JP2006265102A - METHOD FOR CONTROLLING APOPTOSIS DERIVED FROM TGFbeta - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for controlling apoptosis derived from TGFβ by identifying a protein useful in searching an apoptosis inhibitor and an apoptosis promoter and a gene encoding the protein and utilizing the gene. <P>SOLUTION: The method for controlling the apoptosis derived from TGFβ comprises controlling the expression and/or function of an apoptosis-inducing protein Bim. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

【図面の簡単な説明】
【図１】図１は、TGF-β1によるBimの発現誘導の実験結果を示す。
【図２】図２は、Bimのレポーターコンストラクションに対するSmadの効果に関する実験結果を示す。
【図３】図３は、Bimのレポーターコンストラクションの概要を示す。
【図４】図４は、TGF-β1によって誘導されるアポトーシスに対するBimの欠失変異体の効果に関する実験結果を示す。
【図５】図５は、Bimによって誘導されるアポトーシスに対するBimのドミナントネガティブ変異体の効果に関する実験結果を示す。
【図６】図６は、TGF-β1によって誘導されるアポトーシスに対するBimのドミナントネガティブ変異体の効果に関する実験結果を示す。
【図７】図７は、BimのRNAiを発現するvectorの構築の概要を示す。
【図８】図８は、Bimによって誘導されるアポトーシスに対するBim RNAiの効果に関する実験結果を示す。
【図９】図９は、TGF-β1によって誘導されるアポトーシスに対するBim RNAiの効果に関する実験結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for regulating apoptosis derived from TGFβ. More specifically, the present invention relates to a method for regulating apoptosis using a gene that is highly expressed when apoptosis is induced by TGF-β. The present invention also relates to a drug screening method.
[0002]
[Prior art]
Apoptosis refers to the process by which an observed cell dies in programmed cell death in cell and individual development, differentiation, maturation, or in cell death induced under various circumstances. Apoptosis is believed to occur under various physiological conditions. Its morphological characteristics can include lack of contact with surrounding cells, cytoplasmic enrichment, chromatin condensation and nuclear condensation related to endonuclease activity, nuclear segmentation, etc. Disappearance of microvilli, smoothing of the cell surface (cell surface blebbing) is also observed. In addition, the phenomenon of DNA fragmentation due to endonuclease activity has been observed, and it has been discussed as a mechanism by which the final fragment of apoptic cells is phagocytosed by neighboring cells [Immunology Today,7(4), 115-119 (1986)].
[0003]
Apoptosis has been found to have important implications in various diseases, and various attempts have recently been made to diagnose, prevent and treat these diseases by inducing or suppressing apoptosis of cells. Yes.
[0004]
Apoptosis, also called programmed cell death, refers to a type of cell death that occurs in many tissues as a normal physiological process, and is caused by the activation of a genetic program that is inherent in the cell itself. And finally, these apoptotic cells are removed by surrounding phagocytes without causing inflammation (Shigekazu Nagata, (1998) Experimental Medicine, Vol. 16, 1242-1246). Apoptosis is known to be involved in the development of serious diseases such as cancer, autoimmune diseases, and neurodegenerative diseases in addition to normal physiological processes. Therefore, development of a method for inducing or inhibiting apoptosis and a drug useful for these methods are eagerly desired as a treatment method for the above diseases. Regarding the induction mechanism of apoptosis, for example, Fas antigen and tumor necrosis factor receptor ligand bind to those receptors localized on the surface of animal cells to induce apoptosis (Nagata, S., ( 1997) Cell, 88, 355-365), and killing of cancer cells by anti-cancer drugs or X-ray irradiation occurs by activating intracellular pathways that induce apoptosis (Haldar, S., et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4507-4511) was elucidated in detail.
[0005]
[Non-Patent Document 1]
Immunology Today,7(4), 115-119 (1986)
[Non-Patent Document 2]
Shigetaka Nagata, (1998) Experimental Medicine, Vol.16, 1242-1246
[Non-Patent Document 3]
Nagata, S., (1997) Cell, 88, 355-365
[Non-Patent Document 4]
Haldar, S., et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4507-4511
[0006]
[Problems to be solved by the invention]
If a gene that is activated and specifically expressed when apoptosis is induced in a cell is provided, the expression level, structure, and function of each cell, and analysis of the expression product are performed to analyze apoptosis or its related. It becomes possible to elucidate the pathology of the disease, and to establish a diagnosis and treatment method thereof.
[0007]
A first object of the present invention is to identify a protein useful for searching for an apoptosis-inhibiting substance and an apoptosis-promoting substance and a gene encoding the same, and to regulate apoptosis derived from TGFβ by using the gene Is to provide.
A second object of the present invention is to provide a method for screening an apoptosis inhibitor or apoptosis promoting substance that acts by targeting the above gene, and an apoptosis inhibitor or apoptosis promoting substance.
[0008]
[Means for Solving the Problems]
The present inventors have intensively studied in order to solve the above-mentioned problems, and have advanced comprehensive isolation of genes induced by TGF-β using a microarray. In the process, Bim was identified as a gene showing a significant difference in expression level by TGF-β. The present inventors further studied and demonstrated that Bim's RNAi effect can suppress apoptosis induced by TGF-β. The present invention has been completed based on these findings.
[0009]
That is, according to the present invention, the following inventions are provided.
(1) A method for regulating apoptosis derived from TGFβ, comprising regulating the expression and / or function of the apoptosis-inducing protein Bim.
(2) A method for suppressing apoptosis, comprising inhibiting the expression and / or function of the apoptosis-inducing protein Bim.
(3) An apoptosis inhibitor comprising a substance that inhibits the expression and / or function of the apoptosis-inducing protein Bim.
(4) A method for promoting apoptosis, comprising increasing the expression and / or function of the apoptosis-inducing protein Bim.
(5) An apoptosis promoter comprising a substance that increases the expression and / or function of the apoptosis-inducing protein Bim.
[0010]
(6) A diagnostic agent for apoptosis, comprising an oligonucleotide comprising at least 10 consecutive nucleotides in the base sequence of the apoptosis-inducing gene Bim, or an antisense oligonucleotide having a sequence complementary to the oligonucleotide.
