JP2005168492A - Immortalized mast cell strain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immortalized mast cell strain keeping functions and characteristics inherent in a mast cell, to provide an establishing method for establishing the cell strain, and to provide a method for screening a useful substance, using the immortalized mast cell strain. <P>SOLUTION: This immortalized mast cell strain which expresses an FcεRI molecule and a c-kit molecule on a surface of the cell, richly stores granules in cytoplasm of the cell, and has degranulation ability and Ca<SP>2+</SP>concentration increase-inducing ability brought about by activation due to co-crosslinking of the FcεRI is established by treating and hemolyzing a myeloid cell of a transgenic mouse into which a large T antigen gene of a temperature-sensitive mutant strain tsA58 of a VS40 is introduced, then culturing the obtained cell in the presence of an IL-3, so as to differentiate and induce the mast cell, and further repeating successive subculture. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an immortalized mast cell line derived from bone marrow, and in particular, a mast cell derived from the bone marrow of a transgenic mouse (ts SV40 LT Tg mouse) into which a large T antigen gene of SV40 temperature-sensitive mutant tsA58 has been introduced. The present invention relates to an immortalized mast cell line (SVMC) that can be established by subculturing (mast cell), a production method thereof, and use thereof. The immortalized mast cell line of the present invention can be applied to in vitro analysis of mast cells and studies of in vivo allergic reactions and local inflammatory reactions by mast cells.

  Conventionally, animals have been mainly used for research and studies on the safety and efficacy of pharmaceuticals, but instead of using animals from the viewpoint of animal welfare, the efficacy and safety of pharmaceuticals in vitro using cultured cells, etc. Research is being conducted at a practical level of technology for testing and researching. For example, an animal test is performed after testing in advance by a method using a primary cultured cell collected from a living tissue or an immortalized cell (established cell) system that grows indefinitely. However, primary cells proliferate well in the initial stage, but gradually stop with subculture and eventually die (this phenomenon is called cell aging). Furthermore, it has been pointed out that primary cells change with passage in addition to the fear that their characteristics differ each time they are collected from living tissue. In particular, when the growth rate is very slow or when it is derived from a micro-organ, it is said that it is very difficult to obtain primary cells sufficient for the test.

  On the other hand, immortalized cells that have acquired the ability to proliferate indefinitely by avoiding cell aging through repeated passages of primary culture will have stable and uniform characteristics, but many of these immortalized cells Loses some or all of the forms and functions that the cells originally had in the body. Therefore, in tests using such an immortalized cell line, it has been difficult to accurately reflect the original characteristics of the tissue from which the cell line is derived. Therefore, the primary cells were transformed by introducing oncogenes such as ras gene and c-myc gene, adenovirus E1A gene, SV40 virus large T antigen gene, human papillomavirus HPV16 gene, etc. Attempts have been made to establish immortalized cells that retain their active proliferative ability and do not lose their inherent properties by further passage. However, even in such an immortalized cell, depending on the target organ, the primary cell is prepared, and when these oncogene and large T antigen gene are introduced, some functions are already lost. It has been difficult to obtain immortalized cells in a strict sense that retains the original function. In particular, it has been extremely difficult to prepare and establish primary cells when the growth rate is very slow or when they are derived from micro-organs.

  On the other hand, instead of introducing oncogenes and large T antigen genes into individual cells using recently established gene transfer techniques to individual animals, transgenic animals in which these genes are stably integrated into the chromosome And preparing an immortalized cell by substituting a primary cell from an animal organ that already has an oncogene or a large T antigen gene in the cell at the time of development It has been reported. In particular, immortalized cells obtained from the organs of ts SV40 LT Tg mice can be manipulated by changing the temperature of their growth and expression of differentiation traits, and are therefore considered to be very effective (for example, non-patented Reference 1-8).

  On the other hand, mast cells are widely distributed in the boundary with the external world, such as subcutaneous tissue, respiratory tract, esophagus, and stomach / intestinal mucosa, and play an important role in the recognition and elimination of foreign substances such as pathogens. . In addition, it is known that in response to various stimuli, chemical mediators such as histamine, leukotriene, serotonin, heparin, leukocyte attracting factor, glucuronic acid degrading enzyme, hexose amine degrading enzyme are released to cause allergic reactions in the living body. In general, mast cells can be easily induced from bone marrow cells in the presence of cytokine (IL-3), but are difficult to maintain by long-term culture and lose many activities such as mitotic ability and degranulation. Therefore, a mast cell line that can be cultured semi-permanently while maintaining the same properties as the primary culture is considered to be a very useful tool for functional analysis of mast cells. Conventionally, as a mast cell line, rat mast cell line RBL-2H3 (can be maintained in the absence of IL-3, and is used for degranulation experiments because it expresses a gene transfer host and IgE receptor) Mouse mast cell line PT18, mouse cancerous mast cell line P-815 (IL-3 receptor is always activated and can be maintained in the absence of IL-3 in the same manner as RBL-2H3. IL-3 dependent mouse mast cell line MCP-5, mast cell Mcε27 cloned by limiting dilution method from human undifferentiated mast cell line HMC-1 and the like are known.

