JP4527428B2 - Methods for screening for therapeutic components of metabolic syndrome and methods for diagnosing metabolic syndrome - Google Patents

Description

  The invention of this application relates to a screening method for identifying a therapeutic drug component of metabolic syndrome, which is a serious lifestyle-related disease, and a method for diagnosing the onset of metabolic syndrome and its disease state.

  In recent years, metabolic syndrome, including diabetes, hypertension, hyperlipidemia, obesity, and the like, has become a significant increase in the number of affected individuals in developed countries, and is a national disease that can be diagnosed by adults in the United States. Further, in this metabolic syndrome, so-called arteriosclerosis develops as a result of the failure of the metabolic system balance based on obesity and causes serious cardiovascular diseases such as cerebral infarction and myocardial infarction.

  In a healthy person, about 20 to 25% of the body weight is occupied by adipose tissue. Obesity is a state in which fat is excessively accumulated beyond this ratio. Until now, adipose tissue has been considered as a place to simply accumulate surplus energy. However, in recent studies, adipose tissue plays an important role in maintaining the balance of the metabolic system by producing adipopocytokines (such as adiponectin) as the largest endocrine organ in the human body and acting on peripheral and remote organs. It has become clear. In the obese state, adipocytes become enlarged and the function as normal adipocytes is lost, and the expression of adipopocytokines necessary for maintaining a normal metabolic balance decreases, which causes further vicious circles and metabolic syndrome. It is thought to lead to deterioration.

  Therefore, for the prevention of metabolic syndrome or for the fundamental treatment at the initial stage, it is important to correctly diagnose the state of adipocytes and to prevent abnormal transformation of adipocytes, There is a need for effective means for that purpose.

  Various in vivo proteins are known to act as regulators of cell development, differentiation, activation, and the like. KLF (Kruppel-like transcription factor) family proteins are such regulators, and KLF2 is involved in the development of adipocytes (Non-Patent Document 1), and KLF15 regulates insulin-sensitive glucose transport factor GLUT4 (Non-patent document 1). Patent Document 2) is known. KLF5 is also known to be involved in the development of angiogenesis and cardiovascular disease. Furthermore, the inventors of this application have reported that KLF5 is a factor necessary for normal differentiation of adipocytes (Non-patent Document 3). However, the responsible factors for “abnormal transformation” of adipocytes, including these KLF families, have not been known so far.

On the other hand, PPARγ is known to be a nuclear receptor for the insulin resistance improving agent thiazolidine (TZD) in diabetes (Non-patent Document 4). In addition, TZD promotes adipocyte differentiation, and as a result, TZD administration promotes obesity (Non-patent Document 5). Therefore, the action point of TZD and PPARγ in vivo is the precursor of fat. It is thought to be the differentiation of cells into adipocytes and the regulation of gene transcription in mature adipocytes. By the way, transcriptional regulation by nuclear receptors including PPARγ is known to involve a factor called a cofactor that does not bind directly to DNA but acts on a DNA binding regulator through a protein-protein interaction. Thus, for example, Patent Document 1 proposes a method for screening a ligand molecule that acts on a complex between PPARγ and a novel protein PGC2 that is a cofactor as a component substance of a therapeutic agent for diabetes.
JP 2002-58489 A Banerjee et al., J. Biol. Chem. 278: 2581-2584, 2003 Gray et al., J. Biol. Chem. 277: 34322-34828, 2002 Oishi et al., Circ. J. 68: (Supplement) 217, 2003 Lehmann et al., J. Biol. Chem. 270: 12953-12956, 1995 Willson et al., J. Med. Chem. 39: 665-668, 1996

  As noted above, the direct cause of metabolic syndrome is aberrant transformation of adipocytes. Cell transformation is a molecular biological event involving gene expression, and therefore, a molecule involved in such transformation to diagnose, prevent, or treat adipocyte transformation. It is most important to identify biological factors. In this regard, it has been suggested that KLF family proteins and PPARγ play some role, but no in vivo factors have been identified that play a decisive role in abnormal transformation of adipocytes. It is not known at all what the mechanism of action causes transformation of adipocytes.

  The invention of this application was made in view of the circumstances as abnormal, and based on molecular biological events that cause abnormal transformation of adipocytes, component substances of therapeutic agents for metabolic syndrome It is an object of the present invention to provide a screening method and a method for diagnosing metabolic syndrome targeting such molecular biological events.

