JP5789849B2 - Composition for promoting neurogenesis - Google Patents

Description

  The present invention relates to a composition for promoting neurogenesis including nicotine. More specifically, the composition has an effect of promoting differentiation into nerve cells and an effect of inhibiting differentiation of astroglia cells.

Nerve cells are tissues that do not have division ability in the living body, and therefore, when damaged, the damage continues for a long time. On the other hand, the peripheral nerve has a regeneration ability, but the regeneration has a time of several months to one year or more. Furthermore, since regeneration requires a long period of time, nerve cells may die during that time, and function recovery may not occur.
During this recovery period, nervous system cells called astroglial cells change into proliferating cells called reactive astroglial cells, forming glial scars in the tissue. The glial scar becomes an obstacle and prevents reprojection of regenerating nerve axons. Therefore, development of a novel drug capable of inhibiting glial scar formation is desired (see: Patent Document 1).

One of the inventors of the present invention has intensively studied various naturally-derived components having a neuronal cell formation promoting action, and found that the tea component, theanine, has an excellent nerve cell formation promoting action (see Patent Document 2). ).
However, theanine and nicotine are clearly different in structure and are clearly different from the invention of the present application.

On the other hand, nerve growth factor biosynthesis promoters using nicotine have been reported.
Japanese Patent Laid-Open No. 5-201860 (Patent Document 3) discloses a “nerve growth factor biosynthesis promoter containing nicotine as an active ingredient”.
This publication reports that “exposure of neurotrophic factor NGF in astroglia cells is promoted by exposure to nicotine”. However, since NGF has an effect of promoting the growth and maturation of nerve cells, nicotine administration enhances the production of astroglial NGF, and as a result, the possibility of protecting neighboring nerve cells is limited. Advocated.

Neural stem cells are differentiated into neural cells via neural progenitor cells, or differentiated into astroglial cells or oligodendroglial cells via glial progenitor cells. Fetal neural stem cells differentiate into neurons or glial cells with a probability of approximately 50%. On the other hand, almost all adult neural stem cells differentiate into glial cells. Therefore, a component that suppresses differentiation into glial cells and further promotes differentiation into nerve cells can be expected to prevent aging and prevent or treat diseases related to the central nervous system.
Moreover, neurodegenerative diseases represented by Alzheimer's disease, Parkinson's disease and the like are serious diseases that cause nerve loss. However, there is no effective therapeutic agent even now. Furthermore, in psychiatric disorders represented by schizophrenia and depression, neuronal loss is not observed, but neuronal dysfunction is observed. However, many side effects have been reported for currently used therapeutic agents.

  Based on the above situation, development of a therapeutic agent for neuropsychiatric disorders having an effect of promoting differentiation into nerve cells and an effect of inhibiting differentiation into astroglial cells is desired.

International Publication 2006/137377 JP2008-169143 JP 5-201860

  This invention made it the subject which should solve solving the above-mentioned problem. More specifically, an object to be solved is to provide a composition having an effect of promoting differentiation into nerve cells and an effect of inhibiting differentiation into astroglia cells.

  Based on the findings that the present inventors have found that culturing neural progenitor cells in the presence of nicotine promotes differentiation into neurons and suppresses differentiation into astroglial cells. A composition for promoting neurogenesis containing nicotine was completed.

That is, the present invention is as follows.
1. A composition for promoting neurogenesis, comprising nicotine as an active ingredient.
2. 2. The composition for promoting neuronal neogenesis according to item 1 above, which enhances the differentiation ability into a neuron.
3. 3. The neuronal cell neogenesis promoting composition according to item 1 or 2, which suppresses differentiation ability into astroglial cells.
4). 4. The composition for promoting neurogenesis according to any one of 1 to 3 above, which does not induce cell death of neural progenitor cells.
5. 5. The composition for promoting neurogenesis according to any one of 1 to 4 above, which suppresses the proliferation ability of neural progenitor cells.
6). 6. The composition for promoting neurogenesis according to any one of 1 to 5, which is any one of the following therapeutic agents.
(1) Alzheimer's disease (2) Parkinson's disease (3) Stroke (4) Depression (5) Schizophrenia (6) Autism

  According to the present invention, it is possible to provide a neuronal cell neogenesis promoting composition having an effect of promoting differentiation into nerve cells and an effect of inhibiting differentiation into astroglial cells.

