JP2006017692A - Nondestructive procedure for quantitatively determining oxidation degree of tissue cell nucleus dna, pathological examination method using the procedure for disease stemming from dna oxidation, and method for screening therapeutic substances for diseases - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a procedure for quantitatively determining the degree of oxidation of a cell nucleus DNA in a target tissue cell, to provide a method for histopathologically examining the diseases that result from the DNA oxidation by using this procedure, and to provide a method for screening therapeutic substances for the diseases. <P>SOLUTION: 1. Triple staining comprising staining of cell nucleus, 2. double staining of target cell specific substance and double staining of oxidized DNA, 3. after decolorizing of stained nucleus, is carried out, and then the stain degree of a tissue and positions of a cell nucleus and a target cell are analyzed and measured at each stain image, thereby measuring the degree of oxidation of the cell nucleus DNA in the target cell quantitatively. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nondestructive method for quantifying the degree of tissue cell nuclear DNA oxidation. More specifically, a method for examining pathologically a disease caused by DNA oxidation by quantifying oxidized DNA in the cell nucleus while identifying individual cells in a specific tissue cell, and screening for the therapeutic agent for the disease Regarding the method.

  Aerobic cells are always producing respiration, which is a by-product of breathing, at the same time. These active oxygens are erased by the antioxidant action of the cell, but if the action is not sufficient, it leads to an oxidative stress state, causing cell damage and cell death (apoptosis). In recent years, it has been found that products of oxidative stress accumulate in cells in aging and many diseases, and it has become clear that oxidative stress is closely related to aging and diseases.

  For these reasons, establishment of a method for quantitatively measuring the degree and state of oxidative stress is strongly desired, and various methods have already been proposed. For example, glutathione-added hemoglobin as a marker, a method for measuring the amount of glutathione-added hemoglobin in hemoglobin released from red blood cells in blood (see Patent Document 1), monounsaturated fatty acid as a marker, and biological samples such as plasma and blood A method for measuring the amount of monounsaturated fatty acids therein (see Patent Document 2) and the like are known.

In addition, since oxidative stress oxidatively damages DNA in cell nuclei, a method for measuring oxidative stress using oxidative damage products of these DNAs as markers is also known. Examples of such oxidative damage products of DNA include 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine, and the like. Among them, 8-oxoguanine, which is an oxidized form of guanine, is particularly suitable for DNA replication. In addition to cytosine, it is known to form a base pair with adenine to cause C: G → A: T mutation, which is considered to contribute to carcinogenesis and aging. Examples of the method for quantifying 8-oxoguanine include a quantification method by an electrochemical detection method using high performance liquid chromatography (HPLC), a quantification method by a ³² P-post label method, and the like. These oxidatively damaged products of DNA can be quantified using organs and cells, but since they are excreted into the urine via blood in the process of repairing the DNA damage, the urinary concentration of DNA can be determined by quantifying the concentration of urine. A method for measuring the degree of oxidation has been well studied. For example, a method is known in which urine is fractionated through a gel filtration column having the characteristics of a reverse-phase column and an ion-exchange column, the urine sample is purified by passing the fraction through a reverse-phase column, and then quantified by an electrochemical detection method or the like. (See Patent Document 3). In addition, as a method for detecting 8-oxoguanine, 8-oxoadenine and 8-oxodeoxyguanosine in DNA, it is bound to avidin, streptavidin or an anti-biotin antibody, and the binding is detected and analyzed to detect the DNA. A method for quantifying oxidative damage products is known (see Patent Document 4).

The present inventor found that skeletal muscle from a DMD muscular dystrophy patient already accumulated lipofuscin, which is a product of oxidative stress, in the cytoplasm 2 to 7 years after birth. It was found that there was hardly any (see Non-Patent Document 1). In addition, in skeletal muscles of mdx mice, which are model animals of DMD muscular dystrophy, muscle cells that accumulate lipofuscin are observed in the same manner as DMD muscles, and these cells may have undergone cell death (apoptosis). It became clear (refer nonpatent literature 2). Therefore, the relationship between increased oxidative stress and myocyte degeneration in dystrophic muscle was suggested. For these reasons, the method of quantitatively measuring oxidative damage due to oxidative stress in each part or cell of tissue is used in pathological examination of diseases, elucidation of the onset mechanism, efficacy test and screening of antioxidants, etc. It will be a very useful tool.
JP 2000-74923 A JP 2003-83977 A JP 2000-310625 A Japanese National Patent Publication No. 11-507435 Journalof Molecular Histology, (in press) Histochemistry and Cell Biology, 115, 205-214, (2001) HistolHistopathol, 10, 463-479 (1995a) Protein Nucleic Acid Enzyme, 40, 1927-1935 (1995b) Chemistry and Biology, 36, 113−119 (1998)

  However, to measure oxidative stress, the method for quantifying oxidative damage products contained in urine or blood as described above is an index for knowing the degree of oxidative stress in a living body, but it is not suitable for specific tissues. The degree of oxidation cannot be known and cannot be used for pathological examination of the disease. Furthermore, no method has been reported in Japan and abroad at present for quantitatively measuring the degree of oxidation of tissue cell nuclear DNA while identifying individual cells in a specific tissue.

  Accordingly, an object of the present invention is to provide a method for quantitatively measuring the degree of oxidation of nuclear DNA in a specific tissue, and furthermore, by using this method, diseases caused by DNA oxidation can be determined histopathologically. An object of the present invention is to provide a test method and a screening method for the therapeutic agent for the disease.

  As a result of intensive studies to solve the above problems, the present inventor has as follows: Cell nuclear staining with nuclear staining dye, 2. 2. Heavy staining of target cell specific substance, and Decolorization of stained nuclei followed by triple staining of oxidized DNA and analysis or measurement of cell nucleus, target cell position, and tissue staining degree for each stained image, and the degree of oxidation of cell nucleus DNA in target cells It was found that can be measured quantitatively.