(7) An apoptosis inhibitor comprising an antisense oligonucleotide having a sequence complementary to an oligonucleotide comprising at least 10 consecutive nucleotides in the base sequence of the apoptosis-inducing gene Bim.
(8) An apoptosis inhibitor comprising a double-stranded RNA comprising at least 10 consecutive nucleotides in the base sequence of RNA transcribed from the base sequence of the apoptosis-inducing gene Bim, or a DNA encoding the same.
[0011]
(9) A method for screening a substance that suppresses or promotes apoptosis using changes in expression and / or function of the apoptosis-inducing protein Bim as an index.
(10) Cells having a gene encoding the apoptosis-inducing protein Bim are cultured together with TGF-β in the presence and absence of the test substance, and the expression of the apoptosis-inducing protein Bim according to the presence or absence of the test substance and The method according to (9), wherein a substance that suppresses or promotes apoptosis is screened using a change in function as an index.
(11) An RNAi reagent having SEQ ID NO: 6 and SEQ ID NO: 7.
UGGCUCUGUC UGUAGGGAGGG UAGGGGGCC (SEQ ID NO: 6);
GGCCCCUCACC UCCCUACAGA CAGAGCCA (SEQ ID NO: 7)
(12) An RNAi reagent comprising (UGGCUCUGUCUGUAGGGAGGUAGGGGCC (SEQ ID NO: 6))-(linker sequence)-(GGCCCCUCACC UCCCUACGACAGAGCCCA (SEQ ID NO: 7)).
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
(1) Apoptosis-inducing protein (Bim) and apoptosis-inducing gene (Bim) used in the present invention
The apoptosis-inducing protein (Bim) and apoptosis-inducing gene (Bim) used in the present invention used in the present invention are an apoptosis-inducing protein derived from any organism including mammals such as humans and the gene thereof. The sequence of the Bim gene is known in various organisms. For example, O'Connor, L., (1998) EMBO J, 17, 384-395, Mami, U., (2001) FEBS Letters, 509, 135-141 And Michela, M., (2002) Mol. Cell. Biol, 22, 3577-3589. To date, a number of Bcl-2 family molecules have been identified that are involved in the regulation of apoptosis. The structural feature of this family molecule is that it has some of the domains called BH1, BH2, BH3, and BH4 that have high homology to Bcl-2 and is classified into three subfamilies based on its structure and function . Bcl-2 subfamily (Bcl-2, Bcl-xL) with BH1, BH2, BH3, BH4 domains and anti-apoptotic action, Bax subfamily (Bax, with BH1, BH2, BH3 domains and pro-apoptotic action) , Bak), and BH3 only protein (Bim, Bik, Bad, Bid, Hrk / DP5, Noxa, Puma, Bmf) having only BH3 domain and pro-apoptotic action. BH3 only protein promotes apoptosis by binding to Bcl-2 and Bax subfamily via BH3 domain. The apoptotic gene (Bim) used in the present invention belongs to the BH3 only protein.
[0013]
In the present invention, it is particularly preferable to target human-derived apoptosis-inducing protein (Bim). The amino acid sequence and base sequence of human-derived apoptosis-inducing protein (Bim) are known, and examples thereof are shown in SEQ ID NOs: 1 to 4 in the sequence listing. Bim_SDNA base sequence and amino acid sequence are shown in SEQ ID NO: 1 and 2, Bim_ADThe DNA base sequence and amino acid sequence are shown in SEQ ID NOs: 3 and 4.
[0014]
In the present specification, the term “apoptosis-inducing protein (Bim) or apoptosis-inducing gene (Bim)” is not limited to the above-mentioned naturally occurring wild-type protein and wild-type gene, as long as it has an activity to induce apoptosis. All of their variants or homologues are intended to be included.
[0015]
Examples of the mutant protein or homologous protein include the following.
(A) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of a wild-type Bim protein and having an activity of inducing apoptosis; or
(B) a protein comprising an amino acid sequence having a homology of 95% or more with the amino acid sequence of the wild-type Bim protein and having an activity of inducing apoptosis:
[0016]
The range of “1 to several” in the “amino acid sequence in which one or several amino acids are deleted, substituted and / or added” in the present specification is not particularly limited. It means 1 to 10, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably about 1 to 3.
[0017]
The term “amino acid sequence having 95% or more homology” as used herein means that amino acid homology is at least 95% or more, and homology is preferably 96% or more, more preferably 97% or more.
[0018]
The term “protein having an activity to induce apoptosis” as used herein means a protein having an activity capable of promoting apoptosis induction by TGF-β.
[0019]
Whether or not a certain protein has an activity capable of promoting apoptosis induction by TGF-β is determined by, for example, introducing a DNA encoding the protein into a cell in which apoptosis can be induced by TGF-β and transiently overexpressing it. In addition, when TGF-β is added to the cells to induce apoptosis, it can be detected by examining whether or not the overexpression of the DNA increases the number of cells that induce apoptosis.
[0020]
Moreover, the following are mentioned as a mutant gene or a homologous gene.
(A) a gene encoding a protein consisting of a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence of a wild-type Bim gene and having an activity of inducing apoptosis; or
(B) A gene encoding a protein comprising a base sequence that hybridizes with a base sequence of a wild-type Bim gene under stringent conditions and having an activity of inducing apoptosis:
[0021]
The range of “one or several” in the “base sequence in which one or several bases are deleted, substituted and / or added” in the present specification is not particularly limited. It means 1 to 30, preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably about 1 to 5.
[0022]
As used herein, “hybridizes under stringent conditions” means that the probe is labeled under normal conditions (eg, DIG DNA Labeling kit (Cat No. 1175033, Boehringer Mannheim) at 32 ° C. In DIG Easy Hyb solution (Cat No. 1603558 manufactured by Boehringer Mannheim) and the membrane is washed in a 0.5 × SSC solution (containing 0.1% [w / v] SDS) at 50 ° C. Southern hybridization under the conditions (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate) is sufficient as long as it can hybridize to the Bim gene, as described in Molecular Cloning: A laboratory Mannual, 2^nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) And abbreviated as "Current Protocols in Molecular Biology").
[0023]
Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 80% or more, preferably 85% or more, more preferably 90%. More preferably, DNA having homology of 93% or more, particularly preferably 95% or more, and most preferably 98% or more is mentioned.
