JP2001095599A - Measurement of protease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring a protease by which the activity of the protease in a biological sample can accurately be measured and with which the shape of a tissue or cell can easily be observed. SOLUTION: This method for measuring a protease comprises (1) a process for bringing a biological sample containing the protein into contact with a thin film which contains a protease substrate and a film-hardening agent and is formed on the surface of a substrate, (2) a process for dyeing the thin film obtained in the above-mentioned process with a dye selected from the group consisting of a red color dye, a brown color dye, and a yellow color dye (for example, Ponceau 3R) and detecting digested marks formed in the thin film by the action of the protease; and, if necessary, (3) a process for dyeing the biological sample, such as the nuclei of cells in the sample, on the thin film with a dye (for example, hematoxylin) different from the dye used in the process (2).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring a protease. More specifically, it enables accurate diagnosis of cancer malignancy, such as invasive activity and metastatic activity of cancer cells, progression of periodontal disease, such as periodontitis, and destructive lesions in rheumatoid arthritis, etc. The present invention relates to a protease measurement method.

[0002]

BACKGROUND ART In the invasion and metastasis of cancer cells, progression of periodontal disease such as periodontitis, progression of tissue destruction in rheumatoid arthritis, wound healing process, ontogenesis process, etc., matrix metalloproteinase, plasminogen It is known that various proteases such as an activator are involved, and as a method for detecting and quantifying those proteases, an immunoassay using an antibody, an immunoblotting method, an electrophoresis zymography method, and the like are known. . As a method for measuring protease activity in tissues, see The FASEB Journal, Vol. 9, Jul.
The so-called in situ zymography method disclosed in y, pp. 974-980, 1995 or WO 97/32035 is known.

[0003]

SUMMARY OF THE INVENTION The present inventors have made the above-mentioned WO
The in-situ zymography method disclosed in 97/32035 and its application were studied diligently, but in the course of this research, thin films such as gelatin were stained with dyes such as amide black and Coomassie blue to measure protease activity. Then, the problem was recognized that the staining concentration became extremely high, and the thin film was stained dark blue, so that it was sometimes difficult to observe tissues and cells on the thin film. In addition, when staining with amide black or the like was performed, there was also a problem that it was difficult to observe the morphology of nuclei even if the cell nuclei were further stained on the thin film.

Accordingly, an object of the present invention is to provide means for solving these problems. More specifically, it is an object of the present invention to provide a method capable of accurately measuring protease activity and easily observing the morphology of a tissue or a cell on a thin film. An object of the present invention is to provide a method for measuring a protease which can be used in combination with a tissue staining method, a cell nucleus staining method, or the like, and which can analyze in detail a protease activity localized in a specific tissue portion or a specific cell.

[0005]

The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, dyed a thin film of gelatin or the like with one or two kinds selected from the group consisting of red, orange, and yellow. It has been found that staining with the above dye makes it extremely easy to observe tissues and cells on a thin film at the same time as observing traces of digestion, and it is possible to identify cells expressing protease activity. Furthermore, by staining the tissue or cells on the thin film stained as described above with a dye of another color, for example, the morphological observation of cell nuclei can be performed simultaneously with the observation of digestion marks on the same specimen. It has been found that the identification of tissue parts and cells that express protease activity is much easier. The present invention has been completed based on these findings.

That is, the present invention relates to a method for measuring a protease, which comprises the following steps: (1) a step of bringing a biological sample containing a protease into contact with a thin film formed on the surface of a support containing a protease substrate and a hardening agent; And (2) dyeing the thin film obtained in the above step (1) with one or more dyes selected from the group consisting of red, orange, and yellow, and digesting the thin film formed by the action of protease. It is intended to provide a method including a step of detecting a scar.

Another aspect of the present invention relates to a method for measuring a protease according to the present invention, which comprises: (1) a biological sample containing a protease on a thin film formed on a surface of a support containing a protease substrate and a hardening agent; (2) dyeing the thin film obtained in the above step (1) with one or more dyes selected from the group consisting of red, orange, and yellow; Detecting the formed digestion marks; (3) staining the biological sample on the thin film with a dye having a different color from the dye used in the step (2); and (4) obtaining the same in the step (1). There is provided a method including a step of comparing the digestion mark of the obtained thin film with the stained sample obtained in the step (3).