  This application, as a first invention for solving the above-mentioned problems, is a method for screening a therapeutic drug component of metabolic syndrome, wherein the formation of a KLF5-PPARγ complex can be detected by a signal of a reporter molecule There is provided a method characterized by adding a test substance to a system and identifying a KLF5-PPARγ complex activity regulator using the signal amount of a reporter molecule as an indicator.

  In addition, as a second invention, there is provided a method for diagnosing metabolic syndrome, which comprises measuring KLF5 expression level in adipose tissue isolated from a subject.

  That is, the inventors of this application show that KLF5 is not only differentiated in normal adipocytes but also upregulated during abnormal transformation, and that KLF5 is transformed into adipocytes by interacting with PPARγ. I found it to work. The first and second inventions have been completed based on these novel findings.

  In the present invention, the term “therapeutic agent for metabolic syndrome” refers to preventing abnormal transformation of adipocytes, or preventing the development of metabolic syndrome by restoring transformed adipocytes to normal cells, or It is a drug that has the effect of improving or curing symptoms. In addition, the “activity regulating substance” as the component substance is, for example, a substance that inhibits the complex formation of KLF5-PPARγ or a function that inhibits the transcriptional regulatory activity of the KLF5-PPARγ complex. It is a substance that acts on.

  Furthermore, “diagnosis” in the present invention means predicting in advance the possibility that the fat cells of the subject cause abnormal transformation, and quantitatively grasping the degree of transformation of the fat cells.

  Other terms and concepts in the present invention are defined in detail in the description of the embodiments of the invention and the examples. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known documents and the like, except for a technique that clearly indicates the source. For example, the preparation of the drug is in Remington's Pharmaceutical Sciences, 18th Edition, ed.A. Gennaro, Mack Publishing Co., Easton, PA, 1990. Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc.

  In the first invention, a test substance is added to a system that can detect the formation of a KLF5-PPARγ complex by a signal of a reporter molecule, and an activity regulator of the KLF5-PPARγ complex is defined using the signal amount of the reporter molecule as an indicator. It is characterized by specifying.

  The “system capable of detecting the formation of the KLF5-PPARγ complex by a reporter molecule signal” is, for example, a known two-hybrid system or a system using fluorescence resonance energy transfer (FRET).

  For example, in the case of a two-hybrid system, screening can be performed as follows. That is, for example, in the case of the Two-Hybrid system in yeast cells, (1) expressing a fusion protein (DNA-BD / KLF5) of the transcription-activating protein (activator) DNA-binding domain (DNA-BD) and KLF5 (2) a vector that expresses a transcriptional activation domain (AD) of an activator and a PPARγ fusion protein (AD / PPARγ), and (3) a vector that expresses a gene encoding a reporter molecule (reporter gene). Each is introduced into yeast. The reporter gene expression vector has a response factor to an activator and a promoter linked to each other, and is configured to express a reporter molecule by the activator binding.

  Alternatively, the expression vector of (1) and the expression vector of (2) may have opposite fusion partners (ie, DNA-BD / PPARγ expression vector and AD / KLF5 expression vector), or the respective fusion proteins may be used together. A single vector to be expressed (DNA-BD / PPARγ2 · AD / KLF5 expression vector or DNA-BD / KLF5 · AD / PPARγ2 expression vector) may be used.

  As the transcription activator, for example, yeast GAL4 protein or LexA can be used.

  As a DNA binding domain (DNA-BD) in the DNA-BD / KLF5 expression vector, for example, a binding domain of yeast GAL4 protein or a LexA promoter can be used. In this case, the full length of the natural DNA-binding domain is not necessary, and it may be any size that includes at least the DNA-binding region and enables the construction of an expression vector for the fusion protein. For example, in the case of the DNA binding domain of GAL4 protein, a region containing about amino acids 1 to 147 is the DNA binding region.

  As the activation domain (AD) in the AD / PPARγ expression vector, the AD region of the transcriptional activator used as DNA-BD is preferably used. For example, when the DNA binding domain of GAL4 protein is used as DNA-BD, it is the AD region of GAL4 protein. In addition, LexA protein can be used for AD when the LexA operator is DNA-BD. It is not necessary to use the full length of these ADs as long as they contain at least an AD active region.

  Moreover, a two-hybrid system can be performed in mammalian cells by using the herpes simplex virus activation domain as this AD (mammalian two-hybrid system). This mammalian Two-Hybrid system can reproduce programmed changes such as phosphorylation, acetylation, and proteolysis, and is a preferred system for more detailed screening. Therefore, it is also preferable to perform primary screening with the yeast Two-Hybrid system and secondary screening with the mammalian Two-Hybrid system.