[0024]
(2) Acquisition of genes used in the present invention
The method for obtaining the Bim gene used in the present invention is not particularly limited. Appropriate probes and primers are prepared based on the information on the nucleotide sequences and amino acid sequences described in SEQ ID NOs: 1 to 4 in the sequence listing of the present specification, and a human cDNA library (the gene of the present invention is expressed using them). The desired gene can be isolated by selecting a desired clone from a suitable cell prepared according to a conventional method.
[0025]
The gene of the present invention can also be obtained by PCR. For example, chromosomal DNA or cDNA library derived from human cultured cells is used as a template, and PCR is performed using a pair of primers designed to amplify a desired Bim gene.
PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.
[0026]
The above-described procedures such as probe or primer preparation, cDNA library construction, cDNA library screening, and target gene cloning are known to those skilled in the art. For example, Molecular Cloning 2nd Edition, Current Protocols In -It can be performed according to the method described in Molecular Biology etc.
[0027]
A gene (mutant gene) comprising a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence of the Bim gene described above in the present specification and having an activity of inducing apoptosis. ) Can also be made by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques or mutagenesis. For example, mutant DNA can be obtained by using DNA having the base sequence of the Bim gene and introducing mutation into these DNAs. Specifically, it can be carried out using a method of bringing a Bim gene base sequence into contact with a mutagen drug, a method of irradiating with ultraviolet rays, a genetic engineering method, or the like. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular cloning 2nd edition, Current Protocols in Molecular. It can be performed according to the method described in biology and the like.
[0028]
As described above, in the base sequence of the Bim gene, various artificial treatments such as site-directed mutagenesis, random mutation by mutagen treatment, DNA fragment mutation by mutation, deletion, ligation, etc. Even if the DNA sequence is changed, any DNA can be used in the present invention as long as the DNA mutant encodes a protein having an activity of inducing apoptosis.
[0029]
The gene encoding a protein having a base sequence that hybridizes with the Bim gene base sequence described above in this specification under a stringent condition and having an activity of inducing apoptosis is a constant under the above-described conditions. It can be obtained by performing colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like under hybridization conditions.
[0030]
(3) An apoptosis inhibitor using an antibody against apoptosis-inducing protein (Bim)
According to the present invention, an apoptosis inhibitor using an antibody against apoptosis-inducing protein (Bim) is provided. The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it can specifically bind to the apoptosis-inducing protein.
[0031]
Polyclonal antibodies can be prepared by separating and purifying serum obtained from animals immunized with antigen. A monoclonal antibody is a hybridoma produced by fusing an antibody-producing cell obtained from an animal immunized with an antigen and a myeloma cell, and the hybridoma is cultured or administered to an animal to cause ascites tumor. Can be prepared by separating and purifying the culture fluid or ascites fluid.
[0032]
As an antigen, a protein of the present invention is purified from various types of human cultured cells, or a recombinant vector containing a DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 or a mutant sequence thereof or a part thereof is used in Escherichia coli, yeast. The protein can be prepared by introducing it into a host such as an animal cell or an insect cell and expressing the DNA to separate and purify it. The antigen can also be prepared by synthesizing a peptide having a partial sequence of the amino acid sequence shown in SEQ ID NO: 2 using an amino acid synthesizer.
[0033]
As the immunization method, the antigen may be directly administered subcutaneously, intravenously or intraperitoneally to a non-human mammal such as a rabbit, goat, rat, mouse or hamster. Appropriate adjuvants such as hemocyanin, bovine serum albumin, bovine thyroglobulin, etc., combined with a highly antigenic carrier protein, or Freund's complete adjuvant (Complete Freund's Adjuvant), aluminum hydroxide gel, pertussis vaccine, etc. It is also preferred to administer with.
[0034]
The antigen can be administered 3 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and whether or not the serum reacts with the antigen used for immunization is determined by measuring the antibody titer according to an enzyme immunoassay or the like. A non-human mammal whose serum exhibits a sufficient antibody titer against the antigen used for immunization can be used as a source of serum or antibody-producing cells. Polyclonal antibodies can be prepared by separating and purifying the above serum.
[0035]
The monoclonal antibody is produced by fusing the antibody-producing cells and myeloma cells derived from a non-human mammal to produce a hybridoma, and culturing the hybridoma or administering to the animal to cause the animal to undergo ascites tumor, It can be prepared by separating and purifying fluid or ascites. As antibody-producing cells, spleen cells, lymph nodes, antibody-producing cells in peripheral blood can be used, and spleen cells can be used particularly preferably.
[0036]
As myeloma cells, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (P3-U1) strain [Current Topics in Microbiology and Immunology, 18, 1-7 (1978) ], P3-NS1 / 1-Ag41 (NS-1) strain [European J. Immunology, 6,511-519 (1976)], SP2 / 0-Ag14 (SP-2) strain [Nature, 276, 269-270 (1978)] , P3-X63-Ag8653 (653) strain [J.Immunology, 123,1548-1550 (1979)], P3-X63-Ag8 (X63) strain [Nature, 256,495-497 (1975)] Cells can be used.
[0037]
Hybridoma cells can be prepared by the following method. First, antibody-producing cells and myeloma cells are mixed, suspended in a HAT medium [medium in which hypoxanthine, thymidine, and aminopterin are added to a normal medium] and then cultured for 7 to 14 days. After culturing, a portion of the culture supernatant is taken and an enzyme immunoassay or the like is selected that reacts with the antigen and does not react with the protein not containing the antigen. Subsequently, cloning is performed by a limiting dilution method, and a cell having a stable and high antibody titer determined by an enzyme immunoassay is selected as a monoclonal antibody-producing hybridoma cell. Monoclonal antibodies can be prepared by separating and purifying from a culture solution obtained by culturing hybridoma cells or from ascites obtained by administering the hybridoma cells into the peritoneal cavity of an animal to cause ascites cancer.
[0038]
Methods for separating and purifying polyclonal or monoclonal antibodies include centrifugation, ammonium sulfate precipitation, caprylic acid precipitation, or DEAE-Sepharose column, anion exchange column, protein A or G column, or gel filtration column. Examples of the method include a method of processing a single method or a combination of the methods based on lithography.