According to another aspect of the present invention, there is provided a method for measuring a protease according to the present invention, which comprises the steps of (1) substantially removing a biological sample from a thin film containing a protease substrate and a hardening agent and formed on the surface of a support. Contacting one of the two or more sections successively with each other; (2) contacting a thin film formed on a support surface with a protease substrate, a hardening agent, and a protease inhibitor with the other of the above sections. (3) dyeing the thin film obtained in step (1) and step (2) with one or more dyes selected from the group consisting of red, orange, and yellow; Detecting a digestion mark formed on the thin film by the action of a protease; and (4) converting the digestion mark of the thin film obtained in the step (1) with the digestion mark of the thin film obtained in the step (2). A method is provided that includes a contrasting step.

According to the present invention, there is also provided a method for measuring a protease, comprising: (1) two or more substantially continuous biological samples of a biological sample with respect to a thin film formed on the surface of a support containing a protease substrate and a hardener. Contacting one of the above-mentioned sections; (2) contacting another one of the above-mentioned sections with a thin film formed on the surface of the support, including a protease substrate, a hardening agent, and a protease inhibitor. Process; (3)
The thin film obtained in step (1) and step (2) is red, orange,
And staining with one or more dyes selected from the group consisting of yellow and yellow to detect digestion marks formed on the thin film by the action of protease;
Dyeing with a dye different in color from the dye used in (3);
And (5) digestion marks of the thin film obtained in the above step (1), step (2)
And a step of comparing the digestion mark of the thin film obtained in the above with the staining obtained in the step (4).

According to a preferred aspect of the present invention, there is provided the above-mentioned method, wherein the biological sample is a biological sample separated and collected from a mammal, including a human, preferably a patient, a mammal suspected of having a disease, an experimental animal, or the like. Is done. As a biological sample, in addition to a solid sample such as a piece of tissue, a sample containing cells or a piece of tissue collected by suction from tissue, blood, lymph,
A non-solid sample such as saliva can be used. In the method using a continuous section, a tissue section can be used as a biological sample. The above method wherein the protease is a matrix metalloprotease or a serine protease is a preferred embodiment.

According to a further preferred embodiment, the above method wherein the thin film is a gelatin thin film or casein thin film formed on a flat surface of a support;
The above method of staining with a dye of 0 nm to 580 nm; the above method of using Ponceau 3R or Biebrich Scarlet to stain the thin film;
The above method of staining cell nuclei as a stain on a biological sample; the above method of using hematoxylin or methyl green for staining of cell nuclei; the above method of visually determining the detection of digestive scars using a microscope; quantification of digestive scars using an image processing apparatus Alternatively, the above-described method for performing the digitization is provided. In the method using a protease inhibitor, it is preferable that the protease inhibitor is a chelating agent, a matrix metalloprotease inhibitor, or a serine protease inhibitor.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The term measurement method as used herein should be interpreted in the broadest sense, including qualitative and quantitative. The method for measuring a protease of the present invention is a method for measuring a protease by digesting a protease substrate with a protease contained in a sample and forming a digestion mark in a thin film (so-called in situ zyme).
mography method: disclosed in, for example, WO97 / 32035), a thin film on which a digestion mark is formed is red, orange,
And one or more dyes selected from the group consisting of yellow and yellow. According to the method of the present invention, a digestion mark on a thin film can be detected as a portion having a low dye concentration under a microscope, and the presence of protease activity in a sample can be reliably proved. The feature is that the morphological observation of the sample can be easily performed. As the thin film used in the method of the present invention, those described in WO97 / 32035 can be used. As the hardening agent or the protease inhibitor, those described in the above literature can be used. The disclosure of WO 97/32035 is incorporated herein by reference.

[0013] Proteases which are the subject of the present invention include, for example, matrix metalloproteases and serine proteases. These enzymes are described in "Molecular mechanism of cancer metastasis", edited by Takashi Tsuruo, pp.92-. Ten
7, described in detail in Medical View, 1993. Particularly suitable proteases for the method of the present invention include:
For example, matrix metalloproteases such as stromal collagenase (MMP-1), gelatinase A (MMP-2), and gelatinase B (MMP-9); and plasminogen.
Serine proteases such as activators (PA) can be mentioned, but the subject of the method of the present invention is not limited to the above-mentioned specific proteases.