Transgenic Research 4, 215-225, 1995 Genes to Cells, 2, 235-244, 1997 Exp. Cell Res., 197, 50-56, 1991 Exp. Cell Res., 209, 382-387, 1993 Exp. Cell Res., 218, 424-429, 1995 Blood, 86, 2590-2597, 1995 J. Cell. Physiol., 164, 55-64, 1995 Exp. Hematol., 27, 1087-1096, 1999

  Mast cells are cells that can be easily induced from bone marrow cells in the presence of cytokines such as IL-3. However, maintenance by long-term culture is difficult, and many activities such as division ability and degranulation are lost. An object of the present invention is to provide an immortalized mast cell line that retains the functions and characteristics inherent to mast cells, a method for establishing the immortalized mast cell line, and a screening method for useful substances using the immortalized mast cell line.

In order to establish an immortalized mast cell line, the present inventors used SV40 large T antigen transgenic mice produced by Yanai et, al. In this transgenic mouse, a gene encoding a large T antigen involved in immortalization of cells possessed by the SV40 virus was integrated into the genome, and it was considered that a cell line could be established. Therefore, bone marrow cells were collected from the bone marrow of 8-week-old SV40 large T antigen transgenic mice, and 5% CO ² , 33 ° C. in a medium containing 5 ng / ml of IL-3, which is a cytokine that induces mast cell differentiation. Cultivate in incubator for more than 4 weeks, and then pass through and maintain in normal incubator with 5% CO ² , 37 ° C incubator, have immortal growth ability, and release serotonin by FcεRI co-crosslinking And 3 clones in which FcεRI ⁺ and c-kit ⁺ cells were a single population were selected. When these three cell lines were expanded and analyzed in detail, it was confirmed that the properties of the primary cultured mast cells were maintained substantially, and the present invention was completed.

That is, the present invention expresses an immortalized mast cell line derived from bone marrow (Claim 1), FcεRI molecule and c-kit molecule on the cell surface, and abundant accumulation of granules in the cytoplasm, The immortalized mast cell line (claim 2) according to claim 1, which has degranulation ability and Ca ²⁺ concentration increase ability by activation by co-crosslinking of FcεRI, and can grow at 33 ° C. However, the presence of NGF and SCF, or an immortalized mast cell line according to claim 1 or 2, wherein growth is partially suppressed at 37 ° C and growth is stopped at 39 ° C. The immortalized mast cell line according to any one of claims 1 to 3 or the rodent origin, which is differentiated into a connective tissue type mature mast cell by culturing under conditions. Of claims 1 to 4 The immortalized mast cell line according to any one of claims (Claim 5) or the immortalized mast cell line according to Claim 5 (Claim 6), wherein the rodent is a mouse.

The present invention also includes SVMC clone a (FERM AP-20255), SVMC clone c (FERM AP-20256), SVMC clone E (FERM AP-20257), or an SVMC mixture (FERM AP before separation of these clones). The bone marrow cells of an immortalized mast cell line (Claim 7) according to claim 6 or a transgenic mouse into which a large T antigen gene of SV40 temperature-sensitive mutant tsA58 has been introduced. After hemolysis treatment, mast cells are differentiated and induced by culturing the obtained cells in the presence of IL-3, and subculture is repeated to express FcεRI molecule and c-kit molecule on the cell surface. abundantly retained in the cytoplasm, degranulation ability and Ca ²⁺ concentration increased by activation by co-crosslinking of FcεRI A method for producing an immortalized mast cell line characterized by establishing a cell line having a conductivity (Claim 8), and can be grown at 33 ° C., but at 37 ° C., the growth is partially suppressed, The immortalized mast cell line according to claim 7, wherein the cell line is established at 39 ° C to stop growing and to differentiate into connective tissue type mature mast cells by culturing in the presence of NGF and SCF. The present invention relates to a manufacturing method (claim 9).