[0039]
In the present specification, an antibody includes not only a full-length antibody but also an antibody fragment. The antibody fragment is preferably a functional fragment, for example, F (ab ')₂, Fab 'and the like. F (ab ')₂, Fab ′ is produced by treating immunoglobulin with a proteolytic enzyme (for example, pepsin or papain), before and after the disulfide bond existing between two H chains in the hinge region. It is an antibody fragment produced by digestion.
[0040]
When the antibody of the present invention is used for the purpose of administration to humans, it is preferable to use a humanized antibody or a humanized antibody in order to reduce immunogenicity. These humanized antibodies and humanized antibodies can be prepared using mammals such as transgenic mice. Regarding humanized antibodies, for example, Morrison, SL et al. [Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)], Hiroshi Noguchi [Ayumi of Medicine 167: 457-462 (1993)]. 〕It is described in. A humanized chimeric antibody can be prepared by joining the V region of a mouse antibody and the C region of a human antibody by genetic recombination. Humanized antibodies can be produced by substituting regions other than complementarity determining sites (CDRs) from mouse monoclonal antibodies with sequences derived from human antibodies.
Furthermore, among the above, an antibody that can specifically bind to the apoptosis-inducing protein (Bim) identified in the present invention and inhibit its function can be used as an apoptosis inhibitor.
[0041]
When the antibody of the present invention is used as an apoptosis inhibitor in the form of a pharmaceutical composition, the antibody is used as an active ingredient, and further a pharmaceutically acceptable carrier, diluent (for example, an immunogenic adjuvant, etc.) A pharmaceutical composition can be prepared using a stabilizer or an excipient. Apoptosis inhibitors comprising the antibodies of the invention can be sterile filtered and lyophilized and formulated into dosage forms in dosage vials or stabilized aqueous preparations.
[0042]
Administration to a patient can be performed by methods known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. The dose of the antibody as the active ingredient is generally in the range of about 0.1 μg to 100 mg per kg body weight per time. The antibody of the present invention exhibits a therapeutic effect on diseases associated with the promotion of apoptosis by suppressing apoptosis.
[0043]
(4) Oligonucleotides and antisense oligonucleotides
Based on the base sequence information of the Bim gene revealed by the present invention, the expression of the Bim gene in various human tissues, for example, by using a probe or primer having a part or all of the base sequence of the gene. Can be detected. Detection of the expression of the Bim gene can be performed by a conventional method such as Northern blot or RT-PCR. The Bim gene identified in the present invention is a gene involved in the induction of cell apoptosis. As described later, since apoptosis is involved in various diseases, apoptosis-related diseases can be diagnosed by detecting the expression of the Bim gene.
[0044]
When PCR is performed, the primer is not particularly limited as long as it can specifically amplify only the Bim gene, and can be appropriately set based on the known sequence information of the Bim gene. For example, an oligonucleotide containing at least 10 consecutive nucleotides in the base sequence of the Bim gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide can be used as a probe or primer. More specifically, it has an oligonucleotide having a base sequence of 10 to 60 residues, preferably 10 to 40 residues in the base sequence shown in SEQ ID NO: 1, and a sequence complementary to the oligonucleotide. Antisense oligonucleotides can be used.
[0045]
The above-described oligonucleotides and antisense oligonucleotides can be produced by a conventional method using a DNA synthesizer. Examples of the oligonucleotide or antisense oligonucleotide include, for example, a sense primer corresponding to the base sequence on the 5 ′ end side and an antisense primer corresponding to the base sequence on the 3 ′ end side in a part of the base sequence of mRNA to be detected. Can be mentioned. The sense primer and the antisense primer are oligonucleotides whose melting temperature (Tm) and the number of bases do not change extremely, and include those having about 10 to 60 bases, those having about 10 to 40 bases. Is preferred. In the present invention, the above-described oligonucleotide derivatives can also be used, and for example, the methyl form or phosphorothioate form of the oligonucleotide can be used.
[0046]
Furthermore, in the present invention, apoptosis of a cell can be suppressed by introducing the above-described antisense oligonucleotide into the cell to suppress transcription and translation of the Bim gene. That is, according to the present invention, there is provided an apoptosis inhibitor comprising an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 10 consecutive nucleotides in the base sequence of the Bim gene.
[0047]
The method for introducing an antisense oligonucleotide into a cell can be performed in the same manner as the method for introducing a recombinant vector. Cell apoptosis detection methods are not particularly limited, and examples include microscopic observation, TUNEL method, DNA ladder detection method, quantification of DNA fragmentation rate, and cell size distribution measurement.
[0048]
In the microscopic observation, cell shape changes characteristic of apoptosis, for example, concentration of nuclei, condensation of chromatin, atrophy of organelles, formation of apoptotic bodies, contraction of whole cells, etc. are observed. The TUNEL method is a method of detecting a DNA cleavage end caused by apoptosis by labeling with biotin or digoxigenin using terminal deoxynucleotidyl transferase (TdT). The DNA ladder detection method detects the fragmentation of chromatin DNA caused by apoptosis by extracting DNA from cells and performing agarose gel electrophoresis, and the fragmented DNA is detected in a ladder form. . The quantification of the DNA fragmentation rate is a method in which only fragmented low molecular weight DNA is extracted and the DNA is quantified using diphenylamine. The cell size distribution measurement is a method in which cell shrinkage and fragmentation occur due to apoptosis and a small size cell increases using a particle size measuring device such as a Coulter multisizer. By these methods, it is possible to detect whether the cell is undergoing apoptosis.
[0049]
By using the antisense oligonucleotide of the present invention described above to suppress apoptosis by inhibiting transcription and / or translation of the Bim gene, a disease in which promotion of apoptosis is involved in the pathological condition (for example, Alzheimer's disease, etc.) Neurodegenerative diseases and the like). For example, an antisense oligonucleotide is administered by injection or the like to an affected area or whole body of a patient together with an appropriate carrier (for example, liposome or cholesterol) having a high affinity with a cell membrane, which is used to assist cellular uptake. However, the above-mentioned diseases can be treated by suppressing the apoptosis by incorporating into the patient's cells. The dosage of the antisense oligonucleotide, which is an active ingredient, is generally in the range of about 0.1 μg to 100 mg per kg of body weight per time.