Results of immune cell staining of differentiation-induced neural progenitor cells Confirmation that nAChRs are present in neural progenitor cells Measurement results of proliferative ability of neural progenitor cells by nicotine administration Results of MTT measurement of proliferation ability of neural progenitor cells by nicotine administration Measurement results of cell death induction of neural progenitor cells by nicotine administration Measurement results of proliferation ability of neural progenitor cells by administration of nAChRs antagonist Measurement results of the proportion of spontaneously differentiated neural progenitor cells Measurement results of the percentage of neural progenitor cells differentiated in the presence of differentiation-inducing factors

The neuronal cell neoplasia promoting composition of the present invention utilizes the fact that “the effect of promoting differentiation into nerve cells and the effect of inhibiting differentiation into astroglia cells” have been found according to the following examples.
Therefore, the composition for promoting neurogenesis of the present invention can be used as a therapeutic agent for neurodegenerative diseases such as Alzheimer's disease, stroke, Parkinson's disease, and mental diseases such as depression, schizophrenia, and autism.

(Neurogenesis promotion composition)
The “neurogenesis promotion composition” of the present invention contains nicotine as an active ingredient, enhances differentiation ability into nerve cells (differentiation promotion effect), regenerative ability of nerve cells, and into astroglia cells. 1 selected from the group consisting of an inhibitory action on the differentiation ability of cells, an inhibitory action on the proliferation ability of neural progenitor cells (an inhibitory effect on proliferation ability), and an action that does not induce cell death of neural precursor cells (an effect that does not induce cell death) 1 It means what has the above effect | action.
Induction of the ability to enhance differentiation into nerve cells, the ability to regenerate nerve cells, the action to inhibit differentiation into astroglia cells, the action to inhibit proliferation of neural progenitors and the death of neural progenitors The effects of nicotine itself on the enhancement of differentiation into neurons, the regeneration of neurons, the inhibition of differentiation on astroglia cells, the inhibition of proliferation of neural progenitors and the effects of neural progenitors Including the case where it has an action that does not induce cell death, enhancement of differentiation ability into nerve cells by other substances, regeneration ability of nerve cells, inhibition of differentiation ability into astroglia cells, proliferation of neural progenitor cells It is understood that the action of suppressing the ability and the action of not inducing cell death of neural progenitor cells include the case where nicotine is promoted or suppressed catalytically.

(Use of composition for promoting neurogenesis)
The composition for promoting neurogenesis of the present invention can be used for the treatment of Alzheimer's disease, Parkinson's disease, stroke, depression, schizophrenia, autism and the like.

(nicotine)
The “nicotine” of the present invention may be any of D-form and L-form nicotine, and as long as it has a differentiation promoting effect on nerve cells and an inhibitory effect on differentiation to astroglia cells, nicotine salt and free base of nicotine It may be in the form, nicotine derivative, nicotine inclusion complex, or any non-covalent nicotine, as well as mixtures thereof.
For example, nicotine salts include monotartrate, hydrogen tartrate, citrate, malate, and hydrochloride.

(Form of composition for promoting neurogenesis)
The composition for promoting neurogenesis of the present invention can be used alone as food (especially chewing gum). Moreover, this composition can also be used as a pharmaceutical and a quasi drug.
As the form of the composition, it can be dried by freeze-drying or spray-drying, etc. and provided as a dry powder, liquid, tablet, powder, granule, dragee, capsule, suspension, emulsion, ampoule, injection, etc. It can be prepared and provided in any form.
When provided as a pharmaceutical, for example, the active ingredient can be prepared by dissolving it in purified water or physiological saline as it is.
When provided as a quasi-drug, it is preferably provided in a form that is easy to ingest as much as possible, such as a drinking form such as a packaged drink, or a tablet, capsule, granule or the like.