Furthermore, the present invention is characterized by culturing the immortalized mast cell line according to any one of claims 1 to 6 in the presence of a test substance, and measuring and evaluating the degree of expression of a mature marker protein in the cell line. A method for screening a substance for promoting or suppressing cell growth in mast cells (claim 10), or the marker protein is an FcεRI molecule and / or a c-kit molecule. A screening method for a growth promoting or inhibiting substance (Claim 11) or culturing the immortalized mast cell line according to any one of Claims 1 to 6 in the presence of a test substance, and measuring the degree of proliferation of the cell A screening method for a substance that promotes or suppresses cell growth in mast cells (claim 12), or in the presence of a test substance, And co-crosslinking immortalized mast cell line described by FcεRI or Re, degranulation ability and / or Ca ²⁺ activation promoting or inhibiting substances of the mast cells, characterized by the degree of concentration increases inducibility to measure and evaluate The immortalized mast cell line according to any one of claims 1 to 6 is cultured in the presence of NGF and SCF in the presence of a test substance, and in the presence of a test substance. The present invention relates to a screening method for a mast cell maturation promoting or inhibiting substance characterized by observing and evaluating the degree (claim 14).

  According to the present invention, an immortalized mast cell line (SVMC) having the same surface markers and functions as those of primary cultured cells can be established from SV40 large T antigen transgenic mice. Such an immortalized mast cell line has characteristics not found in conventional mast cell lines that differentiate from immature mast cells to mature mast cells, and is useful as a material for mast cell function and differentiation studies.

The immortalized mast cell line of the present invention may be any immortalized mast cell line derived from bone marrow, can be grown at 33 ° C., and the growth is partially suppressed at 37 ° C., Cell lines that stop growing at 39 ° C. are preferred, and those having the properties inherent to mast cells are more preferred except for this temperature sensitivity. For example, cell lines that express FcεRI molecules and c-kit molecules on the cell surface, accumulate abundant granules in the cytoplasm, and have the ability to induce degranulation and Ca ²⁺ concentration increase by activation by co-crosslinking of FcεRI Suitable examples include cell lines that differentiate into connective tissue type mature mast cells by culturing in the presence of NGF (Nerve Growth Factor) and SCF (Stem Cell Factor), and cell lines that have these properties. Can do. Specific examples of such immortalized mast cell lines include three types of immortalized mast cell lines (SVMC) clones a, c, and E. The SVMC clone a has the receipt number FERM AP-20255, the SVMC clone c has the receipt number FERM AP-20256, the SVM clone E has the receipt number FERM AP-20257, and these three types of the SVMC clone (clone) a , C, E before the separation of the SVMC Bulk (SVMC Bulk) as the receipt number FERM AP-20254 on October 19, 2004 National Institute of Advanced Industrial Science and Technology patent biological deposit center (〒305-5466 1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture 6th) The origin of the immortalized mast cell line of the present invention is not particularly limited, but the immortalized mast cell line obtained from an animal such as a rodent such as a mouse has a pathological model rich in mice and has a pharmacological action. It is preferable because it is widely used for the evaluation of the above. Hereinafter, the method for producing an immortalized mast cell line of the present invention will be described taking a method using a mouse as an example.
SVMC bulk represents.

The mouse-derived immortalized mast cell line of the present invention is obtained by, for example, treating bone marrow cells of SV40 large T antigen transgenic mice with ammonium chloride and then treating the resulting cells with 5 ng / mL mouse recombinant IL-3. Incubate with RPMI medium (10% FCS) containing Mast, induce mast cells, repeat subculture for more than a month, express FcεRI molecule and c-kit molecule on the cell surface, abundant granules in the cytoplasm Can be produced by establishing a cell line having degranulation ability and Ca ²⁺ concentration-inducing ability by activation by co-crosslinking of FcεRI.

  In addition, SV40 large T antigen transgenic mice can be prepared as follows. Plasmid pSVtsA58 (−)-2 (OhnoT. Et al., Cytotechnology 7) in which the entire DNA of tsA58ori (−)-2 from which the SV40 replication origin (ori) has been deleted was opened with restriction oxygen BamHI and introduced into pBR322. 165-172, 1991) in a large amount in E. coli according to a conventional method, this amplified plasmid is cleaved with restriction enzyme BamHI to remove the vector site, and a DNA fragment having a large T antigen gene of tsA58 is prepared. . The gene fragment having the large T antigen gene of SV40 temperature-sensitive mutant tsA58 is introduced into all cells by introducing the DNA fragment containing the promoter of the large T antigen gene into a totipotent mouse cell according to a conventional method. Mice, i.e. transgenic mice, can be created. Such transgenic mice express the large T antigen gene of tsA58 in all somatic cells. Specific examples of the totipotent cells include fertilized eggs and early embryos, ES cells having multipotency, and the like. In addition, as a method for introducing DNA into totipotent cells, a known gene introduction method such as a microinjection method, an electric pulse method, a liposome method, or a calcium phosphate method can be used.

  The nucleus of totipotent cells (cultured cells) of the mouse is transferred to an enucleated unfertilized egg and initialized (nuclear transfer) to introduce the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 into the egg. be able to. In addition, an oocyte obtained by microinjecting the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 into the male pronucleus of a pronuclear-stage fertilized egg was transplanted into an oviduct of a foster parent, and then a litter was obtained. A transgenic mouse in which the large T antigen gene of tsA58 has already been integrated into the chromosome of the cell of each tissue at the time of ontogeny, That is, transgenic mice can be produced efficiently.

