JP6128677B2 - Method for determining diseases associated with nerve cell migration disorder and use thereof - Google Patents

Description

The present invention relates to a method for determining a disease associated with nerve cell migration disorder.
The present invention also relates to a method for evaluating the influence of a test substance on the migration activity of nerve cells.

  Synovial dysfunction is one of the congenital genetic diseases of the central nervous system, which is accompanied by a lack of gyrus (cerebral cortical wrinkles) and abnormal layer structure of nerve cells. Yes not yet established. In Japan, it affects 1 in 15,000 newborns. Classic type I spondylosis is thought to be caused by about 60% of the heterozygous mutation in the LIS1 gene present in chromosome 17, and the malfunction of LIS1 due to the mutation causes abnormal migration of neurons. Yes.

  In Non-Patent Document 1, the present inventor restored the expression level of LIS1 protein to the normal level in cells of a spondylosis model (LIS1 gene heterodeficient) mouse by an in vitro cell migration assay using a calpain inhibitor. While improving encephalopathy (like) symptoms at the individual level, it shows almost no side effects on wild-type mice. Based on this, a cell migration assay as described in Non-Patent Document 1 is used for determination of diseases based on the degree of migration of nerve cells and measurement of the effect on drug migration ability, thereby establishing a method for treating spondylosis. Expectation to do is increasing.

Yamada M. et al., Nature Medicine, 2009, vol.15, p.1202-1208

  However, in the conventional nerve cell migration assay, it was difficult to form a uniform aggregate while maintaining the migration activity of nerve cells. Since neuronal cell migration assays usually start from an aggregate state, aggregate formation is an important step in the assay. In addition, in the migration assay, the migration ability of the cell is generally evaluated based on the degree of migration of the cell, but in the conventional assay, the nerve cell known to migrate and the nerve cell not known to migrate are compared. The difference in the degree of migration between the two is not sufficiently large, which affects the reliability of the evaluation results. Therefore, the conventional neuronal migration assay is sufficiently satisfactory in the reliability and reproducibility of the results to be used for disease determination based on the degree of neuronal migration and measurement of the effect on drug migration ability. It was not a thing.

  As a result of intensive studies, the present inventor has found that the above-mentioned problems can be solved by incubating the aggregate of nerve cells in a buffer solution containing purified albumin, and has completed the present invention.

That is, according to the present invention,
Acquiring nerve cells from brain tissue collected from a living body;
A step of forming an aggregate of the obtained nerve cells;
Incubating the formed agglomerates in a buffer solution containing purified albumin to migrate, and measuring the degree of migration of nerve cells,
Determining whether or not the living body is a disease accompanied by a nerve cell migration disorder based on the degree of migration,
A method for determining a disease associated with a neuronal migration disorder is provided.

Moreover, according to the present invention,
Obtaining nerve cells from brain tissue collected from a living body of a disease associated with nerve cell migration disorder;
A step of forming an aggregate of the obtained nerve cells;
Incubating the formed agglomerates in a buffer solution containing the test substance and purified albumin and allowing them to migrate;
Measuring the degree of nerve cell migration;
A step of evaluating the influence of the test substance on the migration activity of nerve cells based on the degree of migration,
A method for evaluating the influence of a test substance on the migration activity of nerve cells is provided.

  Furthermore, the buffer solution itself containing the purified albumin is also within the scope of the present invention.

  The present invention can be used for the determination of diseases based on the degree of migration of nerve cells and the measurement of the effect on drug migration ability of diseases associated with nerve cell migration disorders whose results are sufficiently reliable and reproducible. A determination method and a method for evaluating the influence of a test substance on the migration activity of nerve cells are provided.

FIG. 1A and FIG. 1B are a photograph and a graph showing the effect of a calpain inhibitor on the migration activity of nerve granule cells.

The first step of the measurement method of the present invention is a step of acquiring nerve cells from brain tissue collected from a living body.
The living body is not particularly limited, and examples thereof include mammals (eg, humans, mice, rats, dogs, rabbits). Preferably, the living body is a mammal such as a human or a mouse. As the brain tissue, the cerebellum is particularly preferable. As nerve cells, granule cells are particularly preferred.
The method for preparing cells from the tissue is not particularly limited, and examples thereof include a method of treating with an appropriate buffer containing trypsin and DNase I.

