JP2003259864A - Microglia cell line - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a high purity purified microglia. <P>SOLUTION: This microglia cell line has a property of migrating specifically into the brain by administering it into the blood vessel, and more specifically has the following properties. (a) form: having both or either one of forms of a macrophage like or spherical forms in the presence of a granulocyte- macrophage colony-stimulation factor, or a branched form resembling the branched microglia presenting in the brain in the absence of the factor. (b) functional characteristics: having specific affinity to the brain and also a potent phagocytosis activity. (c) cell proliferating activity: proliferating dependently on granulocyte-macrophage-stimulating factor. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a subcultured established cell line of microglia, a method for separating the same, and its use as a pharmaceutical carrier. Furthermore, the present invention relates to microglia into which a foreign gene or drug has been introduced, a method for introducing the microglia, and a pharmaceutical composition containing the microglia.

[0002]

2. Description of the Related Art There are numerous hereditary diseases related to the nervous system, which are caused by the deficiency of a single enzyme, or various ones whose cause is still unknown. Most of these diseases are currently treated by replacement therapy. On the other hand, a large number of studies have been conducted in Japan and overseas on the selective substance transfer system to the brain, and for gene transfer into the brain at the animal individual level, the adenovirus vector method using a virus with neurotropic properties is used. Devised,
A system capable of introducing a gene into a nerve cell is known (Kozarsky, KF and Wilson, JM, Curr. Opi.
n. Genet. Dev. 3, 499-503, 1993). Also, a method using a retrovirus vector has been devised, and has been successfully introduced into liver artillery or blood cells (Mullingan,
R. C. Science 260, 926-932.1993).

However, the brain has a blood-brain barrier.
Brain Barrier) makes it difficult to carry out replacement therapy and to introduce effective drugs, and also to introduce specifically into the brain even if peripheral substances (such as anticancer drugs and DNA) are administered. I can't. Therefore, conventionally, there has been no method other than direct injection of the substance into the brain by surgery. On the other hand, there is a method of using liposomes as a method that does not involve an intrusive procedure such as surgery, and a group in Japan has developed a method that relatively easily enters the brain by changing the constituent elements of liposomes. However, even with this method, uptake into the brain is about 1% of the injected amount, and it cannot be said that the method is specific to the brain. Thus, since the brain has a blood-brain barrier, there is almost no infiltration of substances or cells from the periphery, and it is difficult to introduce drugs or genes. Actually, infiltration of immune cells such as T cells and macrophages is hardly seen in the normal brain.

[0004] Microglia are central nervous system cells having macrophage-like properties, function as immunocompetent cells in inflammatory reactions and viral infections, act as phagocytes that remove cells, and play a central role in the central nervous system cytokine network. Cells that play a role (Sawada,
M. et al., lnt. J. Dev. Neurosci., 13, 253-264, 1
995). In addition, it has recently been shown to be essential for the expression of higher brain functions such as learning and memory, and is considered to be a specialized cell having a specific role in the brain.
Until now, it was thought that the origin of microglia was that the monocytes invading the brain during the perinatal period specialized and differentiated.
Further, microglia can be obtained by primary culture of brain cells, but in order to perform primary culture, it is necessary to remove the brain and purify it each time it is used, and usually a period of about 2 weeks is required. Therefore, the operation is complicated, and also
Since it is difficult to proliferate in culture and it is almost impossible to subculture, it is extremely difficult to introduce a gene into primary culture microglia and express it.

[0005]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention An object of the present invention is to obtain highly purified microglia, which are considered to be macrophages specific to the brain.

[0006]

[Means for Solving the Problems] The present inventors succeeded in obtaining highly purified microglia in the process of studying microglia which is considered to be a brain-specific macrophage. That is, the present invention relates to microglia capable of subculture, and more particularly to established microglia having the following properties. (A) Morphology: Macrophage-like or globular morphology in the presence of granulocyte-macrophage colony stimulating factor, and / or branched morphology similar to branched microglia existing in the brain in the absence of the factor. It has one of the forms. (B) Functional characteristics: It has a specific affinity for the brain. Also,
Has a strong poor diet. (C) Cell proliferation property: Proliferates depending on granulocyte-macrophage colony stimulating factor.