  The immortalized mast cell line of the present invention retains a permanent growth ability at 33 ° C., partially suppresses growth at 37 ° C., and stops growth at 39 ° C. It has the feature that can be controlled. In addition, this immortalized mast cell line shows good growth at 33 ° C. even when subcultured for 20 months or more, and retains the function as a mast cell. The immortalized mast cell line of the present invention can continue to proliferate stably, and has the ability to induce degranulation and Ca2 + concentration increase by activating the mast cell by co-crosslinking of FcεRI. Research on the function and differentiation of, more specifically, research that inhibits the binding of high affinity IgE receptor (FceRI) and IgE, research that inhibits the release of granules (degranulation) after stimulation of mast cells, It is useful for research that specifically inhibits the biosynthesis of chemical mediators in mast cells, and as shown below, it can be used for screening of useful substances for mast cells. It is also useful as a base.

As a screening method in the present invention, the immortalized mast cell line of the present invention is cultured in the presence of a test substance, and FcεRI molecule, c-kit molecule, tryptase, major basic protein (Major Basic Protein) in the cell line is cultured. ), Screening method for cell growth promoting or inhibiting substance in mast cells to measure and evaluate the expression level of marker protein such as MIST (Mast Cell-specific Immunoreceptor Signal Transductor), and measuring and evaluating the degree of proliferation of the cell line Of mast cells to promote or suppress mast cell activation and mast cell activation by measuring and evaluating the degree of degranulation and Ca ²⁺ concentration increase by co-crosslinking the cell line with FcεRI Alternatively, a screening method for an inhibitory substance, or the cell line is cultured in the presence of NGF, SCF, etc. It can be mentioned screening method etc. maturation promoting agent or an inhibitory substance of the mast cells to observe and evaluate the degree of melanin staining. And the maturation promoting substance in the mast cell obtained by the screening method, the cell growth promoting substance in the mast cell, and the mast cell activation promoting substance are also included in the present invention.

Screening for a substance that promotes or suppresses cell growth in the mast cells is carried out by culturing an immortalized mast cell line in the presence of various concentrations of the test substance, and detecting and measuring the amount of the marker protein expressed after culturing for a certain time. This is carried out by comparing and evaluating with a control cultured in the absence of the test substance. For example, FcεRI molecules, c-kit molecules, and the like, which are marker proteins expressed on the surface of mast cells, can be measured by immunochemical detection using a specific antibody by a conventional method. Moreover, it can also measure by detecting the expression level of mRNA corresponding to these by a conventional method. In other embodiments of mast cells, screening for substances that promote or suppress cell growth involves culturing immortalized mast cell lines in the presence of various concentrations of test substances, and measuring the number of cells and cell morphology after culturing for a certain period of time.・ Analysis is performed by comparing and evaluating with a control cultured in the absence of the test substance. In addition, screening of a mast cell activation promoting or suppressing substance is carried out by binding IgE to FcεRI expressed on the surface of an immortalized mast cell line in the presence of various concentrations of a test substance, followed by anti-IgE antibody Measure the degree of release (degranulation) of chemical mediators cross-linked and retained in the granules, measure the degree of increase in intracellular Ca ²⁺ concentration, and control in the absence of the test substance It is done by comparing and evaluating with. In addition, screening for substances that promote or suppress mast cell maturation involves culturing immortalized mast cell lines in the presence of various concentrations of test substances and NGF or SCF, and observing the degree of safranin staining of the cells. This is carried out by comparing and evaluating with a control cultured in the absence of the test substance.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Creation of transgenic mice)
A transgenic mouse into which DNA of SV40 temperature-sensitive mutant tsA58 was introduced was produced by the following procedure.

(Preparation of transgene)
For microinjection, a genomic DNA of SV40 temperature-sensitive mutant tsA58 was modified by genetic engineering techniques. The genomic DNA of tsA58 was opened with the restriction enzyme BamHI, introduced into the BamHI site of pBR322, the SfiI sequence was converted to SacII, and the DNA clone pSVtsA58ori, which had the SV40 replication origin (ori) deleted as ori (-) ( -)-2 (Ohno T. et al., Cytotechnology, 165-172, 1991), DNA for introduction was prepared according to a conventional method. That is, the plasmid DNA pSVtsA58ori (−)-2 obtained by amplifying in large quantities in E. coli was digested with the restriction enzyme BamHI (Takara Shuzo) and then agarose electrophoresis (1% gel; Boehringer) Then, the gel was dissolved, followed by phenol / chloroform treatment and ethanol precipitation treatment to recover DNA. The recovered purified DNA was dissolved in TE buffer (10 mM Tris-HCl containing 1 mM EDTA; pH 7.6) to obtain a solution containing 170 μg / ml purified DNA. This DNA solution was diluted with an injection buffer (10 mM Tris-HCl containing 0.1 mM EDTA; pH 7.6) to 5 μg / ml to prepare an injection DNA solution. The prepared DNA solution was stored at −20 ° C. until the injection operation.

