JP2002238599A - Method and kit for bladder cancer diagnosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a method for bladder cancer diagnosis, by which the pain of patient is reduced and only bladder cancer can be diagnosed specifically in high sensitivity and a kit for bladder cancer diagnosis. SOLUTION: The method for human bladder cancer diagnosis is characterized in that the amount of CYP4B1 or mRNA encoding the CYP4B1 in human tissue or its treated material is assayed and compared with those of normal health subjects. The kit for bladder cancer diagnosis is characterized in that the kit has at least (a) a forward primer and a reverse primer to be hybridized with a cDNA encoding CYP4B1 and (b) a probe which has a reporter coloring matter and a quencher coloring matter at ends and is hybridized with a template nucleic acid in a domain laid between both the primers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method and a kit for diagnosing human bladder cancer, and more particularly, to CYP4B1 or CYP4 in human tissue or a processed product thereof.
The present invention relates to a method and a kit for diagnosing human bladder cancer, which comprises measuring the amount of mRNA encoding B1.

[0002]

2. Description of the Related Art Bladder cancer is a common malignant tumor in the urological field together with prostate cancer.
９５95% are transitional cell carcinomas. It is known that this tumor develops due to occupational (caring for dyes) chemical carcinogenesis. In addition to this, smoking is presumed to be a factor, and the recurrence rate is high. The feature is to do. Endoscopic surgery can be used for curative treatment for early-stage cancer, but radical cystectomy and urinary diversion are necessary for advanced cancer, and as early as or more than other cancers Needs early treatment.

Conventionally, urinary cytology, urine biochemical analysis and the like have been performed as screening tests for bladder cancer. Urine cytology is a method of observing the presence or absence of exfoliated and detached cancer cells in the urine under a microscope. However, cell degeneration may occur even in cystitis and the like, and thus the reliability of a definitive diagnosis method is poor. Urine biochemical analysis includes measurement of urinary excretion of hyaluronic acid and hyaluronidase (Table 2000-504114).
No.), measurement of telomerase activity (JP-A-9-26210)
No. 0), measurement of telomerase RNA (JP-A-11-24)
No. 3995), an assay for the presence or absence of chromosome 9 in the exfoliated transitional epithelium in urine (Japanese Patent Laid-Open No. 10-68129), and measurement of basic fetoprotein have been reported. In addition, there are problems with reproducibility and specificity. That is, so-called tumor markers with high sensitivity and high specificity, such as prostate specific antigen (PSA) in prostate cancer, have not been found so far in bladder cancer. Therefore, in clinical practice, bladder cancer is diagnosed for the first time in patients with subjective symptoms such as hematuria by performing highly invasive endoscopy, CT scans and MRI that require high capital investment. That is the current situation. In view of the above, it is considered indispensable to establish a simple, highly sensitive and specific diagnostic method using urine or blood as a sample that can predict the onset of bladder cancer and its recurrence and metastasis.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a method for diagnosing bladder cancer which is simple and less sensitive to patients and which can specifically diagnose only bladder cancer with high sensitivity, and a kit for diagnosing bladder cancer used in the diagnostic method. The purpose is to provide.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have developed a biphenyl derivative, DMA, in the bladder of mice and rats.
B (3,2'-dimethyl-4-aminobiphenyl);
It has been found that CYP4B1 exists that activates benzidine derivative DCB (3,3′-dichlorobenzidine) as a carcinogen (Biochemical Pharmacology,
54 (6), 677, 1997). Here, CYP4B1 is one molecular species of cytochrome P450 which is a drug metabolizing enzyme. CYP4B1 also exists in humans, and the cDNA encoding it has been isolated (Nhamburo et al. Biochemi).
stry 28: 8060-8066,1989). The sequence of the mRNA encoding CYP4B1 is also registered in GeneBank with the registration number “N
M 000779 ". However, it has been reported that human CYP4B1 has no activity on 2-aminofluorene and lauric acid, which are typical substrates of CYP4B1, in rats and the like (see the above literature and
Czerwinsi M et al. Cancer Res 51: 4636-4638,199
1). Therefore, the human CYP4B1 gene has a genetic polymorphism and has a different catalytic specificity.
It is described that there will be one variant (see above). Recently, a CYP4B1 mutant different from the previous reports was isolated (Se at 207th).
r), the presence of this type of CYP4B1 in the human bladder, and the possibility of activating carcinogenic amines in the bladder (Imaoka S et al. Bioc.
hem. Biophys. Res, Commun. 277: 776-780, 2000). This result indicates that the expression of CYP4B1 in the bladder may be closely related to bladder cancer.

