JPH104999A - Evaluation of antidementia agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish the evaluation of the suppressing effect of a testing substance on neurocyte toxicity in the presence of β-amyloid protein in high sensitivity by applying a specific substance to a neurocyte in the presence of the testing substance and β-amyloid protein and the establish a method for the search and evaluation of a drug effect on dementia such as Altzheimer's disease. SOLUTION: A substance selected from astroglia cell, cultured liquid of astroglia cell, extract of astroglia cell and product of astroglia cell is applied to a neurocyte in the presence of a testing substance and β-amyloid protein. The presence of the astroglia cell, etc., increases the cytotoxic effect of β-amyloid protein on neurocyte to evaluate with a small amount of testing substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-dementia agent.

[0002]

BACKGROUND OF THE INVENTION Alzheimer's disease is a progressive dementia that occurs after neurodegeneration or neuronal cell death occurs.
Although there are many unclear points in the mechanism of its onset and onset, emphasis has been placed on the relationship with the multiple formation of senile plaques mainly formed by the deposition of β-amyloid protein in the brain parenchyma.

[0003] However, the mechanism by which neuronal degeneration occurs after the deposition of β-amyloid protein is currently unknown, and no fundamental treatment for Alzheimer's disease has yet been established.

Have reported that β-amyloid protein causes neuronal cell death in primary culture of rat-derived nerve cells alone, suggesting that β-amyloid protein may directly damage nerve cells. (Science, Vol. 250, pp. 279-282, 19
90 years).

[0005] In response to this, the mechanism of direct neuronal cell injury by β-amyloid protein has been studied by primary culture of neurons alone, but no clear conclusion has been obtained at present. The present inventor pays attention to and examines the effect of glial cells on neurons of β-amyloid protein.

[0006]

SUMMARY OF THE INVENTION In order to establish a method for searching or evaluating a dementia ameliorating agent, β
It is an object of the present invention to provide an excellent method for evaluating the inhibitory effect of a test substance on neurotoxicity caused in the presence of amyloid protein.

[0007]

The present invention provides the following (1).
To (5). (1) In the presence of a test substance and β-amyloid protein, one of astroglial cells, a culture solution of astroglial cells, a cell extract of astroglial cells, or a product of astroglial cells is interposed in neurons in the presence of a test substance and β-amyloid protein. The present invention relates to a method for evaluating neuronal damage of a test substance, and (2) neuronal damage of the test substance by mixing and culturing neurons and astroglial cells in the presence of the test substance and β-amyloid protein. (3) The present invention relates to (3) a method for evaluating a preventive or therapeutic drug for dementia using the evaluation method according to any of (1) or (2), The present invention relates to a method for evaluating a preventive or therapeutic agent for Alzheimer's disease using the evaluation method according to any one of (1) and (2), and (5) the method described in (1) or (2) above. The present invention relates to a substance that suppresses neurotoxicity obtained by using the evaluation method described in any of the above, and (6) relates to a preventive or therapeutic drug for dementia obtained by using the method described in (3) above. And (7) a prophylactic or therapeutic agent for Alzheimer's disease obtained by using the method described in (4) above.

In order to find a drug effective for dementia such as Alzheimer's disease, it is necessary to establish a method for evaluating an effective drug. Among such evaluation methods, it is desired to establish an evaluation method in which a small amount of a compound is used for the first evaluation, but at present, a reliable evaluation method generally accepted has not been determined. Similarly, no reliable evaluation method has been determined for animal models or in vitro models of human Alzheimer's disease.

The present inventors have studied the method for evaluating neuronal toxicity in the brain, and as a result, have found an evaluation method capable of detecting cell death of nerve cells with high sensitivity, thereby completing the present invention. That is, the present inventors have completed an excellent evaluation method of the present invention by increasing the cytotoxicity of β-amyloid protein to nerve cells by interposing certain glial cells.

[0010] The present invention provides a method for producing neurons in the presence of a test substance and β-amyloid protein by selecting from astroglial cells, culture media of astroglial cells, cell extracts of astroglial cells, products of astroglial cells, and the like. The present invention relates to a method for evaluating neuronal damage of a test substance by interposing one or a combination of two or more selected substances.