(Production of SV40 large T antigen transgenic mice)
Microinjection of the prepared DNA solution for injection into mouse pronuclear fertilized eggs was performed as follows. Sexually mature 8-week-old Wistar mice are bred at a light / dark cycle of 12 hours (4: 0 to 16:00 light time), at a temperature of 23 ± 2 ° C. and a humidity of 55 ± 5%. Observed and selected the day of hormone treatment. First, 150 IU / kg pregnant horse serum gonadotropin (manufactured by Zenyak Co., Ltd .; PMS gonadotropin: PMSG) was intraperitoneally administered to female mice, and 75 IU / kg human chorionic property 48 hours later. After gonadotropin (manufactured by Sankyo Organs Co., Ltd .; human chorionic gonadotropin: hCG) was administered to perform superovulation, mating was carried out by cohabitation with males. Pronuclear fertilized eggs were collected by oviduct perfusion 32 hours after hCG administration. MKRB solution (Toyoda Y. and Chang MC, J. Reprod. Fertil., 36, 9-22, 1974) was used for oviduct perfusion and egg culture. The collected fertilized eggs were subjected to enzyme treatment at 37 ° C. for 5 minutes in mKRB solution containing 0.1% hyaluronidase (Sigma; Hyaluronidase Typel-S) to remove cumulus cells, and then 3 times with mKRB solution. It washed to remove the enzyme, CO ² until DNA injection operation - incubator (5% CO ² -95% of the Air, 37 ° C., saturated humidity) and stored in. The DNA solution was injected into the male pronucleus of the mouse fertilized egg thus prepared. The injected 228 eggs were transplanted to 9 foster parents to give birth and 80 offspring were obtained. For introduction of the injected DNA into the mouse, DNA prepared from the tail obtained by cleaving immediately after weaning was assayed by PCR [Primer used: tsA58-1A, 5′-TCTCATAATTGGCAGTCAGGGTG-3 ′ (from 1365 to 1384 sites). Corresponding: SEQ ID NO: 1), tsA58-1B, 5′-ATGACGAGCTTTGGCACTTG-3 ′ (corresponding to 1571 to 1590 sites: SEQ ID NO: 2)]. As a result, 11 lines of transgenic mice (6 males, 8 females, 6 gender unknown) pups that survived to the age of 12 weeks after the sexual maturation period (20 males, 8 females, 6 gender unknown). Male lines: # 07-2, # 07-5, # 09-6, # 12-3, # 19-5, female lines: # 09-7, # 11-6, # 12-5, # 12-7 , # 18-5, # 19-8). These G0 generation transgenic mice and Wistar mice were crossed, and two lines of male founders (# 07-2, # 07-5) and three lines of female founders (# 09-7, # 11-6, # 19) In -8), gene transfer to the next generation and later was confirmed.

(Separation and preparation of immortalized mast cell lines from mouse bone marrow)
Two strains of 8-week-old SV40 large T antigen transgenic mice and C57BL / 6 mice were used. In addition, a cell (Table 1) containing 5 ng / mL of mouse recombinant IL-3, which is a cytokine that induces mast cell differentiation, was obtained by treating cells obtained by treating erythrocyte lysis with 0.144 M ammonium chloride. Using a T75 flask at a density of 5.0 × 10 ⁵ cells / ml, incubate for 4 weeks or more in a 5% CO ² , 33 ° C. incubator, and then normal primary mast cells in a 5% CO ² , 37 ° C. incubator. Passage and maintenance as well.

  The immortalized mast cell line was established by diluting to 1.0 cell / well, 0.5 cell / well, and 0.2 cell / well and seeding in a 96-well plate. From 1 cell / well and 0.5 cell / well, 18 wells were seeded in 120 wells, and from 0.2 cell / well, cell growth was seen from 18 wells in 240 wells. It was. Eventually, 3 clone a (FERM AP-20255) with excellent proliferative ability, good serotonin release by FcεRI co-crosslinking, and a single population of FcεRI +, c-kit + cells , C (FERM AP-20256), E (FERM AP-20257). When these three types of immortalized mast cell lines (SVMC) were expanded and detailed analysis was performed as shown below, C57BL / 6 mouse primary culture mast cells (C57BL / 6 MC; B6 BMMC) have It turns out that the property is kept almost.