According to the present invention, the techniques and drugs described in (1) to (5) below are provided.
(1) Nondestructive quantification method of tissue cell nuclear DNA oxidation degree, wherein the following steps (a) to (f) are performed in the order of description: (a) After staining tissue cell nuclei with a nuclear staining dye A step of analyzing or measuring at least the position of the nucleus to be stained and the staining degree of the tissue for the image of the stained tissue; (b) the membrane and / or cytoplasm of the target cell in the stained tissue obtained in step (a) (C) a step of decolorizing the stained nuclei in the heavily stained tissue cells obtained in step (b); (d) obtained in step (c). The oxidized DNA in the decolored tissue cells is subjected to heavy staining with a specific binding substance-containing liquid or a specific binding substance-free liquid, and at least the presence and position of the target cells per image of the heavily stained tissue, Analyzing the position of stained DNA to be stained and the staining degree (D) of the tissue or (E) the identity of the position of the cell nucleus analyzed in step (a) and the position of the stained oxidized DNA analyzed in step (d), and the presence and position of the target cell analyzed in step (d) A step of determining the accuracy of the location of the oxidized DNA to be stained; and (f) the staining degree (D) of the tissue obtained by the measurement in step (d) with the specific binding substance-containing liquid of the patient-derived tissue cells. From the degree of staining (Dpt) and the degree of staining of the patient-derived tissue cells with the specific binding substance-free liquid (Dpc), P = Dpt / Dpc, the degree of staining of the healthy tissue-derived tissue cells with the specific binding substance-containing liquid (Dnt) and A step of sequentially calculating N = Dnt / Dnc and then P / N sequentially from the degree of staining (Dnc) of a specific tissue-free solution of tissue cells derived from healthy subjects.
(2) A pathological examination method for diseases caused by DNA oxidation, characterized by using the quantitative method described in (1) above.
(3) A screening method for a therapeutic agent for a disease caused by DNA oxidation, characterized by using the quantitative method described in (1) above.
(4) A therapeutic agent for diseases caused by DNA oxidation, obtained by the screening method according to (3) above.
(5) The nuclear staining dye used in step (a) described in (1) above is neutral red, the specific binding substance used in step (b) is an anti-laminin β2 chain monoclonal antibody, and the specific binding used in step (d) A nondestructive method for quantifying the degree of nuclear DNA oxidation in a muscular dystrophy patient whose substance is an anti-8-oxoguanine monoclonal antibody.
(6) A therapeutic agent for muscular dystrophy obtained by screening using the quantitative method described in (5) above.

  According to the non-destructive quantification method for the degree of tissue cell nuclear DNA oxidation of the present invention, the degree of DNA oxidation of a specific cell can be quantitatively determined. It can be used in screening methods for therapeutic agents. For example, aging, apoptosis, cancer, arteriosclerosis, diabetes, neurodegenerative diseases [eg, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease], ischemia / reperfusion injury, adriamycin cardiomyopathy, arteriosclerosis It can be used for pathological examination of diseases such as gastrointestinal mucosal disorders and screening of therapeutic agents for these diseases.

  The non-destructive method for quantifying the degree of tissue cell nuclear DNA oxidation according to the present invention comprises: Cell nuclear staining with nuclear staining dye, 2. 2. Heavy staining of target cell specific substance, and The dyeing nucleus is decolored and then the oxidized DNA is double-stained, and the position of the cell nucleus and target cells and the staining degree of the tissue are analyzed or measured for each stained image. In this way, by determining the amount of oxidized DNA in individual cell nuclei while identifying the target cells, the degree of oxidation of the cell nuclear DNA in the target cells is quantified. FIG. 1 shows an outline of a nondestructive method for measuring the degree of DNA oxidation in tissue cell nuclei of the present invention. Hereinafter, each step will be described in detail in order.

<Staining of tissue cell nuclei>
Using at least two tissue specimens for each subject, the cell nucleus of the tissue section is stained with a nuclear staining dye. The nuclear dye is not particularly limited as long as it can be decolorized because the dyed nucleus is decolorized in a later step. As such a nuclear dye, neutral red is preferably used.

<Capture and analyze images of stained tissue>
After the tissue cell nucleus is stained as described above, the color image and gray level image of the tissue to be stained are captured. The gray level image is captured using an image analysis system for measuring absorbance described later. By using such an image analysis system for measuring absorbance, it is possible to carry out everything from capturing an image to measuring absorbance (staining degree) in individual cell nuclei. From the captured image, the position of the dyed nucleus and the degree of tissue staining are confirmed.

<Multiple staining of target cell specific protein>
In this step, in order to identify a target cell, a specific protein localized in the membrane and / or cytoplasm of the target cell is selected as a marker, and is overstained with a solution containing a specific binding substance for the marker. The specific binding substance is not particularly limited as long as it is a substance that specifically binds to the marker, but a monoclonal antibody is preferably used. As a method of staining using a monoclonal antibody, a direct method in which a fluorescent substance or enzyme is directly labeled and reacted with the monoclonal antibody, or a monoclonal antibody is reacted as a primary antibody and then labeled with the fluorescent substance or enzyme. An indirect method of reacting a secondary antibody can be used. Specifically, for identifying myocytes, laminin β2 localized in the basement membrane of myocytes is used as a marker, rat anti-laminin β2 (γ ′) chain monoclonal antibody as a primary antibody, and biotin binding as a secondary antibody. The marker laminin can be overstained by using an anti-rat IgG (H + L) antibody followed by the addition of streptavidin-conjugated alkaline phosphatase to form an avidin-biotin complex and finally the chromogenic substrate. In this step, color development by such an alkaline phosphatase reaction is preferable. This is because the coloration by the peroxidase reaction that is often used generally uses the oxidizing hydrogen peroxide as a substrate, and thus affects the quantification of oxidized DNA in the next step. Therefore, it is necessary to select an enzyme-labeled antibody that does not affect the subsequent staining of oxidized DNA. Further, as the chromogenic substrate used in the alkaline phosphatase reaction, NBT / BCIP, Fast Blue B, Fast Red and the like can be raised, and NBT / BCIP is preferably used from the viewpoint of the localization of the pigment precipitation.