  As the reporter gene in the reporter gene expression vector, LacZ gene, HIS3 gene, luciferase gene, chloramphenicol acetyltransferase (CAT) gene, human placenta (secreted) alkaline phosphatase (SEAP) gene and the like can be used. The type of response factor upstream of this reporter gene depends on the DNA-BD used. For example, when the DNA binding domain of GAL4 protein is employed, UASs (upstream activation sites of galactose metabolic genes) can be used. Furthermore, the promoter located downstream of the response factor and upstream of the reporter gene is not particularly limited as long as it enables expression of the reporter gene.

  Each of the above vectors can be prepared by inserting and binding each fusion protein gene or reporter gene to a known plasmid vector, cosmid vector, virus vector or the like. In addition, DNA-BD expression vectors and AD expression vectors suitable for each of the yeast two-hybrid system and mammalian two-hybrid system are known, and the target expression vector is inserted and joined to each of them with the KLF5 gene and the PPARγ gene. It can also be created. For example, a pM vector is commercially available as a DNA-BD vector of the GAL4 protein, and a pV16 vector is commercially available as a V16AD vector. Various reporter gene expression vectors are also commercially available. For example, a pG5SEAP vector that is a SEAP expression vector or a CAT expression vector.

  On the other hand, the KLF5 gene and PPARγ gene constituting the fusion protein gene in these vectors are obtained by screening genomic DNA libraries and cDNA libraries, for example, using a DNA fragment comprising a known nucleotide sequence or a partial sequence thereof as a probe. It can be isolated. The obtained polynucleotide is, for example, a commonly performed gene amplification such as PCR (Polymerase Chain Reaction) method, NASBN (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method and SDA (Strand Displacement Amplification) method. It can be amplified by the method. In the case of cDNA, the target cDNA can also be obtained by RT-PCR using mRNA isolated from cells as a template, using primers synthesized based on known sequences. The cDNA sequence of human KLF5 is known as GenBank / AF287272. Human PPARγ is specifically PPARγ2 (for example, Tontonoz, et al., Cell 79 (7): 1147-1156, 1994), and its cDNA sequence is known as GenBank / NM015015.

  In the FRET system, KLF5 linked with a donor fluorescent protein is reacted with PPARγ linked with an acceptor fluorescent protein. When KLF5 and PPARγ form a complex, the donor fluorescent protein and the acceptor fluorescent protein come close to each other, and excitation energy shifts due to resonance. For example, when cyan fluorescent protein (CFP) is used as the donor fluorescent protein and yellow fluorescent protein (YFP) is used as the acceptor fluorescent protein, CFP emits fluorescence at a wavelength of 480 nm with respect to 440 nm excitation light, but YFP approaches. In this case, the emission wavelength is 535 nm. Therefore, the complex formation between KLF5 and PPARγ can be confirmed by reacting KLF5 and PPARγ, each of which binds such a fluorescent protein, and measuring the change in fluorescence wavelength.

  In the first invention of this application, after confirming the formation of a complex between KLF5 and PPARγ in the system as described above, a test substance is added, and an activity regulator for the KLF5 / PPARγ complex using the signal change of the reporter molecule as an indicator Is identified.

  Test substances to be screened include, for example, organic or inorganic compounds (particularly low molecular weight compounds), proteins, peptides and the like. These substances have known functions and structures and may be unknown.