The protease substrate is not particularly limited as long as it is a high molecular compound that is decomposed as a protease substrate. For example, collagen, gelatin, proteoglycan,
Fibronectin, laminin, elastin, casein, or the like can be used. Preferably, collagen, gelatin, fibronectin, or casein can be used, and more preferably, gelatin or casein can be used. When gelatin is used, the kind of gelatin is not particularly limited, and examples thereof include alkali-treated bovine bone, alkali-treated pig skin gelatin, bovine acid-treated gelatin, bovine phthalated gelatin, and pig skin acid-treated gelatin. Can be used. One of the above-mentioned protease substrates may be used, or two or more may be used in combination.

By using two or more different protease substrates in combination, the type of protease contained in a biological sample may be accurately specified in some cases. For example, one of the substantially continuous sections in a biological sample may be contacted with a thin film containing a certain protease substrate to detect digestion marks, and the other section may be contacted with a thin film containing a different protease substrate. Digestion marks on the thin film can be detected and the results can be compared. For comparison, two or more thin films containing different protease substrates may be used. As the thin film containing the protease substrate, it is preferable to use a thin film having a thickness of 1 to 10 μm, preferably 4 to 7 μm after drying.

The biological sample used in the method of the present invention includes:
Biological samples separated and collected from mammals including humans can be used. For example, a biological sample separated and collected from an affected mammal, a mammal suspected of having a disease, or an experimental animal can be used. The form of the biological sample is not particularly limited, but a solid sample such as a tissue section or a non-solid sample such as a body fluid can be used. As the non-solid sample, for example, a sample containing cells or a piece of tissue collected by suction from a tissue, or a bodily fluid such as blood, lymph, or saliva can be used. For example, it is separated from cancer tissues such as lung cancer, stomach cancer, esophagus cancer, colon cancer, breast cancer, uterine cancer, ovarian cancer, thyroid cancer, liver cancer, oral cancer, prostate cancer, kidney cancer, and bladder cancer by surgery or histological examination. Tissues separated and collected by surgery or histological examination from tissues such as collected cancer tissues, lymph nodes, periodontal disease tissues, synovium and bone tissues of rheumatoid arthritis, atherosclerotic tissues, gingival crevicular fluid, destruction Fluids contained in sexually pathological tissues (for example, synovial fluid of rheumatic lesions or alveolar pyorrhea tissue extract), pleural effusion, ascites, cerebrospinal fluid, abnormal mammary gland secretion, ovarian storage, sputum and the like can be used.

When the sample is a tissue, for example, the thickness of the sample is rapidly frozen in liquid nitrogen using a cryosectioning apparatus.
By preparing a section of 1 to 10 μm, preferably 4 to 6 μm, and attaching the section to the thin film, the sample can be brought into contact with the thin film. A non-solid sample containing cells or tissue pieces collected by fine needle aspiration can also be mixed with a molding material such as a compound, rapidly frozen with liquid nitrogen, and similarly used to prepare a slice. When a non-solid sample containing cells or tissue pieces collected from a tissue by puncture suction is used as it is, the sucked sample may be discharged onto a thin film, and the cells may be adhered to the thin film in a dispersed state. Furthermore, when the biological sample is a piece of tissue, the sample and the thin film can be brought into contact by gently wiping the water of the collected tissue and then allowing the sample to stand on the thin film containing the protease substrate for about 1 to 30 minutes. it can.

When a non-solid sample such as synovial fluid collected from a patient with rheumatoid arthritis is used, the sample is diluted to an appropriate concentration and / or subjected to necessary pre-treatment, and then subjected to about 1 to 50 μL, preferably about 1 to 20 μL, may be dropped on the thin film. When using gingival crevicular fluid for periodontal disease as a sample, a method of inserting a filter paper into the gingival sulcus, collecting about 5 to 10 μL of gingival crevicular fluid, and applying the filter paper to a thin film is adopted. be able to. After collecting the gingival crevicular fluid, if necessary, use distilled water or an appropriate buffer (eg, 50 mM Tris
-HCl, pH 7.5, 10 mM CaCl ₂ , 0.2 M NaCl, etc.) to extract the gingival crevicular fluid from the filter paper, and the extract may be dropped on the thin film.

