cells (CBNU-NSC) , Especially in the experimental group in which the immortalized neural stem cells (CBNU-NSC.ChAT) into which the ChAT gene was introduced showed a higher MDA reduction (see FIG. 4A). Similarly, it was confirmed that the concentration of nitric oxide in cerebrospinal fluid increased in cerebral artery occlusion-induced cerebral infarction group was effectively inhibited by immortalized neural stem cells (CBNU-NSC.ChAT) or immortalized neural stem cells (CBNU-NSC.ChAT) with ChAT gene (See FIG. 4B).

In Experimental Example 4 of the present invention, the cognitive restoration effect of the immortalized stem cell (CBNU-NSC) or the immortalized neural stem cell (CBNU-NSC.ChAT) into which the ChAT gene was introduced was examined in an animal model of cerebral infarction. As a result, it was confirmed that cognitive dysfunction caused by cerebral artery occlusion-induced cerebral infarction was effectively restored by administration of immortalized neural stem cells (CBNU-NSC) or immortalized neural stem cells (CBNU-NSC.ChAT) into which a ChAT gene was introduced (see FIG. 5) .

Further, in Experiment 5 below, the effect of immortalized stem cell (CBNU-NSC) or ChAT gene-introduced immortalized neural stem cells (CBNU-NSC.ChAT) on the cerebral infarction animal model was examined, (CBNU-NSC) or immortalized neural stem cells (CBNU-NSC.ChAT) to which the ChAT gene has been introduced, as well as a decrease in activity (see FIG. 6A) and activity (see FIG. 6B) according to cerebral artery occlusion- Administration was confirmed to be restored.

In Experimental Example 6 below, the effect of the immortalized stem cell (CBNU-NSC) or ChAT gene-introduced immortalized neural stem cell (CBNU-NSC.ChAT) on the cerebral infarcted animal model was evaluated. As a result, it was confirmed that administration of the immortalized neural stem cells (CBNU-NSC.ChAT) into which the ChAT gene was introduced effectively reduced the inflammatory cytokine IL-1β, which is increased by the induction of cerebral artery occlusion (see FIG. 7).

In Experimental Example 7 of the present invention, the neural stem cell protein (MAP) in the cerebral tissue following administration of the immortalized stem cell (CBNU-NSC) or immortalized neural stem cell (CBNU-NSC.ChAT) -2, the expression of GFAP, an astrocytic skeletal protein, and ICAM-1, an adhesion molecule. As a result, the content of the protein decreased with the induction of cerebral artery occlusion in immortalized neural stem cells CBNU-NSC) or the immortalized neural stem cells (CBNU-NSC.ChAT) into which the ChAT gene was introduced (see FIG. 8).

Therefore, it has been objectively proved that the composition of the present invention comprising an immortalized neural stem cell, a culture solution thereof or an extract thereof as an active ingredient can effectively prevent or treat cerebral infarction or stroke.

The cerebral infarction of the present invention may be cerebral infarction caused by focal cerebral ischemia.

In the present invention, &quot; treatment &quot;, as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term &quot; treatment &quot; as used herein refers to an act of treating when &quot; treating &quot; is defined as described above. Thus, in mammals, &quot; treatment &quot; or &quot; therapy &quot; of cerebral infarction, stroke and related diseases may include one or more of the following:

(1) inhibiting the development of cerebral infarction, stroke and related diseases,

(2) preventing the spread of cerebral infarction, stroke and related diseases,

(3) relieving cerebral infarction, stroke and related diseases,

(4) preventing recurrence of cerebral infarction, stroke and related diseases, and

(5) palliating symptoms of cerebral infarction, stroke and related diseases

In the present invention, a disease associated with cerebral infarction or stroke refers to a disease caused or accelerated by cerebral infarction or stroke.

The composition of the present invention is a pharmaceutical composition comprising an immortalized neural stem cell, a culture thereof or an extract thereof as an active ingredient, and may be prepared using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients, A disintegrant, a sweetening agent, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. 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. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition of the present invention is a pharmaceutical composition for topical administration wherein the route of administration is selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral injection, intracranial injection, intranasal administration, acupoint injection injection, and the like.

The injectable buffer and other adjuvant components used to formulate the compositions of the present invention as injectable preparations are well known in the art. The injectable preparation for the composition of the present invention may contain, in addition to the buffer for injection, other adjuvants such as, for example, solubilizers, pH adjusters, suspensions and the like. For example, physiological saline or the like may be used as the injectable buffer solution.

The dosage of the immortalized stem cells varies depending on the condition and the weight of the patient, the degree of disease, the period of administration, and the like, but may be, for example, at least once in a single dose of 1 × 10 ³ to 1 × 10 ⁹ cells, It may be administered 3 to 5 times. In the following examples of the present invention, rats were administered with 1 x 10 ⁶ cells / 200 μl saline.

The pharmaceutical composition of the present invention may contain the above-mentioned immortalized stem cells as an active ingredient, and may include a pharmaceutically acceptable carrier, and may be formulated in the form of a liquid injection according to a conventional method. The pharmaceutically acceptable carriers include aqueous diluents or solvents such as phosphate buffered saline (PBS), purified water, sterilized water, etc., and may contain propylene glycol, polyethylene glycol, olive And a non-aqueous diluent or solvent such as olive oil. If necessary, preservatives, stabilizers, and the like may be included.