  Further, the “combinatorial chemical library” is an effective means as a test substance group for efficiently specifying a target substance. A combinatorial chemical library is a collection of various chemical compositions generated by combining many chemical “building blocks”, such as reagents, by chemical or biological synthesis. For example, a linear combinatorial chemical library, such as a peptide library, is formed by combining a set of building blocks (amino acids) in all possible ways for a given compound length (ie, peptide size). The A number of chemical compositions can be synthesized through such combinatorial mixing of chemical building blocks. For example, systematic combinatorial mixing for 100 commutative chemical building blocks results in 100 million tetramer compounds or 10 billion pentamer compounds (eg, Gallop et al., (1994) 37 (9): 1233-1250). The preparation and screening of combinatorial chemical libraries is well known in the art (see, eg, US Pat. Nos. 6,004,617; 5,985,365). Such combinatorial chemical libraries are for example peptide libraries (eg, US Pat. No. 5,010,175; Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al., (1991). Nature, 354: 84-88), peptoids (see eg WO91 / 19735), encoded peptides (see eg WO93 / 20242), random biooligomers (eg see WO92 / 00091), benzodiazepines (see eg For example, diversomers, vinylogous, such as hydantoin, benzazepine, and dipeptides (see, eg, Hobbs (1993) Proc. Natl. Acad. Sci. USA 90: 6909-6913), US Pat. No. 5,288,514) Polypeptides (see, eg, Hagihara (1992) J. Amer. Chem. Soc. 114: 6568), non-peptidic peptidomimetics with a β-D-glucose backbone (eg, Hirschmann (1992) J. Amer. Chem. Soc 114: 9217-9218), low Similar organic synthesis of child compound libraries (see, eg, Chen (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (eg, Cho (1993) Science 261: 1303), and / or peptidyl phosphonate ( For example, see Campbell (1994) J. Org. Chem. 59: 658). Also, nucleic acid libraries (see eg Gordon (1994) J. Med. Chem. 37: 1385), peptide nucleic acid libraries (see eg US Pat. No. 5,539,083), antibody libraries (eg Vaughn (1996) Nature biotechnology 14: 309). -314), carbohydrate libraries (see eg Liang et al., (1996) Science 274: 1520-1522, US Pat. No. 5,593,853), isoprenoid libraries as organic small molecule libraries (see eg US Pat. No. 5,569,588) ); Thiazolidinone and metathiazonone libraries (see US Pat. No. 5,549,974); pyrrolidine libraries (see US Pat. Nos. 5,525,735 and 5,519,134); morpholino compound libraries (see US Pat. No. 5,506,337), benzodiazepine libraries (See US Pat. No. 5,288,514) or the like. Furthermore, commercially available libraries (for example, libraries made by ComGenex, USA, Asinex, USA, Tripos, Inc., ChemStar, Ltd, Russia, 3D Pharmaceuticals, Martek Biosciences, etc.) Can also be used.

  Next, the second invention uses the expression level of KLF5 in adipose tissue isolated from a subject as an indicator of abnormal transformation of adipocytes, so that the subject is at risk of suffering from metabolic syndrome or has already It is a method of diagnosing whether it is affected or how much it is affected.

  Specifically, the expression of KLF5 can be quantified by measuring the transcript of the KLF5 gene. When measuring mRNA as a transcript of the KLF5 gene, in accordance with known genetic engineering and molecular biological techniques, techniques known in the art for detecting and measuring the expression of specific genes, such as in situ hybridization, are used. , Northern blotting, dot blot, RNase protection assay, RT-PCR, Real-Time PCR (Journal of Molecular Endocrinology, 25, 169-193 (2000) and references cited therein), DNA array analysis method (edited by Mark Shena) , "Microarray Biochip Technology", Eaton Publishing, 2000) etc., and can be carried out by detecting and measuring the expression level of KLF5 mRNA.

For example, a method for detecting the amount of KLF5 mRNA using an oligonucleotide probe (in the case of Northern blot analysis, at least the following steps:
(a) preparing RNA from a biological sample of a subject;
(b) electrophoretically separating the RNA prepared in step (a);
(c) hybridizing the RNA separated in step (b) with an oligonucleotide probe under stringent conditions;
(d) using the labeled amount of the oligonucleotide probe hybridized to RNA in step (e) as an indicator of the expression level of KLF5 mRNA, and comparing it with the result of a normal biological sample; and
(e) a step of using a KLF5 mRNA expression level that is significantly higher than that of a normal biological sample as an indicator of the degree of further transformation of adipocytes or the risk thereof,
It is characterized by including.

  Since the oligonucleotide probe hybridizes with KLF5 mRNA under stringent conditions (for example, the conditions described in JP-T-10-508186 and JP-T9-511236), the oligonucleotide probe accurately matches any region of mRNA. A DNA sequence consisting of complementary sequences is used. Such a DNA sequence can also be obtained, for example, by cleaving KLF5 cDNA with an appropriate restriction enzyme. Alternatively, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47: 411-418; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acid Res. 25: 3440-3444 Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. : 109; Beaucage (1981) Tetra. Lett. 22: 1859; can be synthesized in vitro by well-known chemical synthesis techniques as described in US Pat. No. 4,458,066.

The oligonucleotide probe is labeled by a radioisotope (RI) method or a non-RI method, but it is preferable to use a non-RI method. Examples of the non-RI method include a fluorescence labeling method, a biotin labeling method, a chemiluminescence method, and the like, but it is preferable to use a fluorescence labeling method. As the fluorescent substance, those capable of binding to the base moiety of the oligonucleotide can be appropriately selected and used. However, cyanine dyes (eg, Cy Dye ^TM series Cy3, Cy5 etc.), rhodamine 6G reagent, N-acetoxy- N ² -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like can be used. As the labeling method, methods known in the art (for example, random prime method, nick translation method, DNA amplification by PCR, labeling / tailing method, in vitro transcription method, etc.) can be appropriately selected and used. For example, a functional group (for example, primary aliphatic amino group, SH group, etc.) is introduced into the HRF oligonucleotide, and the above-mentioned label is bound to such a functional group to prepare a labeled oligonucleotide probe.