Therefore, the present inventors have proposed that human bladder cancer and C
As a result of repeated studies on the effects of YP4B1 on the action or expression level, etc., it was found that C
It was found that the expression level of YP4B1 was higher than that in healthy individuals. Furthermore, the present inventors also found that the expression level of CYP4B1 was increased in normal tissues other than cancer tissues of the bladder mucosa. According to this finding, it is not necessary to directly collect cancer tissue on the bladder mucosa for diagnosis. For example, blood or bladder mucosa (other than cancer tissue) that is easy to collect
By examining such factors, bladder cancer can be diagnosed. Therefore, at the time of diagnosis, pain and the like of the patient can be reduced. In addition, the present inventors have found that in patients with cancer other than bladder cancer (eg, prostate cancer), CYP
It was also found that the expression level of 4B1 was at a low level as in healthy subjects. Based on this finding, the expression level of CYP4B1 can be an index specific to bladder cancer, and only bladder cancer can be specifically diagnosed.

That is, the present invention relates to (1) CYP4B1 or CYP4B in human tissue or a processed product thereof.
A method for diagnosing human bladder cancer, comprising measuring the amount of mRNA encoding 1 and comparing it with that of a healthy person;
(2) Measurement of the amount of mRNA encoding CYP4B1
(A) obtaining an RNA fraction containing mRNA encoding CYP4B1 from a human tissue or a processed product thereof;
(B) producing a cDNA using mRNA encoding CYP4B1 as a template, (c) amplifying the cDNA by PCR, and (d) measuring the amount of the amplified cDNA. Said (1)
(3) a cDNA prepared using mRNA encoding CYP4B1 as a template,
(A) a forward primer capable of hybridizing to the cDNA, (b) a reverse primer capable of hybridizing to the cDNA,
And (c) having the reporter dye and the quencher dye at the ends and having the cD
Using a probe that hybridizes with NA, 5 ′ →
PCR with a DNA polymerase having 3 ′ exonuclease activity is performed, and the step of measuring the amount of the amplified cDNA further comprises the step of measuring the intensity of fluorescence emitted by the reporter dye of the hydrolyzed probe by irradiation with excitation light. The method for diagnosing bladder cancer according to (2), wherein the measurement is performed; (4) nucleotide 5 of cDNA encoding CYP4B1 as a forward primer
An oligonucleotide that hybridizes to the region of nucleotides 4 to 74, an oligonucleotide that hybridizes to the region of nucleotides 112 to 132 of the cDNA is used as a reverse primer, and a probe hybridizes to the region of nucleotides 77 to 101 of the cDNA. (5) The method for diagnosing bladder cancer according to the above (3), which comprises using a soybean oligonucleotide; (5) The following nucleotide sequence or a nucleotide sequence having 80% or more homology with each of the following nucleotide sequences: (4) The method for diagnosing bladder cancer according to the above (4), comprising using a forward primer, a reverse primer, and a probe comprising an oligonucleotide comprising: a forward primer: 5′-tgggctgatcttggtcttagg-
3 'reverse primer: 5'-gtccatagccttagccaacgt-3' probe: 5'-ttctcaagctcatccacctgctgct-3 '(6) (a) forward primer capable of hybridizing to cDNA encoding CYP4B1, (b) CY
A reverse primer capable of hybridizing to the cDNA encoding P4B1, and (c) a probe which has a reporter dye and a quencher dye at its ends and which hybridizes to the cDNA encoding CYP4B1 within a region sandwiched by both primers. (7) a forward primer, wherein the forward primer is an oligonucleotide that hybridizes to the region of nucleotides 54 to 74 of the cDNA encoding CYP4B1, and the reverse primer is nucleotides 112 to 74 of the cDNA. 132 is an oligonucleotide that hybridizes to the region 132, and the probe is the cD
The diagnostic kit for bladder cancer according to the above (6), which is an oligonucleotide that hybridizes to a region of nucleotides 77 to 101 of NA, and (8) a forward primer, a reverse primer and a probe, Each base sequence or each base sequence below and 80
%. The kit for diagnosing bladder cancer according to the above (7), which comprises an oligonucleotide containing a base sequence having homology of at least%, forward primer: 5′-tgggctgatcttggtcttagg-
3 ′ reverse primer: 5′-gtccatagccttagccaacgt-3 ′ probe: 5′-ttctcaagctcatccacctgctgct-3 ′

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In a diagnostic method according to the present invention, a patient's tissue or a processed product thereof (hereinafter referred to as a “test sample”)
That. ), But the tissue need not be bladder cancer itself, eg, blood, bladder mucosa (other than cancer tissue),
Oral mucosa, hair and the like can be used. In particular, blood and bladder mucosa are easy to collect and CYP4B1
(Or its mRNA) is also preferred because it is easy to detect.
On the other hand, examples of the “processed tissue” include those obtained by freezing mucous tissue with liquid nitrogen to prevent enzymatic degradation of CYP4B1 and its mRNA. Alternatively, blood may be processed by a method known per se, and components obtained from blood may be taken out as test samples. Among them, commercially available kits from whole peripheral blood (for example, VACUTAINER CRT (trade name: Beeton Dickinso
lymphocytes), and the lymphocytes are suspended in a physiological saline or the like, and then centrifuged (for example, 3000).
rpm, for 3 minutes) to form pellets.