Here, "intervening" means, for example, a mixed culture of nerve cells and astroglial cells, that is, a mixed culture in which both cells can be brought into direct contact with each other. A state in which each of the cells is separated by adjacent different chambers (partitions),
In the case of culture in a state where cells cannot move but humoral substances or protein factors secreted by cells can pass therethrough, or selected from cell cultures of astroglial cells, cell extracts of the cells, or products of the cells Or the case of adding or treating one or more of them.

The phrase "in the presence of β-amyloid protein" preferably refers to the case where β-amyloid protein is added.
In addition, in the case of mixed culture with cells that produce a sufficient amount of β-amyloid protein, in the case of culturing in a medium containing the cell itself that has produced a sufficient amount of β-amyloid protein or a treatment solution of the produced cell, or The meaning includes the case of treating with a liquid (solution) containing β-amyloid protein.

In the present invention, the term "neural cells" or "altroglia cells" refers to primary cultured cells isolated from mammalian brain tissue, cells subcultured from the primary cultured cells,
Primary cells isolated from the transgenic mouse brain,
It includes passaged cells and cell lines. In the case of neurons, in particular, a mammalian fetal or neonatal brain is used, and in the isolation of astroglial cells, adult animals are also included in addition to mammalian neonates and fetuses.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Preparation of a primary culture of nerve cells : The mammal for obtaining the nerve cells used in the present invention may be a mammal. For example, a rat or the like which is a kind of mammal and is usually used as an experimental animal can be used. By removing the brain tissue of such a mammalian animal and using it as a material for primary cultured cells, primary cultured cells can be completed.
If such primary culture cells can be obtained, a mixed culture system of a neural cell culture system and a microglial cell, or a mixed culture system of a nerve cell and an astroglial cell can be prepared.

Preparation of a single cell culture system for nerve cells : The brain of a mammalian fetus is removed, the meninges are removed, the cerebral cortex and the hippocampus are separated, and the separated brain tissue is subjected to cell digestive juice and / or DNase
For example, a cell suspension can be obtained by treating with a nuclease such as DNase 1 and dispersing the mixture by pipetting or the like.

Astroglial cells and microglial cells
Separation and culturing of cells: In the same manner as in the preparation of a single neuronal cell culture system, a fetal or mammalian brain or fetal brain tissue of a mammal is dispersed and a cell suspension is cultured for a certain period from a culture vessel such as a flask. After the cells detached by means such as towels are adhered to the incubator again, washing and culturing are repeated, and the cells obtained by separating only the adhered cells can be used as microglia cultured cells.

On the other hand, cells obtained by washing the cells remaining without being peeled off from the incubator prepared as described above can be used as astroglial cultured cells.

Preparation of mixed culture system with nerve cells: For preparation of a mixed culture system of astroglial cells and nerve cells,
A cell suspension obtained by stripping the astroglial culture cells obtained as described above with a trypsin solution was prepared, and the cells cultured until reaching a saturated cell density were added to the nerves derived from fetal brain used in the nerve cell alone culture system. A mixed culture system can be prepared by seeding and culturing the cell suspension in the same manner.

As for the preparation of a mixed culture system of microglia cells and nerve cells, it is preferable to further inoculate microglia cells previously separated on neurons cultured for a certain period of time to prepare a mixed culture system. it can.

Furthermore, for the preparation of a mixed culture system of established astroglial cells and neurons, an astroglial cell line isolated and established from the brain of a transgenic mouse into which a temperature-sensitive oncogene has been introduced can be used. As the transgenic mouse, a transgenic mouse expressing the SV40 temperature-sensitive T antigen by the SV40 self-promoter (Yanai, Exp. Cell Res. 197: 50-5
6, 1991) can be used.

Nerve cells in various nerve cell culture systems
Of the purity of glial and glial cells : It is necessary to test the purity of nerve cells, astroglial cells, and microglial cells contained in the nerve cell culture system. The method can be performed as follows. First, after fixation with ethanol or the like, the microtubule-associated protein 2 (MAP, a neuron-specific protein)
2) Indirect immunostaining for glial fibrillary acidic protein (GFAP), a protein specific to astroglial cells, and Fc receptor (FcR), a protein specific to microglial cells, for each culture system Can be used to calculate the number of positive cells for immunostaining and determine the average density.