After the tissue section containing the protease substrate is attached to the thin film, or the sample containing the protease is brought into contact with the thin film by such means as dropping a liquid sample,
The thin film is incubated at a temperature suitable for the expression of the protease activity, for example, at a saturated humidity of 37 ° C. for a time necessary for digestion of the substrate. The time required varies depending on the type of sample or thin film, but is preferably 1 to 4 at 37 ° C for a tissue section or a non-solid sample containing cells or tissue pieces obtained by suction.
Incubate for 8 hours, more preferably for 6 to 30 hours, for liquid samples such as exudates, for 0.5 to 24 hours, preferably for 1 to 6 hours, and allow proteases in the sample to form digestion marks in the thin film. Thereafter, the thin film is stained with a dye selected from the group consisting of red, orange and yellow, and digestion marks can be observed with an optical microscope.

[0020] One of the two or more substantially continuous sections in the biological sample is attached to a thin film containing no protease inhibitor, and one of the other sections is attached to a thin film containing a protease inhibitor. By comparing the digestion marks of both thin films, it becomes possible to specify the type of protease. Although the type of the protease inhibitor is not particularly limited, for example, a chelating agent, a matrix metalloprotease inhibitor, a serine protease inhibitor, or the like can be suitably used.

The staining of the thin film containing the protease substrate can be carried out using a dye selected from the group consisting of red, orange and yellow dyes. The terms red, orange or yellow as used herein should be interpreted in the broadest sense and not in any sense in a limiting sense. Such dyes generally have an absorption maximum in the range of 400 nm to 580 nm.

Preferred red dyes include, for example, Acid
Red 1 (CI18050), Acid Red 4 (CI14710), Acid Re
d 8 (CI14900), Acid Red 37 (CI17045), Acid Red
40 (CI18070), Acid Red 44, Acid Red 106 (CI181
10), Acid Red 183 (CI18800), Xylidin Ponceau 2R
(CI16150), Mordant Red 19, Nitro Red, Biebrich S
carlet and Ponceau 3R, especially Bieb
Rich Scarlet or Ponceau 3R is preferred. Examples of orange dyes include, for example, Methyl Orange, Ethyl Orange, Cro
cein Orange G, Orange II, Orange G, Acid Orange 8
(CI15575), Acid Orange 74 (CI18745), and examples of the yellow dye include Mordant Yellow 1
0, Mordant Yellow 7, Acid Yellow 99 (CI13900), Ac
id Yellow 65 (CI14170), Acid Yellow 17 (CI1896
5), Nitrazine Yellow (CI14890) can be preferably used.

For example, in the method disclosed in WO97 / 32035 in which gelatin is stained using amide black, the gelatin is stained dark blue, and only the portion where the gelatin has been digested by the protease has a reduced staining concentration, and a white portion appears. . For example, when the sample is a tissue section, the tissue with a digestion mark can be observed faintly due to the low staining concentration, but it may be difficult to observe the tissue other than the digestion mark. Further, even if nuclear staining is performed to enable observation of a tissue, it is usually difficult to observe typical dyes for nuclear staining such as hematoxylin and methyl green because the hue is close to amide black.

In accordance with the present invention, for example,
When stained red with R, the maximum absorbance of the membrane usually becomes 2 or more, but the tissue can be observed over the entire thin film, and a microscope is used to specify which part of the tissue has a digestion mark. It becomes easier. Furthermore, the biological sample on the thin film may be tissue-stained with a dye of another color tone, and the digestion scar and the stained tissue may be compared. For example, when the cell nucleus of a cell contained in a biological sample is stained with hematoxylin or methyl green, a morphological change of the nucleus and a digestion mark can be simultaneously observed as a signal of another color under an optical microscope. Furthermore, by combining this tissue staining method with a method using a protease inhibitor, the presence of a protease localized in each cell and identification of its type can be simultaneously performed.