The present invention also provides the use of the above immortalized neural stem cells, a culture thereof or an extract thereof as an active ingredient for the manufacture of a medicament for the prevention or treatment of cerebral infarction, stroke and diseases associated therewith. The composition of the present invention comprising an immortalized neural stem cell, a culture thereof or an extract thereof as an active ingredient can be used for the preparation of a medicament for the prevention or treatment of cerebral infarction, stroke and related diseases.

The present invention also provides a method of preventing or treating cerebral infarction, stroke and related diseases comprising administering to a mammal a therapeutically effective amount of a pharmaceutical composition of the present invention.

As used herein, the term &quot; mammal &quot; refers to a mammal that is the subject of treatment, observation, or experimentation, preferably a human.

The term &quot; therapeutically effective amount &quot; as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal, or human, as contemplated by a researcher, veterinarian, physician, pharmacist or other clinician , Which includes an amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

The therapeutic method of the present invention includes a step of administering the immortalized neural stem cells, the culture thereof or the extract thereof through a parenteral route in a conventional manner. Preferably, it is administered intravenously, intracerebrally, intrathecally, or intranasally to a patient having cerebral infarction or stroke disease so that the stem cells can reach the brain injury site parenterally.

In one embodiment of the present invention, the stem cells may be contained in the composition at 1 × 10 ³ to 1 × 10 ⁹ cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example >

Statistical analysis

All results were statistically analyzed using one way analysis of variance (ANOVA). When significance was recognized, a significance test was conducted at 95% confidence level using Turkey's test.

< Example  1>

Choline Acetyltransferase  Gene-introduced immortalization Neural stem cell line  Produce

<1-1> Neural stem cells  Ready

Brain tissue from the human embryo aborted at 15 weeks of gestation (approved by the Institutional Review Board of the University of British Columbia Hospital in Canada) was minced using a scalpel and rinsed with HBSS. The washed brain tissue was incubated with 0.25% trypsin containing 40 μg / mL of DNase I for 5 minutes. DMEM containing 10 mL of 10% FBS was added to inhibit the activity of typsin. Followed by centrifugation for 10 minutes. After the supernatant was removed, the cells were washed with HBSS, and the tissue was loosely packed using a pipette. Single cells were obtained by filtration through 0.04 μm nylon mesh (Millipore). The cells were washed once with HBSS and used a medium consisting of DMEM containing 5% fetal bovine serum (FBS), 20 μg / mL gentamycin, 5 μg / mL amphotericin B and high glucose And cultured. The medium was replaced every three days and cultured for one week to prepare neural stem cells.

<1-2> Establishment of immortalized neural stem cell line

An immortalized neuronal cell line was established using a v-myc oncogene-encoding retroviral vector to obtain the same culture medium stably by maintaining the characteristics of the prepared neural stem cells (Lindvall et al., 2006); Kim et al., 2009); Lee et al., 2007). NSC was composed of DMEM containing 10 μg / mL insulin, 10 μg / mL transferrin, 30 nM sodium selenate, 50 nM hydrocortisone, 0.3 nM triiodothyronine and 20 μg / mL gentamycin and high glucose concentration And then cultured using serum-free medium. Recombinant human bFGF (10 ng / mL; PeproTech, Rocky Hill, NJ, USA) was supplemented with DM4 during normal medium exchange. All compounds except bFGF were purchased from Sigma (St Louis, MO, USA).

In detail, we infected the PA317 amphotropic packaging cell line (American Type Culture Collection #CRL 9078) using the pLSN.v-myc retroviral vector and obtained a PA317 producer cell line, PASK1.2, with successfully infected retroviral particles. The PASK1.2 was cultured in 0.5 μg / mL puromycin for 72 hours. After 72 hours, colonies formed puromycin-resistant clones were isolated and resuspended in 10 μg / mL insulin, 10 μg / mL transferrin, 30 nM sodium selenate, 50 nM hydrocortisone, 0.3 nM triiodothyronine, and 20 μg / mL The cells were cultured in serum-free medium containing DMEM containing high concentration of glucose containing gentamicin. Recombinant human bFGF (10 ng / mL; PeproTech, Rocky Hill, NJ, USA) was supplemented with DM4 during normal medium exchange. All compounds except bFGF were purchased from Sigma (St Louis, MO, USA). Afterwards, the established cell lines were confirmed to be stem cells by immunostaining for nestin and CD133.

The immortalized neural stem cells of the present invention established through the above process are briefly referred to as 'CBNU-NSC'.

<1-3> Choline Acetyltransferase  Gene-introduced immortalization Neural stem cell  Establishment of transformant

Human choline acetyltransferase (ChAT) full-length cDNA was obtained from human small-cell marathon-Ready cDNA (Clontech, Mountain View, Calif.) According to the method previously described in the known literature for rat peripheral nerve type ChAT (Matsuo et al., 2005) , &Lt; / RTI &gt; CA, USA). Sequence analysis of the plasmid DNA was performed using an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, Calif., USA). ChAT cDNA was first ligated to the multiple cloning site of the retroviral vector pLPCX in order to produce ChAT overexpressed human NSC cell lines (see Figure 1).

