JPH10206424A - Wound progress measurement inspection method, measurement kit, and wound surface treatment method determination method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To evaluate the state of a wound surface objectively and easily by adsorbing exudate from a wound surface by an adsorption film and detecting constituents in the liquid by the immunological chemical method. SOLUTION: A measurement inspection method adsorbs an exudate from a wound surface to an adsorption film and measures wound progress on the wound surface for detecting constituents in the liquid by the immunological chemical method. The adsorption film is preferably nitrocellulose film that is hydrophobic adsorption film, cellulose acetate film, fluorine vinylidene film, or nylon film. The immunological chemical method is preferably the dot blot method, the western blot method, or the northern blot method. More specifically, the exudate from the wound surface is adsorbed, for example, by nitrocellulose film and is blocked by protein for blocking. Then, a primary antibody treatment is performed by a specifical antibody and a color development enzyme label anti-primary antibody (secondary antibody) treatment is performed, thus developing color. Also, this method allows the degree of wound cure to be measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for testing the degree of wound development on a wound surface, a kit for measuring and testing the same, a method for measuring and testing the degree of wound healing using the same, and a method for treating a wound surface using the same. It relates to an inspection method for determining.

[0002]

2. Description of the Related Art Conventionally, a method for treating a wound surface such as a pressure ulcer or a burn has been performed by a physician through a visual observation and an empirical judgment based thereon. As an attempt to objectively evaluate the wound surface based on test data, a method has been used in which the exudate exuding from the wound surface is stored for a certain period of time, and various physiologically active substances and growth factors contained therein are measured. I was

[0003]

The external force of the wound means that the surface of the body or organs covered with skin or mucous membranes becomes pathological due to tissue damage (edited by Koji Uchizono and Kunori Kosaka) , Dictionary of Nursing, 4th edition, 1994
Year). Such wound treatments include local and systemic therapies, the former involving pressure on the wound, suturing, hemostasis, and disinfection to prevent infection, suturing, application of chemicals, and cooling to control inflammation. Measures have been taken. In addition, drainage and debridement (removal of crushed and necrotic tissue) are performed to discharge bleeding and purulent fluid, to remove wound crush, and to promote wounds in the direction of healing at an early stage.

As a systemic therapy, a chemotherapeutic drug is used for preventing infection and suppressing inflammation (Koji Uchizono, Kunori Kosaka, Ed., Dictionary of Nursing, 4th ed., 1994). In the treatment of such a wound, the above-described treatment method has conventionally been adopted based on the gross observation by the doctor and the empirical judgment of the doctor. However, understanding the changes over time in each process of a normal wound is an important key in performing a treatment for positively promoting wound healing.

[0005] For this purpose, it is important to use empirical means as described above. In addition to this, it is necessary to obtain objective data and use it as a guideline for diagnosis to treat wounds. However, a diagnostic method for performing such an evaluation has not been known so far. Exudate from the wound surface has been stored for a certain period of time, and various physiologically active substances contained therein have been measured.However, the time-dependent changes in each process of wound healing or the distribution at the wound site are objectively and directly measured. No attempt has been made to use it for diagnosis of wounds.

[0006]

It is known that various growth factors and physiologically active substances are released from wound sites such as pressure sores and burns, and a series of wound healing processes gradually progress. However, many of these studies evaluate exudates that have been stored over a period of time, and therefore cannot be said to directly evaluate the economic changes in each stage of wound healing. In order to actively promote healing to promote wound healing, it is important to understand the changes over time on the wound surface such as cytokines and cell adhesion factors in these exudates during each process of normal wound healing It seems to hold an important key.

[0007] The present inventors have recognized that it is important to quickly and objectively know the progress of a wound and the state of healing from objective data for wound diagnosis and wound treatment, and that it must be a simple method in practice. Based on repeated studies, he learned that components such as various physiologically active substances in the exudate from the wound surface were closely related to the progress of the wound and the healing status, and completed the present invention. Then, the research was advanced with the development of a measuring method for evaluating the exudate immediately after being released from the wound surface, and a simple measuring method was successfully developed, thereby completing the present invention. Furthermore, a method for objectively and simply evaluating the state of the wound surface by this measurement method has been established, whereby a change over time of components in the exudate during the wound healing process, and a method for detecting differences and characteristics in various wounds can be detected. Established. Further, the present inventors have completed a measurement and inspection kit for performing the above-described method.