[0050]
(5) Inhibition of apoptosis by RNAi
Furthermore, in the present invention, it is also possible to suppress apoptosis using RNAi instead of using the antisense oligonucleotide described in (4) above.
RNAi (RNAinterference) is a phenomenon in which the expression of a target gene is suppressed when RNA (double stranded RNA: dsRNA) in which a part of mRNA encoding a part of a target gene is double-stranded is introduced into a cell. .
[0051]
That is, according to the present invention, a double-stranded RNA comprising at least 10 consecutive nucleotides in the base sequence of RNA transcribed from the base sequence of the apoptosis-inducing gene (Bim gene) of the present invention, or a DNA encoding the same. Provided. Examples of DNA encoding double-stranded RNA include DNA having an inverted repeat sequence of an apoptosis-inducing gene (Bim gene) or a partial sequence thereof.
By introducing a DNA having such an inverted repeat sequence into a mammalian cell, the inverted repeat sequence of the target gene can be expressed in the cell, whereby the target gene (Bim gene) can be expressed by the RNAi effect. It becomes possible to suppress the expression.
Specific examples of the RNAi reagent of the present invention include RNAi reagents having SEQ ID NO: 6 and SEQ ID NO: 7, particularly RNAi reagents comprising (SEQ ID NO: 6)-(linker sequence)-(SEQ ID NO: 7).
[0052]
The inverted repeat sequence refers to a sequence in which the target gene and the reverse sequence thereof are arranged in parallel via an appropriate sequence. Specifically, when the target gene has a double strand consisting of n base sequences shown below,
5'-X₁X₂. . . . . . X_n-1X_n-3 '
3'-Y₁Y₂. . . . . . Y_n-1Y_n-5 '
The reverse sequence has the following sequence.
5'-Y_nY_n-1. . . . . . Y₂Y₁-3 '
3'-X_nX_n-1. . . . . . X₂X₁-5 '
(Here, in the base represented by X and the base represented by Y, those with the same subscript number are complementary bases)
[0053]
The inverted repeat sequence is a sequence in which the above two sequences are arranged through appropriate sequences. As the inverted repeat sequence, there are two cases where the target gene sequence is upstream of the reverse sequence and when the reverse sequence is upstream of the target gene sequence. The inverted repeat sequence used in the present invention may be any of the above, but preferably the inverted sequence is present upstream of the sequence of the target gene.
[0054]
The sequence existing between the sequence of the target gene and its reverse sequence is a region that forms a hairpin loop when transcribed into RNA. The length of this region is not particularly limited as long as a hairpin loop can be formed, but is generally 0 bp to 700 bp, preferably about 0 to 300 bp, more preferably about 0 to 100 bp. A restriction enzyme site may be present in this sequence.
[0055]
In the present invention, the inverted repeat sequence of the target gene can be expressed in the mammalian cell by incorporating the inverted repeat sequence of the target gene downstream of the promoter sequence operable in the mammal. The promoter sequence used in the present invention is not particularly limited as long as it is operable in mammals.
[0056]
Furthermore, according to the present invention, an apoptosis inhibitor containing the above-described double-stranded RNA or DNA is provided. By using the above-described double-stranded RNA or DNA to suppress the transcription and / or translation of the Bim gene to suppress apoptosis, a disease in which the promotion of apoptosis is involved in the pathological condition (for example, nerves such as Alzheimer's disease) Degenerative diseases) can be treated. For example, the above-mentioned double-stranded RNA or DNA is used in the affected area or whole body of a patient together with an appropriate carrier (for example, liposome, cholesterol, etc.) having a high affinity for the cell membrane, which is used for assisting cellular uptake. The above diseases can be treated by administering by injection or the like and taking them into the cells of the patient to suppress apoptosis. The dosage of the double-stranded RNA or DNA that is an active ingredient is generally in the range of about 0.1 μg to 10 mg per kg of body weight per time.
[0057]
(6) Control of apoptosis using the apoptosis-inducing protein of the present invention
As described in (3) Apoptosis inhibitor using antibody against apoptosis-inducing protein (Bim), (4) Oligonucleotide and antisense oligonucleotide, and (5) Inhibition of apoptosis by RNAi in the present specification, the present invention It is possible to suppress apoptosis by inhibiting the expression and / or function of the apoptosis-inducing protein (Bim). Thus, all methods for suppressing apoptosis by inhibiting the expression and / or function of the apoptosis-inducing protein of the present invention are within the scope of the present invention.
[0058]
Specific examples of methods for suppressing apoptosis include treatment of diseases associated with promotion of apoptosis, and methods for suppressing cell death due to apoptosis. For example, the following (a) production of useful proteins, and ( b) Examples of use described in transplanted cells / transplanted organs.
[0059]
(A) Production of useful proteins
Cell lines that produce useful proteins such as antibody-producing cell lines (hybridomas) and virus vector-producing cell lines may cause cell death due to the cytotoxicity of the proteins produced during long-term culture. Moreover, in these cell lines, protein production can be performed more efficiently by removing serum from the culture solution, but there is a problem that cell death is induced in the absence of serum. Many of these cell deaths are known to be due to apoptosis. Therefore, it is possible to increase the production efficiency of useful proteins by suppressing apoptosis by the method of the present invention.
[0060]
(B) Transplanted cells / transplanted organs
In the treatment of Parkinson's disease, a technique of transplanting dead fetal nerve cells and dopaminergic cells into the patient's brain is used. In addition, cell transplantation therapy is also performed in which autologous or other cells are introduced into patients, such as blood transfusion, bone marrow transplantation, and ex vivo methods in gene therapy. Transplanted cells generally cause cell death due to apoptosis or the like, and are difficult to maintain for a long time. Therefore, by suppressing apoptosis by the technique of the present invention, an effect can be achieved in organ transplantation and cell transplantation therapy.
[0061]
Furthermore, an apoptosis inhibitor containing a substance that inhibits the expression and / or function of the apoptosis-inducing protein of the present invention is also within the scope of the present invention. Specific examples of the substance that inhibits the expression and / or function of the apoptosis-inducing protein of the present invention include the antibody described in (4) above, the antisense oligonucleotide described in (5), and (6) The double-stranded RNA described in 1) or DNA encoding the same is included, but is not limited thereto.