The PA317 amphotropic packaging cell line was infected with a recombinant retroviral vector and the supernatant of the packaging cells was taken and added to neural stem cells (CBNU-NSC). Stably transformed colonies were screened by resistance to furomycin. ChAT mRNA expression was forward: 5'-CTGTGCCCCCTTCTAGAGC-3 '; reverse: reverse transcriptase (RT) PCR using a primer with 5'-CAAGGTTGGTGTCCCTGG-3 sequence. The expression of ChAT protein was confirmed by immunocytochemistry using human ChAT (1: 100, rabbit polyclonal, AB143, Chemicon, Temecula, CA, USA) The immortalized neural stem cells into which the ChAT gene of the present invention prepared through the above process is briefly referred to as' CBNU-NSC.ChAT ', and CBNU-NSC.ChAT of the present invention is referred to as' (KCTC), and deposited a grant number (KCTC 12885BP) on the date of 01.01.205.

<1-4> immortalization Neural stem cells  Preparation of culture medium

Cultures of immortalized neural stem cells (CBNU-NSC) were cultured in DMEM containing 10% fetal bovine serum (FBS), 20 μg / mL gentamycin and 5 μg / mL amphotericin B and containing high glucose Lt; / RTI &gt; All of the products used in the medium were Gibco (Grand Island, NY, USA). The culture solution (raw solution) used in the test was centrifuged to remove cell debris, and 30 mL of each culture was frozen and stored in a 50 mL tube. The culture solution obtained by concentrating 10-fold was prepared by lyophilizing the tube containing the stock solution of the above culture solution, storing the solution in the freezer and concentrating it 10 times by adding 3 mL of PBS immediately before use.

<1-5> ChAT  Gene-introduced immortalization Neural stem cells  Preparation of culture medium

The culture medium of the immortalized neural stem cells (CBNU-NSC.ChAT) to which the ChAT gene was introduced contained 10% fetal bovine serum (FBS), 20 μg / mL gentamycin and 5 μg / mL amphotericin B, DMEM. &Lt; / RTI &gt; All of the products used in the medium were Gibco products. The culture solution (raw solution) used in the test was centrifuged to remove cell debris, and 30 mL of each culture was frozen and stored in a 50 mL tube. The culture solution obtained by concentrating 10-fold was prepared by lyophilizing the tube containing the stock solution of the above culture solution, storing the solution in the freezer and concentrating it 10 times by adding 3 mL of PBS immediately before use.

< Example  2>

Stroke animal models and immortalization Neural stem cells  administration

<2-1> Preparation of experimental animals

Animals used in the model of cerebral ischemia due to occluded cerebral artery occlusion were purchased from BioLink (Negative, Korea) with a male rat (Spraque-Dawley) weighing 260-280 g at 7 weeks of age. During the experimental period, feed and water were supplied unrestrictedly. The temperature was maintained at 23 ± 3 ℃, the relative humidity was 50 ± 10%, and the darkness was maintained for 12 hours. All animal studies were conducted in accordance with the standard work guidelines of the Chungbuk National University Laboratory Animal Research Support Center and were approved by the Animal Experimental Ethics Committee.

<2-2> Middle cerebral artery occlusion, MCAO )

Schmid-Elsaesser et al. (1998), a silicone-coated yarn was prepared. Silicone was infused into polyethylene tubing (Intramedic, Batavia, IL, USA) with an internal diameter of 0.28 mm, and 5 mm of 4/0 monofilament nylon suture (Aille, Busan, Korea) was inserted into one end of the silicone tube al., 2012). A point 0.5 mm from the end of the nylon suture was cut with a razor and then the silicon was dried for 24 hours. Immediately prior to surgery, the polyethylene tube was removed from the nylon suture and the length of the silicone was kept constant at 5 mm. As a local cerebral ischemia model, middle cerebral artery occlusion was performed by Longa et al. (Park et al., 2012a), which was modified by the proposed method (1989). After anesthesia was induced with isoflurane in anesthesia box, anesthesia was maintained with oxygen and isoflurane during the operation. After the induction of anesthesia, the rats were calibrated to the sternal position and the neck was incised along the midline of the neck. The right common carotid artery (CCA), the internal carotid artery (ICA) and the external carotid artery ). The lower part of ICA and CCA was clipped and the upper part of ECA was ligated. Next, a hole was made in the central part of the ECA, a silicone-coated thread was inserted, and the lower part of the ligated ECA was cut off so that the direction of the thread could be reversed. The needle is inserted 18 mm in the ICA direction and placed at the origin of the middle cerebral artery. After closure, the thread was ligated together with the blood vessels to prevent movement. Two hours after closure of the middle cerebral artery, reperfusion was induced by removing the thread.

<2-3> immortalization Neural stem cells  administration

Immediately after reperfusion, immortalized neural stem cells (control) and immortalized neural stem cells (experimental cells) (1 × 10 ⁶ cells / 200 μL saline / rat) with ChAT gene introduced were administered subcutaneously.