The method for diagnosing bladder cancer according to the present invention comprises the steps of (a)
A method for measuring the amount of CYP4B1 itself;
The method can be broadly classified into a method for measuring the amount of mRNA encoding 4B1. First, the following method can be shown as a representative example of the method of (a) measuring the amount of CYP4B1 itself. That is, first, a crude protein is extracted from a test sample. As the extraction method, a method known per se may be used,
For example, when the test sample is bladder mucosa, a method of homogenizing and then preparing a microsomal fraction by ultracentrifugation is preferable. Then the crude protein, preferably the microsomal fraction, was separated by electrophoresis,
The CYP4B1 in the gel is transferred to a membrane such as a nitrocellulose membrane or a nylon membrane by capillary action of a buffering solution (blotting solution), and then labeled anti-CYP4B1
This is a method for measuring the band intensity of CYP4B1 by an antigen-antibody reaction using an antibody. This method is called Western blotting, and detailed conditions have been well established in the past. For example, Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratories (Cold Spring Harbor Laboratories)
atory) (1989).

[0010] "Measurement of band intensity of CYP4B1"
Specifically, for example, rabbit anti-CY
An ECL kit (trade name: Amersham) using P4B1 antibody
(Manufactured by Sharp Corporation). In addition, using a rabbit anti-CYP4B1 antibody, a goat antibody to the rabbit antibody labeled with biotin, and an avidin-biotin-oxidase complex, a method of measuring the color development with hematoxylin by microscopic examination with an optical microscope ( Hsu
SM, Raine L and Fanger H. J Histochem Cytochem 29:
577-580, 1981).

Next, (b) m encoding CYP4B1
A method for diagnosing bladder cancer, which is characterized by measuring the amount of RNA, is described below. The diagnosis method includes (b1) a method of measuring the amount of mRNA encoding CYP4B1 in a test sample, and (b2) preparing a cDNA using the mRNA encoding CYP4B1 in the test sample as a template. c
The method is broadly classified into a method in which DNA is amplified by PCR and the amount of amplified cDNA is measured. In the present invention, it is particularly preferable to use the latter method, which can measure with high sensitivity even when the amount of the test sample is very small.

In the above diagnostic method, (b1) mRNA encoding CYP4B1 in a test sample (hereinafter referred to as “CYP4B1”)
The method for measuring the amount of “4B1-mRNA”) is described below. As the method, a method known per se may be used, but it is measured by hybridization using an oligonucleotide probe that hybridizes to CYP4B1-mRNA by hydrogen bonding, for example, by dot blot hybridization or Northern blot hybridization. be able to. That is, total mRNA was extracted from the test sample, separated by electrophoresis, and then buffered (blotti).
ng liquid) by the capillary action of CYP4B1-m in the gel.
Transferring RNA to a membrane such as a nitrocellulose membrane or a nylon membrane, immersing the membrane in a probe solution, incubating, and measuring the amount of CYP4B1-mRNA using a probe that hybridizes to CYP4B1-mRNA on the membrane. it can. Detailed conditions are described in Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory (Cold Spring)
Harbor Laboratory) (1989) may be used. As a method for extracting total mRNA from a test sample, a method known per se such as a phenol method or a guanidine thiocyanate method may be used. In addition, commercially available reagents for RNA extraction can be appropriately used. Further, CYP4B1-mR is obtained by directly hybridizing RNA in tissue without extracting RNA.
The amount of NA can also be measured.

As a method for producing a probe capable of hybridizing to the above-mentioned CYP4B1-mRNA, for example, a known method such as chemical synthesis and production according to a known method per se in accordance with the reported base sequence of the CYP4B1-cDNA fragment is used. Can be. CYP4B1-mRN
More specifically, as a probe that can hybridize to A, 5′-ccagtaccataatgacttca-3 ′ (rat CYP
Nucleotides 689-7 of mRNA encoding 4B1
08) and 5'-tagaggcggaagcactcctt-3 '(region of nucleotides 1113-1132 of mRNA encoding rat CYP4B1).
The probes are usually labeled for detection. Examples of such a labeling substance include biotin, digoxigenin and the like in addition to radioactive isotopes such as [P ³² ]. The method for labeling the probe may be a method known per se, for example, a 3′-end labeling method, a 3′-end tailing method and the like.