[0062]
Further, contrary to the above, a method of promoting apoptosis by increasing the expression and / or function of apoptosis-inducing protein (Bim) is also within the scope of the present invention, and the expression and / or expression of apoptosis-inducing protein (Bim) is also within the scope of the present invention. Alternatively, pro-apoptotic agents including substances that increase function are also within the scope of the present invention.
Examples of a method for increasing the expression and / or function of apoptosis-inducing protein (Bim) include a method of introducing a gene encoding Bim into a cell, a factor that acts on the promoter region of the gene and increases its expression, or encoding the same For example, a method of introducing a gene into a cell.
[0063]
In addition, the presence or absence and the extent of apoptosis suppression or promotion are detected by techniques such as microscopic observation, TUNEL method, DNA ladder detection method, DNA fragmentation rate determination, and cell size distribution measurement described above in this specification. be able to.
[0064]
(7) Screening method for apoptosis regulator
By using changes in the expression and / or function of the apoptosis-inducing protein (Bim) identified by the present invention as an index, a substance that suppresses or promotes apoptosis can be screened. As an example of screening, cells having a gene encoding Bim are cultured together with TGF-β in the presence and absence of a test substance, and the expression and / or function of the Bim depending on the presence or absence of the test substance Using these changes as indicators, substances that suppress or promote apoptosis can be screened.
[0065]
Measurement of the expression level of the apoptosis-inducing protein at the mRNA level can be performed by a conventional method such as Northern blot or RT-PCR, and the measurement of the expression level at the protein level is described in (3) above. It can be performed by ordinary immunoassay such as Western blotting or ELISA using antibodies. Specifically, it can be performed by a conventional method known to those skilled in the art described in Molecular Cloning 2nd Edition or Current Protocols in Molecular Biology.
[0066]
The change in the function of the apoptosis-inducing protein can be analyzed by measuring whether or not apoptosis induction is suppressed or promoted. The above-described measurement of apoptosis induction can be performed by the above-described microscopic observation, TUNEL method, DNA ladder detection method, quantification of DNA fragmentation rate, cell size distribution measurement, and the like.
[0067]
Any substance can be used as a test substance to be subjected to the screening method of the present invention. The kind of the test substance is not particularly limited, and may be an individual low molecular synthetic compound, a compound present in a natural product extract, or a synthetic peptide. Alternatively, the test compound can also be a compound library, a phage display library or a combinatorial library. The test substance is preferably a low molecular compound, and a compound library of low molecular compounds is preferable. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.
[0068]
In addition to normal physiological processes, apoptosis is also involved in the development of serious diseases such as cancer, autoimmune diseases (eg systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, etc.), neurodegenerative diseases, etc. It is known that For example, malignant transformation of cancer cells is thought to be caused by inhibition of the process of apoptosis in cancer cells and the continued growth of cells that should be killed (Harris, CC, (1996) J. Natl. Cancer Instit., 88, 1442-1445). Autoimmune diseases develop because immune cells that respond to their own antigens, which should be removed by apoptosis during development, are no longer removed due to abnormal regulation of apoptosis (Rieux-Laucat). F. et al., (1995) Science, 268, 1347-1349). Neurodegenerative diseases (eg Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea) are thought to be caused by abnormally elevated apoptosis (Wolozin, B., et al., ( 1996) Science, 274, 1710-1713).
[0069]
Therefore, the above-mentioned apoptosis inhibitor of the present invention is considered to be particularly effective as a therapeutic agent for neurodegenerative diseases. Apoptosis-promoting substances are considered to be particularly effective as therapeutic agents for various cancers and autoimmune diseases such as rheumatism.
[0070]
The therapeutic agent for diseases involving apoptosis, which contains the apoptosis-suppressing substance and apoptosis-promoting substance according to the present invention as an active ingredient, can be administered orally or parenterally systemically or locally. Examples of parenteral administration methods include intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage varies depending on the age, administration route, and number of administrations, and can be appropriately selected by those skilled in the art.
[0071]
Examples of the dosage form suitable for parenteral administration include those containing additives such as stabilizers, buffers, preservatives, tonicity agents, and further containing pharmaceutically acceptable carriers and additives. But you can. Examples of such carriers and additives include water, organic solvents, polymer compounds (collagen, polyvinyl alcohol, etc.), stearic acid, human serum albumin (HSA), mannitol, thurbitol, lactose, surfactants, and the like. However, it is not limited to these.
[0072]
The following examples further illustrate the present invention, but the present invention is not limited to the examples.
[0073]
【Example】
Example 1: Screening of TGF-β target gene
2 × 10⁶To A549 cells, 10 ml of Opti MEM medium (0.02% FBS) was added, and after 24 hours, TGF-β1 (R & D) was added to 1 ng / ml and left for 1 hour. RNeasy Mini kit (QIAGEN) was used to collect total RNA, and Oligotex-dT30 <super> (JRS) was used to prepare poly (A) + RNA. Then, subtraction is performed by subtracting cDNA prepared from Poly (A) + RNA of A549 cells not treated with TGF-β1 from cDNA prepared from Poly (A) + RNA of A549 cells treated with TGF-β1 for 1 hour. went. Subtraction is CLONTECH PCR-Select^TM This was performed according to the protocol of cDNA Subtraction Kit (CLONTECH). A library obtained by subtraction was subjected to PCR to prepare a microarray. As a probe, Poly (A) + RNA recovered from A549 cells treated with TGF-β1 for 1 hour was used as a probe. Poly (A) + RNA recovered from cDNA labeled with Cy5 and A549 cells not treated with TGF-β1 Using a cDNA labeled with Cy3, the genes whose expression changes were analyzed by hybridizing both probes to one microarray. As a result, Bim was identified as a novel gene showing a significant difference in the expression level by TGF-β1.