< Experimental Example  1>

ChAT  Gene-introduced immortalization Neural stem cell  Functional protein production analysis

In this experiment, the functional protein produced by the immortal neural stem cells into which the ChAT gene of the present invention prepared in Example 1 was introduced was analyzed.

<1-1> Immunocytochemistry ( Immunocytochemisry ) immortalization Nervous tension Identification of Functional Protein Expression in Cells

The expression of ChAT gene in immortalized neural stem cells into which choline acetyltransferase gene was introduced was analyzed by immunocytochemical staining.

Immortalized neural stem cells (CBNU-NSC), neural stem cells (NSC), and immortalized neural stem cells (CBNU-NSC.ChAT), into which ChAT gene was introduced were cultured on Aclar plastic coverslips coated with poly-L- And fixed with 4% paraformaldehyde at room temperature for 5 minutes. Immobilized cells were treated with human ChAT (diluted 1: 100; rabbit polyclonal antibody, Chemicon, Temecula, CA, USA) for 24 h at 4 ° C. The secondary antibody, Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) was then treated for 1 hour at room temperature. Next, 4,6-diamino-2-phenylindole (DAPI; Sigma, St. Louis, Mo., USA) was treated to stain the nuclei.

As a result, it was confirmed that nestin (stem cell indicator) and ChAT (functional gene) were doubly stained in the cells as shown in FIG. 2A, and it was confirmed that the cholin acetyltransferase gene immortalized neural stem cells CBNU-NSC.ChAT) expresses the ChAT gene.

<1-2> Western Blat  through immortalization Neural stem cell Brain protection  Protein expression analysis

Western blotting was used to analyze growth factors / neurotrophic factors, which are protective proteins in immortalized neural stem cells with immortalized neural stem cells and choline acetyltransferase genes.

Cells were treated with RIPA solution (Sigma-Aldrich, St. Louis, Mo., USA) to extract proteins and centrifuged at 14,000 g for 10 min at 4 ° C to obtain proteins. Proteins were denatured by heating in a protein-loading buffer (Fermentas, Hanover, MD, USA) at 100 ° C for 10 minutes, and then the proteins were separated into their molecular sizes using 15% SDS gel. After electrophoresis, the gel protein was transferred to a polyvinylidene difluoride membrane in 25 mM Tris buffer. Membrane was blocked with 5% skim milk dissolved in Tris-buffered saline (TBS-T) containing Tween-20 for 1 hour at room temperature. Then, brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (diluted 1: 500; rabbit monoclonal antibody) for 4 hours at 4 ° C for each of the neuron-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF). The membranes were washed with TBS-T and reacted with goat anti-rabbit IgG conjugated horseradish peroxidase antibody (diluted 1: 2,000; Santa Cruz Biotechnology) for 1 hour at room temperature and electrochemiluminescence solution (Thermo, Rockford, .

The results showed that the expression of growth factors and neurotrophic factors such as GDNF, VEGF, CNTF, and NGF related to neuroprotection and regeneration of the brain was significantly higher than that of immortalized stem cells (CBNU -NSC.ChAT) increased significantly. In particular, VEGF and NGF were hardly expressed in immortalized neural stem cells (CBNU-NSC), but these factors were highly expressed in immortalized stem cells (CBNU-NSC.ChAT) transfected with ChAT gene.

< Experimental Example  2>

Effect on cerebral infarction

Forty-eight hours after the cognitive function test, the brain was quickly removed and brain tissue was cut into 2-mm-thick sections using a brain matrix. Brain sections were placed on a 24-well plate and reacted with 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37 ° C for 15 minutes (Park et al., 2012a, 2012b). After staining, the TTC solution was removed and fixed with 10% neutral formalin solution. The infarct area of the brain slice was measured by image analysis software (ImageJ; National Institutes of Health, Bethesda, Maryland, USA). The total volume of cerebral infarction was calculated by multiplying the infarct area of each brain slice by the thickness of the slice (2 mm).

As a result, as shown in FIG. 3, the infarction volume of the MCAO-induced group was 29.1% of the total brain tissue. In contrast, the infarcted neural stem cells (CBNU-NSC) showed a significant decrease in cerebral infarct volume (5.9%). In particular, the infusion of immortalized neural stem cells (CBNU-NSC.ChAT) In the group, the volume of cerebral infarction was 3.1%, which was significantly decreased compared to the induced group.

< Experimental Example  3>

Effects on lipid peroxidation and NO production

Malondialdehyde (MDA) assay was performed to assess the level of lipid peroxidation by identifying thiobarbituric acid-reactive substances (TBARS) in brain tissue (Ohkawa et al., 1979; Callaway et al., 1998). The rats were systemically perfused with physiological saline and brain was rapidly removed. The tissue was homogenized by adding PBS 10 times the brain weight, and centrifuged at 4,000 rpm for 5 minutes to obtain supernatant. Sodium dodecylsulfate (SDS; 500 μL of 8.1% w / v solution) and 1 mL of 20% acetic acid (pH 3.5) were added to the supernatant, followed by centrifugation. The same amount of thiobarbituric acid solution (0.8% w / v) was added to the supernatant obtained by centrifugation and reacted at 95 ° C for 30 minutes. After completion of the reaction, the reaction solution was rapidly cooled and the absorbance was measured at 532 nm.