By measuring the protease activity contained in a sample by the method of the present invention, the condition of the living body from which the sample has been separated, for example, the metastasis of cancer or the progress of rheumatism, can be examined. It can be used for various purposes such as diagnosis of diseases, clinical tests, animal experiments and the like. To determine the digestive strength of digestion marks, a method of visual judgment under an optical microscope, a method of measuring the optical density of digestive marks by a spectroscope, and a method of analyzing the image obtained by an optical microscope into a computer and analyzing the image Thus, a method of digitizing various types of digestion marks can be used.

[0026]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples. Example 1 Measurement of Protease Activity of Breast Cancer Tissue A breast cancer sample, which has been removed by surgery and frozen, is sliced into a thickness of 4 μm at −25 ° C. using a cryosection preparation apparatus, and is subjected to WO97 / 320.
It was adhered to a gelatin film disclosed in No. 35. Gelatin having a thickness of 6 μm was used. Apply this gelatin thin film to 37
The mixture was incubated at 100 ° C. and a relative humidity of 100% for 16 to 30 hours, and air-dried. As a staining solution, a solution prepared by dissolving Ponceau 3R in a 6% aqueous solution of trichloroacetic acid at a concentration of 0.8% was used, and immersion was performed at room temperature for 60 seconds to perform staining. After washing with water for 5 minutes, the cells were immersed in Mayer's hematoxylin solution for 2 minutes to perform nuclear staining, washed with water for 10 minutes, and immersed in ethanol for 20 seconds and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the tissue section, and the breast cancer section was enclosed. This film was held on a plastic mount shown in FIG.

This mount comprises a substrate 4 and a holding plate 5, and the holding plate 5 is attached to two arms of the holding plate 5 respectively.
It is fixed to the substrate 4 by an adhesive at an adhesive portion 1 provided at each location. The outer edge of the film is held in a slight gap formed between the substrate 4 and the holding plate 5, and the film is inserted along the guide 3 until it comes into contact with the stopper, so that the film can be attached and detached. Secured to the mount where possible. Observation of the film fixed on the mount using an optical microscope revealed that the breast cancer tissue section showed gelatin digestion at the site where cancer cells are thought to exist due to the nucleus morphology, indicating that it had protease activity. Was.

Example 2 Measurement of Protease Activity of Thyroid, Uterine, and Ovarian Cancer Tissues The above-mentioned cancer samples, which were removed by surgery and frozen, were thinned to a thickness of 4 μm at −20 ° C. using a cryosection preparation device. Cut, WO97 / 3
Adhered to a gelatin film disclosed in 2035. Gelatin having a thickness of 7 μm was used. Apply this gelatin film to 37
The mixture was incubated at 60 ° C. and a relative humidity of 100% for 6 to 16 hours, and further immersed in a mixed solution of methanol: acetic acid: water = 7: 1: 2 for 15 minutes to be fixed. As a staining solution, a solution obtained by dissolving Ponceau 3R in a 6% aqueous solution of trichloroacetic acid so as to have a concentration of 0.4% was used, and immersion was performed at room temperature for 60 seconds to perform staining.
After washing with water for 1 minute, the cells were immersed in Meyer's hematoxylin solution for 1 minute for nuclear staining, washed with water for 5 minutes, immersed in ethanol for 20 seconds, and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the tissue section, and the section was enclosed. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Gelatin digestion was observed at the site where cancer cells were present in the cancer tissue section, and it was revealed that there was protease activity.

Example 3 Measurement of Protease Activity of Esophageal Cancer, Gastric Cancer, and Colorectal Cancer Tissues The above-mentioned cancer sample, which has been removed by surgery and frozen, is sliced into a 4 μm-thick plate at −20 ° C. using a cryosectioning apparatus. Then, a continuous section was prepared, and one piece was adhered to a gelatin thin film disclosed in WO97 / 32035. Gelatin having a thickness of 4 μm was used. This gelatin film is heated at 37 ° C and 100% relative humidity for 16 hours.
Incubate for 30 hours and air dry. As a staining solution, a solution prepared by dissolving Ponceau 3R in a 6% aqueous solution of trichloroacetic acid at a concentration of 0.8% was mixed with an equal amount of ethanol, and the solution was immersed at room temperature for 60 seconds to perform staining. After washing with water for 20 seconds, it was immersed in ethanol for 20 seconds and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the tissue section, and the section was enclosed. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Gelatin digestion was observed at the site where cancer cells were present in the cancer tissue section, and it was revealed that there was protease activity. The images were captured by a digital camera attached to an optical microscope into a computer, and the area, area ratio, optical density, etc. of the digestion site were quantified by image processing software.