  In addition to the specific protein (marker) for identifying the target cell, as a tumor marker, α-fetoprotein, S-100 protein, intermediate filament (keratin, vimentin, desmin, neurofilament protein) localized in the cytoplasm , GFAP) and the like, such as epithelial membrane antigen localized in the cell membrane. In nerve tissue, MAP2, which is localized in neuronal cells, neurofilament protein, myelin basic protein, various neuropeptides, glial fibrillary acidic protein (GFAP) which is localized in astrocytes, F4 which is present in microglia / 80 etc. are raised as markers. Other muscle cell markers include actin, myosin, dystrophin, and the like. Examples of secretory cell markers include endocrine hormones such as gastrin, insulin, glucagon, ACTH, calcitonin, and other secretory proteins such as amylase. Examples of apoptotic cell markers include proteins such as Bax, Bcl-2, Bcl-x, and Fas. In the present invention, these cells are used as markers, and target cells can be identified by multiple staining with a specific binding substance such as that described above, for example, a labeled antibody or a specific staining dye.

  When the method of the present invention is used for a pathological examination of a disease caused by DNA oxidation, for example, in order to conduct a pathological examination of muscular dystrophy, a myocyte is selected as the target cell, and the degree of oxidation of the nuclear DNA of the myocyte is quantified. . Hepatocytes are selected as target cells for hepatocellular carcinoma pathology, lung epithelial cells for lung cancer pathology, squamous cells for head and neck squamous cell carcinoma, and squamous cells for esophageal cancer pathology. For pathological examination of epithelial cells and diabetes, monocytes, amyotrophic lateral sclerosis (ALS), neuropathy for neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, etc. Pathological examination of nerve cells, ischemia / reperfusion injury In the present invention, cardiomyocytes, cardiomyocytes for pathological examination of adromycin cardiomyopathy, and vascular smooth muscle cells can be selected as target cells for pathological examination of arteriosclerosis and used in the present invention.

  Here, the target cell-specific protein overstaining in this step must be performed before the oxidized DNA overstaining step described later. This is because, in the process of heavy staining of oxidized DNA, it is necessary to perform a protease treatment in advance to expose the DNA in the cell nucleus. However, when this protease treatment is performed, not only the DNA-binding protein but also the target cell is identified. This is because even specific proteins are degraded and cannot be detected. Therefore, in the present invention, it is essential that the target cell-specific protein is first heavily stained and then the oxidized DNA is heavily stained.

<Decolorization of dyed nuclei>
In the next step, double staining of oxidized DNA stains cell nuclei, and thus the stained nuclei stained in the previous step must be decolorized. When neutral red is used as the nuclear staining dye in the nuclear staining of the previous step, neutral red is completely released from the nucleus to be stained by acid treatment with hydrochloric acid or glacial acetic acid, or alkali treatment such as NaOH or KOH. be able to. Preferably, hydrochloric acid is used. The concentration of the acid and alkali is about 0.1 to 2N and can be sufficiently decolored. At this time, if the decolorization treatment is performed using 2N hydrochloric acid, the denaturation of the DNA in the next step can be performed simultaneously, which is very efficient.

  Fig. 2 shows a nuclear stained image of 1% neutral red tissue of a 2-year-old boy with Duchenne muscular dystrophy (DMD) (A), and complete release of neutral red by 2N hydrochloric acid treatment of the same nuclear stained section. It is an image (B). As shown in FIG. 2 (A), when the fixed tissue sections were incubated with 1% neutral red for 30 seconds at room temperature, all cell nuclei stained red. Further, when this stained tissue section is treated with 2N hydrochloric acid, neutral red is completely released from the tissue section as shown in FIG. 2 (B). Therefore, it can be said that the immuno-staining performed subsequently by the decolorization treatment by such a method has no influence of nuclear staining with neutral red. In addition, neutral red is completely liberated by decolorization by acid treatment, but formazan, which is a final reaction product of alkaline phosphatase that is preferably used for the target cell-specific protein heavy staining in the previous step, is hardly decolorized. Therefore, since the presence and position of the target cell to be stained can be confirmed from the image after the subsequent double staining of oxidized DNA, it is not necessary to take an image before decolorization and confirm the presence and position of the target cell.

<Double staining of oxidized DNA>
In this step, the oxidized DNA in the tissue cell nucleus decolorized in the above step is double-stained with a specific binding substance-containing solution or a non-specific binding substance-containing solution for the oxidized DNA. Since the nuclear density of cell nuclei differs between individual nuclei and their cut surfaces, the degree of oxidation of tissue cell nucleus DNA cannot be accurately quantified by comparing the staining degree using different tissue sections. Therefore, in the method of the present invention, after staining a tissue section with a nuclear staining dye, the stained nucleus is decolored, and the same tissue section is subjected to multiple staining with oxidized DNA, and the intensity of nuclear staining with the nuclear staining dye in the same cell nucleus is determined. And the staining intensity of oxidized DNA can be accurately quantified. Furthermore, in this step, when the specific binding substance-free solution is also heavily stained, and this heavily stained image is used as a control in subsequent image analysis, the degree of oxidation of tissue cell nuclear DNA can be quantified more accurately.