In the immunostaining indirect method, a monoclonal antibody derived from a mouse specific to each protein is used as a primary antibody, a horseradish peroxidase-labeled anti-mouse Ig antibody is used as a secondary antibody, and DAB (diaminobenzidine) is used at the end of the reaction. And nuclear staining can be performed with a methyl green solution or the like.

Neurocytotoxicity test : For each of these culture systems, an appropriate concentration of β-protein solution was added when the nerve cells reached a specified cell density. By staining, the number of surviving nerve cells can be calculated with a light microscope. The immunostaining used herein refers to a so-called MAP2 protein (Microtuble associated protein) which is a protein specific to living neurons.
This refers to a technique for performing immunostaining using an antibody against protein 2).

Similarly, the number of glial cells mixed in each culture system is determined by the number of glial fibrillary acidic protein (G) which is a protein specific to astroglial cells and microglial cells, respectively.
FAP), an immunostaining using an antibody against the Fc receptor. By using these immunostainings, counting under a light microscope and calculating the number of surviving neurons, a neurotoxicity test can be performed. it can.

The present invention also relates to a substance selected by evaluating a test substance by mixing and culturing astroglial cells and nerve cells in the presence of β-amyloid protein. The substance selected by such an evaluation method is a substance that suppresses a neuropathy effect, and the substance that inhibits or suppresses the damage to nerve cells is a prophylactic or therapeutic drug for anti-dementia, or Alzheimer's disease. It is useful as a prophylactic or therapeutic agent for diseases.

Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited to these examples.

[0027]

【Example】

[Example 1] (1) Preparation of primary cultured neurons : Various neural cells were separated from the fetal brain of SLC rats (SLC: Wistar strain, Nippon SLC) 18 days after gestation, and a neural cell culture system was prepared. A mixed culture of glial and neuronal cells was also prepared. The medium used was DMEM (Dulbecco's modified Eagle medium, manufactured by Nissui Pharmaceutical Co., Ltd.), L-glutamine 0.0584 g / l, glucose 6 g / l, penicillin G potassium 20,000 units / l, streptomycin sulfate 0.05 g / l And Funginson 250 μg / l, and adjusted the pH to about 7.4 with sodium bicarbonate as a basal medium. To maintain cells, add fetal bovine serum (F
CS) and non-immobilized horse serum (HS, manufactured by GIBCO) were used in a serum-supplemented medium supplemented with 10% each. At the time of the experiment, serum-free medium was prepared by adding a B27 additive factor (Gibco) to the basal medium at a 1/50 dose. Medium was used. Plates for seeding cells were coated in advance with polyethyleneimine. For treatment with polyethyleneimine, polyethyleneimine diluted 500-fold with 0.15 M borate buffer was added to the plate and incubated overnight.
Incubate at ℃ C, then plate
Washing was performed twice with (-) and then twice with DMEM.

(2) Preparation of single cultured neurons : A fetus was excised from a pregnant rat, the brain was further removed, the meninges were removed under a stereoscopic microscope, and the cerebral cortex and hippocampus were separated. This separated brain tissue was used for cell digestion (papain 90 U, DL-cysteine 2 mg, bovine serum albumin 2 mg, glucose 50
(10 mg of PBS (-) containing 1 mg of DNase 1) and then removing the supernatant, followed by pipetting with 10 ml of DMEM supplemented with 1 mg of DNase 1 instead. Cell dispersion was performed.
The dispersed cells were resuspended at a concentration of 10 ⁵ cells / ml in a medium supplemented with 10% FCS and HS (horse serum), and 100 μl / well (well, hereinafter referred to) in a 16-well slide chamber (manufactured by Nunk). ) And cultured in a carbon dioxide gas incubator (5% carbon dioxide, 95% air, 37 ° C). In order to suppress glial cell contamination after 2 days of culture, 1 μM of cytosine arabinoside (manufactured by Sigma) was added to the medium, and the next day, 3 days after culture, the cells were used in the experiment.

(3) Astroglial cells and microglia
Separation and culture of cells: In the same manner as in the preparation of a single neuron culture system, a rat fetal brain was dispersed to prepare a cell suspension, which was placed in a culture flask (Falcon) for 1 cell per cell. A cell suspension from the fetal brain of each animal was inoculated and cultured in a serum-supplemented medium for 2 weeks. Thereafter, the culture flask is
After shaking by hand for a minute, transfer the detached cells to a Petri dish (manufactured by FALCON) together with the medium, incubate for 30 minutes in a carbon dioxide incubator, wash twice with serum-supplemented medium, and remove only the cells attached to the Petri dish. This was used as a microglial cell culture system. On the other hand, after the above culture flask was shaken vigorously by hand for about 3 minutes, the cells remaining in the Petri dish without peeling were further removed by 5 mM
Washed three times with TA-containing PBS (-) was used as astroglial cell culture.