The present invention also provides for IL-3 and / or purification, preferably in the presence of cytokines, preferably colony stimulating factors (CSF), more preferably granulocyte-macrophage colony stimulating factors (GM-CSF). The present invention relates to a method for separating subcultured established microglia from microglia cells in the presence of astrocytes.

Furthermore, the present inventors have found that macrophages and microglia are decisively different in terms of affinity to the brain and whether they can infiltrate the brain. Furthermore, when they were stained by a method capable of distinguishing the two and examined their distribution, they found that microglia existed in the brain from the early stage of development. Therefore, microglia are not derived from monocytes that differentiate and mature in bone marrow, but a group of cells with a specific affinity for the brain invades into the brain in the early stage of development, leading to higher-order functions such as brain morphogenesis and memory learning. It will be adjusted. Therefore, the present inventors compared the isolated macrophages and microglia into the rat peripheral arteries and compared whether or not they have a selective orientation to the brain.As a result, when microglia labeled with a fluorescent dye were injected, He had many fluorescent cells in his brain, but few in his liver. On the other hand, when macrophages were injected, almost no fluorescent cells were found in the normal brain, but many fluorescent cells were found in the liver.

[0009] Next, it was examined whether or not the microglial cell line established by the present inventors was introduced with the lacZ expression vector into the bloodstream of the rat to selectively express the gene in the brain. Then, the activity of β-galactosidase could be detected in the rat brain slice injected with the lacZ-expressing cells. From the above results, microglia, unlike macrophages, are cells with a specific affinity for the brain, and by utilizing this affinity, it is possible to specifically introduce a specific substance or gene into the brain from the peripheral blood flow. all right.

That is, the present invention relates to a pharmaceutical carrier (carrier) comprising the above-mentioned microglia and having a specific affinity for the brain. The present invention also relates to the established cell line microglia introduced with a gene or a drug. The present invention also provides the above-mentioned microglia into which a gene or drug has been introduced,
And a pharmaceutical composition comprising a pharmaceutical carrier, particularly a therapeutic agent for brain diseases. Further, the present invention provides
The present invention relates to a screening method and a production method for microglia introduced with a foreign gene, which comprises introducing the gene into the microglia using the foreign gene and a gene expressing a fluorescent protein, preferably a fluorescent protein derived from jellyfish. As the microglia in this method of the present invention, conventional microglia can be used, but it is preferable to use the above-mentioned established microglia of the present invention. The present invention also relates to a method for treating a brain disease, which comprises delivering a drug or gene specifically to the brain using the pharmaceutical composition.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The established microglia of the present invention is obtained by primary culturing mouse or rat brain cells to purify the microglia, and further isolating the established microglia clone of the present invention from the purified microglia by the following method. can do. Further, since the established cell line microglia of the present invention is easy to handle and has an affinity for the brain, a gene or drug is introduced into the established cell line microglia, and the gene is specifically expressed in the brain by injecting into peripheral blood vessels. , Can also deliver drugs.

The method for producing the established microglia of the present invention will be described in more detail. (1) Purification of microglia First, the brain membrane of the collected mouse or rat brain is peeled off,
Separate into single cells using a pipette or nylon mesh. The mouse and rat are preferably newborn. Examples of the mouse include C57BL6, C3H, ICR,
Ba1b / c and the like, and examples of the rat include Fisher and Wistar.
r), SD and the like, but not limited thereto.

The obtained cells are seeded in a usual animal cell culture medium (10% FCS or CS-containing EMEM etc.) and cultured for 10 to 14 days. The medium is replaced every 3 to 4 days. Next, cells to be established are selected from the obtained cultured cells. Here, there are types of microglia called Type I and Type II, which are detached from the culture dish by mechanically stimulating the primary culture (spraying the medium with a pipette, shaking the culture dish, etc.). It is a floating cell. On the other hand, type II is a cell (adherent cell) that does not float by the mechanical stimulation. Since the microglia of the present invention belongs to type II, purified microglia can be selected as follows. The adherent cells that do not float due to the above mechanical stimulation are further treated with trypsin-EDTA to obtain single cells, which are then seeded and adhered on a plastic dish that has not been treated at all (referred to as a non-coated plastic dish). This is because the culture dish is generally treated with a drug so as to have a positive charge, but in order to obtain adherent cells, it is necessary to use a dish that has not been treated. After heating in a CO2 incubator at 37 ° C for 1 hour, mechanical stimulation is applied to remove cells floating in the medium, and cells that can grow while adhering to the dish are collected with a rubber policeman or the like, and the same procedure is repeated. The cells obtained by repeating twice can be obtained as purified microglia for cell line establishment. The purified microglia obtained as described above has sufficient purity, but a cell sorter or the like may be used in order to further raise the purification purity.