Using the label of the probe, CYP4B1
-The amount of CYP4B1-mRNA can be measured by measuring the amount of probe to which mRNA has been hybridized. The amount of the probe hybridized with CYP4B1-mRNA may be measured according to a method known per se. For example, when a radiolabeled probe is used, a method of measuring by autoradiography may be mentioned. Alternatively, when a probe labeled with biotin or digoxigenin is used, the probe is allowed to react sequentially with a conjugate of an avidin or anti-digoxigenin antibody having a biotin-binding ability and a detection enzyme, and a substrate that develops or emits light with the enzyme. And a method of measuring the amount of probe hybridized to CYP4B1-mRNA from the amount of color development or luminescence.

In the diagnostic method according to the present invention, (b2) C
A cDNA using YP4B1-mRNA as a template is prepared,
The cDNA is amplified by PCR, and the amplified cDNA
The method for measuring the amount of is described below. Where C
CDNA prepared using YP4B1-mRNA as a template
Is referred to as "cDNA encoding CYP4B1" (hereinafter abbreviated as CYP4B1-cDNA). In the above method, a method known per se such as PCR or a method for improving PCR may be used, but PCR (for example, Scienc
e, 230, 1350 (1985)), RT-PCR (Reverse T
ranscription PCR) (Genome Res., 6 (10), 986 (1996)
), Real-time detection methods (eg, TaqMan PCR, ABI
PRISMTM 7700 SEQUENCE DETECTIONSYSTEM, Applied Bio
systems, Ver1, June 1996).

More specifically, total mRNA is extracted from the test sample in the same manner as described above, and cDNA complementary thereto is prepared by using mRNA as a template according to a conventional method, and then complemented with CYP4B1-cDNA. A method of performing PCR using a unique primer is exemplified. Combining the production of the cDNA and amplification of the cDNA by PCR,
RT-PCR in which these are performed in the same system may be used.
PCR and RT-PCR can be basically performed according to a known method. These reaction conditions can also be determined according to a known method. Specifically, it is preferable to employ the conditions described in the examples below.

A more specific embodiment will be described below. CYP4
As a method for preparing B1-cDNA, there is a method for preparing cDNA from mRNA extracted from a test sample using reverse transcriptase. Reverse transcriptase is an RNA-dependent D
Refers to an enzyme that catalyzes the synthesis of NA. For example, a genetically modified reverse transcriptase derived from an RNA tumor virus can be used. Then, the CYP4B1-cDNA was
Amplify in CR. Specific examples of PCR include, for example,
The above-prepared CYP4B1-cDNA, two types of oligonucleotides (primers) complementary to the sequence at both ends of the site to be amplified, ie, capable of specifically binding to the sequence, thermostable DNA polymerase and DNA synthesis The required four nucleotides (dNTPs) are mixed.
At a temperature of about 55 ° C., each primer is annealed to its complementary sequence on the single-stranded template. Next, the temperature is adjusted to about 72 ° C., an extension reaction is performed using a DNA polymerase, and a new DNA strand is synthesized. Each newly synthesized DNA strand functions as a template for the next cycle. The mixture is heated to about 94 to 95 ° C. to denature the double-stranded DNA into single-stranded DNA. By repeating such an amplification cycle, CYP4B1-c
A preferred method is a method in which a DNA fragment is amplified exponentially. DNA used in the present invention
As the polymerase, a DNA polymerase having high thermostability is desirable. For example, a genetically modified thermostable DNA polymerase derived from Thermus aquaticus is preferably used. In the present invention, the treatment of the above two steps is
RT-PCR performed continuously in one system may be used.

As described above, the primer pair used in the PCR or RT-PCR is CYP4B1-c
It is selected from oligonucleotides that can hybridize to DNA. Here, “to hybridize” means PC
It means to hybridize under the conditions of R or RT-PCR. More specifically, the primer pair includes a sense primer which is an oligonucleotide capable of hybridizing to the 5 'end of the sequence to be amplified as an upstream primer, and a 3' end of the sequence to be amplified as a downstream primer An antisense primer, which is an oligonucleotide capable of hybridizing to the above, is used. The number of nucleotides of the primer oligonucleotide is at least about 15, usually about 15 to 50, and preferably about 20 to 40. When the number of nucleotides of these primers and probes is too large, hybridization to the template DNA becomes difficult, and when it is too small, the specificity of hybridization decreases. Therefore, the above range is preferable.

Each of the oligonucleotides as the above primers can be easily synthesized according to a conventional method using an automatic synthesizer, for example, a DNA synthesizer (Perkin Elmer), and the obtained oligonucleotides are further commercially available, if desired. It can also be purified using a purification cartridge or the like.