  Moreover, the neuronal cell neoplasia promoting composition of the present invention may contain a vitamin agent, an antibacterial agent, another substance having an effect of promoting neuronal neurogenesis (particularly theanine), and the like in addition to nicotine, which is an active ingredient.

(Administration mode of the composition for promoting neurogenesis)
The administration route of the composition for promoting neuronal neogenesis of the present invention is not particularly limited, and oral administration or parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration) It may be administered by any route of administration (internal administration, local administration to the affected area, skin administration, etc.). Formulation forms suitable for oral administration include solid or liquid forms, and preparation forms suitable for parenteral administration include injections, drops, suppositories, external preparations, eye drops, nasal drops and the like. Can be mentioned.

(Dose of composition for promoting neurogenesis)
The dose of the composition for promoting neurogenesis of the present invention is not particularly limited as long as the effect of the composition is not impaired. For example, it is preferable to administer nicotine, which is 0.5 mg to 5.0 mg of active ingredient per kg of body weight of the subject of administration, and in particular, administration of nicotine as active ingredient of 0.5 mg to 2.0 mg per kg of body weight of the subject of administration is further performed. preferable.
For example, assuming that the composition of the present invention is administered to an adult and the body weight is 40 kg to 100 kg, it is preferable to administer 20 mg to 500 mg of nicotine as an active ingredient, and 20 mg to 200 mg of nicotine as an active ingredient. More preferably, is administered.

  For example, it is preferable to adjust the content of nicotine, which is an active ingredient of 3.0 mg to 300 mg, and the content of nicotine, which is an active ingredient of 30 mg to 120 mg. Is more preferable.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples. In the following examples, the test is performed based on the Kanazawa University animal experiment guideline.

(Culture of neural progenitor cells and confirmation of proliferation and differentiation ability of the cells)
Neural stem cells and neural progenitor cells have a self-replicating ability capable of repeating proliferation and multipotency capable of differentiating into cells constituting the central nervous system. Therefore, it was confirmed whether the neural progenitor cells used in Example 2 and later have self-renewal ability and differentiation ability.

(Animal used)
Wistar female pregnant rats (Sankyo Labo Service) were allowed to eat from 8:45 to 20:45 at free time and under water at a temperature of 25 ± 1 ℃ and humidity of 55 ± 2%. The animals were bred under a light / dark cycle with a dark period from 45 hours to 8:45 hours.

(Acquisition of rat fetal cerebral cortex)
The uterus was completely removed from the female rat on the 18th day of pregnancy bred with the above “0024”, and the fetus was removed from the uterus in a clean bench. The head of the fetus was cut from the back to the neck and the whole brain was removed. Under a stereomicroscope, excise the cerebral cortex from the whole brain, and use Phosphate buffer saline (PBS) special (PBS without Ca ²⁺ and Mg ²⁺ , containing 33 mM Glucose, 100 U / mL Penicillin and 100 mg / mL Streptomycin ) Was collected in a Petri dish filled with a) and allowed to stand in a CO ₂ incubator until cell dispersion.