(2) Separation of established cell line microglia In order to separate the subcultured cell line culture of the present invention which can be subcultured from the purified microglia obtained as described in (1), the purified microglia is prepared by using a cytokine, Preferably colony stimulating factor (CSF), more preferably granulocyte-macrophage colony stimulating factor (GM-CSF)
It can be carried out by culturing in the presence of, and cloning it. As a cytokine to be present in the culture, G
Although M-CSF may be used alone, GM-CSF may be further present with a supernatant derived from IL-3 and / or purified astrocytes. The cytokine used may be natural or recombinant.
More specifically, for example, it can be performed as follows.

Seed on a petri dish of about 10 cm, and recombinant gene-type granulocyte-macrophage colony stimulating factor (rG
M-CSF) for 7-10 days. After the culturing, the cells are collected and further cultivated in the presence of rGM-CSF by the so-called limiting dilution method for 4 to 10 weeks. Then, the cloned cells are selected and separated by releasing the cells forming one colony per well of the test plate with rubber policeman, and finally the established cell line microglia is obtained.

The established cell line microglia thus obtained has the following properties. (A) Morphology As a result of observing the morphology under a fluorescence microscope after staining with a fluorescent dye or under a phase contrast microscope without staining, r
It has a macrophage-like or globular morphology in the presence of GM-CSF, or a branched morphology similar to the branched microglia present in the brain in the absence of rGM-CSF. Alternatively, it has both of the above forms. (B) Functional characteristics When the established microglia of the present invention is administered to mouse arteries, it migrates specifically to the brain, and thus has specific affinity to the brain. In addition, stimulation of microglia with lipopolysaccharide produces interleukin-1 (IL-1) and interleukin-6 (IL-6). Also,
When stimulated with interferon γ, it produces IL-5.
This property is different from macrophages that do not express IL-5 in IFN-γ. Further, as a result of a test for phagocytosis using the incorporation of a fluorescent dye as an index, the cell line of the present invention has a strong phagocytosis. The established microglia of the present invention has a phagocytic ability several hundred to several thousand times that of astrocytes. (C) Cell Proliferation Proliferation is not observed when an experiment for removing rGM-CSF from the medium is carried out, and thus the established cell line microglia of the present invention proliferates in a GM-CSF-dependent manner. First, the morphology and characteristics of the established microglia of the present invention
4 to 4 illustrate a specific example.

(3) Introduction of gene into established cell line of microglia Introducing gene into established cell line of microglia is as follows.
It is important for the specific expression of the gene in the brain. The target gene can be obtained by a known cloning method, or a commercially available gene can be used, and the kind of gene is not particularly limited.

For example, there is a method of using DOTAP (manufactured by Boehringer-Mannheim) as a method for highly efficiently introducing a gene into established microglia. That is, DOTA
There is a method of culturing established microglia with a target gene in a P-containing medium for gene transfer. After culturing at 37 ° C. for 16 to 24 hours in a CO 2 incubator, it is further cultivated for 30 to 72 hours, preferably 48 hours in a microglial culture medium together with rGM-CSF.
In addition, as a method for introducing a gene into microglia,
In addition to the above methods, for example, calcium phosphate method, DE
Known methods such as the AE dextran method, lipofection method, electroporation method, and particle gun method can also be used.

By the way, the most commonly used method of separating the stably expressing strains of the transgene is to introduce the drug resistance gene into cells at the same time as the gene of interest, to express it, and to incorporate it into chromosomal DNA. This is a method in which cells other than the cells whose expression is stable and constitutive are killed by the drug treatment and eliminated from the culture to be separated.
When this screening method is applied to microglia, the microglia that have become capable of stable and constitutive expression may recognize dead cells and express strong phagocytosis, and at the same time, they may be activated and lose proliferative capacity. For this reason, a preferred method for introducing the gene of the present invention is to use a fluorescent protein instead of the drug resistance gene, preferably green jellyfish-derived green fluorescent protein (green fl).
Examples include a method of separating microglia that stably and constitutively express target cells by a difference in fluorescence intensity, using an expression vector modified to express ureorescent protein (GFP) in higher animals.