With such a technique, the amplified cDN
The amount of A is measured by a method known per se. For example, the amplified cDNA is separated by electrophoresis, and the cDNA in the gel is transferred to a membrane such as a nitrocellulose membrane or a nylon membrane by capillary action of a buffering solution (blotting solution), and then hybridized with a labeled probe. And the corresponding band intensity is measured.
Blotting and the like. The band intensity may be measured according to a method known per se. DNA is 2
Since light having a wavelength of 50 to 270 nm is absorbed, a change in absorbance at the wavelength can be measured, and it can also be measured by a staining method known per se such as ethidium bromide staining. In addition, glyceraldehy
The above PCR or RT-PCR is performed using de-3-phosphate dehydrogenase (hereinafter referred to as GAPDH), and the resulting PCR product is measured.
The amount of CYP4B1-mRNA can be measured more accurately based on the ratio of both B1-cDNA and GAPDH-cDNA.

In the present invention, the PCR or RT
-Instead of PCR, a real-time detection method, which is an improved method of PCR, may be used. The use of the real-time detection method has the advantage that amplification of CYP4B1-cDNA by PCR and measurement of the amount of the amplified CYP4B1-cDNA can be performed continuously in one process. Therefore, the operation is simple and short (usually several hours). In addition, the use of this method facilitates quantitative measurement. This method also has the advantage that multiple analytes can be measured at once.
Therefore, in the diagnostic method according to the present invention, it is more preferable to employ the real-time detection method.

As a real-time detection method, there is a method of measuring a nucleic acid using a probe in which a reporter dye and a quencher dye are bound. In this method, a site on the template sandwiched between a site on the template to which a primer (forward primer) used in PCR hybridizes and a site on the template to which another primer (reverse primer) hybridizes, A probe in which a reporter dye and a quencher dye are hybridized, and PCR is performed using a DNA polymerase having 5 ′ → 3 ′ exonuclease activity.

FIG. 3 shows a specific embodiment of the real-time detection method.
This will be described with reference to FIG. For example, in FIGS. 3A to 3D, when the forward primer is extended by the action of DNA polymerase and hits the probe, the oligonucleotide constituting the probe becomes 5 ′ → 3 ′ exonuclease possessed by DNA polymerase. It is degraded by the action of the activity, followed by the generation of extension products of the forward primer. In this case, while the reporter dye 1 and the quencher dye 2 are bound to the probe, the reporter dye 1 does not emit fluorescence due to the interaction between the two, but the oligonucleotide constituting the probe becomes longer with the extension of the primer. When decomposed, the reporter dye 1 and the quencher dye 2 are cut off, and the reporter dye 1 does not receive the action of the quencher dye 2 and emits fluorescence when irradiated with ultraviolet light. Therefore, CYP4B1-
By selecting primers and probes that specifically hybridize to the cDNA,
YP4B1-cDNA can be selectively measured. This method has already been widely used as Taq ManPCR (trademark) or the like. Therefore, the detailed conditions of the present invention can basically follow the conditions of a method known per se.

In the present invention, the probes and primers used in the real-time detection method preferably satisfy the following requirements. (A) The amplification product is about 50 to
(B) that the primer and the probe are as close as possible, (c) that the ratio of G (guanine) and C (cytosine) contained in the sequence is around 50% as much as possible. (D) that G (guanine) is not continuous four or more, (e) the Tm value of the probe is around 70 ° C., and the Tm value of the primer is 60
℃. As described above, the number of nucleotides of each of the primer and probe oligonucleotides is at least about 15, usually about 15 to 5
It is preferably in the range of about 0, preferably about 20-40.

When the real-time detection method according to the present invention is used, preferred examples of primers and probes include CYP4B1-
oligonucleotides that hybridize to the region of nucleotides 54 to 74 of the cDNA, oligonucleotides that hybridize to the region of nucleotides 112 to 132 of the cDNA as a reverse primer, and nucleotides 77 to 10 of the cDNA that serve as the probe.
Oligonucleotides that hybridize to region 1. More specifically, the forward primer
5'-tgggctgatcttggtcttagg-3 ', reverse primer
5'-gtccatagccttagccaacgt-3 ', probe is 5'-ttctcaag
Those that are ctcatccacctgctgct-3 ′ are more preferred. In addition, the primer and the probe are the template strand (CYP4B
1-cDNA), can hybridize with the template DNA even if there is a small number of mismatches, and can function as primers and probes for PCR. Therefore, the primers and probes in the present invention are not limited to those having the specific nucleotide sequence described above, but include those as a part thereof, and, for example, 4 or less, preferably 2 or less in this sequence. It may be an oligonucleotide having a sequence modified by nucleotide substitution, deletion and / or addition. In addition, about 80% or more, preferably about 85%
Above, more preferably about 90% or more, and most preferably about 95% or more of oligonucleotides containing a base sequence having a homology of about 95% or more can also be used as primers and probes. Each oligonucleotide as the primer and the probe can be obtained by a method known per se, such as chemical synthesis according to a conventional method as described above.