[0074]
Example 2: Induction of Bim expression by TGF-β1
In order to confirm that Bim is induced by TGF-β treatment, the Bim ORF (Bim shown in SEQ ID NO: 3 in the Sequence Listing) is used._ADNorthern hybridization was carried out using the region of ORF) as a probe. For RNA recovery, A549 cells were cultured in Opti MEM medium, and HaCaT cells, Hep3B cells, and HUH7 cells were cultured in DMEM medium. 2 × 10⁶10 ml of an appropriate medium (0.02% FBS) was added to these cells, and 24 hours later, TGF-β1 (R & D) was added to 1 ng / ml and left for 1 hour. RNeasy Mini kit (QIAGEN) was used to collect total RNA, and Oligotex-dT30 <super> (JRS) was used to prepare poly (A) + RNA. For Northern hybridization, Poly (A) + RNA 2 μg recovered from cells treated with TGF-β1 for 1 hour and untreated cells were used, and the Bim ORF region was used as a probe. G3PDH was used as a loading control and PAI-1 was used as a positive control for TGF-β1 response.
[0075]
The results are shown in FIG. From the results shown in FIG. 1, it was shown that Bim expression was induced by TGF-β1 in all cells.
[0076]
Example 3: Bim Reporter Construction
BIM ORF was searched with httg of NCBI BLASTN, and BAC clone RP11-438K19 was selected. It was confirmed that RP11-438K19 was not abnormal compared to the DNA base sequence of NCBI human genome, and a DNA fragment (base sequence is shown in SEQ ID NO: 5 in the sequence listing) from Bim ORF to p5.7 A reporter introduced into promoter vector (Promega) was created. Figure 3 shows an overview of Bim's reporter construction.
[0077]
Example 4: Effect of Smad on Bim's reporter construction
1x10 Bim reporter construction^FiveEffectene on Hep3B cells^TM Gene transfer was performed using Transfection Reagent (QIAGEN). PGL3 basic vector was used for control, 3TPLux was used for positive control, and pRLTK was used for internal control. For gene transfer, 0.1 μg of reporter, 0.1 μg of expression vector, and 0.001 μg of internal control were used per well. 18 hours after introducing the reporter, TGF-β1 (R & D) was added to 10 ng / ml and left for 24 hours. Luciferase was measured using Dual-Luciferase Repoter Assay system (Promega). A luciferase assay was performed. The results are shown in FIG. The results in FIG. 2 suggest that Bim activates transcription depending on Smad3 and Smad4.
[0078]
Example 5: Effect of Bim deletion mutant on apoptosis induced by TGF-β1
(1) Construction of Bim deletion mutants
The deletion mutant is Bim_AD The cDNA was prepared by PCR using the cDNA as a template.
BMprimer 1 5'-ATATGAATTCATGGCAAAGCAACCTTCTG-3 '(SEQ ID NO: 8)
  BMprimer 2 5'-ATATGAATTCGGGGCCCCTACCTCCCTACA-3 '(SEQ ID NO: 9)
  BMprimer 3 5'-ATATGAATTCATGCGCCCAGAGATATGGA-3 '(SEQ ID NO: 10)
  BMprimer 4 5'-ATATGAATTCTTGCGGCGTATCGGAGACG-3 '(SEQ ID NO: 11)
  BMprimer 5 5'-ATATCTCGAGGTTAAACTCGTCTCCGATAC-3 '(SEQ ID NO: 12)
  BMprimer 6 5'-ATATCTCGAGTCAATGCATTCTCCACACCAG-3 '(SEQ ID NO: 13)
[0079]
Bim_SΔN30: Bim_SAmino acid sequence with Leu Gly Lys added to the C-terminus of the 31st to 76th amino acid sequences
Bim_SΔN51: Bim_SAmino acid sequence in which Leu Gly Lys is added to the C-terminal of the amino acid sequence from the 52nd position to the 76th position in the amino acid sequence of
Bim_SΔN61: Bim_SAmino acid sequence with Leu Gly Lys added to the C-terminus of the amino acid sequence from the 62nd to 76th amino acid sequence
Bim_SΔC70: Bim_S1st to 70th amino acid sequence in the amino acid sequence of
BH3 : Bim_SThe amino acid sequence from the 62nd to the 70th in the amino acid sequence of
[0080]
(2) Effect of deletion mutant of Bim on apoptosis induced by TGF-β1
Each Bim deletion mutant constructed in (1) above with LacZ expressing plasmid is 1 × 10^FourEffectene on Hep3B cells^TM Gene transfer was performed using Transfection Reagent (QIAGEN). For gene transfer, 0.1 μg of expression vector and 0.025 μg of LacZ expression plasmid were used. 18 hours after the transfection, the medium was replaced with DMEM medium (0% FBS), and TGF-β1 was added to a concentration of 5 ng / ml and cultured for 24 hours. After 24 hours of treatment with TGF-β1, the cells were fixed with 1% glutaraldehyde and stained with X-gal. In this study, all blue-stained cells on the well were counted, and blue-stained cells were regarded as surviving cells. The results are shown in FIG. From the results of FIG. 4, the apoptosis induced by TGF-β1 is Bim_SIt was shown to be suppressed by ΔN51 gene transfer.
[0081]
Example 6: Effect of dominant negative mutant of Bim on apoptosis induced by Bim
0.025μg LacZ expression plasmid and 0.05μg Bim expression plasmid and 0.01μg, 0.025μg, 0.05μg, 0.1μg Bim_SΔN51 expression plasmid 1 × 10^FourEffectene on Hep3B cells^TM Gene transfer was performed using Transfection Reagent (QIAGEN). 24 hours after gene transfer, the cells were fixed with 1% glutaraldehyde and stained with X-gal. The results are shown in FIG. From the results of FIG. 5, apoptosis induced by Bim is Bim_SSuppressed by ΔN51 dose dependent, Bim_SIt was suggested that ΔN51 is a dominant negative mutant of Bim.
[0082]
Example 7: Effect of Bim's dominant negative mutant on apoptosis induced by TGF-β1
0.01μg, 0.025μg, 0.05μg, 0.1μg Bim with 0.025μg LacZ expression vector_SΔN51 expression vector 1 × 10^FourEffectene on Hep3B cells^TM Gene transfer was performed using Transfection Reagent (QIAGEN). 18 hours after the transfection, the medium was replaced with DMEM medium (0% FBS), and TGF-β1 was added to a concentration of 5 ng / ml and cultured for 24 hours. After 24 hours of treatment with TGF-β1, the cells were fixed with 1% glutaraldehyde and stained with X-gal. The results are shown in FIG. From the results of FIG. 6, the apoptosis induced by TGF-β1 is Bim_SIt was suppressed by ΔN51 dose dependent, suggesting that Bim plays an important role in apoptosis induced by TGF-β1.