Measurement of nitrogen monoxide (NO) in cerebrospinal fluid was performed by ether anesthesia, sacrificed animals, and cerebrospinal fluid was collected through the cervical cisterna membrane to prevent blood from mixing. The final product of nitric oxide (NO), nitrite, was reacted with Griess reagent (Promega, Madison, WI, USA) and absorbance was measured at 540 nm.

As a result, the content of malondialdehyde (MDA) was increased to 54.2 nmol / g in the brain tissue of the animal causing ischemic brain injury compared with the normal animal, whereas the content of immortalized neural stem cells (CBNU- In the animals treated with NSC, the content of MDA decreased to 38.6%. Especially, the content of MDA in the brain tissues of animals treated with immortalized neural stem cells (CBNU-NSC.ChAT) 33.4%).

Similarly, the concentration of NO in the cerebrospinal fluid of animals that induced ischemic stroke was 28.3 μmol / L, which is higher than that of normal animals, whereas the immortalized neural stem cells (CBNU-NSC) and immortalized neural stem cells (CBNU-NSC) In the experimental group treated with ChAT, NO production was suppressed to 26.5 μmol / L (see FIG. 4B).

< Experimental Example  4>

Recovery effect on cognitive function

Morris water-maze test was performed to confirm the effect of immortalized neural stem cells and immortalized neural stem cells with ChAT gene. A circular water tank measuring 180 cm in diameter and 27 cm in height is used for the Morris water maze. The water tank is divided into four quarters, and a platform with a diameter of 10 cm and a height of 25 cm is installed at the center of one quadrant. To prevent the rats from seeing the platform in these tanks, water was filled to locate the platform 2 cm below the surface of the water, and styrofoam with a diameter of 5 mm was placed on the surface of the water. The spatial cues were always kept constant so that the rats could find the platform with spatial cues around the underwater maze. The cognitive function confirmation end time was set to 300 seconds, and the time it took for the rat to find the platform was recorded.

As a result, as shown in FIG. 5, it took 11 seconds to reach the platform in normal animals, but in the MCAO-induced group, the time required for the cognitive function was prolonged to 115 seconds. On the other hand, in the rats receiving the immortalized neural stem cells and the immortalized neural stem cells transfected with the ChAT gene, the time required for the improvement of cognitive function was shorter than that of the induced group. In the CBNU-NSC group The time was reduced to 63 seconds, and the cognitive function improved in the group treated with the immortalized neural stem cells (CBNU-NSC.ChAT) into which the ChAT gene was introduced.

< Experimental Example  5>

Physical vitality (Exercise function and activity)

<5-1> Rotation bar motion (ROI)

Twenty - four hours after middle cerebral artery occlusion, swimmer exercise tests were performed to evaluate motor function and balance. The rotation speed of the rotating rod was gradually increased from 4 rpm to 40 rpm for 5 minutes, and the staying time was measured by positioning the animal on the rotating rod. Three consecutive runs were performed to calculate the average value.

As a result, as shown in FIG. 6A, in the normal group, the time to stay on the rotating bar was 163 seconds, whereas in the MCAO-induced group, the time required for the rotating bar to measure the cooperative function was greatly reduced to 50 seconds. However, with the administration of immortalized neural stem cells (CBNU-NSC) and immortalized neural stem cells (CBNU-NSC.ChAT) transduced with ChAT gene, The effect was better in the immortalized neural stem cells treated with the ChAT gene than in the simple immortalized neural stem cells without the introduction of the ChAT gene.

<5-2> Measurement of locomotor activity

We measured 24 hours after middle cerebral artery occlusion in order to evaluate self - issued equivalence as a measure of physical activity. Animals were placed in the middle of a square test box (50 x 50 x 30 cm), adapted to the environment in the box for 15 seconds, then resting, resting, slow-moving and fast moving -moving time (Habig et al., 1974; Park et al., 2012b).

As a result, as shown in FIG. 6B, the moving time (slow motion + fast movement) was relatively long in the normal animal (black bar), whereas in the MCAO-induced group (red bar) The rest time ratio was 80%, slow motion and fast motion time were 17% and 3%, respectively. However, by administering immortalized neural stem cells (CBNU-NSC, green bar) and immortalized neural stem cells (CBNU-NSC.ChAT, yellow bar) with ChAT gene, the resting time is reduced and the moving time is increased, The function was recovered.

< Experimental Example  6>

Inflammatory cytokine-reducing effect

Total RNA was extracted from damaged brain of rats using TRIzol (Invitrogen, Carlsbad, CA, USA) to analyze the gene expression of inflammatory cytokines. CDNA was synthesized by inserting oligodT primer and Moloney Murine Leukemia virus reverse transcriptase (Promega, Madison, WI, USA) 40 U into 1 ㎍ of isolated RNA. The product synthesized from cDNA was isolated by 1.5% agarose gel containing ethidium bromide. The sequence of each primer used for RT-PCR is shown in Table 1 (Bioneer, Daejeon, Korea).