(4) Mixing neurons with astroglial cells
Preparation of a combined culture system :
The cells were detached with PBS (-) containing 1% trypsin, and the centrifuged precipitate (2000 rpm, 10 minutes) was suspended in a medium supplemented with serum, seeded in a 16-well slide chamber, and cultured until a saturated cell density was reached. Into the slide chamber previously laid with astroglial cells, the nerve cell suspension derived from the fetal rat brain used in the nerve cell alone culture system was seeded in the same manner, and cultured in a serum-containing medium for 3 days. It was used for.

(5) Mixed culture of microglial cells and nerve cells
Preparation of nutrient : According to the method described in the above section "Separation and culture of astroglial cells and microglial cells", cells attached to the dish are peeled off by shaking or the like to prepare a cell suspension, and seeded on cultured neurons. As a result, a mixed culture of microglial cells and nerve cells was prepared.

(6) Mixing of established astroglia and nerve cells
Preparation of culture : temperature-sensitive oncogene-introduced transgenic mouse (transgenic mouse expressing SV40 temperature-sensitive T antigen by SV40's self-promoter; created by Otori et al. (1991); Yanai, Exp. Cell Res. 19
7: 50-56, 1991), astroglial cell lines isolated and established from the brain were seeded in advance in a 16-well slide chamber,
Culture was performed until a saturated cell density was reached. Into the slide chamber on which the established astroglial cells were spread, the nerve cell suspension derived from the rat fetal brain used in the nerve cell alone culture system was seeded in the same manner, and cultured in a serum-added medium for 3 days. Used for experiments.

(7) Nerve cells in various nerve cell culture systems
Assay of purity of glial cells and glial cells : The purity of nerve cells, astroglial cells, and microglial cells contained in various nerve cell culture systems prepared in a 16-well slide chamber was assayed. In the assay, after fixing the chamber with 95% ethanol (-20 ℃, 15min), the microtubule-associated protein 2 (MAP2), a neuron-specific protein (Neuroscience 4: 151-160,1)
984), immunostaining for glial fibrillary acidic protein (GFAP), a protein specific for astroglial cells, and Fc receptor (FcR), a protein specific for microglial cells, was performed by indirect immunostaining for each culture system. The number of positive cells for immunostaining for the above-mentioned protein was determined at 200 times magnification using a microscope equipped with an eyepiece with a grid pattern engraved by selecting any three visual fields in one well. Was calculated, and the average density was calculated. The indirect immunostaining method uses a mouse-derived monoclonal antibody specific to each protein as the primary antibody, horseradish peroxidase-labeled anti-mouse 1g antibody for the secondary antibody, and DAB at the end of the reaction.
(Diaminobenzidine, manufactured by Sigma), and nuclear staining was performed with 1% methyl green solution (Keiji Shimizu, Manual of Neurobiochemistry (Yodosha), 1990, 253-263).
page).

[Example 2] Neurotoxicity test : The following four culture systems were constructed using these cells and maintained in a 16-well slide chamber (Nunc, manufactured by Nunc).

The plates of the first group were separated according to a conventional method, and cultured neurons were seeded at 5 × 10 ⁴ cells / well. This group is a cell line consisting of a cell group in which most cells are composed of nerve cells and some astroglial cells are mixed.

The plate of the second group is a cell line obtained by further adding ⁴ × 10 ⁴ cells / well to the first group of microglia cells previously separated, and is a mixed culture system of glial cells and neurons, mainly microglia cells. It is.

The third group is a mixed culture system of glial cells, mainly astroglial cells. This group was pre-seeded with cells derived from Wistar rats, and was set to a saturated cell density (confl.
This is a culture system in which nerve cells are seeded in a chamber cultivated to be uent). This is a mixed culture system of glial cells and nerve cells, mainly astroglial cells.