[0083]
Example 8: Construction of a vector expressing Bim RNAi
The vector expressing Bim RNAi is Bim_SSynthetic oligos (Bim RNAi F1 and Bim RNAi R1) were prepared based on the 92nd to 123rd DNA base sequences. For annealing, 48 μl annealing buffer [100 mM potassium acetate, 30 mM HEPES-KOH (pH 7.4), 2 mM Mg acetate], 1 μl Bim RNAi F1 (100 pmol) and 1 μl Bim RNAi R1 (100 pmol) ) And treated at 95 ° C. for 4 minutes and 70 ° C. for 10 minutes, and then allowed to stand at room temperature for 30 minutes. Thereafter, the annealed synthetic oligo was inserted into the Bse RI-Bam HI site of pSHAG (pSHAG-BimRNAi). FIG. 7 shows an outline of construction of a vector expressing Bim RNAi.
[0084]
Synthetic oligo
Bim RNAi F1
5'-tggctctgtctgtagggaggtaggggccgaagcttgggcccctacctccctacagacagagccacaatttttt -3 '(SEQ ID NO: 14)
Bim RNAi R1
5'-gatcaaaaaattgtggctctgtctgtagggaggtaggggcccaagcttcggcccctacctccctacagacagagccacg -3 '(SEQ ID NO: 15)
[0085]
Example 9: Effect of Bim RNAi on apoptosis induced by Bim
0.05 × g Bim expression plasmid and 0.01 μg, 0.025 μg, 0.05 μg, 0.1 μg pSHAG-BimRNAi together with 0.025 μg LacZ expression plasmid 1 × 10^FourEffectene on Hep3B cells^TM Gene transfer was performed using Transfection Reagent (QIAGEN). 24 hours after gene transfer, the cells were fixed with 1% glutaraldehyde and stained with X-gal. The results are shown in FIG. As can be seen from the results in FIG. 8, apoptosis induced by Bim was suppressed by Bim RNAi.
[0086]
Example 10: Effect of Bim RNAi on apoptosis induced by TGF-β1 0.1 × g pSHAG-BimRNAi with 0.025 μg LacZ expression plasmid 1 × 10^FourEffectene on Hep3B cells^TM Gene transfer was performed using Transfection Reagent (QIAGEN). 18 hours after the transfection, the medium was replaced with DMEM medium (0% FBS), and TGF-β1 was added to a concentration of 5 ng / ml and cultured for 24 hours. After 24 hours of treatment with TGF-β1, the cells were fixed with 1% glutaraldehyde and stained with X-gal. The results are shown in FIG. The results of FIG. 9 suggested that apoptosis induced by TGF-β1 was suppressed by Bim RNAi, and that Bim plays an important role on apoptosis induced by TGF-β1.
[0087]
【The invention's effect】
According to the present invention, a novel apoptosis-inducing gene has been identified. By using the gene of the present invention, the expression of this gene in each tissue can be detected, and an apoptosis-inducing protein can be produced by genetic engineering. Furthermore, by using the gene of the present invention, it is possible to diagnose an apoptosis-related disease and screen for an apoptosis inhibitor or promoter. Furthermore, the gene of the present invention is extremely useful for gene therapy for the purpose of suppressing apoptosis and for the development of methods for preventing and treating diseases using antisense oligonucleotides.
[0088]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the experimental results of the induction of Bim expression by TGF-β1.
FIG. 2 shows the experimental results regarding the effect of Smad on Bim's reporter construction.
FIG. 3 shows an overview of Bim's reporter construction.
FIG. 4 shows the experimental results on the effect of Bim deletion mutants on apoptosis induced by TGF-β1.
FIG. 5 shows the experimental results regarding the effect of Bim's dominant negative mutant on Bim-induced apoptosis.
FIG. 6 shows the experimental results regarding the effect of dominant negative mutant of Bim on apoptosis induced by TGF-β1.
FIG. 7 shows an outline of construction of a vector expressing Bim RNAi.
FIG. 8 shows the experimental results regarding the effect of Bim RNAi on Bim-induced apoptosis.
FIG. 9 shows the experimental results regarding the effect of Bim RNAi on apoptosis induced by TGF-β1.

Claims (12)

  A method of regulating apoptosis derived from TGFβ, comprising regulating the expression and / or function of the apoptosis-inducing protein Bim.   A method for suppressing apoptosis, comprising inhibiting the expression and / or function of an apoptosis-inducing protein Bim.   An apoptosis inhibitor comprising a substance that inhibits the expression and / or function of the apoptosis-inducing protein Bim.   A method of promoting apoptosis comprising increasing the expression and / or function of the apoptosis-inducing protein Bim.   A pro-apoptotic agent comprising a substance that increases the expression and / or function of the apoptosis-inducing protein Bim.   A diagnostic agent for apoptosis, comprising an oligonucleotide comprising at least 10 consecutive nucleotides in the base sequence of the apoptosis-inducing gene Bim, or an antisense oligonucleotide having a sequence complementary to the oligonucleotide.   An apoptosis inhibitor comprising an antisense oligonucleotide having a sequence complementary to an oligonucleotide comprising at least 10 consecutive nucleotides in the base sequence of the apoptosis-inducing gene Bim.   An apoptosis inhibitor comprising a double-stranded RNA comprising at least 10 consecutive nucleotides in the base sequence of RNA transcribed from the base sequence of the apoptosis-inducing gene Bim, or a DNA encoding the same.   A method of screening for a substance that suppresses or promotes apoptosis using changes in the expression and / or function of the apoptosis-inducing protein Bim as an index.   Cells having a gene encoding the apoptosis-inducing protein Bim are cultured together with TGF-β in the presence and absence of the test substance, and the expression and / or function of the apoptosis-inducing protein Bim depending on the presence or absence of the test substance The method according to claim 9, wherein a substance that suppresses or promotes apoptosis is screened using a change in the above as an index.   An RNAi reagent having SEQ ID NO: 6 and SEQ ID NO: 7.   An RNAi reagent comprising (SEQ ID NO: 6)-(linker sequence)-(SEQ ID NO: 7).

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