(Culture of neural progenitor cells)
The cerebral cortex obtained in paragraph “0025” was dispersed with a Pasteur pipette whose tip was heated and thinned, and centrifuged at 400 × g for 5 minutes. After removing the supernatant, the cerebral cortex is incubated with an enzyme treatment solution containing 2.5 U / mL papain, 250 U / mL Deoxyribonuclease (DNase) and 1 U / mL Neutral protease at 37 ° C for 30 minutes to bind cells. Relaxed. Subsequently, medium with serum {dulbecco's modified eagle medium: Nutrient mixture F-12 (1: 1) (DMEM / F12) containing 10% fetal bovine serum, 0.11% (w / v) Sodium bicarbonate, 100 U / mL Penicillin, After washing with 100 μg / mL Streptomycin, 16.2 mM Glucose and 250 μM N-acetyl-L-cysteine}, the mixture was centrifuged at 400 × g for 5 minutes, and the resulting cells were redispersed with Medium with serum. This cell dispersion was mixed at a ratio of dispersion: 10 x PBS: Percoll = 10: 1: 9, then centrifuged at 20,000 xg for 30 minutes at 18 ° C to create a density gradient, 1.065 g / mL Cells present in the density layer of ˜1.075 g / mL were collected.
The collected cells are washed with Medium with serum and centrifuged at 400 × g for 5 minutes, and the obtained cells are added to Growth medium {DMEM / F12 containing 0.11% Sodium bicarbonate, 100 U / mL Penicillin, 100 μg / mL Streptomycin. , 16.2 mM Glucose, 250 μM N-acetyl-L-cysteine, 20 nM Progesterone, 30 nM Sodium selenite, 60 μM Putrescine, 25 μg / mL Insulin, 100 μg / mL Apo-transferrin and 10 ng / mL Epidermal Growth Factor (EGF)} Suspended.
Next, the proportion of viable cells was calculated using Typan blue staining and seeded on a 24-well culture dish (NUNC: multi-dish 24 well diameter: 15.5 mm) at a concentration of 1.5 × 10 ⁴ cells / mL. These cells were allowed to stand in a CO ₂ incubator (SANYO, MC0-17AIC type: 37 ° C., 5% CO ₂ /95% O ₂ ) until used in the experiment.

(Measurement of proliferation ability in neural progenitor cells)
The formation of nerve spheres (Neurosphere), which is one of the characteristics shown under the culture conditions of neural progenitor cells, was measured. Details are as follows.
A phase contrast microscopic image of the nerve mass of neural progenitor cells cultured for 12 days in paragraph “0026” was acquired every 2 days from the 4th to 10th day of culture, and from the obtained images using image analysis software The area was measured.

(Induction of differentiation of neural progenitor cells)
Cells cultured for 12 days in Paragraph “0026” were seeded in a dish pre-coated with Poly-L-lysine, and left in a CO ₂ incubator for 1 hour. After cell adhesion, remove Medium, Differentiation medium (DMEM / F12 containing 1% FBS, 0.11% (w / v) Sodium bicarbonate, 250 μM N-acetyl-L-cysteine, 100 U / mL penicillin and 100 μg / mL streptomycin) Replaced with At this time, ATRA, which induces differentiation into neurons, was added to the differentiation medium as a differentiation inducer at a concentration of 100 ng / ml and CNTF, which induces differentiation into astroglia cells, at a concentration of 20 ng / ml. These cells were left in a CO ₂ incubator until the experiment. Medium was replaced with a new Differentiation medium on the 4th day of culture after cell attachment. The cells were used after adhering and culturing in Differentiation medium for 6 days.

(Immune cell staining of differentiation-induced neural progenitor cells)
The neural progenitor cells induced to differentiate in paragraph “0028” were seeded in a dish that had been previously coated with Poly-L-lysine, and allowed to stand in a CO ₂ incubator for 1 hour to allow the cells to adhere to the dish. After cell adhesion, the cells were washed with PBS and fixed in 4% paraformaldehyde (dissolved in PBS) for 15 minutes. Then, it was washed with PBS, and reacted for 1 hour at room temperature in a blocking reagent in which normal serum of an animal in which a secondary antibody was prepared and 0.1% Triton X-100 were mixed with PBS (blocking reaction). Subsequently, it was washed with PBS, and antigen-antibody reaction was performed overnight at 4 ° C. using various antibodies (anti-MAP2 antibody, anti-GFAP antibody, anti-Nestin antibody) as primary antibodies (primary antibody reaction). This was washed with PBS, and then an antigen-antibody reaction was performed at room temperature for 1 hour using a secondary antibody labeled with a fluorescent substance (secondary antibody reaction). Subsequently, the fluorescently labeled sample was washed with PBS and 0.1 M Phosphate buffer (PB), and then an image was obtained using an epi-illuminated inverted fluorescence microscope (KEYENCE: BZ-8100).
MAP2 is a neuronal marker protein, GFAP is an astroglial marker protein, and Nestin is a neural progenitor cell marker protein.