[0008] That is, the present invention relates to a method for measuring and estimating the degree of progress of a wound on a wound surface, wherein the exudate from the wound surface is adsorbed on an adsorbent film, and the components in the exudate are detected by an immunochemical method. Therefore, it is preferable that the adsorption film is a hydrophobic adsorption film. The hydrophobic adsorption film is preferably a nitrocellulose film, a cellulose acetate film, a vinylidene fluoride film or a nylon film, and particularly preferably a nitrocellulose film. The immunochemical method is preferably dot blotting, Western blotting or Northern blotting. The component in the wound exudate is collagen, cytokine, fibrin, fibronectin or laminin, and the cytokine is TG.
F-β or PDGF.

Further, the present invention relates to a method for measuring and estimating the degree of wound development on a wound surface, wherein the exudate from the wound surface is adsorbed on a nitrocellulose membrane, blocking is carried out with a blocking protein, and a specific antibody is used. Performs a secondary antibody treatment, performs a chromogenic enzyme-labeled anti-primary antibody (secondary antibody) treatment,
Color development is performed, and the method of measuring and estimating the degree of wound healing is a method of measuring and estimating the degree of wound healing, and the method of measuring and estimating the degree of wound elongation is a method for determining a method of treating a wound.

The present invention further provides at least a hydrophobic membrane,
A kit for measuring and testing the degree of progress of a wound on a wound surface comprising a blocking protein, a primary antibody, a secondary antibody, and a composition of a coloring composition, wherein the hydrophobic membrane is a nitrocellulose membrane, and the blocking protein is rabbit anti-human I Type collagen antibody, rabbit anti-human type III collagen antibody, anti-TGF-
The β antibody and / or anti-PDGF antibody, the secondary antibody is a biotinylated anti-mouse / anti-rabbit IgG antibody, and the coloring composition is a streptavidin-biotinylated peroxidase complex and diaminobenzidine.

That is, according to the present invention, a nitrocellulose membrane which has been conventionally used for the purpose of protein adsorption in various studies to measure the exudate immediately after being released from the wound surface is used as an exudate adsorbent. Used as
The example of the adsorption membrane is not limited to a nitrocellulose membrane, and any membrane that can be used for adsorption may be used. Generally, a hydrophobic membrane generally used for adsorption of proteins and nucleic acids is used. Examples of the hydrophobic membrane include a nitrocellulose membrane (nitrocellulose filter), a cellulose acetate membrane, a vinylidene fluoride membrane, and a nylon membrane (nylon filter). A nitrocellulose membrane is generally and most suitable.

First, in order to measure the degree of wound development on the wound surface, the exudate produced and exuded at the wound site is adsorbed on the adsorbent film surface. Adsorption takes a small amount of wound exudate,
It is adsorbed on the adsorption film. In order to measure the degree of wound development, it is necessary to collect exudate from the wound over time (daily), to collect exudate exuding from the wound over a certain period of time, or to use a different wound surface. It is possible to diagnose the chronological (day-to-day) progress and healing status of the wound surface, as well as to diagnose changes in the wound surface over a period of time and the two-dimensional distribution of wound progression by collecting exudate from the affected area. Becomes
In order to measure and diagnose the two-dimensional distribution (planar distribution) of a wound or to collect exudate, it is possible to directly adsorb the exudate by attaching an adsorption film to the wound surface. It is possible.

The exudate from the wound surface shows quantitative and qualitative changes in proteins, growth factors, cytokines, cell adhesion factors and other physiologically active substances contained in the exudate from the wound surface during the progress of the wound and the healing process. It is known. Further, knowing such a change can be considered as an index for judging the degree of wound development or the degree of healing on the wound surface. Therefore, proteins in the exudate produced from the wound surface adsorbed on the adsorption membrane, growth factors,
Physiologically active substances such as cytokines and cell adhesion factors, or genes or DNAs thereof, are measured.