To confirm whether or not the cells into which the gene has been introduced have reached the brain, and to confirm the expression of the gene, the introduced cells are previously stained with a fluorescent dye specific for phagocytic cells, and the brain is removed. After freeze-drying, a section of about 8 microns thick is prepared, and cells having fluorescence are identified under a fluorescence microscope, or the section is subjected to activity staining with a substrate of a gene to be introduced. The above confirmation can also be performed by magnetic resonance imaging (MRI), positron emission tomography (PET), or the like. For example, cells in which a contrast agent for MRI or the like has been incorporated can be injected into an animal and the trend thereof can be observed. According to these methods, the animals can be observed non-invasively and easily without killing them.

[0021]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1: Isolation of established microglial clones (1) Isolation of microglia Brains of newborn mice (C57BL6, op / op) and newborn rats (Fisher) were removed and ice-cooled microglia culture medium (Mi medium). Say: Eagle's MEM,
10% bovine serum, 0.2% glucose and 5 μg / ml
Brain membranes were dissected in bovine insulin). The cells were made into single cells with a Pasteur pipette or nylon mesh, and the cells were cultured in the above Mi medium. For mouse brain, cells for one brain per 20 ml of Mi medium and for rat brain, cells for one brain per 40 ml of Mi medium were cultured in two 10 cm culture dishes and four 10 cm culture dishes, respectively. did. Culturing was carried out in a CO2 incubator (5% CO2, 95% air) at 37 ° C for 10 to 10.
I went on the 14th. The medium was replaced every 3 to 4 days. Cells that show bright phase contrast images (PBs)
RC) appeared, after removing PBRC by mechanical shaking, 200 U / ml trypsin-0.02% E
Peel off cells with DTA and in an uncoated plastic dish, 3
Incubated for 30 minutes at 7 ° C. The cells adhered to the uncoated plastic dish were washed twice with Mi medium, and the cells were detached and collected. Repeat the same process two more times,
Purified microglia was obtained.

(2) Isolation of clones The purified microglial cells obtained in (1) above were 10 cm.
1 × 10 5 cells were inoculated on a petri dish, and 7 in Mi medium in the presence of rGM-CSF (manufactured by Genezyme).
Cultured for a day. The cells were collected, counted, and clones were separated by the limiting dilution method. That is, in each well of a 96-well plate (manufactured by Falcon),
Seed at a cell concentration of 0.5 cells / well (in 100 μl) and 2 ng / ml of mouse recombinant GM-CSF.
Approximately 3 in the presence of (Genezyme)
Cultured for a week. The presence or absence of cloning was confirmed for each well, and the target clone was isolated. As a result, 5 types (Ra2, GMI-M6-
1, GMI-M6-3, GMI-M5-2, GMI-M
F11), one kind from rat brain (GM
The established microglia of I-R1) were obtained. this house,
The established microglia Ra2 and GMI-R1 are referred to as "mouse microglia Ra2" for Ra2 by the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, and as "FERM P-16109" and "rat microglia GMI" for GMI-R1. -R1 "
And are deposited as FERM P-16110, respectively.

The properties of the obtained clones were examined. (A) Morphology GMI-R1 was cultured in the presence and absence of rGM-CSF and observed with a phase contrast microscope.
It showed a macrophage-like or spherical shape in the presence of M-CSF (Fig. 1A), and was branched in the absence of rGM-CSF (Fig. 1B).