(Measurement results of proliferation ability in neural progenitor cells)
Nerve mass formation, one of the characteristics of neural progenitor cells under culture conditions, was observed from day 4 of culture. Furthermore, the surface area of this nerve mass increased with the passage of days and was observed until the 12th day of culture.
From the above, it was confirmed that the neural progenitor cells used in this example had a proliferative ability.

(Results of immune cell staining of differentiation-induced neural progenitor cells)
On day 12 of culture, many Nestin positive cells were observed, but MAP2 and GFAP positive cells were not observed. However, Nestin positive cells were not observed in the cells on the 18th day of culture, whereas many MAP2 and GFAP positive cells were observed (see FIG. 1).
Based on the above, it was confirmed that the neural progenitor cells used in this example exhibited not only proliferative ability but also pluripotency.

(Confirmation that nAChRs are present in neural progenitor cells)
It was confirmed whether nAChRs (nicotinic acetylcholine receptors) are present in neural progenitor cells. Details are as follows.

(Extraction of total RNA)
The cells cultured in paragraph “0026” were collected, and then centrifuged at 400 × g for 5 minutes. After removing Medium, 500 μL of RNA extraction reagent ISOGEN (NIPPON GENE Co., Tokyo) was added to the obtained cells and suspended. Next, 100 μL of Chloroform was added to the resulting suspension, and the mixture was allowed to react at room temperature for 3 minutes after inversion stirring. Thereafter, centrifugation was performed at 4 ° C. and 12,000 × g for 15 minutes, and 250 μL of the supernatant was recovered. 250 μL of Isopropanol was added to the collected solution, and the mixture was allowed to react at room temperature for 10 minutes after inversion stirring. Subsequently, the mixture was centrifuged at 4 ° C., 12,000 × g for 10 minutes, the supernatant was removed, 70% Ethanol was added, and the mixture was centrifuged again at 4 ° C., 12,000 × g for 5 minutes. The precipitate (total RNA) obtained after complete removal of Ethanol after centrifugation was dissolved in Dietary pyrocarbonate (DEPC) -treated water and stored at −80 ° C. until just before use.

(Preparation of cDNA)
Add 1 μL of 50 μM Oligo (dT) ₁₈ primer (Sigma) and 3.6 μL of 2.5 mM dNTP mixture (TaKaRa) to 1 μg of total RNA obtained in paragraph “0033”, and add DEPC-treated water to a total volume of 18 μL. The mixture was reacted at 65 ° C. for 5 minutes and then rapidly cooled on ice for 2 minutes. Next, add 5μ First-strand buffer (Invitorogen) 6μL, 0.1M DTT (Invitrogen) 3μL, 200 U / μL M-MLV reverse transcriptase (Invitrogen) 0.5μL, and add DEPC treated water to a total volume of 30μL. The mixture was further reacted at 37 ° C. for 50 minutes. Then, it was made to react at 70 degreeC for 15 minutes in order to stop reaction. The obtained cDNA was stored at −20 ° C. until just before use.

(PCR operation)
10 × Buffer (TaKaRa) {100 mM Tris-HCl (pH 8.3) containing 500 mM KCl and 15 mM MgCl ₂ } 2.5 μL, dNTP mixture (Each 2.5 mM) 2 μL, cDNA obtained in paragraph “0034” 1 μL, 10 μM Sense primer 1 μL, Antisense primer 1 μL, 5 U / μL Recombinant Taq DNA polymerase (TaKaRa) 0.125 μL was added, and the total volume was adjusted to 25 μL with sterilized purified water, followed by PCR reaction. The obtained PCR product was subjected to electrophoresis on a 1.5% (w / v) agarose gel containing 0.005% (v / v) Ethidium bromide solution, and UV detection was performed. One cycle of the PCR reaction was constituted by denaturation 94 ° C., 1 minute, annealing 1 minute, extension reaction 72 ° C., 1 minute, and the annealing reaction temperature was set to the optimum temperature of various Primers. The number of cycles was 40 cycles. PCR used the primers shown in Table 1 below.
That is, nAChRs mRNA expression analysis was performed by RT-PCR using cells immediately after collection (0 DIV) and on the 12th day of culture (12 DIV).