Similarly, another one of the serial sections was replaced with WO97 / 3203.
5 was adhered to a gelatin film containing the matrix metalloprotease inhibitor disclosed in 5, (1,10-phenanthroline). Gelatin having a thickness of 4 μm was used. This gelatin film was incubated at 37 ° C. and 100% relative humidity for 16 to 30 hours, and air-dried. 6% for staining solution
A solution obtained by dissolving Ponceau 3R in an aqueous solution of trichloroacetic acid at a concentration of 0.8% was mixed with an equal amount of ethanol, and immersed at room temperature for 60 seconds for dyeing. After washing with water for 20 seconds,
It was immersed in ethanol for 20 seconds and air-dried. After drying,
A cover aid film (manufactured by Sakura Seiki) was attached using xylene so as to cover the tissue section, and the section was enclosed.
This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Many samples in which gelatin digestion was not observed at the site of the cancer cells in the cancer tissue section were observed, but protease activity remained in some samples of gastric cancer. For a sample in which protease activity was observed in a gelatin film containing no matrix metalloprotease inhibitor and no protease activity was observed in a gelatin film containing an inhibitor, it is possible that this activity is derived from the matrix metalloprotease. it was thought.

Example 4 Measurement of Protease Activity of Prostate, Bladder, and Kidney Cancer Tissues The above-mentioned cancer sample, which was removed by surgery and frozen, was thinned to a thickness of 6 μm at -25 ° C. using a cryosection preparation device. Cut, WO97 / 3
Adhered to a gelatin film disclosed in 2035. Gelatin having a thickness of 6.9 μm was used. Apply this gelatin film to 37
The mixture was incubated at 100 ° C. and 100% relative humidity for 24 to 40 hours, and allowed to dry naturally. As a staining solution, a solution in which Acid Yellow 17 was dissolved to a concentration of 1% in a 50% aqueous ethanol solution was used, and the sample was immersed at room temperature for 5 minutes to perform staining. After washing with water for 2 minutes, nuclear staining was performed with 1% methyl green solution for 15 minutes, and after gently washing with water, immersed in ethanol for 20 seconds and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the tissue section, and the cancer section was enclosed. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Gelatin digestion was observed at the site where cancer cells were present in the cancer tissue section, and it was revealed that there was protease activity.

Example 5 Measurement of Protease Activity of Prostate Cancer Puncture Aspirate Sample Prostate cancer aspirate aspirated, mixed with a compound, and frozen at -25 ° C. using a cryosection preparation apparatus.
With a thickness of 4 μm, and adhered to a gelatin thin film disclosed in WO97 / 32035. Gelatin having a thickness of 6.9 μm was used. The gelatin film was incubated at 37 ° C. and 100% relative humidity for 24 to 40 hours and allowed to dry naturally. As a staining solution, a solution obtained by dissolving Ponceau 3R in a 6% aqueous solution of trichloroacetic acid so as to have a concentration of 0.8% was mixed with an equal amount of ethanol. 1
After washing with water for 2 minutes, the cells were immersed in Mayer's hematoxylin solution for 2 minutes to perform nuclear staining, washed with water for 5 minutes, and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was applied using xylene to cover the tissue section, and the prostate cancer section was enclosed. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Gelatin digestion was observed at the site where cancer cells were present in the puncture-aspirated sample, which revealed that it had protease activity.