The amount of KLF5 mRNA can also be measured by using a DNA microarray. This method comprises at least the following steps:
(a) preparing RNA from a biological sample of a subject;
(b) a step of preparing labeled cDNA from the RNA prepared in step (a),
(c) contacting the labeled cDNA prepared in step (b) with a DNA microarray;
(d) a step of comparing the amount of labeled cDNA hybridized with the capture probe of the DNA microarray in step (c) as an index of the amount of KLF5 mRNA and comparing the result with a normal biological sample; and
(e) a step of using a KLF5 mRNA amount significantly higher than that of a normal biological sample as an indicator of the degree of risk of further transformation of adipocytes or the risk thereof,
It is characterized by including.

  The DNA microarray used in this method includes an oligonucleotide that hybridizes with cDNA synthesized from KLF5 mRNA as a capture probe. As a method for producing a microarray, a method of directly synthesizing oligonucleotides on the surface of a solid support (on-chip method) and a method of immobilizing a prepared oligonucleotide or polynucleotide on the surface of a solid support are known. . The microarray used in the present invention can be produced by any of these methods. As an on-chip method, a combination of the use of a protective group that is selectively removed by light irradiation and the photolithography technology and solid-phase synthesis technology used in semiconductor manufacturing can be combined in a predetermined area of a minute matrix. It can be performed by a method of performing selective synthesis (masking technique: for example, Fodor, SPA Science 251: 767, 1991). On the other hand, when immobilizing oligonucleotides or polynucleotides prepared in advance on the surface of a solid support, synthesize oligonucleotides with functional groups introduced, spot the oligonucleotides on the surface of the solid support and treat them covalently. (Eg, Lamture, JB et al. Nucl. Acids Res. 22: 2121-2125, 1994; Guo, Z. et al. Nucl. Acids Res. 22: 5456-5465, 1994). Oligonucleotides or polynucleotides are generally covalently bonded to a surface-treated solid support via a spacer or a crosslinker. A method is also known in which polyacrylamide gel fragments are aligned on a glass surface and a synthetic oligonucleotide is covalently bound thereto (Yershov, G. et al. Proc. Natl. Acad. Sci. USA 94: 4913, 1996). ). In addition, an array of microelectrodes was prepared on a silica microarray, an agarose permeation layer containing streptavidin was provided on the electrode as a reaction site, and this site was charged positively to immobilize the biotinylated oligonucleotide. There is also known a method that enables precise hybridization at high speed by controlling the site charge (Sosnowski, RG et al. Proc. Natl. Acad. Sci. USA 94: 1119-1123, 1997). When measuring the amount of KLF5 mRNA using this microarray, for example, cDNA is synthesized and amplified by PCR using mRNA isolated from a subject's cells as a template. At that time, labeled dNTP is incorporated into labeled cDNA. The labeled cDNA is brought into contact with the macroarray, and the cDNA hybridized with the capture probe of the microarray is detected. Hybridization can be performed by dispensing a 96- or 384-well plastic plate and spotting a labeled cDNA aqueous solution on the microarray. The amount of spotting can be about 1 to 100 nl. Hybridization is preferably performed in the temperature range of room temperature to 70 ° C. for 6 to 20 hours. After completion of hybridization, washing is performed using a mixed solution of a surfactant and a buffer to remove unreacted labeled cDNA. As the surfactant, sodium dodecyl sulfate (SDS) is preferably used. As the buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good buffer solution, or the like can be used, and a citrate buffer solution is preferably used.

Furthermore, the KLF5 mRNA level can also be measured by the RT-PCT method. This method comprises at least the following steps:
(a) preparing RNA from a biological sample of a subject;
(b) using the RNA prepared in step (a) as a template and synthesizing cDNA using a primer set;
(c) using the amount of cDNA synthesized in step (b) as an indicator of the amount of KLF5 mRNA and comparing it with the result of a normal biological sample; and
(d) a step of using a KLF5 mRNA amount that is significantly higher than that of a normal biological sample as an indicator of the degree of transformation of adipocytes or the risk thereof,
It is characterized by including.