(Confirmation result that nAChRs are present in neural progenitor cells)
Expressions of α6 and β3 subunits were not observed in the samples immediately after collection, but α2 to α5, α7, α9, β2 and β4 subunits were observed. In the sample on day 12 of culture, expression of β3 subunit was not observed, but expression of α2-7, α9, β2, and β4 subunits was observed (see: FIG. 2).
This confirmed that nAChRs were expressed in neural progenitor cells.

(Confirmation of proliferation ability of neural progenitor cells by nicotine administration)
It was confirmed whether nicotine administration affects the proliferative ability of neural progenitor cells. Details are as follows.

(Measurement of proliferation ability of neural progenitor cells by nicotine administration)
The neural progenitor cells cultured in paragraph "0026" are continuously exposed to nicotine at a concentration range of 1-1000 μM for 12 days from the beginning of the culture, and cell proliferation ability is determined according to the proliferation ability measurement method in paragraph "0027". As an index, the nerve mass area was measured every 2 days from the 4th day to the 10th day of culture.

(Measurement result of proliferation ability of neural progenitor cells by nicotine administration)
When cells were cultured with nicotine added to the medium, a significant decrease in the nerve mass area was observed depending on the nicotine concentration from the 6th day to the 10th day of culture (see: FIG. 3).
From the above, it was confirmed that the neural progenitor cells administered with nicotine had suppressed self-proliferation ability. In other words, nicotine has an effect of suppressing the ability of neural progenitor cells to proliferate.

(Confirmation of inhibition of proliferation of neural progenitor cells by administration of nicotine and confirmation of cell death induction)
In Example 2, nicotine was confirmed to suppress the self-proliferating ability of neural progenitor cells. In this example, an MTT test for confirming in detail the proliferation ability of the cells by nicotine and an LDH test for confirming the cell death of the cells were performed. Details are as follows.

(Measurement of proliferation ability of neural progenitor cells by nicotine administration)
The neural progenitor cells cultured in paragraph "0026" are continuously exposed for 12 days at a concentration range of 1-1000 μM of nicotine from the beginning of the culture, and further seeded on a dish coated with Poly-L-lysine, and CO ₂ The cells were allowed to stand in the incubator for 1 hour to adhere the cells on the dish. After cell adhesion, Medium was removed, and the cells were washed once with PBS special, and then 0.5 mg / mL MTT (dissolved in PBS special) was added and reacted for 1 hour in a CO ₂ incubator. Subsequently, the same amount of 0.04 N HCl / isopropanol as 0.5 mg / mL MTT solution was added to lyse the cells, and the lysate was dispersed with a sonicator and stirred until the dye was uniform. The change in absorbance at 550 nm was measured with a microplate reader (Molecular Devices: THERMOmax Kinetic Microplate Reader) using the amount of formazan in the lysate produced by MTT reduction as an indicator of mitochondrial activity.

(Measurement of cell death induction of neural progenitor cells by nicotine administration)
The neural progenitor cells cultured in paragraph “0026” were continuously exposed to nicotine at a concentration range of 1-1000 μM for 12 days from the beginning of the culture, and the culture medium of the cells was collected. A 0.1 mM NADH solution (dissolved in 0.1 M KH ₂ PO ₄ , stored at room temperature, prepared at the time of use) and a serum-free medium were placed in a cuvette, and the absorptiometer was stabilized at an OD of 340 nm for an appropriate time. After stabilization, the culture solution collected in each culture day was reacted with a 0.1 mM NADH solution. Na pyruvate was added 20 seconds after the start of measurement, and measurement was performed for 1 minute. The above operation was performed at 37 ° C., and the change in absorbance decrease rate before and after addition of Na pyruvate was calculated.