  The specific binding substance for overstaining oxidized DNA in tissue cell nuclear DNA is not particularly limited as long as it is a substance that specifically binds to oxidized DNA. Examples of the oxidized DNA include oxidatively damaged bases, oxidatively damaged deoxynucleosides, oxidatively damaged deoxynucleotides, and the like. Specific examples include 8-oxoguanine, 2,6-diamino-4hydroxy-5-formamide. Pyrimidine, 8-oxoadenine, 4,6-diamino-5-formamide pyrimidine, uracil glycol, 5-hydroxycytosine, thymine glycol, 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 6-hydroxy Oxidatively damaged bases such as -5,6-dihydrothymine, deoxynucleosides containing these oxidatively damaged bases, and deoxynucleotides are raised. As specific binding substances for these various oxidized DNAs, the above-mentioned oxidatively damaged bases and monoclonal antibodies against deoxynucleosides and deoxynucleotides containing these oxidatively damaged bases can be preferably used. As such monoclonal antibodies, anti-8-hydroxydeoxyguanosine monoclonal antibody N45.1 (Niken Zile Co., Ltd., Japan Aging Control Laboratory, Shizuoka), mouse 8-oxoguanine monoclonal antibody (MAB3560; Chemicon International, Temecula, California, USA), anti-8-hydroxydeoxyguanosine & 8-hydroxyguanosine monoclonal antibody (QED bioscience, San Diego, California, USA), FITC-labeled anti-8-oxoguanine antibody (Biotrim International, Dublin, Ireland), etc. Can be used.

  Among the various oxidized DNAs described above, 8-oxoguanine is caused by low oxidative stress, and the mutation caused by 8-oxoguanine is suggested to be involved in cancer and aging. It is considered very important to quantify the amount of 8-oxoguanine in the medium. For these reasons, it is preferable to use a monoclonal antibody against 8-oxoguanine in DNA as the specific binding substance. Particularly preferably, the mouse anti-8-oxoguanine monoclonal Fab166 used in this experimental example (special gift from Dr. Ivan Bespalov, University of Vermont, USA; Soultanakis et al., Free Radical Biology & Medicine, 28, 987-998, 2000) is used. To do.

<Uploading images of heavily stained tissue>
After double staining of oxidized DNA as described above, a color image and a gray level image are taken in the heavily stained tissue. The gray level image is captured using an image analysis system for measuring absorbance described later, as in the stained image of the tissue cell nucleus. By using such an image analysis system for measuring absorbance, it is possible to carry out everything from capturing an image to measuring absorbance (staining degree) in individual cell nuclei. From the captured image, the presence and position of the target cell, the position of the oxidized DNA to be stained, and the staining degree (D) of the tissue are confirmed.

<Image analysis>
Using the ARGUS-100 image analysis system for measuring absorbance described later, the absorbance at 451.2 nm of each tissue cell nucleus is measured from the gray level image of the target cell-specific substance and oxidized DNA double-stained obtained in the above step. To do. In the same manner, the absorbance of individual tissue cell nuclei is also measured for a heavy-stained image of a continuous tissue section in the absence (control) of a specific binding substance for oxidized DNA obtained in the above step. At this time, it is necessary to confirm the identity of the tissue cell nucleus whose absorbance is measured by comparing it with the position of the cell nucleus stained with the nuclear staining dye, and further confirm that it is the tissue cell nucleus of the target cell.

<Calculation of DNA oxidation degree>
Regarding the staining degree (D) of the tissue obtained above, the staining degree (Dpt) of the patient-derived tissue cells with the specific binding substance-containing liquid and the staining degree (Dpc) of the patient-derived tissue cells with the specific binding substance-free liquid From P = Dpt / Dpc, the staining degree (Dnt) of a tissue cell derived from a healthy person with a specific binding substance-containing liquid and the staining degree (Dnc) of a tissue cell derived from a healthy person with no specific binding substance N = Dnt / By calculating Dnc and then P / N sequentially, the degree of DNA oxidation for healthy patients can be obtained. This calculation formula was derived from this experimental example described later.

  By calculating the degree of DNA oxidation for a healthy patient using the above calculation formula P / N, a pathological examination of a disease caused by DNA oxidation can be performed. That is, from the heavily stained image of oxidized DNA obtained by the method described above, the absorbance of 50 to 300 tissue cell nuclei of the target cells was measured for each patient and healthy subject, and the value of the calculation formula P / N was 1.1 to When 1.3, it can be determined that the patient is suffering from a disease caused by DNA oxidation.

<Device>
For the capture of an image in each step and analysis of the captured image, an ARGUS-100 image analysis system for absorbance measurement developed by the present inventor (see Non-Patent Documents 3 to 5) (hereinafter abbreviated as “present system”) Is preferably used. Hereinafter, the ARGUS-100 image analysis system for measuring absorbance will be described in detail. This system consists of an optical microscope (Nikon, Tokyo), a Plumbicon camera (Hamamatsu Photonics, Hamamatsu), an ARGUS-100 image processing device (Hamamatsu Photonics), a monitor (Hamamatsu Photonics), a printer (Hamamatsu Photonics), and a stabilized power supply (Takasago). Manufacturing, Kawasaki), and software. The software was created by the inventor presenting measurement principles, mathematical formulas, and measurement procedures, and requesting Hamamatsu Photonics to program them. The system can perform from the acquisition of a gray level image of a tissue section to the measurement of the absorbance of individual cell nuclei in the captured image.

  In addition to this system, software for measuring absorbance in tissue images is not commercially available. However, if the intensity of transmitted light from an ND filter with a known transmittance is measured with the system used, the intensity of incident light can be determined in advance. The absorbance can be calculated from the intensity of the transmitted light of the specimen. The following AQUA-C / IMAGING microscope imaging system (Hamamatsu Photonics, Hamamatsu), DXM 1200F system (Nikon, Tokyo), etc. are listed as commercially available equipment that seems to be capable of such measurement. Instead of this system, it is also possible to use a system as described above.

  Next, the present invention will be specifically described with reference to experimental examples and examples, but the scope of the present invention is not limited thereto.

(Experimental example 1)
In the experimental examples, the method of the present invention was applied to skeletal muscle and healthy muscle of muscular dystrophy, and the influence of oxidative stress on nuclear DNA in muscular dystrophy was clarified.
<Organization>
Obtained informed consent from the National Mental and Nerve Center (Kodaira), 3, 4 and 7 year old Duchenne muscular dystrophy (DMD) 3 male patients and 2, 3 and 6 year old 3 healthy boys A portion of a frozen specimen of a biceps biopsy was used.