The above-mentioned fluorescent-labeled probe for the real-time detection method generally has a reporter dye bound to one end, eg, the 5′-end, and a quencher dye bound to the other end, eg, the 3′-end. . The reporter dye is
For example, a substance that emits fluorescence when irradiated with excitation light,
It is preferable that the quencher dye has a function of quenching the generation of fluorescence by acting on the reporter dye when present in a distance from the reporter dye. Examples of the reporter dye include, for example, a fluorescein fluorescent dye, specifically, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4. ,
5-dichloro-6-carboxyfluorescein (JO
E), hexochloro-6-carboxyfluorescein (HEX) and the like. Examples of the quencher dye include, for example, a rhodamine fluorescent dye, specifically, 6-carboxy-tetramethyl-rhodamine (TAMRA).

The fluorescent-labeled probe can be prepared by binding a reporter dye and a quencher dye to a probe oligonucleotide having a sequence satisfying the above conditions. The method and means for binding may be those known per se. For example, the 5 ′ side of the probe usually has several methylene chains as linkers, and a phosphate group at the end is linked with a reporter dye such as a FAM molecule. It can be attached in the form of an acid ester. In addition, a quencher dye such as a TAMRA molecule can be bound to the 3 ′ side via an amide bond via a structural unit shown below.

[0028]

Embedded image Wherein TAMRA is 6-carboxy-tetramethyl-
Represents rhodamine. )

In the real-time detection method, as described above, the amount of CYP4B1-cDNA can be measured by the intensity of the fluorescence emitted from the reporter dye. That is, only the probe hybridized to the amplified CYP4B1-cDNA is hydrolyzed, and the reporter dye emits fluorescence without being interfered by the quencher dye. Therefore, the amount of the amplified CYP4B1-cDNA can be measured by measuring the fluorescence intensity emitted by the reporter dye. The measurement of the fluorescence intensity emitted by the reporter dye is basically performed in accordance with a conventional method, for example, by using an argon laser beam by PCR.
The reaction can be performed by irradiating the reaction solution and detecting the emitted fluorescence using a CCD camera.

In the diagnostic method according to the present invention, as described above, CYP4B1 or m coding CYP4B1 is used.
The amount of RNA is measured, and if the measured value is significantly higher than that of a healthy person, it can be diagnosed that the patient has bladder cancer or is highly suspicious.

As described above, in the diagnostic method according to the present invention, the PCR real-time detection method is suitably employed. Thus, in the present invention, a combination of a primer pair and a probe used in the method is provided as a bladder cancer diagnostic kit. Specific examples and preferred examples of the kit of the present invention are as described above. In addition, as in the case of a general PCR detection kit, it can be provided as a product of the type in which, for example, a primer pair and a probe are lyophilized individually or in a mixed state and then diluted with distilled water or a buffer when used. it can.

[0032]

EXAMPLES [Sample Collection] Human bladder tissue samples were obtained by total cystectomy or transurethral resection at Osaka City General Medical Center and Osaka City University Hospital. Of course, all tissues were collected after obtaining informed consent from patients and their families. First, bladder cancer patient 2
The bladder mucosa was collected from five patients at the cancer site and at the normal site separated from the cancer site. Furthermore, samples of the bladder mucosa at normal sites were obtained from 13 other patients with bladder cancer. On the other hand, from 21 patients who performed surgery other than bladder cancer,
A sample of normal bladder mucosa was obtained. These patients include those with prostate cancer and benign prostatic hyperplasia. The bladder cancer patients included 33 men and 5 women (mean age 65 years old at examination, width 45 to 86 years), and the patients without bladder cancer were 18 men and 3 Women (average age 6
3 years old, 38-72 years old). And these patients have not received any type of therapy for bladder cancer prior to surgery. A small piece of tissue containing the bladder mucosa obtained above was frozen with liquid nitrogen immediately after excision.

[Isolation of total RNA and preparation of cDNA] The tissue fragments obtained above were homogenized, and
Total RNA was purified from mg using Isogen (Nippon Gene Co., Ltd.). In addition, in order to remove the contamination of the gene DNA, the treatment was performed with DNase (manufactured by Nippon Gene Co., Ltd.). Next, the above total RNA
Was converted to cDNA using RNA PCR Kit (Takara Shuzo Co., Ltd.). The synthesized cDNA is PC
Stored at -20 ° C until R reaction.