A primer sequence for the analysis of the inflammatory cytokines TNF-alpha (tumor necrosis factor-alpha) and IL-1 beta (interleukin-1 beta) gene primer sequence Accession No. TNF-a Forward 5'-GACCCTCACACTCAGATCATCTTCT-3 ' NM_012675 Reverse 5'-TGCTACGACGTGGGCTACG-3 ' IL-1? Forward 5'-CCCTGCAGCTGGAGAGTGTGG-3 ' NM_031512 Reverse Gt;

As shown in FIG. 7, the TNF-α and IL-1β genes were expressed in the ischemic stroke-induced group compared with the normal group, as shown in FIG. 7, as a result of measuring gene expression of inflammatory cytokines such as TNF-α and IL- However, IL-1β was decreased in the experimental group treated with the immortalized neural stem cells (CBNU-NSC.ChAT) into which the ChAT gene was introduced. However, administration of immortalized neural stem cells (CBNU-NSC) did not affect TNF-α gene expression.

< Experimental Example  7>

Effect on protein content in brain tissue

The production of cytoskeletal protein, MAP-2, astrocytes skeletal protein, GFAP, and ICAM-1, an adhesion molecule, in brain tissue was analyzed by Western blotting. Proteins were extracted from the brain tissue by treatment with RIPA solution (Sigma-Aldrich, St. Louis, Mo., USA) and centrifuged at 14,000 g for 10 minutes at 4 ° C to obtain proteins. Proteins were denatured by heating in a protein-loading buffer (Fermentas, Hanover, MD, USA) at 100 ° C for 10 minutes, and then the proteins were separated into their molecular sizes using 15% SDS gel. After electrophoresis, the gel protein was transferred to a polyvinylidene difluoride membrane in 25 mM Tris buffer. Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS-T) containing tween-20 for 1 hour at room temperature and then incubated with monoclonal antibodies against MAP-2, GFAP and ICAM- Dilution ratio 1: 500; rabbit monoclonal antibody) overnight at 4 ° C. The membranes were washed with TBS-T and reacted with a secondary antibody, goat anti-rabbit IgG conjugated horseradish peroxidase antibody (dilution 1: 2,000; Santa Cruz Biotechnology) for 1 hour at room temperature, and electrochemiluminescence solution (Thermo, Rockford, .

As shown in FIG. 8, the content of MAP-2 protein in the ischemic brain injury-induced group was significantly lower than that in the normal group, but the immortalized neural stem cells (CBNU-NSC) and the immortalized neural stem cells CBNU- NSC.ChAT), respectively, showed that MAP-2 production was restored. In addition, the contents of GFAP and ICAM-1, which are involved in inflammation induction, were significantly increased in the induced group, but the immortalized neural stem cells (CBNU-NSC) and the immortalized neural stem cells (CBNU-NSC.ChAT) And it was confirmed that it recovered to a similar level.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

MCAO: middle cerebral artery occlusion
TNF-α: tumor necrosis factor-α
IL-1 [beta]: interleukin-1 [beta]
GDNF: glial cell line-derived neurotropic factor
VEGF: Vascular endothelial growth factor
CNTF: Ciliary neurotrophic factor
NGF: Nerve gowth factor
MAP-2: microtubule-associated protein 2
GFAP: glial fibrillary acidic protein
ICAM-1: intercellular adhesion molecule-1