(Form observation of SVMC)
In order to observe the morphology of SVMC, Wright-Giemsa staining, Toluidine blue staining and Alcian blue / Safranin staining were performed. SVMC and C57BL / 6 MC were each adhered to the slide glass by cytospin, and the light Giemsa method (Light stain solution and Giemsa stain solution, both manufactured by Merck), Toluidine blue method (Toluidine blue stain solution manufactured by Chroma), and Alshan It was stained and visualized by the blue / safranin method (Arshan blue staining solution manufactured by Pierce, Safranin staining solution manufactured by Chroma). The results are shown in FIG. 1 (Reference Photo 1). In FIG. 1, SVMC bulk (FERM AP-20254) represents an SVMC mixture before separating three types of SVMC clones a, c, and E. Mast cells abundantly store granules containing various chemical mediators involved in allergies and inflammation in the cytoplasm. When three types of SVMC were attached to a slide glass with cytospin and stained, Wright-Giemsa staining showed abundant retention of granules (Fig. 1a), and Toluidine blue staining showed mast cell characteristics that are characteristic of mast cells (Fig. 1b) In Alcian blue / Safranin staining, typical characteristics of Alcian blue-positive mucosal mast cells were observed (FIG. 1c).

(Confirmation from SV40 large T antigen transgenic mouse)
When the SV40 large T antigen gene was amplified by PCR, bands were detected from three types of SVMC clones and confirmed to have a transgene (FIG. 2a; Reference Photo 2). For PCR, a primer sequence consisting of forward 5′-GGAGGAGGATAGAATGTTGAG-3 ′ (SEQ ID NO: 3) and reverse 5′-GTGTTGATGCAAGTTACGC-3 ′ (SEQ ID NO: 4) was used, and 94 ° C. for 30 seconds, 54 ° C. for 30 seconds, 72 ° C. for 30 minutes. The second was defined as one cycle, and the PCR was performed under 30 cycles of PCR. The SV40 large T antigen gene was not detected in the primary culture C57BL / 6 MC and the negative control rat mast cell line (RBL-2H3) and mouse mast cell line (P815 mastocytoma).

  In addition, the large T antigen protein of SVMC and C57BL / 6 MC was obtained by Western blotting (Experimental Medicine separate volume manual UP series "Cancer Research Protocol by Molecular Biological Approach" pages 108-115, Yodosha, 1995) Detected. Each cell line of SVMC and C57BL / 6 MC was washed with PBS, solubilized with 1 mL of solubilizing solution (1% SDS, 10 mM Tris, 1 mM EDTA, 10% glycerin), and then at 100 ° C. for 10 minutes. After heating, the insoluble fraction was removed by centrifugation (10000 rpm for 10 minutes), and then the total protein mass was quantified by the Floodford method (using BCA protein assay reagent A manufactured by PIERCE). 10 μg of each protein was separated by SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Anti-SV40 large T antigen mouse antibody (Ab-2; manufactured by Oncogene) as the primary antibody and HRP-labeled anti-mouse IgG antibody (produced by Birmingham) as the secondary antibody on the nitrocellulose membrane blocked with 3% skim milk solution A reaction specific to large T antigen was detected using an ECL Western blotting detection system (RPN2106M1) manufactured by Amersham. As a result, bands were detected from the three types of SVMC, and the presence of SV40 large T antigen was confirmed at the protein level (FIG. 2b; Reference Photo 3). Expression of SV40 large T antigen protein was not observed in C57BL / 6 MC as the primary culture and RBL-2H3 and P815 masstocytoma as the negative controls.

(Surface marker for SVMC)
It is known that mast cells highly express FcεRI and c-kit on the cell surface. Therefore, the expression of FcεRI and c-kit on SVMC was examined by flow cytometry. The cells were adjusted to 1.0 × 10 ⁶ cells / ml, 5 ng / ml mouse IgE (BD Biosciences) was added, cultured in a 37 ° C., 5% CO ² incubator for 2 hours, and IgE was bound to FcεRI on the cell surface. I let you. Thereafter, the cells were subjected to FACS buffer [9.8 g / l HANKS (NISSUI), 0.25 g / l NaHCO ³ (Wako Pure Chemical Industries), 2.0 g / l bovine serum albumin (SIGMA), 10 mM HEPES (pH 7.4) (sum) Photopure drug), 0.02% sodium azide (SIGMA)], and washed twice, 0.5 μg / 100 μl anti-mouse IgE antibody (BD Biosciences) and 0.2 μg / 100 μl PE-anti-mouse CD117 (c-kit) antibody ( BD Biosciences) for 10 minutes in ice. After washing twice with FACS buffer, the expression of FcεRI and c-kit was examined by flow cytometry BD-LSR (BD Biosciences). As a result, all three types of SVMC clones expressed FcεRI and c-kit at high levels (FIG. 3; Reference Photo 4).