Aggregate formation, which is the second step of the measurement method of the present invention, can be performed, for example, by culturing the nerve cells obtained as described above in a medium.
The medium is not particularly limited as long as it can cultivate neurons.Basal Medium Eagle (BME) medium (Gibco, Sigma, Cat. 21010), 10% horse serum (Horse serum (HS), invitrogen) Examples thereof include 10% HS / BME medium.
The culture temperature is preferably 30 to 40 ° C, more preferably 35 to 37 ° C.
The culture time is preferably 7 to 10 hours, more preferably 8 to 9 hours.
The formation of the aggregate is preferably performed using a cell adhesion molecule in order to facilitate adhesion between cells. Such cell adhesion molecules are not particularly limited as long as they are used as scaffolds in culture in the art, and include poly-L-lysine, poly-D-lysine, laminin, collagen, and the like.

Purified albumin means albumin purified by a purification method known to those skilled in the art, preferably serum albumin substantially free of globulin.
Albumin is not particularly limited, and those known to those skilled in the art can be used. Albumin may be a known commercial product, or may be prepared from mammals (eg, human, cow, horse, goat, etc.) serum.
The purification method is not particularly limited as long as albumin can be purified. Precipitation using an organic solvent (for example, alcohol precipitation), alcohol fraction (for example, low temperature alcohol fraction of corn), heat treatment and low temperature treatment, low Examples include at least one method selected from pH treatment, crystallization, chromatography (ion exchange chromatography, affinity chromatography, etc.), electrophoresis, and charcoal treatment.
Purified albumin may be a commercially available product. Examples of such products include albumin essentially globulin-free (Sigma, A3156).

The buffer solution is obtained by dissolving the above purified albumin in an appropriate solvent.
The solvent is not particularly limited as long as it does not affect the survival and activity of nerve cells, and any solvent known to those skilled in the art can be used, but Mason's Medium is preferable.
The content of purified albumin in the buffer solution can be appropriately set according to the state of the obtained cells, etc., but is usually 5 to 25 mg / mL, preferably 8 to 20 mg / mL, more preferably 10 to 15 mg / mL. is there.
The buffer solution may optionally contain other components. Examples of such components include nutrient stock solutions such as N2-Supplement (Gibco, Cat. 17502-048), antibiotics such as penicillin and streptomycin.
The content of other components in the buffer solution can be appropriately set by those skilled in the art.

The incubation and migration step, which is the third step of the measurement method of the present invention, is performed by incubating the nerve cells in the buffer solution containing the purified albumin.
The temperature at the time of incubation is 30 to 40 ° C, preferably 35 to 38 ° C, more preferably 36 to 37 ° C.
The incubation time is 1 to 24 hours, preferably 3 to 12 hours, more preferably 6 to 9 hours.
The incubation / migration process is preferably performed on a solid support in order to facilitate the measurement of the following migration distance. The solid support is not particularly limited, and examples thereof include a cover glass (for example, Matsunami; 24 mm × 24 mm), a slide glass, and a culture dish.

  Thereafter, optionally, before the fourth step described below, a step of introducing a factor capable of affecting the migration activity of the nerve cell, such as a gene or RNA, may be performed. The method for introducing such a factor is not particularly limited, and includes any transduction method known to those skilled in the art, such as electroporation using Neon (registered trademark), and a method using RNA interference.

The fourth step of the determination method of the present invention is a step of measuring the degree of nerve cell migration.
In the present specification, the “degree of migration” intends data indicating the migration ability of a cell obtained from the number of migrated cells and the migration distance of the cells or an area calculated based on the migration distance. Examples of the distance include a distance from the edge of the aggregate, a distance from the center of the aggregate, and the like. Examples of the area include an area of a region where each cell has migrated, an area of a region including all migrated cells, and the like.