Example 6 Measurement of Protease Activity of Breast Cancer Aspiration Sample (Part 1) A sample obtained by breast cancer aspiration was blown from a syringe needle onto a thin gelatin film disclosed in WO97 / 32035.
The cells were allowed to adhere in a dispersed state. Gelatin film thickness 5 μm
Was used. This gelatin film is kept at 37 ° C and 100% relative humidity.
And allowed to air dry for 8-20 hours. As a staining solution, the concentration is 0.
A solution prepared by dissolving Ponceau 3R so as to have a concentration of 8% was mixed with an equal amount of ethanol, and immersed at room temperature for 60 seconds for dyeing. After washing for 1 minute, add 2 portions to Mayer's hematoxylin solution.
The nucleus was stained by immersion for 10 minutes, washed with water for 10 minutes, and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the cell sample dispersed on the film, and the breast cancer cells were encapsulated. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Gelatin digestion was observed at the site where cancer cells were present in the puncture-aspirated sample, which revealed that the sample had protease activity.

Example 7 Measurement of Protease Activity of Breast Cancer Aspiration Sample (Part 2) A sample collected by breast cancer aspiration was dispersed in PBS and washed by repeating centrifugation and dispersion two to three times. Then WO
Cells were adhered in a dispersed state on the gelatin thin film disclosed in 97/32035 using cytospin. Gelatin having a thickness of 6.9 μm was used. This gelatin film was incubated at 37 ° C. and 100% relative humidity for 8 to 20 hours, and air-dried. As a staining solution, a solution obtained by dissolving Ponceau 3R in a 6% aqueous trichloroacetic acid solution at a concentration of 0.8% was mixed with an equal amount of ethanol, and immersed at room temperature for 60 seconds to perform staining. After washing with water for 1 minute, the cells were immersed in Mayer's hematoxylin solution for 2 minutes to perform nuclear staining, washed with water for 10 minutes, and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the cell sample dispersed on the film, and the breast cancer cells were encapsulated. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Gelatin digestion was observed at the site where cancer cells were present in the puncture-aspirated sample, which revealed that the sample had protease activity.

Example 8: Measurement of Protease Activity of Synovial Fluid of Rheumatic Patient About 20 μL of rheumatic synovial fluid was dropped on a gelatin thin film disclosed in WO97 / 32035. Gelatin film thickness 6 μm
Was used. This gelatin film is kept at 37 ° C and 100% relative humidity.
And allowed to dry for 1 to 3 hours.
As a staining solution, a solution in which Ponceau 3R was dissolved in a 6% aqueous solution of trichloroacetic acid to a concentration of 0.8% was used.
Dipped for 30 seconds to stain. After washing with water for 20 seconds, the sample was immersed in ethanol for 20 seconds and air-dried, and a cover-aid film (manufactured by Sakura Seiki) was applied using xylene to cover the sample dropping portion, thereby protecting the gelatin film. When this film was observed with the naked eye, gelatin digestion was observed at the portion where the sample was dropped, and the presence of protease activity became apparent.

Example 9: Measurement of Protease Activity of Breast Cancer Tissue A breast cancer sample, which has been removed by surgery and frozen, is sliced to a thickness of 4 μm at -25 ° C. using a cryosection preparation apparatus and published internationally.
It was adhered to a gelatin film disclosed in WO97 / 32035. A gelatin film having a thickness of 5 μm was used. This gelatin thin film was incubated at 37 ° C. and 100% relative humidity for 14 hours and allowed to air dry. As a staining solution, a solution prepared by dissolving Biebrich Scarlet in a 6% aqueous solution of trichloroacetic acid at a concentration of 0.8% and an equal volume of a mixture of ethanol were used, and immersion was performed at room temperature for 3 minutes to perform staining. After washing with water for 10 minutes, nucleus staining was carried out by immersion in Mayer's hematoxylin solution for 2 minutes, followed by washing with water for 10 minutes and air drying. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the tissue section, and the breast cancer section was enclosed. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. In the breast cancer tissue section, gelatin digestion was observed at the site where cancer cells are considered to exist due to the nucleus morphology, and it was revealed that there was protease activity.