  The primer set to be used can be designed based on a known KLF5 cDNA sequence (GenBank / AF287272) and prepared through each step of synthesis and purification. The size of the primer (number of bases) is 15-40 bases, preferably 15-30 bases in consideration of satisfying specific annealing with the template DNA. However, when performing LA (long accurate) PCR, at least 30 bases are effective. In order to prevent the primer or pair (two pairs) consisting of the sense strand (5 'end) and antisense strand (3' end) from annealing to each other, avoid complementary sequences between the primers and within the primer. In order to prevent the formation of hairpin structures, self-complementary sequences should also be avoided. Further, in order to ensure stable binding to the template DNA, the GC content is set to about 50% so that GC-rich or AT-rich is not unevenly distributed in the primer. Since the annealing temperature depends on Tm (melting temperature), in order to obtain highly specific PCR products, primers that have similar Tm values of 55-65 ° C are selected. It is also necessary to pay attention to adjusting the final concentration of primers used in PCR to be about 0.1 to about 1 μM. Also, commercially available software for primer design such as Oligo ™ [manufactured by National Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)], etc. can be used.

  Another method for measuring the expression of the KLF5 gene is to quantify the amount of KLF5 protein as a transcript of the KLF5 gene. Such diagnostic methods are known in the art according to known genetic engineering and molecular biological techniques, such as in situ hybridization, Western blotting, various immune tissues known for detecting and measuring a specific amount of protein. It can be carried out by detecting and measuring the amount of KLF5 protein by a scientific method.

Specifically, it can be carried out using an antibody that specifically recognizes KLF5 protein. The antibody is a polyclonal antibody or a monoclonal antibody, and includes all the whole molecules capable of binding to the epitope of KLF5 protein, Fab, F (ab ′) ₂ , Fv fragment, and the like. For example, in the case of a polyclonal antibody, such an antibody can be obtained from serum after immunizing an animal using a protein or a partial fragment thereof as an immunogen. Alternatively, it can be prepared by introducing the expression vector for eukaryotic cells into animal muscle or skin by injection or gene gun, and then collecting serum. As the animal, mouse, rat, rabbit, goat, chicken and the like are used.

  Monoclonal antibodies are known monoclonal antibody production methods ("monoclonal antibodies", Kamei Nagamune, Hiroaki Terada, Yodogawa Shoten, 1990; "Monoclonal Antibody" James W. Goding, third edition, Academic Press, 1996) Can be manufactured according to

It is also used as an antibody labeled with a labeling substance. As the labeling substance, an enzyme, a radioisotope, or a fluorescent dye can be used. The enzyme is not particularly limited as long as it satisfies the conditions such as a large turnover number, stability even when bound to an antibody, and specific coloring of a substrate, and is used for normal EIA. Enzymes such as peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase and the like can also be used. Moreover, an enzyme inhibitor, a coenzyme, etc. can also be used. These enzymes and antibodies can be bound by a known method using a crosslinking agent such as a maleimide compound. As the substrate, a known substance can be used according to the type of enzyme used. For example, when peroxidase is used as an enzyme, 3,3 ′, 5,5′-tetramethylbenzidine can be used, and when alkaline phosphatase is used as an enzyme, paranitrophenol or the like can be used. As the radioisotope, those used in usual RIA such as ¹²⁵ I and ³ H can be used. As the fluorescent dye, those used in usual fluorescent antibody methods such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) can be used.

  Methods using such antibodies can be performed, for example, by immunostaining, eg, tissue or cell staining, immunoelectron microscopy, immunoassay, eg, competitive or noncompetitive immunoassay, radioimmunoassay (RIA), fluorescence Immunoassay (FIA), luminescent immunoassay (LIA), enzyme immunoassay (EIA), ELISA, etc. can be used, and the measurement may be performed with or without BF separation. it can. RIA, EIA, FIA, and LIA are preferable, and a sandwich type assay is also included. The sandwich type assay may be a simultaneous sandwich type assay, a forward sandwich type assay or a reverse sandwich type assay.

  One embodiment of the diagnostic method using such an antibody is a method for detecting the binding between the antibody and the KLF5 protein in a liquid phase system. For example, the labeled antibody is brought into contact with the biological sample to bind the labeled antibody and the KLF5 protein, and this conjugate is separated. Separation can be performed by known separation means (chromatography, solid phase method, etc.). A method according to a known Western blot method can also be employed. When an enzyme is used as a label, the label signal is measured by adding a substrate that develops color by enzymatic action and optically measuring the amount of degradation of the substrate to determine the enzyme activity. The amount of antibody is calculated by conversion and comparison with the standard value. When using a radioactive isotope, the radiation dose emitted by the radioactive isotope is measured with a scintillation counter or the like. In addition, when a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope.