  In a preferred embodiment of the present invention, an aggregate of nerve cells placed in a substantially circular shape is assumed to be a circle, the circumference thereof is set to 0 μm, and the radius of the assumed circle is set to r μm. ) Obtained by subtracting the number of post-migration neurons existing in a concentric circle having a radius of (r + 20 (n−1)) μm from the number of post-migration neurons existing in a concentric circle having a radius of μm. The degree of migration is expressed as a ratio (%) of the number of cells showing the migration distance of (20n-10) μm to the total number of cells. However, n is an integer of 1 to 11, and the number of cells obtained when n ≧ 12 is included in the number of cells when n = 11.

  In another preferred embodiment of the present invention, the number of stained cell nuclei is counted as the number of cells in each concentric compartment drawn at a radius of 20 μm using a known confocal laser microscope and image analysis software. The degree of migration is expressed as a percentage (%) of the total number of cells. Examples of the image analysis software include analysis software mounted on a TCS-SP5 confocal laser microscope (Leica).

When measuring the degree of migration, cells may optionally be stained using a staining reagent. Such a reagent is not particularly limited, and reagents for staining cell nuclei known to those skilled in the art such as Hoechst and DAPI, as well as enzymes, fluorescent substances, luminescent substances, radioisotope elements, biotin and avidin known to those skilled in the art. And a labeled antibody labeled with.
The microscope used for measuring the degree of migration is not particularly limited, and examples thereof include a confocal microscope, such as a TCS-SP5 confocal laser microscope (Leica), and an inverted microscope.

The fifth step of the determination method of the present invention is a step of determining whether or not the living body has a disease accompanied by a nerve cell migration disorder based on the degree of migration.
As a determination method, when the degree of migration is small, it is determined that the living body has a disease accompanied by nerve cell migration disorder. This determination can be made empirically by accumulating data relating to the degree of migration of nerve cells, but in order to determine with higher accuracy, it is preferable to make a determination by comparison with a predetermined threshold. Such a threshold can be appropriately determined based on, for example, the degree of migration of cells that are known to migrate and cells that are known not to migrate. In a preferred embodiment of the present invention, based on the migration distance obtained in the fourth step, the number of cells showing a migration distance of 10 to 210 μm is set as 100%, and the number of cells showing a migration distance of 10 μm is 50% or more. When it is preferably 60% or more, more preferably 70% or more, it is determined that the living body has a disease associated with a migration disorder of nerve cells.

Another aspect of the present invention is:
Obtaining nerve cells from brain tissue collected from a living body of a disease associated with nerve cell migration disorder;
A step of forming an aggregate of the obtained nerve cells;
Incubating the formed agglomerates in a buffer solution containing the test substance and purified albumin and allowing them to migrate;
Measuring the degree of nerve cell migration;
A step of evaluating the influence of the test substance on the migration activity of nerve cells based on the degree of migration,
This is a method for evaluating the influence of a test substance on the migration activity of nerve cells.

The disease associated with the neuronal migration disorder is not particularly limited, and is a gene having a function associated with migration (for example, LIS1 present in human chromosome 17, doublecortin (DCX) on X chromosome, Reln, 14 -3-3ε, etc.) due to mutations, deletions and substitutions. Examples of such diseases include spondylosis, cerebellar gyrus, periventricular ectopic gray matter, cobblestone cortical dysplasia, and the like.
In one embodiment of the present invention, the disease associated with a neuronal migration disorder is spondylosis caused by a heterozygous mutation (haploinsufficiency) in the LIS1 gene present in human chromosome 17.

The first step of the evaluation method of the present invention is the same as the first step of the above measurement method, except that the living body is limited to those suffering from diseases associated with the above-mentioned nerve cell migration disorder.
The second step of the evaluation method of the present invention is the same as the second step of the measurement method.

The third step of the evaluation method of the present invention is a step in which the formed aggregate is incubated and migrated in a buffer solution containing the test substance and purified albumin.
The test substance is not particularly limited, and includes any compound known to those skilled in the art or a salt thereof, a polynucleotide, a polypeptide, and the like, and is added to evaluate the effect on nerve cell migration disorder. The test substance is preferably a cysteine protease inhibitor or a calpain inhibitor, more preferably a calpain inhibitor.
Thereafter, a step of introducing a factor capable of affecting the migration activity of the nerve cell may optionally be performed.
The fourth step of the evaluation method of the present invention is the same as the fourth step of the above determination method.