(Growth factor requirement of SVMC)
Many immune system cells need to be cultured in a medium containing a specific growth factor when isolated outside the body. It is known that mast cells are required for IL-3 to maintain proliferation and function. Since growth factors are expensive, if they can be reduced as much as possible, it will lead to cost savings. SVMC has been cultured in a medium containing 5.0 ng / ml IL-3 from the beginning of induction from bone marrow cells, but it was cultured under conditions where this was reduced or eliminated, and [ ³ H] -thymidine incorporation was observed. We examined the proliferation of SVMC as an index. Cells were adjusted to a concentration of 0.6 × 10 ⁶ cells / ml in RPMI medium without IL-3. To a 96-well microplate, add 100 μl of medium with IL-3 concentration adjusted to 0, 0.2, 2.0, 10.0 and 20.0 ng / ml, and dispense 100 μl of cell suspension into each well. The final concentrations of IL-3 were 0, 0.1, 1.0, 5.0 and 10.0 ng / ml, respectively, and the cells were cultured at 37 ° C. in a 5% CO 2 incubator for 16 hours. Thereafter, 20 μl of 1 MBq / ml [ ³ H] -thymidine (Amersham) was dispensed into each well and again cultured at 37 ° C. in a 5% CO 2 incubator for 16 hours to incorporate [ ³ H] -thymidine into the cells. Thereafter, the cells were transferred to filter paper, and after sufficient drying, the specific activity was measured with MATRIX 9600 Direct Beta Counter (Packard instrument company), and the proliferation ability of the cells at each IL-3 concentration was examined using the amount of DNA synthesis as an index. . As a result, it was suggested that SVMC could be maintained at a lower concentration of IL-3 than normal primary cultured mast cells (B6BMMC) (FIG. 4; Reference Photo 5).

(Degranulation ability in SVMC)
FcεRI is composed of an α chain that binds IgE, and a β chain and a γ chain that are involved in the transmission of stimuli. Upon entry of an antigen, an IgE antibody against the antibody is produced in the body, and IgE binds to the α chain of FcεRI. (Sensitized state). When the same antigen enters again, adjacent FcεRI bound with IgE is cross-linked by the antigen to form a bridge structure (co-crosslinking). Co-crosslinking becomes a stimulus, β- and γ-chains are phosphorylated, stimulus-transmitting factors present in cells are mobilized, and intracellular proteins are phosphorylated to transmit stimuli in a relay state, some of which are cells Increases internal Ca2 + concentration, causing release of intracellular granules. As described above, it is known that mast cells are activated by co-crosslinking of FcεRI and release chemical mediators stored in the granules (degranulation). Accordingly, the degranulation ability of SVMC was examined using the release of serotonin labeled with ³ H as an index. As a result, all three types of SVMC clones retained higher degranulation ability than the primary mast cells (B6BMMC) (FIG. 5; Reference Photo 6).

(Intracellular Ca ²⁺ dynamics in SVMC)
It is known that FcεRI co-crosslinking of mast cells activates molecules that transmit various intracellular signals and induces an increase in intracellular Ca ²⁺ concentration. Therefore, the dye Indo-1AM (manufactured by SIGMA) that emits fluorescence by binding to Ca ²⁺ is incorporated into SVM, and the intracellular Ca ²⁺ concentration is increased by using the change in the fluorescence intensity of the co-crosslinking of FcεRI as an indicator. I investigated. Normally (Ca ²⁺ free) emits fluorescence around 500 nm, but in the Ca ²⁺ binding state, it emits fluorescence around 400 nm, and the increase in intracellular Ca ²⁺ concentration was measured using the change as an index. As a result, all of the three types of SVMC clones showed an increase in intracellular Ca ²⁺ concentration higher than that of primary cultured mast cells (C57BL / 6 MC) (FIG. 6; Reference Photo 7).

(Induction of connective tissue type mature mast cells)
It is known that when mast cells are cultured in the presence of growth factors such as NGF (Nerve Growth Factor) and SCF (Stem Cell Factor), they differentiate into connective tissue type mast cells positive for Safranin. Therefore, the cells were cultured for 3 weeks in the presence of IL-3, NGF and SCF to try to induce differentiation into connective tissue type mast cells. Using RPMI-1640 medium supplemented with 5.0 ng / ml recombinant IL-3, 50 ng / ml recombinant-NGF (R & D Systems, Inc.) and 100 ng / ml recombinant SCF (Peprotech EC. Ltd.), 2 The cells were cultured at a cell concentration of 0 × 10 ⁵ cells / ml. As soon as it became confluent, an equal amount of fresh medium was added, and the cells were transferred in the order of 35 mm dish, 100 mm dish, and T75 flask to grow the cells. A part of the cultured cells was collected at 4, 8, 12, 16 and 20 days after the start of culturing and stained with Alcian blue / safranin to examine the presence or absence of safranin positive cells. As a result, mast cells in primary culture (C57BL / 6 MC) were found to have connective tissue type mast cells positive for safranin after about 2 weeks in 1 clone (clone a) of 3 types of SVMC clones after about 1 week. (FIG. 7; Reference Photo 8).