Korea Biotechnology Research Institute KCTC12885BP 20150827

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Composition for the prevention or treatment of cerebral          infarction and stroke comprising immortalized neural stem cells <130> PN1507-220 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 748 <212> PRT <213> Homo sapiens <400> 1 Met Gly Leu Arg Thr Ala Lys Lys Arg Gly Leu Gly Gly Gly Gly Lys   1 5 10 15 Trp Lys Arg Glu Glu Gly Gly Gly Thr Arg Gly Arg Arg Glu Val Arg              20 25 30 Pro Ala Cys Phe Leu Gln Ser Gly Gly Arg Gly Asp Pro Gly Asp Val          35 40 45 Gly Gly Pro Ala Gly Asn Pro Gly Cys Ser Pro His Pro Arg Ala Ala      50 55 60 Thr Arg Pro Pro Leu Pro Ala His Thr Pro Ala His Thr Pro Glu  65 70 75 80 Trp Cys Gly Ala Ala Ser Ala Glu Ala Ala Glu Pro Arg Arg Ala Gly                  85 90 95 Pro His Leu Cys Ile Pro Ala Pro Gly Leu Thr Lys Thr Pro Ile Leu             100 105 110 Glu Lys Val Pro Arg Lys Met Ala Ala Lys Thr Pro Ser Ser Glu Glu         115 120 125 Ser Gly Leu Pro Lys Leu Pro Val Pro Pro Leu Gln Gln Thr Leu Ala     130 135 140 Thr Tyr Leu Gln Cys Met Arg His Leu Val Ser Glu Glu Gln Phe Arg 145 150 155 160 Lys Ser Gln Ala Ile Val Gln Gln Phe Gly Ala Pro Gly Gly Leu Gly                 165 170 175 Glu Thr Leu Gln Gln Lys Leu Leu Glu Arg Gln Glu Lys Thr Ala Asn             180 185 190 Trp Val Ser Glu Tyr Trp Leu Asn Asp Met Tyr Leu Asn Asn Arg Leu         195 200 205 Ala Leu Pro Val Asn Ser Ser Pro Ala Val Ile Phe Ala Arg Gln His     210 215 220 Phe Pro Gly Thr Asp Asp Gln Leu Arg Phe Ala Ala Ser Leu Ile Ser 225 230 235 240 Gly Val Leu Ser Tyr Lys Ala Leu Leu Asp Ser His Ser Ile Pro Thr                 245 250 255 Asp Cys Ala Lys Gly Gln Leu Ser Gly Gln Pro Leu Cys Met Lys Gln             260 265 270 Tyr Tyr Gly Leu Phe Ser Ser Tyr Arg Leu Pro Gly His Thr Gln Asp         275 280 285 Thr Leu Val Ala Gln Asn Ser Ser Ile Met Pro Glu Pro Glu His Val     290 295 300 Ile Val Ala Cys Cys Asn Gln Phe Phe Val Leu Asp Val Val Ile Asn 305 310 315 320 Phe Arg Arg Leu Ser Glu Gly Asp Leu Phe Thr Gln Leu Arg Lys Ile                 325 330 335 Val Lys Met Ala Ser Asn Glu Asp Glu Arg Leu Pro Pro Ile Gly Leu             340 345 350 Leu Thr Ser Asp Gly Arg Ser Glu Trp Ala Glu Ala Arg Thr Val Leu         355 360 365 Val Lys Asp Ser Thr Asn Arg Asp Ser Leu Asp Met Ile Glu Arg Cys     370 375 380 Ile Cys Leu Val Cys Leu Asp Ala Pro Gly Gly Val Glu Leu Ser Asp 385 390 395 400 Thr His Arg Ala Leu Gln Leu Leu His Gly Gly Gly Tyr Ser Lys Asn                 405 410 415 Gly Ala Asn Arg Trp Tyr Asp Lys Ser Leu Gln Phe Val Val Gly Arg             420 425 430 Asp Gly Thr Cys Gly Val Val Cys Glu His Ser Pro Phe Asp Gly Ile         435 440 445 Val Leu Val Gln Cys Thr Glu His Leu Leu Lys His Met Thr Gln Ser     450 455 460 Ser Arg Lys Leu Ile Arg Ala Asp Ser Val Ser Glu Leu Pro Ala Pro 465 470 475 480 Arg Arg Leu Arg Trp Lys Cys Ser Pro Glu Ile Gln Gly His Leu Ala                 485 490 495 Ser Ser Ala Glu Lys Leu Gln Arg Ile Val Lys Asn Leu Asp Phe Ile             500 505 510 Val Tyr Lys Phe Asp Asn Tyr Gly Lys Thr Phe Ile Lys Lys Gln Lys         515 520 525 Cys Ser Pro Asp Ala Phe Ile Gln Val Ala Leu Gln Leu Ala Phe Tyr     530 535 540 Arg Leu His Arg Arg Leu Val Pro Thr Tyr Glu Ser Ala Ser Ile Arg 545 550 555 560 Arg Phe Gln Glu Gly Arg Val Asp Asn Ile Arg Ser Ala Thr Pro Glu                 565 570 575 Ala Leu Ala Phe Val Ala Val Thr Asp His Lys Ala Ala Val Pro             580 585 590 Ala Ser Glu Lys Leu Leu Leu Leu Lys Asp Ala Ile Arg Ala Gln Thr         595 600 605 Ala Tyr Thr Val Ale Ile Thr Gly Ale Ile Asp Asn His Leu     610 615 620 Leu Ala Leu Arg Glu Leu Ala Arg Ala Met Cys Lys Glu Leu Pro Glu 625 630 635 640 Met Phe Met Asp Glu Thr Tyr Leu Met Ser Asn Arg Phe Val Leu Ser                 645 650 655 Thr Ser Gln Val Pro Thr Thr Thr Glu Met Phe Cys Cys Tyr Gly Pro             660 665 670 Val Val Pro Asn Gly Tyr Gly Ala Cys Tyr Asn Pro Gln Pro Glu Thr         675 680 685 Ile Leu Phe Cys Ile Ser Ser Phe His Ser Cys Lys Glu Thr Ser Ser     690 695 700 Ser Lys Phe Ala Lys Ala Val Glu Glu Ser Leu Ile Asp Met Arg Asp 705 710 715 720 Leu Cys Ser Leu Leu Pro Pro Thr Glu Ser Lys Pro Leu Ala Thr Lys                 