The fifth step of the evaluation method of the present invention is a step of evaluating the influence of the test substance on the degree of migration of nerve cells based on the measured distance.
As an evaluation method, when the degree of nerve cell migration with the addition of the test substance is increased compared to the degree of nerve cell migration without the addition of the test substance, the test substance becomes the nerve cell migration. It is evaluated that it has influenced the degree of. In a preferred embodiment of the present invention, the number of cells exhibiting a migration distance of 10 to 210 μm is set as 100% based on the migration distance obtained in the fourth step, compared with the case where no test substance is added. When the test substance is added, when the number of cells having a migration distance of 10 μm is reduced by 30% or more, preferably 40% or more, more preferably 50% or more, the test substance affects the degree of migration of nerve cells. Evaluate that given.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

Example 1: In vitro nerve cell migration activity measurement method (material) using granule cell aggregates derived from mouse cerebellum
・ Mouse; 2-6 days after birth (C57BL / 6)
・ CF-HBSS
HBSS (Hank's Balanced Salt Solution; Sigma, Cat.H8264) 200 ml
1M MgCl ₂ (final concentration 5 mM) 1 ml
Glucose (final concentration 4mg / ml) 0.8 g
Penicillin / Streptomycin (Gibco, Cat. 15140-122) 2 ml

・ Trypsin-HBSS 10 ml
CF-HBSS 10 ml
2.5% trypsin (final concentration 0.025%) 100 μl
10 mg / ml DNaseI (final concentration 0.0013%) 6.3 μl

・ DNase Medium 5 ml
BME (Basal Medium Eagle; Gibco, Sigma, Cat. 21010) 4.5 ml
10% Horse Serum (HS) 0.5 ml
2.5% trypsin (final concentration 0.025%) 100 μl
10 mg / ml DNaseI (final concentration 25 μg / ml) 12.5 μl

・ Mason's Medium 100 ml
Nutrient Stock (N2-Supplement; Gibco, Cat. 17502-048) 1.0 ml
Bovine serum-derived purified albumin (Albumin essentially globulin-free (Sigma, A3156
))
1.0g
Penicillin / Streptomycin 1 ml

Cover glass coating The cover glass (Matsunami; 24 mm X 24 mm) was washed with 1 mM hydrochloric acid, rinsed with a large amount of running water, dipped in 70% EtOH and dried. The cover glass was coated with 0.0002% Poly-D-lysine (Sigma; Cat. P4707) for 1 hour at room temperature, then with Laminin (Sigma, Cat; L2020) for 1 hour at room temperature, and then shaken twice. Washed a total of 10 times including washing.

(Method)
The cerebellum was extracted from a newborn mouse of a normal or spondylosis model (LIS1 gene heterozygous mutation model) and made into about 10 pieces. These cerebellum pieces were transferred to a tube (15 ml) so as not to collapse. The suspension was suspended in CF-HBSS (5 ml), centrifuged at 500 rpm x 1 minute, and the supernatant was removed. Trypsin-HBSS (10 ml) was added and incubated at 37 ° C. for 20 minutes with occasional tapping, followed by centrifugation at 500 rpm for 1 minute to remove the supernatant. Washed twice (centrifuged at 500 rpm) with 5 ml of BME medium (10% HS / BME) containing 10% horse serum (HS, invitrogen). DNase Medium (2 ml) was added and pipetted about 30 times. DNase Medium (3 ml) was added and left for 1 minute. A 70 μm mesh (Cell strainer, Becton Dickinson) was passed. Incubation was carried out at 37 ° C. for 20 minutes on an uncoated culture dish. In order not to break up a lump, it was again transferred to a tube (15 ml) and centrifuged at 1,500 rpm for 1 minute to remove the supernatant. 10% HS / BME (5 ml) was added and centrifuged at 500 rpm for 1 minute, and the supernatant was removed. An appropriate amount (several ml) of 10% HS / BME was added and incubated on an uncoated culture dish at 37 ° C. for 8 hours to reconstitute the aggregate. The suspension was carefully transferred to a microtube (1.5 ml), allowed to stand for 1 minute, and then the supernatant was removed. Add 1.0-1.2 ml of Mason's Medium, place the cell mass on 35 mm Glass Base Dish (IWAKI) coated with Poly-D-Lysin (SIGMA) and Laminin (SIGMA), and add 200 μM calpain inhibitor. Cultured at 37 ° C. for 12 hours with or without addition. Thereafter, Hoechst was added for nuclear staining, medium was exchanged for L15 (Leibivitz; Gibco), and observation was performed with a confocal laser microscope (TCS-SP5, Leica). Using the analysis software of a confocal laser microscope (TCS-SP5, Leica), the number of nuclei in each concentric compartment drawn with a radius of 20 μm is counted as the number of cells and expressed as a percentage (%) of the total number of cells. did.