<Test method>
(1) Preparation and fixation of tissue sections From frozen specimens of biceps biopsy, 7 μm frozen sections were prepared using a cryostat (Bright, Huntingdon, UK), and 0.01% poly-L- in advance. Attached to a glass slide coated with lysine (Sigma Chemical, St. Louis, Missouri, USA). Tissue sections were fixed by immersing in methanol at −20 ° C. for 30 minutes after air drying, and immediately cooled in ice with 0.1 M Tris-buffered saline (TBS) (pH 7.4) and 0.1 M acetate buffer (pH 4). 8), the sections were washed for 5 minutes each. Six tissue sections were used per subject.

(2) Staining of cell nuclei Tissue sections were stained with 1% neutral red (Wako Pure Chemical Industries, Osaka) dissolved in 0.1 M acetate buffer (pH 4.8) at room temperature for 30 seconds and then for 5 minutes each with the same buffer. Washed 3 times at room temperature. The tissue section was sealed with the same buffer, and the periphery of the cover glass was sealed with nail polish. Immediately, a gray-level image and a color image of the nuclear staining image were captured using an ARGUS-100 image analysis system for measuring absorbance described later and an optical microscope equipped with a digital camera. In order to perform subsequent immuno-staining, the preparation was soaked in TBS, the nail polish and cover glass turned to milky white were removed, and the tissue section was exposed again.

(3) Immunostaining of laminin Subsequently, in order to distinguish the nucleus (muscle nucleus) in the muscle fiber (cell) from the outer nucleus, it is localized in the basement membrane covering the outer surface of the muscle fiber cell membrane. The enzyme immunohistochemical staining of laminin β2 chain was performed as follows. However, since the muscle satellite cell usually exists between the muscle cell membrane and the basement membrane, the nucleus and the muscle cell nucleus cannot be distinguished. First, the tissue sections that were subjected to nuclear staining in (1) were washed three times for 5 minutes each with ice-cold TBS, and then the water around the tissue sections was wiped off, surrounded by a PAP pen (Daido Sangyo, Tokyo), and avidin solution Laboratories, South San Francisco, California, USA) were placed on the sections and incubated for 10 minutes. After washing with ice-cold TBS three times for 5 minutes each, it was incubated with d-biotin solution (Zymed) for 10 minutes and washed in the same manner. Next, in order to block non-specific antibody binding sites, sections are incubated with 5% normal serum (Sigma) of rabbit dissolved in TBS (TBST) containing 0.05% Tween20 (Katayama Chemical, Osaka) for 30 minutes. did. Subsequently, rat anti-laminin β2 (γ ′) chain monoclonal primary antibody (MAB1914; Chemicon International, Temecula, California, USA) diluted 200-fold with the normal serum was incubated at 4 ° C. overnight. After washing with ice-cold TBS three times for 5 minutes each, rabbit biotin-conjugated anti-rat IgG (H + L) secondary antibody (61-9540; Zymed) and tissue sections diluted 2500 times with TBST containing 5% normal rabbit serum were incubated at room temperature. And incubated for 2 hours. After washing 3 times for 5 minutes each with ice-cold TBS, it was incubated with streptavidin-alkaline phosphatase (Zymed) for 1 hour at room temperature. After washing the sections twice for 5 minutes each with ice-cold TBS, 0.1 mM Tris-HCl buffer containing 1 mM levamisole chloride (inhibitor of endogenous alkaline phosphatase; Nacalai Tesque, Kyoto), 0.1 M sodium chloride and 50 mM magnesium chloride The solution (pH 9.5) was incubated at room temperature for 5 minutes. Next, alkaline phosphatase BCIP / NBT substrate solution (Zymed) containing 1 mM levamisole chloride and 2% polyvinyl alcohol (soluble in boiling water; Sigma) and tissue sections were observed under a microscope for the deposition of purple formazan, the final reaction product. The reaction was allowed to proceed for 35 minutes at room temperature. The reaction was stopped by applying TBS containing 2% polyvinyl alcohol to the section, and then washed twice with TBST for 5 minutes.

(4) Decolorization of stained nucleus and immuno-staining of 8-oxoguanine Tissue sections subjected to immuno-staining of laminin were incubated with 0.1% TritonX-100 (Sigma) dissolved in TBS for 15 minutes at room temperature. Washed 3 times for 5 minutes each with ice-cold TBS. Next, the sections were treated with 20 μg / ml RNase (DNase free; Nippon Gene, Toyama) dissolved in 0.01 M Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and 0.4 M sodium chloride at 37 ° C. for 1 hour. And washed 3 times for 5 minutes each with ice-cold TBS. Further, it was treated with 10 μg / ml proteinase K (PCR grade; Roche Diagnostics, Mannheim, Germany) dissolved in 0.1 M Tris-HCl buffer (pH 8.0) containing 50 mM EDTA for 10 minutes at room temperature, and sectioned with ice-cold TBS. Was washed for 3 minutes. Tissue sections were soaked in 2N hydrochloric acid for 5 minutes for DNA denaturation and complete release from neutral red tissue sections. Sections were washed for 5 minutes each in a mixture of 2N hydrochloric acid and 1M Tris (1: 2.5) and in ice-cold TBS. In order to block the non-specific antibody binding site, the treatment with avidin / biotin as described in (3) above was performed, followed by incubation with a room temperature BEAT blocker solution 1A (Zymed) for 1 hour. After washing twice with ice-cold TBS for 5 minutes each, it was further blocked with BEAT blocker solution 1B (Zymed) at room temperature for 20 minutes and washed twice with ice-cold TBS for 5 minutes each. As a test for detecting 8-oxoguanine, 0.02 μg / ml mouse anti-8-oxoguanine monoclonal primary antibody Fab166 dissolved in TBST containing 5% goat normal serum (Sigma) (special gift from Dr. Ivan Bespalov, University of Vermont, USA; Soultanakis) et al., Free Radical Biology & Medicine, 28, 987-998, 2000), and TBST containing only 5% goat normal serum as controls was placed on serial sections and incubated overnight at 4 ° C. (6 tissue sections / one subject were reacted in the presence of anti-8-oxoguanine monoclonal primary antibody and 3 in the absence of anti-8-oxoguanine monoclonal primary antibody). After washing the sections 3 times for 5 minutes each with TBS, normal serum of rat (benefit from Dr. Nagakatsu Harada, Tokushima University), rabbit, human (benefit from Yasuko Sato, University of Tokushima), and goat normal serum each at a concentration of 1% And incubated with a goat biotin-conjugated anti-mouse IgG (Zymed) secondary antibody solution for 2 hours at room temperature. Sections were washed 3 times for 5 minutes each with ice-cold TBS and incubated with streptavidin-peroxidase (HRP; Zymed) for 1 hour at room temperature. The sections were washed twice for 5 minutes each with ice-cold TBS, and then incubated with 10 mM sodium azide (inhibitor of endogenous alkaline phosphatase; Nacalai Tesque) dissolved in TBS for 5 minutes at room temperature. Next, the tissue section was reacted with a peroxidase DAB substrate solution (Zymed) containing 10 mM sodium azide for 50 seconds at room temperature while observing with a microscope. After washing the sections three times for 5 minutes each with TBS, they were sealed with GVA mounting solution (Zymed), and the periphery of the cover glass was sealed with nail polish. Immediately, the gray level image and the color image of the immuno-stained image were captured using an ARGUS-100 image analysis system for measuring absorbance described later and an optical microscope equipped with a digital camera.