(B-1) Functional Characteristics (Brain Affinity) The established microglia GMI-RI of the present invention is attached to a plastic petri dish, and a phagocyte staining solution (Di
luent B. : Fluorescent dye PKH26 (manufactured by Zynaxis) (Zynaxis (Z
(Yenaxis) and 10% serum were mixed at a ratio of 1: 1 and added to the dish, and fluorescent staining was performed at 37 ° C. for 15 minutes (I
shihara, S., Sawada, M. et al., Exp, Neurol ,. 12
4, 219-230, 1993). Cells were harvested and 2 × 10 6 cells were injected into 5 week old syngeneic (Fisher) rat axillary arteries.
48 hours after the injection, and 1, 2 and 3 weeks after the injection, the organs of the rat were removed, and OCT (Tissue Tech (Ti
Sue Tek)) solution. Macrophages isolated by washing the peritoneal cavity of microglia GMI-R1 and syngeneic rats (Fisher) as a comparison with cold PBS were woven with fluorescent dyes specific for phagocytic cells and injected into rat axillary arteries. The tissue orientation was examined by making tissue slices. As a result, when the established microglia of the present invention was injected, many fluorescent cells were found in the normal brain (Fig. 2A), but not in the liver (Fig. 2B). On the other hand, when macrophages were injected, almost no fluorescent cells were found in the normal brain (Fig. 2C), whereas many fluorescent cells were found in the liver (Fig. 2D). Therefore, the established microglia of the present invention had a brain-specific affinity.

(B-2) Functional characteristics (IL-1 and IL
-6 productivity) 1 × 10 ⁶ Ra2 cells were seeded on a 6 cm culture dish and stimulated with lipopolysaccharide for 12 hours, and unstimulated total RNA was extracted with RNeasy (manufactured by Qiagen). Then, a mixed cDNA was obtained using reverse transcriptase (BRL) from 2 μg of the mixture. Using this cDNA as a template, PCR was carried out using IL-1 specific synthetic primers and IL-6 specific synthetic primers having the following sequences. IL-1 specific synthetic primer (Sawada et al., Lnt. J. Dev. Neurosci. 13, 253-264, 1995): sense strand: 5′-ATGGCAACTGTTCCTGAACTCAACT-3 ′ (SEQ ID NO: 1) antisense strand: 5 '-CAGGACAGGTATAGATTCTTTCCTTT-3' (SEQ ID NO: 2) IL-6-specific synthetic brimer (Sawada et al., Brain Res. 583, 296-299, 1992): sense strand: 5'-ATGAAGTTCCTCTCTGCAAGAGACT-3 '(SEQ ID NO: 2) 3) Antisense strand: 5′-CACTAGGTTTGCCGAGTAGATCTC-3 ′ (SEQ ID NO: 4) PCR was performed at 55 ° C. for 1 minute, 72 ° C. for 2 minutes and 94 ° C. for 1 minute.
This was carried out for 30 cycles (using HYBAID's Omnigene), with each minute reaction being one cycle. PCR
Then, the amplified product was subjected to agarose gel electrophoresis to examine the expression of the gene.
-1 and IL-6 expression was found to be increased (3rd
Figure, "LPS" lane). In FIG. 3, M
Represents a molecular weight marker, and cont represents a control (unstimulated).
In addition, it was confirmed by ELISA that IL-1 and IL-6 were produced. Further, the culture supernatant of the established cell line microglia of the present invention after stimulation with lipopolysaccharide is added to MH60 cells that proliferate in an IL-6-dependent manner or D10 cells that proliferate in an IL-1-dependent manner, and cultured to give MH60 cells and D10 cells.
The presence or absence of cell proliferation was examined. As a result, it was found that all cells proliferate in the presence of the culture supernatant of the cell line microglia of the present invention stimulated with lipopolysaccharide.

(C) Cell Proliferation 5 × 10 ⁴ GMI-RI cells were seeded on a 96-well test plate, and 1000 μg of 2 μg / ml rGM-CSF was used.
One-fold, 5000-fold, and 10000-fold dilutions were added, and the mixture was cultured for 4 days and subjected to MTT assay.
Human M-CSF (Green cross) 400 u / m as a control
1 diluted 1000 times, or PMA (phorbol myristate acetate) 0.1 mg / ml to 1000
A one-fold or 5000-fold dilution was used (Fig. 4A). On the other hand, rGM-CSF diluted 5000 times and 100 u / ml each of mouse IL-3, IL-4 and IL-6 (all of which are Genezyme).
Company). MTT assay was performed 2 days and 4 days after the addition of each reagent (FIG. 4B). As a result, it was found that GMI-R1 proliferates depending on rGM-CSF.