The results are shown in FIGS. 1A and 1B.
When no drug is added, migration of normal neural granule cells is observed, while the migration distance of LIS1 heterozygous cells is small, and most of them exhibit a migration distance of 10 μm. From this, it was found that the in vitro cell migration assay of the present invention can be used for the determination of diseases associated with nerve cell migration disorders.
In addition, when the drug was added, there was not much change in the migration distance in normal cells, whereas in the LIS1 heterozygous mutant cells, the number of migrated cells (n) increased and the number of cells showing a migration distance of 10 μm. Reduced. From this, it was found that the in vitro cell migration assay of the present invention can be used to evaluate the influence of the test substance on the migration activity of nerve cells.
A similar experiment was performed 10 times. These experiments stably reproduced results similar to FIGS. 1A and 1B.

  Since the in vitro cell migration assay of the present invention has sufficient reliability and reproducibility of results, it can be used for disease determination based on the degree of migration of nerve cells and measurement of the effect on drug migration ability. .

Claims (7)

Acquiring nerve cells from brain tissue collected from a living body;
A step of forming an aggregate of the obtained nerve cells;
Incubating the formed aggregates in a buffer solution containing purified albumin and allowing them to migrate;
Assuming that the aggregate of nerve cells placed in a substantially circular shape is a circle and its circumference is 0 μm, and the radius of the assumed circle is r μm, it is within a concentric circle having a radius of (r + 20) μm. The ratio of the number of cells showing a migration distance of 10 μm to the total number of cells obtained by subtracting the number of neurons after migration existing in a concentric circle having a radius of r μm from the number of neurons after migration is present. Determining that the living body is a disease accompanied by nerve cell migration disorder when it is 50% or more, and determining that the living body is not a disease accompanied by nerve cell migration disorder when it is less than 50%. It is characterized by
A method for determining a disease associated with a neuronal migration disorder. Obtaining nerve cells from brain tissue collected from a living body of a disease associated with nerve cell migration disorder;
A step of forming an aggregate of the obtained nerve cells;
Incubating the formed agglomerates in a buffer solution containing the test substance and purified albumin and allowing them to migrate;
Measuring the degree of nerve cell migration;
A step of evaluating the influence of the test substance on the migration activity of nerve cells based on the degree of migration,
A method for evaluating the influence of a test substance on the migration activity of nerve cells.   The purified albumin is albumin purified by at least one method selected from precipitation using an organic solvent, alcohol fractionation, heat treatment and low temperature treatment, low pH treatment, crystallization, chromatography, electrophoresis, and charcoal treatment. The method according to claim 1 or 2.   The method according to any one of claims 1 to 3, wherein the purified albumin is serum albumin substantially free of globulin.   The method according to any one of claims 1 to 4, wherein the disease accompanied by a neuronal migration disorder is caused by a mutation of a gene having a function associated with migration.   The method according to any one of claims 1 to 5, wherein the disease associated with a neuronal migration disorder is caused by a mutation in the LIS1 gene.   The method according to any one of claims 1 to 6, wherein the disease accompanied by migration disorder of nerve cells is spondylosis.