(5) Image analysis About the gray level image of the tissue section taken in (2) and (4) above, a frame inscribed in each cell nucleus was set and the absorbance was measured. The absorbance was measured using the aforementioned ARGUS-100 image analysis system for measuring absorbance. A transmission optical microscope (Nikon, Tokyo) included in the system was equipped with a 451.2 nm interference filter (Nihon Vacuum Optics, Tokyo) and a 40 × objective lens. On the other hand, color images of tissue sections were obtained from Fujix digital camera (HC-2500 3CCD; Fuji Photo Film, Tokyo) attached to a Zeiss Axioskop optical microscope (Oberkochen, Germany) with Photograb-2500 version1.0 (Fuji Photo Film). Used to capture. The digital image processing shown in FIGS. 2 to 4 was performed using Photoshop version 3.0J (Adobe Systems, Mountain View, California, USA).

<Result>
(Neutral staining with nuclear red and decolorization of stained nuclei)
FIG. 2 shows a nuclear staining image (A) with 1% neutral red in a 2-year-old male biceps tissue section of DMD among the tissue sections prepared in (1), and 2N hydrochloric acid of the same nuclear staining section. It is the complete free image (B) of neutral red by processing. As shown in FIG. 2, when the methanol-fixed tissue sections were incubated with 1% neutral red for 30 seconds at room temperature, all cell nuclei stained red. Since it is known that the cytoplasm of immature muscle fibers shows base preference, small muscle fibers whose cytoplasm is lightly stained with neutral red were identified as immature muscle fibers (FIG. 2A). The nuclei of immature muscle fibers are circular and larger than those of mature muscle fibers. The arrowhead indicates the nucleus of juvenile muscle fibers, and the arrow indicates the cytophilic cytoplasm. The bar in the figure is 20 μm. When the nuclear-stained section of FIG. 2A was treated with 2N hydrochloric acid, neutral red was completely released from the tissue section (FIG. 2B). Therefore, subsequent immuno-staining was not affected by nuclear staining with neutral red.

(Immunostaining of laminin and 8-oxoguanine)
FIG. 3 shows a nuclear-stained image (A, D) obtained by neutral red obtained in (1) above in a biceps brachii muscle section of a 3-year-old healthy boy, basement membrane laminin and 8-oxo obtained in (4) above. An immuno heavy staining image (B, L) of guanine and an immuno heavy staining image (C, F) performed in the absence of anti-8-oxoguanine antibody are shown. A to C are color images, and D to F are gray level images respectively corresponding to A to C captured by the ARGUS-100 image analysis system. In addition, the nuclear staining image by neutral red corresponding to C and F is not carried. The bar in the figure is 20 μm. FIG. 4 shows a nuclear red stained image (A, D) obtained in (1) above of a biceps brachii section of a 4-year-old DMD boy, laminin and 8-oxoguanine obtained in (4) above. The immuno-stained image (B, E) and the immuno-stained image (C, F) performed in the absence of the anti-8-oxoguanine antibody are shown. A to C are color images, and D to F are gray level images corresponding to A to C captured by the ARGUS-100 image analysis system. In addition, a nuclear-stained image (G) obtained by neutral red obtained in (1) above of a biceps brachii section of a 2-year-old DMD boy, and immunostaining of laminin and 8-oxoguanine obtained in (4) above. An image (H) is also shown. In addition, the nuclear staining image by neutral red corresponding to C and F is not carried. The bar in the figure is 20 μm. In FIGS. 3 and 4, the arrowhead indicates 8-oxoguanine intensely stained muscle fiber nucleus, * indicates 8-oxoguanine attenuated muscle fiber nucleus, and the arrow indicates basement membrane laminin. The black marks represent mature muscle fiber nuclei and the gray marks represent juvenile muscle fiber nuclei.