The plate of the fourth group is a culture system in which the established astroglial cells (Clone No. 3) were seeded in advance and the nerve cells were seeded in a chamber in which the cells were cultured to a saturated cell density. This is a mixed culture system that does not contain cells other than astroglial cells and neurons.

In each of the first to fourth groups, β-protein (25-35, Sigma Co., Ltd.) previously dissolved at a concentration of 1 mM in pure water at the time when the neurons were cultured for 3 days. 2) solution was added to a final concentration of 25 μM, and two days after the addition, the cells were collected and fixed. Then, MAP2 (Microtuble assoc), a protein specific to living neurons,
The number of surviving neurons was calculated under light microscopy by immunostaining for iated protein), and the ratio of surviving neurons in the administration group to the non-beta protein administration group (neural cell survival rate) was determined in each culture system. In addition, the number of glial cells mixed in each culture system is determined by glial fibrillary acidic protein (hereinafter referred to as G), which is a protein specific to astroglial cells and microglial cells.
Glial fibrillary acidic protein)
Immunostaining for Fc receptors was performed and calculated under light microscopy.

Table 1 shows the glial cell tissues contained in each cell line constructed this time. By using the established astroglial cells, it was possible to construct a mixed culture system of astroglial cells and neurons in which the microglial cells were almost excluded in the fourth group. The administration of β protein resulted in a significant decrease or a tendency to decrease in the number of surviving neurons in all groups, but the degree of the decrease in the second group was not significantly different from that in the first group. And in group 4 there was a markedly stronger reduction in the percentage of viable neurons compared to group 1 (Table 2). In each group, no cytotoxicity to astroglial cells or microglial cells due to β protein was observed.

In this test, the possibility of astroglial cells enhancing the neurotoxicity of β-protein could be directly shown for the first time by the examination using a mixed culture system of neurons and various glial cells. In this test, in the first group, in which glial cells were scarcely mixed and almost consisted of nerve cells, a slight decrease in the rate of surviving nerve cells was observed by the addition of β protein (Table 2).

In microglial cells, production of nitric oxide (hereinafter referred to as NO) and various cytokines is induced by β protein (BBRC: 199: 984-991, 1994: Brain R).
esearch, 692: 207-214, 1995: Brain Research 569: 14
1-145, 1992). However, the second group, which is a culture system in which microglia cells were mixed with neurons, did not show a significant difference compared to the first group, and the results showing the enhancement of neuroglial toxicity of β protein mediated by microglia were Not obtained (Table 2).

[0043]

[Table 1]

In a mixed culture of glial cells and nerve cells, mainly astroglial cells (Group 3), and a mixed culture of established astroglial cells and nerve cells (Group 4), β
The remarkable enhancement of neuronal cytotoxicity due to the protein shows the effect of enhancing neuronal cytotoxicity by astroglial cells, which indicates that astroglial cells are involved in the pathogenesis of Alzheimer's disease. Suggested to be involved.

[0045]

[Table 2]

[0046]

The method of the present invention is useful as a method for evaluating the inhibitory or inhibitory effect of a test substance on the cytotoxicity of nerve cells by β-amyloid protein.

Such an evaluation method is useful as a method for evaluating or searching for a therapeutic drug for Alzheimer's disease. Also,
By evaluating various compounds and other substances using the method of the present invention, the selected substances are identified as anti-Alzheimer's
It is useful as a drug for treating diseases or an anti-dementia drug.

Claims (7)

[Claims] Claims: 1. An astroglial cell, a culture solution of astroglial cells, a cell extract of astroglial cells, or a product of astroglial cells, which is added to a neuron in the presence of a test substance and β-amyloid protein. A method for evaluating neuronal damage of a test substance by intervening. 2. In the presence of a test substance and β-amyloid protein,
A method for evaluating neuronal damage of a test substance by co-culturing neurons and astroglial cells. 3. A method for evaluating a preventive or therapeutic drug for dementia using the evaluation method according to claim 1 or 2. 4. A method for evaluating a preventive or therapeutic agent for Alzheimer's disease using the evaluation method according to claim 1 or 2. 5. A substance which inhibits neurotoxicity obtained by using the evaluation method according to claim 1 or 2. 6. A prophylactic or therapeutic agent for dementia obtained by using the method according to claim 3. 7. A preventive or therapeutic agent for Alzheimer's disease obtained by using the method according to claim 4.