  Another method for diagnosis in a liquid phase system is to contact a primary antibody and a biological sample (adipocyte) to bind the primary antibody and the KLF5 protein, bind a labeled secondary antibody to the conjugate, The label signal in the human conjugate is detected. Alternatively, in order to further enhance the signal, an unlabeled secondary antibody may first be bound to the primary antibody + KLF5 protein conjugate, and a labeling substance may be bound to the secondary antibody. Such binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylating the secondary antibody and avidinizing the labeling substance. Alternatively, an antibody (tertiary antibody) that recognizes a partial region (for example, Fc region) of the secondary antibody may be labeled and the tertiary antibody may be bound to the secondary antibody. The primary antibody and the secondary antibody may both be monoclonal antibodies, or one of the primary antibody and the secondary antibody may be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be performed as described above.

  Still another embodiment in the case of using an antibody is a method for testing the binding between the antibody and the KLF5 protein in a solid phase system. This method in the solid phase system is a preferable method for detecting a very small amount of KLF5 protein and simplifying the operation. That is, in this solid phase system, the antibody is immobilized on a resin plate or membrane, the KLF5 protein is bound to this immobilized antibody, unbound protein is washed away, and then the antibody + KLF5 protein conjugate remaining on the plate In this method, a labeled anti-antibody is bound to and the signal of the labeled antibody is detected. This method is a so-called “sandwich method”. When an enzyme is used as a marker, this method is widely used as an “ELISA (enzyme linked immunosorbent assay)”. Both of the two types of antibodies can be monoclonal antibodies, or one of them can be a polyclonal antibody.

  In the second invention of this application, a more accurate diagnosis method can be obtained by combining two or more of the methods exemplified above.

  Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.

The expression of KLF5 in the adipocyte differentiation stage was confirmed as follows.
(1) Materials and methods After differentiation of mouse adipose precursor cell 3T3-L1 cultured cells, differentiation induction stimuli including insulin, dexamethasone, and isobuthylmethylxantihe were added. Cells were recovered and total RNA was extracted at each time passage shown in FIG. RT-PCR was used to examine gene expression at the mRNA level.
(2) Results
In an experimental system for adipocyte differentiation in vitro using 3T3-L1 cells, KLF5 was highly expressed in the early stages of differentiation, followed by C / EBPδ. In addition, expression of PPARγ2, an adipocyte-specific transcription factor, was induced after KLF5 expression.

  From the above results, it was confirmed that KLF5 is expressed at an early stage of adipocyte differentiation, and that KLF5 controls adipocyte differentiation by interaction with PPARγ2.

It was confirmed as follows that the expression of KLF5 is decreased during abnormal fat cell hypertrophy.
(1) Materials and methods Total RNA was obtained from white adipose tissue of ob / ob mice with advanced morbid obesity, IRS-2 knockout mice (homomo) with moderate obesity, and wild type C57BL6 mice without obesity. Extracted and purified. RT-PCR was used to examine the expression of KLF5 at the mRNA level.
(2) Results As the degree of obesity is higher, fat cell hypertrophy is observed pathologically. As shown in FIG. 2, the expression of KLF5 was decreased in ob / ob mice and IRS-2 knockout mice exhibiting morbid obesity and adipocyte hypertrophy compared to wild-type mice.

It was confirmed that KLF5 protein is also involved in fat accumulation in brown adipocytes and hepatocytes as follows.
(1) Materials and methods
KLF5 knockout mouse (hetero) and littermate wild-type mouse 6 (both male, 6
A high fat diet was loaded for 20 weeks. After the end of loading, the tissue was dissected and brown adipose tissue and liver tissue were collected. Each tissue was fixed with formalin, embedded in paraffin, and sliced to prepare a hematoxylin-eosin stained specimen.
(2) Results Weight gain due to high-fat diet load was almost the same between KLF5 hetero knockout mice and wild-type mice. As shown in FIG. 3, pathologically, a high degree of fatty liver was observed in the liver of wild-type mice, but in KLF5 hetero knockout mice, fatty liver was mild and liver tissue was relatively normal. It was kept in.

  Further, as shown in FIG. 4, in the brown adipose tissue of the wild type mouse, many cells were changed to a shape containing a large single lipid droplet, and white adipogenesis was apparent. On the other hand, in brown adipose tissue of KLF5 hetero knockout mice, many cells showed cytoplasm-rich traits including a plurality of small lipid droplets peculiar to brown adipocytes, and white adipogenesis was hardly observed.