  In this experimental example, as described in (3) above, laminin β2 chain localized in the basement membrane covering the outer surface of the cell membrane of muscle fibers was double-stained in order to identify myocytes. From FIG. 3 and FIG. 4, it was confirmed that the basement membrane in which laminin was localized was dyed light purple. However, since the basement membrane covers both the muscle fibers and the muscle satellite cells attached to them, in this experimental example, it is impossible to distinguish between the muscle fiber nucleus and the nucleus of the muscle satellite cell. Therefore, the muscle fiber nucleus described below includes the muscle satellite cell nucleus. 3B, E, and FIGS. 4B, E, H show that the 8-oxoguanine immunostaining of the DMD muscle fiber nucleus (brown deposition of DAB oxidative polymerization compound) compared to the muscle fiber nucleus of healthy muscle. Qualitatively strong. Furthermore, in the DMD muscle (FIGS. 4G and H), a large number of regenerated muscle fibers having an 8-oxoguanine-stained central nucleus were detected. In contrast, regenerated muscle fibers were not found in healthy muscles without muscle cell disruption. Further, in the absence of anti-8-oxoguanine antibody, nonspecific staining was slightly observed (FIGS. 3C and F and FIGS. 4C and F).

(Calculation of relative content of 8-oxoguanine by image analysis)
As is clear from the variety of nuclear staining properties with neutral red (FIGS. 3A and 3D and FIGS. 4A, 4D, and 4G), the nucleic acid density of cell nuclei differs between individual nuclei and their cut surfaces. In the present experimental example, the relative tissue content of 8-oxoguanine with respect to the cell nucleus DNA is obtained from the ratio of the intensity of immunostaining of 8-oxoguanine in the same cell nucleus and the intensity of nuclear staining with neutral red in the same cell nucleus. The method for calculating the relative content of 8-oxoguanine is described below.

  Using the gray level images obtained in (1) and (4) above, the staining intensity (absorbance) was analyzed. First, the absorbance at 451.2 nm of the same myofiber nucleus was described above from the gray level image of the nuclear-stained section by neutral red and the gray-level image of laminin and 8-oxoguanine immunostained using the same section. Each measurement was performed with an ARGUS-100 image analysis system for measuring absorbance. Similarly, the absorbance of the same myofiber nuclei was measured in a gray level image of neutral red nuclear staining and immunostaining in the absence (control) of anti-8-oxoguanine antibody. DAB oxidation showing the localization of 8-oxoguanine in the muscle fiber nucleus when the absorbance of neutral red bound to the muscle fiber nucleus of DMD patient-derived muscle tissue cells was Dpn, and immuno-staining of 8-oxoguanine and laminin was performed. The absorbance of the polymerized compound is Dpt, the absorbance of DAB oxidatively polymerized compound in immunostaining in the absence of 8-oxoguanine is Dpc, and the absorbance of neutral red bound to the muscle fiber nucleus of healthy tissue-derived muscle tissue cells is Dnn, The absorbance of DAB oxidatively polymerized compound showing the localization of 8-oxoguanine in the muscle fiber nucleus when immunostaining with 8-oxoguanine and laminin is expressed as Dnt, and DAB in immunostaining in the absence of 8-oxoguanine. The absorbance of the oxidative polymerization compound was defined as Dnc. With respect to the absorbance measured in this way, Dpt / Dpn (◯ in the figure) and Dnt / Dnn (● in the figure) are plotted against Dpn or Dnn in FIG. FIG. 5B shows a plot of Dpn (Δ in the figure) and Dnc / Dnn (black Δ in the figure) against Dpn or Dnn. Then, in all cases, a curve relationship that was approximately approximated by a power was shown.

  Next, when the data in FIG. 5 was plotted in the common logarithm, a linear relationship as shown in FIG. 6 was obtained. ○ is log (Dpt / Dpn) = 0.704 (−logDpn) −0.301 [Formula 1], ● is log (Dnt / Dnn) = 0.580 (−logDnn) −0.363 [Formula 2], Δ is log (Dpc / Dpn) = 0.596 (−logDpn) −0.417 [Formula 3], and black Δ can be approximated by log (Dnc / Dnn) = 0.552 (−logDnn) −0.477 [Formula 4]. In order to correct the absorbance of the immunostaining in the presence of the anti-8-oxoguanine antibody with the absorbance in the absence of the anti-8-oxoguanine, the anti-8-oxoguanine calculated using the measured value Dpn or Dnn and the above formulas 2 and 4 The log (Dpc / Dpn) value or log (Dnc / Dnn) value in the absence of antibody is subtracted from the measured value log (Dpt / Dpn) or log (Dnt / Dnn) in the presence of anti-8-oxoguanine antibody. As a result, log (Dpt / Dpc) values were obtained for DMD muscle, and log (Dnt / Dnc) values were obtained for healthy muscle.

  Further, when the obtained values of Dpt / Dpc = P and Dnt / Dnc = N are plotted against Dpn or Dnn, almost constant P values (DMD muscles) and N values (healthy muscles) independent of Dpn and Dnn are obtained. It was found (Fig. 7). The symbols in FIG. 7 indicate the P values in the marginal nucleus (◯) and central nucleus (×) of the mature muscle fiber and the nucleus (Δ) of the juvenile muscle fiber, respectively, of the biceps brachii muscles of the DMD boy. ● indicates the N value in the muscle fiber nucleus of the biceps brachii muscles of a healthy child. P and N values indicate the relative content of 8-oxoguanine and do not depend on the nucleic acid density of the nucleus. Furthermore, the nuclei of mature muscle fibers (marginal and central nuclei) and juvenile muscle fibers in the biceps brachii muscles of DMD boys and the nuclei of mature muscle fibers of biceps brachii muscles of 2-6 year old healthy boys The average value and standard error (SEM) of P, N, Dpn and Dnn were determined for (marginal nucleus and central nucleus). The results are shown in Table 1. The numbers in parentheses in the table are the number of muscle fiber nuclei whose absorbance was measured.

  For DMD muscles, the average P value was 1.57, and for healthy muscles, the average N value was 1.38. There is a significant difference between these two values (p <0.0001), indicating that the degree of oxidation of guanine contained in the myofiber nucleus DNA of DMD muscle is about 14% higher than that of healthy muscle ( P / N = 1.137). According to the present invention, the effect of oxidative stress on nuclear DNA in muscular dystrophy could be quantitatively clarified for the first time. Moreover, it can be seen from Dpn / Dnn = 1.066 that the nucleus (DNA) density of DMD muscle is about 7% higher than that of healthy muscle (there is a significant difference, p <0.02). The significant difference test was performed by a normal test (Z test).