[Preparation of Primers and Probes] Using a DNA / RNA synthesizer manufactured by ABI as an automatic synthesizer, oligonucleotides having the following sequences were synthesized using a substrate (dNTP) and specified reagents. Primers and probes for CYP2D6 for comparison and primers and probes for β-actin for standardization were known in the literature (Genomics, 2, 174 (1988) and N.
ucleic Acids Res., 12, 1687 (1984)), designed as follows, and synthesized in the same manner as above. (A) For CYP4B1 ・ Forward primer: 5'-tgggctgatcttggtcttagg
-3 '-Reverse primer: 5'-gtccatagccttagccaacgt-3'-Probe: 5'-Fam-ttctcaagctcatccacctgctgct-Tamra
-3 '(b) For CYP2D6 ・ Forward primer: 5'-tagtggtggctgacctgttct
ct-3 '・ Reverse primer: 5'-tcgtcgatctcctgttggaca-3' ・ Probe: 5'-Fam-ctcctgctcatgatcctacatccgga-Tamr
a-3 '(c) For β-actin ・ Forward primer: 5'-ggtcatcaccattggcaatg-
3 '・ Reverse primer: 5'-cgtcacacttcatgatggagttg-
3 'Probe: 5'-Fam-atggagtcctgtggcatccacgaaactac-T
amra-3 'In the above sequence, Fam represents 6-carboxy-fluorescein, and Tamra represents 6-carboxy-tetramethyl-rhodamine.

[PCR] 0.1 μg of the sample, 0.1
μg and 0.01 μg, respectively, CYP4B1
10p for CYP2D6 and β-actin
mol of the forward and reverse primers and 5 pmol of the fluorescent probe, 0.2 mM of each deoxyribonucleotide and 1.25 units of
Amplitaq DNA polymerase (manufactured by Applied Biosystems) containing 50 μL / tube and ABI
PCR was performed using RISM ^™ 7700 Sequence Detection System (manufactured by Applied Biosystems).
The temperature conditions were as follows: after keeping the temperature at 95 ° C for 10 minutes,
A cycle of 5 seconds and 1 minute at 60 ° C. was performed 40 times, and the fluorescence intensity was measured for each cycle. As a standard, all regions of CYP4B1, CYP2D6 and β-actin cDNA (Biochemistry, 28, 8060 (1989), Gen
omics, 2, 174 (1988) and Nucleic Acids Res., 12,
1687 (1984)), and PCR was performed in the same manner.
The copy number of the cDNA in the sample was determined by comparing the fluorescence intensity with the sample. And each mRNA
The amount was expressed as a ratio to the value of β-actin in order to normalize the original total mRNA amount of each sample.

[Results] Samples of patients other than bladder cancer (n
on-BT), a sample of normal tissue of a bladder cancer patient (BT
FIG. 1 shows the results of measurement of CYP4B1 for normal and bladder cancer patient cancer tissue samples (BT tumor). From FIG. 1, it can be confirmed that, in the bladder cancer patient, the expression level of CYP4B1 is higher than usual not only in the cancer tissue but also in the normal tissue (29.64 ± 4.17).
And 29.73 ± 6.18; both mean ± standard deviation). In addition, in the group containing cancer patients other than bladder cancer, the expression level of CYP4B1 is found to be low (10.15 ± 0.97; mean ± standard deviation). In addition, the horizontal bar in FIG. 1 shows an average value. On the other hand, Romkes-Sparks et al. Reported the relationship between CYP2D6-mRNA levels and human bladder cancer (C
arcinogenesis, 15, 1955 (1994)). Therefore, the correlation between the CYP2D6 mRNA level and the CYP4B1 mRNA level measured above was examined. As a result, no correlation was found between the two (FIG. 2 (r ² = 0.301)).
It was confirmed that only the CYP4B1 mRNA level could be used as an indicator of bladder cancer.

[0037]

According to the method and the kit for diagnosing bladder cancer of the present invention, the pain given to the patient can be minimized and the bladder cancer can be specifically diagnosed with high sensitivity. Moreover,
The technique is simple. Such a simple and specific method for diagnosing bladder cancer and a diagnostic kit are useful in diagnosing bladder cancer in which the recurrence rate is high and early detection and early treatment are important. In addition, according to the diagnostic method of the present invention, since the bladder mucosal tissue or blood that is easy to collect is to be diagnosed, the patient's pain at the time of diagnosis is reduced. In particular, the bladder mucosal tissue is not limited to a cancer tissue, and is a normal tissue to be diagnosed. Therefore, it is easy to collect a test sample from a patient, and the burden on the patient is small.

Furthermore, if a real-time detection method is used as the diagnostic method and diagnostic kit according to the present invention,
Amplification of CYP4B1-cDNA by PCR does not require two steps of measuring the amount of the cDNA, and the amount of CYP4B1-cDNA amplified by PCR can be continuously measured in one treatment, and can be quantitatively measured. it can. Therefore, a more rapid and simple diagnosis of bladder cancer can be performed with high sensitivity.