The binding of KLF5 and PPARg was confirmed by the Two-Hybrid system using mammalian cells.
(1) Materials and Methods Yeast Gal4DNA-BD / PPARγ ₂ expression vector or a yeast Gal4DNA-BD / empty control vector, the Gal4 binding sequence -Luciferase vector as VP16 AD / KLF5 expression vector or VP16 AD / empty control vector and reporter culture It was introduced into smooth muscle cells. Luciferase activity was quantified 48 hours after the introduction.
(2) Results As shown in FIG. Although Gal4DNA-BD / PPARγ ₂ expression vector and VP16 AD / empty control vector and VP16 AD / KLF5 luciferase activity when introduced into a cell with a combination of expression vectors and yeast Gal4DNA-BD / empty control is hardly observed, Gal4DNA- when BD / PPARy ₂ expression vector and VP16 AD / KLF5 expression vector together cell transfer is higher luciferase activity was observed. This result, KLF5 and PPARy ₂ is able to bind intracellularly was confirmed.

Interactions in cells of KLF5 and PPARy _2, was confirmed using a reporter assay.
(1) Materials and methods
KLF5 expression vector or PPARy ₂ expression vector, and the apolipoprotein AII promoter -Luciferase vector as a reporter for PPARg ₂ were introduced into cultured smooth muscle cells. Some cells 12 hours after introduction were added Trogrlitazone 2 mM is a ligand of PPARy ₂ in the culture supernatant. Luciferase activity was quantified 48 hours after the introduction.
(2) Results As shown in FIG. That, KLF5 is alone does increase the apolipoprotein AII promoter activity was synergistically increased the apolipoprotein AII promoter activity in the presence of PPARy _2. This effect was further enhanced by the addition of PPARy ₂ ligand.

Activity regulators for the KLF5-PPARγ complex were screened by the Two-Hybrid system using mammalian cells.
(1) Materials and Methods Yeast Gal4DNA BD / PPARγ2 ・ VP16 AD / KLF5 expression vector or Gal4DNA-BD / KLF5 ・ VP16 AD / PPARγ2 expression vector and Gal4 binding sequence-SEAP vector as a reporter were introduced into cultured smooth muscle cells . PBS was added as a test substance and a control as a control after 12-24 hours from the introduction, and 100 microliters of each culture supernatant was collected 12/12 hours after the addition, and SEAP activity was quantified.
(2) Results A high level of stable SEAP activity was obtained in cultured smooth muscle cells into which the above-described vectors were introduced. This is a result showing that PPARγ2 and KLF5 are forcibly highly expressed in cells, and that the state where both are bound on the Gal4 binding sequence can be reproduced. In addition, the addition of PBS as a control did not change the SEAP activity, but the test substance (compound A) further increased the SEAP activity, and conversely, the test substance (compound B) had a SEAP activity below the baseline.

  As described in detail above, according to the invention of this application, a method for screening a component substance of a therapeutic agent for metabolic syndrome based on a molecular biological event that causes abnormal transformation of fat cells, and such a molecule A method for diagnosing metabolic syndrome in a biological event is provided. These inventions enable effective prevention of metabolic syndrome, improvement of symptoms, and cure of symptoms.

This is an RT-PCR result showing that KLF5 is expressed at an early stage of adipocyte differentiation and that KLF5 regulates adipocyte differentiation through interaction with PPARγ2. It is the result of RT-PCR which shows that the expression of KLF5 is falling in the fat tissue of an obesity model mouse. It is a microscope image which shows that the onset of fatty liver is suppressed in KLF5 hetero knockout mouse. It is a microscope image which shows that white fat organization of a brown adipose tissue by KLF5 hetero knockout mouse | mouth is suppressed by high fat diet addition. It is the result confirmed by the mammalian cell Two-Hybrid system which KLF5 and PPARγ2 bind in cells. It is the result of confirming that KLF5 and PPARγ2 interact in cells by a reporter assay.

Claims (2)

A method for screening therapeutic components of metabolic syndrome, wherein a test substance is added to a system in which the formation of a KLF5-PPARγ complex can be detected by a reporter molecule signal, and the signal level of the reporter molecule is used as an indicator A method characterized by identifying a substance that enhances the activity of a PPARγ complex. A method for using a decrease in KLF5 expression in an adipose tissue isolated from a subject as an indicator of metabolic syndrome.