  The clinical findings at 2 and 4 years of age under informed consent were the same as in the above experiment using biceps biopsies obtained from 2 Duchenne muscular dystrophy (DMD) boy patients and 1 3 year old healthy boy. The method was used for heavy staining and image analysis. The results are shown in Table 2. From Table 2, P / N = 1.163 was calculated for DMD patient 1 and P / N = 1.148 for DMD patient 2, and both of the two patients suffered from muscular dystrophy histopathologically. It was determined that

  In the above examples, the pathological examination of muscular dystrophy was performed by quantifying the degree of oxidation of muscle cell nuclear DNA. However, the nondestructive method for quantifying the degree of tissue cell nuclear DNA oxidation according to the present invention is not limited to muscle cells. It can also be applied to cultured cells, and is expected to be used in a wide range of fields such as pathological examination and research of diseases caused by DNA oxidation, research on aging, sports medicine, and forensic medicine.

  For example, the non-destructive quantification of tissue cell nuclear DNA oxidation degree according to the present invention includes aging, apoptosis, and cancer, arteriosclerosis, diabetes, neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease). It can be used for pathological examination of diseases caused by DNA oxidation such as ischemia / reperfusion injury, adriamycin cardiomyopathy, arteriosclerosis, gastrointestinal mucosal injury, and screening of therapeutic agents for these diseases.

FIG. 1 is a flow chart showing an outline of a nondestructive measurement method for the degree of DNA oxidation in tissue cell nuclei of the present invention. FIG. 2 shows a nuclear-stained image (A) of a 2-year-old DMD boy's biceps brachii muscle with neutral red and a neutral red completely free image (B) of the same nucleus-stained slice by hydrochloric acid treatment. FIG. 3 shows a nuclear red stained image (A, D), a laminin and 8-oxoguanine immunostained image (B, E), and anti-8-oxoguanine of a 3-year-old healthy male biceps section It is an immuno-stained image (C, F) in the absence of an antibody, and D to F are gray level images respectively corresponding to A to C captured by the image analysis system. FIG. 4 shows a nuclear red stained image (A, D), a laminin and 8-oxoguanine immunostained image (B, E), and anti-8-oxoguanine of a 4-year-old DMD boy biceps brachii muscle section. Immuno-stained images (C, F) performed in the absence of antibody. D to F are gray level images respectively corresponding to A to C captured by the image analysis system, and further, a nuclear-stained image (G) of a 2-year-old DMD boy's biceps brachii muscle with neutral red, laminin and 8- It is an immuno-stained image (H) of oxoguanine. FIG. 5 (A) shows a value obtained by dividing the absorbance (Dpt, Dnt) of DAB oxidation polymerization compound of 8-oxoguanine immunostaining in the muscle fiber nucleus by the absorbance (Dpn, Dnn) of neutral red bound to the muscle fiber nucleus. And (Dpn, Dnn) are graphs showing the relationship between (Dpn, Dnn), and (B) is a graph showing the same relationship in immunostaining in the absence of anti-8-oxoguanine antibody. FIG. 6 is a graph plotting the data of FIGS. 5A and 5B in common logarithm. FIG. 7 shows the neutral red in which the absorbance ratio (Dpt / Dpc, Dnt / Dnc) of the DAB oxidation polymerization compound of 8-oxoguanine immunostaining in the presence and absence of anti-8-oxoguanine antibody is bound to the myofiber nucleus. It is the graph which was plotted with respect to the light absorbency (Dpn, Dnn).

Claims (6)

The following steps (a) to (f) are performed in the order of description, and the method for nondestructive quantification of the degree of tissue cell nuclear DNA oxidation: (a) After staining tissue cell nuclei with a nuclear staining dye, A step of analyzing or measuring at least the position of the nucleus to be stained and the degree of staining of the tissue for the image of the stained tissue; (b) the membrane and / or cytoplasm of the target cell in the stained tissue obtained in step (a), (C) a step of decolorizing the nucleus to be stained in the heavily stained tissue cell obtained in step (b); (d) a step of decolorization obtained in step (c); Oxidized DNA in tissue cells is subjected to heavy staining with a specific binding substance-containing solution or a specific binding substance-free solution, and then at least the presence and position of the target cells in the image of the heavily stained tissue, the stained oxidation Analyze or measure DNA location and tissue staining (D) Step (e) The identity of the position of the cell nucleus analyzed in Step (a) and the position of the oxidized DNA to be stained analyzed in Step (d), and the presence and position of the target cell analyzed in Step (d) and the staining A step of determining the accuracy of the location of the oxidized DNA; and (f) a staining degree of the tissue-derived tissue cell obtained by the measurement in the step (d) with a specific binding substance-containing liquid. (Dpt) and the staining degree (Dpc) of a patient-derived tissue cell with a specific binding substance-free solution from P = Dpt / Dpc, the staining degree (Dnt) of a healthy person-derived tissue cell with a specific binding substance-containing liquid A step of calculating N = Dnt / Dnc and then P / N sequentially from the degree of staining (Dnc) of the tissue-derived cells not containing a specific binding substance. A pathological examination method for diseases caused by DNA oxidation, characterized by using the quantification method according to claim 1. A method for screening for a therapeutic agent for a disease caused by DNA oxidation, comprising using the quantitative method according to claim 1. The therapeutic agent of the disease resulting from DNA oxidation obtained by the screening method of Claim 3. The nuclear staining dye used in step (a) according to claim 1 is neutral red, the specific binding substance used in step (b) is an anti-laminin β2 chain monoclonal antibody, and the specific binding substance used in step (d) is anti-8. A non-destructive method for quantifying the degree of oxidation of nuclear DNA in muscular dystrophy patients, which is an oxoguanine monoclonal antibody. A therapeutic agent for muscular dystrophy obtained by screening using the quantitative method according to claim 5.