(Summary)
From the above results, it was found that the three established immortalized mast cell lines SVMC almost have the properties of primary cultured mast cells. These mast cell lines SVMC have properties closer to primary mast cells than previously reported mast cell lines, and can induce differentiation from immature to mature mast cells. Therefore, the mast cell line SVMC of the present invention seems to be very useful for the study of mast cell function / differentiation.

It is a figure which shows the result of Wright-Giemsa dyeing | staining, Toluidine blue dyeing | staining, and Alcian blue / Safranin dyeing | staining of the immortalized mast cell line SVMC of this invention. It is a figure which shows the result of PCR of an immortalized mast cell line SVMC of this invention, and a Western blot. It is a figure which shows the expression result of Fc (epsilon) RI and c-kit on the immortalized mast cell line SVMC of this invention by flow cytometry. ^[3 H] - immortalized mast cell lines SVMC of the present invention by thymidine incorporation is a diagram showing that the IL-3 requirement. ³ is a graph showing the results of examining the degranulation ability of immortalized mast cell lines SVMC of H-labeled serotonin release invention as an index. It is a figure which shows the result of having investigated the raise of the intracellular Ca2 ⁺ density | concentration of the immortalized mast cell line SVMC of this invention which used the change of fluorescence intensity as a parameter | index. It is a figure which shows the result of the safranine staining which shows that the immortalized mast cell line SVMC of this invention is induced | guided | derived to a connective tissue type mature mast cell.

Claims (14)

An immortalized mast cell line characterized by being derived from bone marrow. It is characterized by expressing FcεRI and c-kit molecules on the cell surface, storing abundant granules in the cytoplasm, and having the ability to degranulate and induce Ca ²⁺ concentration increase by activation by co-crosslinking of FcεRI. The immortalized mast cell line according to claim 1. The immortalized mast cell line according to claim 1 or 2, wherein the cell line can grow at 33 ° C, but the growth is partially suppressed at 37 ° C, and the growth stops at 39 ° C. The immortalized mast cell line according to any one of claims 1 to 3, which is differentiated into a connective tissue type mature mast cell by culturing in the presence of NGF and SCF. The immortalized mast cell line according to any one of claims 1 to 4, which is of rodent origin. The immortalized mast cell line according to claim 5, wherein the rodent is a mouse. SVMC clone a (FERM AP-20255), SVMC clone c (FERM AP-20256), SVMC clone E (FERM AP-20257), or SVMC mixture (FERM AP-20254) prior to isolation of these clones. The immortalized mast cell line according to claim 6. After hemolysis of bone marrow cells of transgenic mice introduced with the large T antigen gene of SV40 temperature-sensitive mutant tsA58, the resulting cells are cultured in the presence of IL-3 to differentiate and induce mast cells. , Repeated subculture, expresses FcεRI and c-kit molecules on the cell surface, stores abundant granules in the cytoplasm, and activates FcεRI by cross-linking to induce degranulation and increase Ca ²⁺ concentration A method for producing an immortalized mast cell line, comprising establishing a cell line having Can grow at 33 ° C, but growth is partially suppressed at 37 ° C, growth stops at 39 ° C, and differentiates into connective tissue type mature mast cells by culturing in the presence of NGF and SCF The method for producing an immortalized mast cell line according to claim 8, wherein a cell line is established. In the presence of a test substance, the immortalized mast cell line according to any one of claims 1 to 7 is cultured, and the degree of expression of a mature marker protein in the cell line is measured and evaluated. Screening method for cell growth promoting or inhibiting substance. The method for screening a substance for promoting or suppressing cell proliferation in mast cells according to claim 10, wherein the marker protein is an FcεRI molecule and / or a c-kit molecule. Culturing the immortalized mast cell line according to any one of claims 1 to 7 in the presence of a test substance, and measuring or evaluating the degree of proliferation of the cell, promoting or inhibiting cell proliferation in mast cells Substance screening method. Co-crosslinking the immortalized mast cell line according to any one of claims 1 to 7 with FcεRI in the presence of a test substance, and measuring and evaluating the degree of degranulation ability and / or Ca ²⁺ concentration increase inducing ability A screening method for a mast cell activation promoting or inhibiting substance. The immortalized mast cell line according to any one of claims 1 to 7 is cultured in the presence of NGF and SCF in the presence of a test substance, and the degree of safranin staining of the cell is observed and evaluated. Screening method for mast cell maturation promoting or inhibiting substance.