725 730 735 Glu Lys Ala Thr Arg Pro Ser Gln Gly His Gln Pro             740 745 <210> 2 <211> 2458 <212> DNA <213> Homo sapiens <400> 2 aaatgctgag ctaggggcag gaggcatggg cgggacagtg ttctgtgccc ccttctagag 60 cctaaatttg ttgcccgagt tcctccggga agcgctccgg gtagattctg ggggccggga 120 gctgagatcc ctgggcgggg agctggggaa gggatggggc tgaggacagc gaagaagagg 180 gggcttgggg gaggggggaa atggaagaga gaggagggag gaggtacaag aggaaggaga 240 gaagtgcggc cagcttgctt tctccagtcg ggtggccgcg gggacccggg cgacgtcgga 300 ggccctgccg ggaacccagg ctgcagcccc cacccccgcg ctgcgacacg ccccccaccc 360 cttccggctc acacccccgc ccacactcct gagtggtgcg gtgcagcgtc ggccgaggca 420 gcagagccga ggagagcagg tccacacctc tgcatccctg caccaggact caccaagacg 480 cccatcctgg aaaaggtccc ccgtaagatg gcagcaaaaa ctcccagcag tgaggagtct 540 gggctgccca aactgcccgt gcccccgctg cagcagaccc tggccacgta cctgcagtgc 600 atgcgacact tggtgtctga ggagcagttc aggaagagcc aggccattgt gcagcagttt 660 ggggcccctg gtggcctcgg cgagaccctg cagcagaaac tcctggagcg gcaggagaag 720 acagccaact gggtgtctga gtactggctg aatgacatgt atctcaacaa ccgcctggcc 780 ctgcctgtca actccagccc tgccgtgatc tttgctcggc agcacttccc tggcaccgat 840 gaccagctga ggtttgcagc cagcctcatc tctggtgtac tcagctacaa ggccctgctg 900 gacagccact ccattcccac tgactgtgcc aaaggccagc tgtcagggca gcccctttgc 960 atgaagcaat actatgggct cttctcctcc taccggctcc ccggccatac ccaggacacg 1020 ctggtggctc agaacagcag catcatgccg gagcctgagc acgtcatcgt agcctgctgc 1080 aatcagttct ttgtcttgga tgttgtcatt aatttccgcc gtctcagtga gggggatctg 1140 ttcactcagt tgagaaagat agtcaaaatg gcttccaacg aggacgagcg tttgcctcca 1200 attggcctgc tgacgtctga cgggaggagc gagtgggccg aggccaggac ggtcctcgtg 1260 aaagactcca ccaaccggga ctcgctggac atgattgagc gctgcatctg ccttgtatgc 1320 ctggacgcgc caggaggcgt ggagctcagc gacacccaca gggcactcca gctccttcac 1380 ggcggaggct acagcaagaa cggggccaat cgctggtacg acaagtccct gcagtttgtg 1440 gtgggccgag acggcacctg cggtgtggtg tgcgaacact ccccattcga tggcatcgtc 1500 ctggtgcagt gcactgagca tctgctcaag cacatgacgc agagcagcag gaagctgatc 1560 cgagcagact ccgtcagcga gctccccgcc ccccggaggc tgcggtggaa atgctccccg 1620 gaaattcaag gccacttagc ctcctcggca gaaaaacttc aacgaatagt aaagaacctt 1680 gacttcattg tctataagtt tgacaactat gggaaaacat tcattaagaa gcagaaatgc 1740 agccctgatg ccttcatcca ggtggccctc cagctggcct tctacaggct ccatcgaaga 1800 ctggtgccca cctacgagag cgcgtccatc cgccgattcc aggagggacg cgtggacaac 1860 atcagatcgg ccactccaga ggcactggct tttgtgagag ccgtgactga ccacaaggct 1920 gctgtgccag cttctgagaa gcttctgctc ctgaaggatg ccatccgtgc ccagactgca 1980 tacacagtaca tggccataac agggatggcc attgacaacc acctgctggc actgcgggag 2040 ctggcccggg ccatgtgcaa ggagctgccc gagatgttca tggatgaaac ctacctgatg 2100 agcaaccggt ttgtcctctc cactagccag gtgcccacaa ccacggagat gttctgctgc 2160 tatggtcctg tggtcccaaa tgggtatggt gcctgctaca acccccagcc agagaccatc 2220 cttttctgca tctctagctt tcacagctgc aaagagactt cttctagcaa gtttgcaaaa 2280 gctgtggaag aaagcctcat tgacatgaga gacctctgca gtctgctgcc gcctactgag 2340 agcaagccat tggcaacaaa ggaaaaagcc acgaggccca gccagggaca ccaaccttga 2400 ctcctgccac taggtttcac ctcccaaacc cagcctctag aacagccaga ccctgcag 2458

Claims (7)

A pharmaceutical composition for the prevention and treatment of cerebral infarction or stroke, comprising an immortalized neural stem cell, a culture solution thereof or an extract thereof as an active ingredient. The method according to claim 1,
Wherein the immortalized neural stem cells are transgenic immortalized neural stem cells into which the choline acetyltransferase gene has been introduced. 3. The method of claim 2,
Wherein said choline acetyltransferase has the amino acid sequence of SEQ ID NO: 1. 3. The method of claim 2,
Wherein the choline acetyltransferase gene comprises the polynucleotide sequence of SEQ ID NO: 2. 3. The method of claim 2,
Wherein the transformed immortalized neural stem cells into which the choline acetyltransferase gene has been introduced is the accession number KCTC 12885BP. 6. The method according to any one of claims 1 to 5,
Wherein the neural stem cells are contained in the composition in an amount of 1 × 10 ³ to 1 × 10 ⁹ cells. 6. The method according to any one of claims 1 to 5,
Wherein said cerebral infarct is caused by focal cerebral ischemia.

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