Example 2 Introduction of Gene into Microglia of the Present Invention Escherichia coli-derived lacZ gene expression vector ptkβ (Clonetech) and DOTAP lipid (Boehringer Mannheim (Boehrin)
ger-Mannheim)), and the final concentration is 1 μm.
g / ml. This was mixed with a medium containing serum to prepare, added to the established microglia of the present invention, and treated for 16 hours. As controls, established microglia with no gene introduced and macrophages obtained in the same manner as in Example 1 were used. Next, normal medium (EMEM: 10% FC
Further, the cells were cultured for 48 hours in Example 1 (b-).
The fluorescent staining described in 1) was performed to examine whether the gene was transferred to the brain and expressed. That is, under adult Nembutal anesthesia of a mature rat (250 to 300 g), the left axillary artery was exposed, and after the hemostatic treatment, a cannula was inserted to obtain 1-2.
× 10 ⁶ cells were injected. After injection, suture the incision,
The rat was allowed to recover. Then, 48 hours after the cell injection, the rat brain was excised, three consecutive frozen sections of about 8 μm were prepared, and each was observed with a fluorescence microscope. Further, the activity staining of β-galactosidase and the quantification of the activity were performed as follows. One of the above three sections was fixed with 0.5% glutaraldehyde and
Et al. (BioTechniques 7, 576-579, 1989)
Was used as a substrate for activity staining. To quantify the activity, one section was homogenized with 100 μl of lysis buffer by sonication, and a commercially available kit (GalactoLight; Boehringer-M) was used.
anheim). As a result, when the gene was introduced into the established microglia of the present invention, the presence of lacZ-positive cells could be confirmed from a rat brain section (Fig. 5).
In addition, as a result of quantifying the β-galactosidase activity by a chemiluminescence method, a much higher activity was detected in a rat brain section injected with established microglia in which an Escherichia coli-derived gene lacZ expression vector was introduced, than that in the case where no gene was introduced ( (Fig. 6).

Example 3: Introduction of chemical substances into microglia of the present invention and specific introduction into brain The fluorescent dye PKH26 used in Example 1 produces granules in Diluent B. The microglial cell line of the present invention specifically takes up these granules, retains them inside the cells, and transfers them to the brain. Therefore, the fluorophore PKH26
Was used as a model for chemicals (anti-cancer agents). As a result, when the established microglia of the present invention was injected, many fluorescent cells were found in the normal brain, but not in the liver. Therefore, it can be said that the microglia of the present invention carries a chemical substance (drug) in a brain-specific manner.

Example 4 Gene Transfer Method Using GFP 1 × 10 6 GMI-R1 cells were seeded in a petri dish, and 16 hours later, a GFP expression vector pEGFP10 μ
g (manufactured by Clontech) was added to the medium for gene transfer, this medium was exchanged and the cells were cultured in a CO 2 incubator at 37 ° C. for 24 hours, and then for 7 days together with rGM-CSF in the medium for microglia. Cultured. After culturing for 7 days, the cells are suspended and the fluorescent activated cell sorter (fluorescent activated cell sorte
r, FACS, Becton-Dickinson (Becto
n-Dickinson) FACS Calibu
In r), the cells having fluorescence intensity 100 times or more that of the non-transfected cells were fractionated and concentrated.
Culture was continued with GM-CSF. Do the same operation 7
Repeated twice more each day with almost 90% or more cells
Since it could be collected in a fraction having about 00 times fluorescence, this was seeded into the TP96 test plate at the rate of 1 cell / well by the cell limiting dilution method, and rGM-was added to the medium for microglia.
It was further cultured with CSF. As a result, it was possible to isolate the microglia that stably and constitutively express the transgene pEGFP. An example of the results of FACS analysis of cells showing how they could be separated by this method is shown in the figure. FIG. 7 shows FA of cells into which no gene has been introduced.
Fig. 8 shows the results of CS analysis, and Fig. 8 shows FA of GFP-introduced cells.
It is a result of CS analysis.

[0031]

INDUSTRIAL APPLICABILITY The present invention provides established cell line microglia having a specific affinity for brain. The established microglia of the present invention is useful not only as a carrier (carrier) for introducing a gene into the brain, but also as a carrier (carrier) for brain-specific introduction of a chemical substance such as a drug.