[0039]

[Sequence Listing] Sequence Listing <110> OTSUKA PHARMACEUTICAL FACTORY, INC. <120> A method and a kit for diagnosis of bladder cancer <130> DO01J297 <160> 3 <210> 1 <211> 21 <212> DNA <213 > Artificial sequence <220> <221> <222> <223> Forward primer <400> 1 tgggctgatc ttggtcttag g 21 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <220> <221> <222 > <223> Reverse primer <400> 2 gtccatagcc ttagccaacg t 21 <210> 3 <211> 25 <212> DNA <213> Artificial sequence <220> <221> <222> <223> Probe <400> 3 ttctcaagct catccacctg ctgct 25

[Brief description of the drawings]

FIG. 1 shows a sample of a patient other than bladder cancer (“non” in the figure).
-BT "), a sample of a normal tissue of a bladder cancer patient (represented by" BT normal "in the figure) and a sample of a cancer tissue of a bladder cancer patient (" BT tumo in the figure ")
r)) shows the measurement results of the amount of CYP4B1-mRNA. In addition, the horizontal bar in a figure shows an average value.

FIG. 2 shows a correlation between the mRNA level encoding CYP2D6 and the mRNA level encoding CYP4B1.

FIG. 3 shows the principle of the real-time detection method.

Continued on the front page F term (reference) 2G054 AA06 AA07 CA22 CE02 EA03 GA04 4B024 AA12 CA04 CA09 HA12 HA15 4B063 QA01 QA19 QQ03 QQ53 QQ58 QR08 QR33 QR42 QR56

Claims (8)

[Claims] Claims: 1. C in human tissue or a processed product thereof
A method for diagnosing human bladder cancer, comprising measuring the amount of mRNA encoding YP4B1 or CYP4B1, and comparing it with that of a healthy person. 2. The method for measuring the amount of mRNA encoding CYP4B1 comprises the steps of: (a) determining the amount of CYP4B1
A step of obtaining an RNA fraction containing mRNA encoding 4B1; (b) a step of preparing a cDNA using the mRNA encoding CYP4B1 as a template;
R) and (d) amplified cDN
The method for diagnosing bladder cancer according to claim 1, comprising a step of measuring the amount of A. 3. The step of amplifying, by PCR, a cDNA prepared using mRNA encoding CYP4B1 as a template, comprises: (a) a forward primer capable of hybridizing to the cDNA; and (b) a primer hybridizing to the cDNA. 5 ′ → 3 ′ exonuclease activity using the obtained reverse primer and (c) a probe having a reporter dye and a quencher dye at the ends and hybridizing with the cDNA in a region sandwiched by both primers. The method according to claim 1, wherein the step of measuring the amount of the amplified cDNA comprises measuring the intensity of the fluorescence emitted by the reporter dye of the hydrolyzed probe by irradiation with excitation light. The method for diagnosing bladder cancer according to claim 2. 4. The method according to claim 1, wherein the forward primer is CYP4B1.
Is an oligonucleotide that hybridizes to the region of nucleotides 54 to 74 of the cDNA encoding
The method for diagnosing bladder cancer according to claim 3, wherein the oligonucleotide is an oligonucleotide that hybridizes to a region of 132, and the probe is an oligonucleotide that hybridizes to a region of nucleotides 77 to 101 of the cDNA. 5. The method according to claim 1, wherein a forward primer, a reverse primer and a probe are used, each of which is composed of an oligonucleotide containing the following nucleotide sequence or a nucleotide sequence having 80% or more homology with each of the following nucleotide sequences. 5. The method for diagnosing bladder cancer according to 4. Forward primer: 5'-tgggctgatcttggtcttagg-
3 'reverse primer: 5'-gtccatagccttagccaacgt-3' probe: 5'-ttctcaagctcatccacctgctgct-3 ' 6. (a) cDN encoding CYP4B1
A forward primer capable of hybridizing to A,
(B) a reverse primer capable of hybridizing to cDNA encoding CYP4B1, and (c) a cD having a reporter dye and a quencher dye at its ends and encoding CYP4B1 within a region sandwiched by both primers.
A bladder cancer diagnostic kit comprising at least a probe that hybridizes with NA. 7. The method according to claim 7, wherein the forward primer is CYP4B1.
Is an oligonucleotide that hybridizes to the region of nucleotides 54 to 74 of the cDNA encoding
7. The bladder cancer diagnostic kit according to claim 6, wherein the oligonucleotide is an oligonucleotide that hybridizes to a region of 132, and the probe is an oligonucleotide that hybridizes to a region of nucleotides 77 to 101 of the cDNA. 8. The forward primer, the reverse primer and the probe are each composed of an oligonucleotide containing the following nucleotide sequence or a nucleotide sequence having 80% or more homology with each of the following nucleotide sequences. A kit for diagnosing bladder cancer according to claim 7. Forward primer: 5'-tgggctgatcttggtcttagg-
3 'reverse primer: 5'-gtccatagccttagccaacgt-3' probe: 5'-ttctcaagctcatccacctgctgct-3 '