Comparative Example: Measurement of Protease Activity of Breast Cancer Tissue (Amid Black Staining) In the same manner as in Example 1, a breast cancer sample was adhered to a gelatin film, and
The mixture was incubated at 100 ° C. and a relative humidity of 100% for 16 to 30 hours, and air-dried. The amide black solution is prepared by adding amide black 10 to a mixture of 70% methanol, 10% acetic acid and 20% water.
B was added to a concentration of 1%, stirred overnight to dissolve, and then filtered through filter paper before use. The film treated as described above was immersed in this amide black dyeing solution at room temperature for 15 minutes to be dyed. After being immersed in a mixed solution of 70% methanol, 10% acetic acid, and 20% water for 10 minutes to decolorize, it was immersed in ethanol for 20 seconds and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the breast cancer section, and the section was enclosed. This film was held on a plastic mount shown in FIG. 1 and observed using an optical microscope. Gelatin digestion was observed in part of the thin film,
Protease activity was revealed, but it was unknown which cells of the breast cancer sections had the activity.

[0038]

According to the method of the present invention, it is possible to observe the tissue or cells on the thin film simultaneously with the observation of the digestion marks, and it is easy to specify the tissue parts and cells expressing the protease activity. For example, by performing cell nucleus staining together with thin film staining, it is possible to observe the morphological information of the nucleus and digestion marks in the same specimen, so that it is possible to determine whether abnormal cells that have undergone morphological changes express protease activity. It can be easily determined.

[Brief description of the drawings]

FIG. 1 is a view showing a plastic mount used in Examples 1 to 7 and 9 in an embodiment.
(a) is a plan view of the holding plate, (b) is a side view of the holding plate, (c) is a plan view of the board, (d) is a side view of the board, and (e) is a perspective view of the mount. .

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Guide 2 Adhesion part 3 Stopper 4 Substrate 5 Holding plate

Claims (8)

[Claims] 1. A method for measuring a protease, comprising:
Contacting a biological sample containing a protease with a thin film formed on the surface of a support containing a protease substrate and a hardener; and (2) red-orange the thin film obtained in the step (1), One or two selected from the group consisting of and yellow
A method comprising the steps of staining with at least one kind of dye and detecting a digestion mark formed on the thin film by the action of a protease. 2. A method for measuring a protease, comprising:
Contacting a biological sample containing a protease with a thin film formed on the support surface containing a protease substrate and a hardening agent; (2) the thin film obtained in the above step (1) is red, orange, and A step of staining with one or more dyes selected from the group consisting of yellow and detecting a digestion mark formed on the thin film by the action of a protease; (3) the biological sample on the thin film is subjected to the step (2). A step of dyeing with a dye having a different color from the dye used; and (4) a step of comparing the digestion mark of the thin film obtained in the above step (1) with the dyeing obtained in the above step (3). . 3. The method according to claim 2, wherein the cell nucleus is stained in the step (3). 4. A method for measuring a protease, comprising:
Contacting one of two or more substantially continuous sections of a biological sample with a thin film formed on a support containing a protease substrate and a hardening agent; (2) a protease substrate; Contacting another of the above sections with the thin film formed on the surface of the support containing the agent and the protease inhibitor; (3) the step (1) and the step
Dyeing the thin film obtained in (2) with one or more dyes selected from the group consisting of red, orange, and yellow, and detecting digestion marks formed on the thin film by the action of protease; And (4) a method comprising the step of comparing the digestion mark of the thin film obtained in the above step (1) with the digestion mark of the thin film obtained in the above step (2). 5. A method for measuring a protease, comprising:
Contacting one of two or more substantially continuous sections of a biological sample with a thin film formed on a support containing a protease substrate and a hardening agent; (2) a protease substrate; Contacting another of the above sections with the thin film formed on the surface of the support containing the agent and the protease inhibitor; (3) the step (1) and the step
Dyeing the thin film obtained in (2) with one or more dyes selected from the group consisting of red, orange, and yellow, and detecting digestion marks formed on the thin film by the action of protease; (4) a step of dyeing the section on the thin film with a dye having a different color from the dye used in step (3); and (5) the step (1).
A method comprising comparing the digestion mark of the thin film obtained in the step, the digestion mark of the thin film obtained in the step (2), and the staining obtained in the step (4). 6. The method according to claim 5, wherein the cell nucleus is stained in the step (4). 7. The method according to claim 3, wherein hematoxylin or methyl green is used for staining the biological sample. 8. The method according to claim 1, wherein Ponceau 3R is used for dyeing the thin film.