(Measurement result of proliferation ability of neural progenitor cells by nicotine administration)
When cells were cultured with nicotine added to the medium, a significant decrease in mitochondrial activity was confirmed depending on the concentration of nicotine (see: FIG. 4).

(Measurement results of cell death induction of neural progenitor cells by nicotine administration)
There was no significant difference in the LDH activity of the nicotine-exposed group at any concentration compared to the LDH activity of the control group (see FIG. 5). That is, it was confirmed that nicotine did not induce cell death of neural progenitor cells.

  From the above two measurement results, it was found that nicotine suppresses only proliferation ability without inducing cell death. The theanine of Patent Document 2 has the promotion of proliferation ability and the promotion of differentiation of nerve cells, whereas the nicotine of the present invention has the ability of inhibiting proliferation of neural progenitor cells and the ability of differentiation and proliferation into neurons.

(Confirmation of the effect of nAChRs antagonists on the proliferation ability of neural progenitor cells)
It was confirmed whether administration of the nAChRs antagonist Mecamylamine (mecamylamine: nicotine receptor blocker) affects the proliferative ability of neural progenitor cells. Details are as follows.

(Measurement of proliferation ability of neural progenitor cells by administration of nAChRs antagonist)
The neural progenitor cells cultured in paragraph “0026” are cultured for 12 days in the presence of 10 μM nicotine and / or 10 μM Mecamylamine from the beginning of the culture, and as an indicator of cell proliferation ability according to the proliferation ability measurement method in paragraph “0027” The nerve mass area was measured every 2 days from the 4th day to the 10th day of culture.

(Measurement results of proliferation ability of neural progenitor cells by administration of nAChRs antagonist)
Reduction of nerve mass area by exposure to 10 μM nicotine was significantly suppressed by simultaneous exposure to 10 μM nicotine and 10 μM Mecamylamine. In addition, a significant increase in the area of the nerve mass was confirmed from the 8th day to the 12th day of culture by exposure to 10 μM Mecamylamine alone (see FIG. 6).
As described above, from the results of Example 2 and the results of this example, it was found that suppression of proliferation ability of neural progenitor cells by administration of nicotine is a mechanism mediated by nAChRs.

(Confirmation of the effect of nicotine administration on the differentiation potential of neural progenitor cells)
It was confirmed whether nicotine administration affects the differentiation ability of neural progenitor cells. Details are as follows.

(Measurement of percentage of spontaneously differentiated neural progenitor cells)
The neural progenitor cells cultured in the presence of 10 μM nicotine and / or 10 μM Mecamylamine for 12 days from the beginning of the culture (see paragraph “0026”) in the differentiation induction method of paragraph “0028” in a differentiation medium that does not contain a differentiation-inducing factor. Spontaneous differentiation was induced. Then, the neural progenitor cells induced to spontaneously differentiate were exposed to a solution containing 10 μg / ml Hoechst and reacted in a CO ₂ incubator for 10 minutes. Furthermore, according to the cell staining method in paragraph “0029”, immunostaining is performed using a primary antibody against MAP2 and GFAP, and the number of Hoechst positive cells is defined as the total number of cells, and the number of MAP2 and GFAP positive cells relative to the number of cells. The percentage of was measured.

(Measurement of the proportion of neural progenitor cells differentiated in the presence of differentiation-inducing factors)
In the differentiation induction method of paragraph “0028”, a neural progenitor cell (see paragraph “0026”) cultured for 12 days in the presence of 10 μM nicotine and / or 10 μM Mecamylamine from the beginning of the culture is differentiated with a differentiation inducer (100 mg / ml ATRA or Differentiation was induced with a differentiation medium containing 20 ng / ml CNTF). The differentiation-induced neural progenitor cells were exposed to a solution containing 10 μg / ml Hoechst and reacted in a CO ₂ incubator for 10 minutes. Furthermore, in accordance with the cell staining method of paragraph “0029”, immunocell staining was performed using primary antibodies against MAP2 and GFAP, and the ratio of the number of MAP2 and GFAP positive cells to the number of Hoechst positive cells was measured.