[0032]

[Sequence list]                          SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Established microglia cell lines <130> PC903595 <160> 4 <170> PatentIn version 3.1 <210> 1 <211> 25 <212> DNA <213> Artificial <220> <223> IL-1 sense primer <400> 1 atggcaactg ttcctgaact caact 25 <210> 2 <211> 26 <212> DNA <213> Artificial <220> <223> IL-1 antisense primer <400> 2 caggacaggt atagattctt tccttt 26 <210> 3 <211> 25 <212> DNA <213> Artificial <220> <223> IL-6 sense primer <400> 3 atgaagttcc tctctgcaag agact 25 <210> 4 <211> 24 <212> DNA <213> Artificial <220> <223> IL-6 antisense primer <400> 4 cactaggttt gccgagtaga tctc 24

[Brief description of drawings]

FIG. 1 is a photograph (morphology of an organism) showing the morphology of established microglia of the present invention.

FIG. 2 is a photograph showing a difference in tissue specificity between the established microglia of the present invention and macrophages (morphology of organism).

FIG. 3 is an electrophoretic photograph showing the expression of cytokines stimulated by lipopolysaccharide.

FIG. 4 is a diagram showing GM-CSF-dependent proliferation of established cell line microglia of the present invention.

FIG. 5 is a photograph showing gene expression in rat brain (morphology of organism).

FIG. 6 is a diagram showing gene expression in rat brain.

FIG. 7: FACS of cells to which no gene has been introduced
It is a figure which shows the result of analysis.

FIG. 8 is a diagram showing the results of FACS analysis of GFP-introduced cells.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 C12Q 1/02 4C087 A61P 25/00 C12N 5/00 B C12Q 1/02 A61K 37/02 / / C12N 15/09 ZNA C12N 15/00 ZNAA F term (reference) 4B024 AA01 AA11 CA02 DA02 EA04 FA10 GA11 HA11 4B063 QA01 QQ08 QQ13 QQ79 QR32 QR77 QR80 QS36 QS38 QX02 4B065 AA90Y AA92X76A08B4ABC0BBD AC01 BD20 AC08B4ABC0BAB0AC4BAD4AC50B02AC08B4AC01 AA13 AA16 MA66 NA14 ZA022 4C087 AA01 AA02 AA03 BB45 MA66 NA14 ZA02

Claims (14)

[Claims] 1. An established microglia having the property of specifically migrating to the brain when administered to a blood vessel. 2. The established microglia according to claim 1, which further has the following properties. (A) Morphology: Macrophage-like or globular morphology in the presence of granulocyte-macrophage colony stimulating factor, and / or branched morphology similar to branched microglia existing in the brain in the absence of the factor. It has one of the forms. (B) Functional characteristics: It has a specific affinity for the brain. It has a strong phagocytosis. (C) Cell proliferation property: Proliferates depending on granulocyte-macrophage colony stimulating factor. 3. A method for separating the established microglia of claim 1 or 2 from the microglia in the presence of a cytokine. 4. The method according to claim 3, wherein the cytokine is GM-CSF. 5. The method according to claim 4, wherein GM-CSF is a recombinant type. 6. The method according to claim 3, which is further carried out in the presence of IL-3 and / or purified astrocyte culture supernatant. 7. The established microglia according to claim 1, wherein a gene or drug is introduced. 8. The established cell line microglia according to claim 7, wherein a gene has been introduced. 9. The microglia according to claim 7 or 8,
And a pharmaceutical composition comprising a pharmaceutical carrier. 10. The pharmaceutical composition according to claim 9, which is a therapeutic agent for brain diseases. 11. A method of introducing a foreign gene and a gene expressing a fluorescent protein into the established cell line of microglia according to claim 1 or 2, and screening the gene-introduced microglia with the fluorescent protein. 12. The method according to claim 12, wherein the gene that expresses the fluorescent protein is derived from jellyfish. 13. A method for producing a cell line microglia according to claim 8, wherein a gene is introduced into the cell line microglia according to claim 1 or 2 by introducing a gene expressing a foreign gene and a fluorescent protein. . 14. The method according to claim 13, wherein the gene that expresses the fluorescent protein is derived from jellyfish.