(Measurement result of each cell ratio of spontaneously differentiated neural progenitor cells)
In the 10 μM nicotine-exposed group, a significant increase in the number of MAP2-positive cells and a significant decrease in the number of GFAP-positive cells were confirmed compared to the control group. The increase in the number of MAP2-positive cells by 10 μM nicotine was significantly suppressed by simultaneous exposure to 10 μM Mecamylamine. Furthermore, the decrease in the number of GFAP positive cells by 10 μM nicotine was significantly antagonized by simultaneous exposure to 10 μM Mecamylamine (see FIG. 7).
Based on the above, nicotine has the effect of promoting differentiation of spontaneously differentiated neural progenitor cells into neurons and the effect of inhibiting differentiation into astroglial cells, and that both effects are mediated by nAChR according to the results of Example 2. confirmed.

(Measurement result of each cell ratio of neural progenitor cells differentiated in the presence of differentiation-inducing factor)
In the presence of both ATRA and CNTF, a significant increase in the number of MAP2-positive cells and a significant decrease in the number of GFAP-positive cells were confirmed in the 10 μM nicotine-exposed group compared to the control group (see: FIG. 8).
As described above, nicotine has an effect of promoting differentiation of neural progenitor cells differentiated in the presence of a differentiation-inducing factor into nerve cells and an effect of suppressing differentiation into astroglial cells, and both effects are nAChR based on the results of Example 2. Confirmed through.

(Confirmation of the effect of nicotine using mouse-derived neural progenitor cells)
Similar to rat-derived neural progenitor cells, nicotine was confirmed to have an effect of promoting differentiation of mouse-derived neural progenitor cells into neurons and an effect of inhibiting differentiation into astroglial cells. Furthermore, it was confirmed whether nAChR is present in mouse-derived neural progenitor cells.

(Measuring method)
In the same manner as in the above Examples, the proliferation ability of neural progenitor cells and the ratio of each cell of spontaneously differentiated neural progenitor cells were measured. Further, as in the above Examples, nAChRs mRNA expression analysis was performed by RT-PCR using cells immediately after collection (0 DIV) and cells on day 10 of culture (10 DIV).

(Measurement result)
It was confirmed that nicotine has an effect of promoting differentiation of neural progenitor cells derived from mice into nerve cells and an effect of suppressing differentiation into astroglia cells. Furthermore, it was confirmed that nAChR was present in mouse-derived neural progenitor cells.

(General)
From the results of the above examples, it was confirmed that the composition for promoting neurogenesis containing nicotine of the present invention has an effect of promoting differentiation into neurons and an effect of inhibiting differentiation into astroglia cells. Furthermore, it was confirmed that nicotine suppresses only proliferation ability without inducing cell death. In addition, these effects were confirmed to be a mechanism mediated by nAChRs.

  It is possible to provide a composition for promoting neurogenesis, which has an effect of promoting differentiation into nerve cells and an effect of inhibiting differentiation into astroglial cells. Furthermore, the composition can be used as a therapeutic agent for diseases related to neurodegenerative diseases such as Alzheimer's disease, stroke, Parkinson's disease, and mental diseases such as depression, schizophrenia, and autism.

Claims (5)

A composition for promoting neurogenesis , which comprises nicotine as an active ingredient and is added during culture of neural progenitor cells to enhance differentiation of neural progenitor cells into neural cells . The composition for promoting neurogenesis according to claim 1, wherein the composition is capable of suppressing differentiation into astroglial cells. 3. The composition for promoting neurogenesis according to claim 1 or 2 , which does not induce cell death of neural progenitor cells. The composition for promoting neuronal cell neogenesis according to any one of claims 1 to 3 , which suppresses the proliferation ability of neural progenitor cells. A method for differentiating neural cells from neural progenitor cells, comprising culturing neural progenitor cells in the presence of nicotine.