The measuring method may be a method for measuring proteins, nucleic acids and the like which are found in ordinary literature textbooks. Generally, measurement by an immunochemical method is the simplest and preferable. The immunochemical measurement may be performed directly on the adsorption membrane, or may be performed after the components are separated by means such as electrophoresis. Among the immunochemical methods, a method by an immunoblot method, for example, a dot blot method, a western blot method, and a northern blot method are preferable. The specific method of the immunoblot method can be performed according to a method described in a textbook of the literature (for example, edited by The Japanese Biochemical Society, Shinsei Kagaku Kenkyusho I, Protein, 1990).

A simple measurement test kit used for the above-mentioned method for measuring and testing the degree of wound progression or healing of the wound surface is composed of, for example, reagents and materials suitable for the immunoblotting method described above. Can be done. An example of a measurement kit for the analysis and measurement of wound exudate by immunoblotting is described below.

-Adsorption membrane for wound exudate adsorption: nitrocellulose membrane-Primary antibody: anti-fibronectin monoclonal antibody, anti-TGF-β antibody, anti-PDGF antibody, anti-collagen antibody, etc.-Secondary antibody: biotinylated antibody Mouse antibody, peroxidase-labeled antibody, alkaline phosphatase-labeled antibody, glucose oxidase, radioactive iodine-labeled protein, etc. -buffer -diaminobenzidine and the like.

Next, a method for measuring exudate from a wound surface will be specifically described. 1) Cut the nitrocellulose membrane into a suitable size. 2) A small amount of wound exudate is spotted on the nitrocellulose membrane surface, or the exudate is spotted on the membrane surface by directly applying the nitrocellulose membrane to the wound surface washed with sterile physiological saline. 3) The surface of the nitrocellulose membrane is coated with phosphate buffered saline (PBS) containing skim milk or bovine serum albumin at 4 ° C. for 24 hours to perform blocking. 4) Primary antibody (rabbit anti-human collagen I etc.) diluted with 3% bovine serum albumin / PBS, moist
Treat for 2 hours at room temperature in ure chamber.

5) PB containing 0.05% Tween 20
Wash twice with S for 5 minutes. 6) Treat with a secondary antibody (biotinylated mouse antibody). 7) Wash. 8) A color development reaction is performed by treatment with an avidin-biotinylated chromogenic enzyme / avidin complex (ABC reaction). 9) Wash. 10) Treat with diaminobenzidine. 11) Dry.

By such a treatment step, a qualitative measurement of the physiologically active substance in the wound exudate becomes possible, and it is possible to know the degree of progress of the wound and the degree of healing. can do. Furthermore, the measurement test kit enables objective evaluation of the condition of the wound site at the bedside, outpatient, or the like, whereby the healing process can be grasped and an appropriate treatment method can be selected.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited thereto. Example 1 The exudate from 8 wound surfaces after collecting the separated skin was subjected to nitrocellulose membrane (Nitro plus 2000, Micr).
on Separation Inc. MI20202
0). After drying the adsorption membrane, 10% skim milk (Bacto Ski Milk, DIFCO
0037-17-3) PBS (physiological saline buffer, powder for instant phosphate buffer, IATORON®)
EAGENT RH102-5 NaCl Nakarai Co
(using de313-20), the excess protein portion on the membrane was treated at 4 ° C. for 24 hours to perform blocking.

Rabbit anti-human type I collagen antibody (SAN
BIO PS47), rabbit anti-human type III collagen antibody (SANBIO PS49), anti-fibronectin monoclonal antibody (CBL181), anti-TGF-β (T
transformationgrowth facto
r-β) antibody, anti-PDGF (Platelet der)
Primary antibody treatment was performed for 2 hours at room temperature in a Moisture chamber using an activated growth factor antibody, an anti-fibrin antibody and an anti-laminin antibody.

These primary antibody-treated membranes were washed twice using a rotary shaker with 0.05% Tween 20 PBS for 5 minutes and further twice with PBS for 5 minutes. Next, a biotinylated anti-mouse antibody (Biotinyla) diluted with 10% skim milk PBS solution and 5% calf serum was used.
ted anti-mouse / Anti rabbi
t IgG (H + L), VECTOR BA-120
Using 0), primary antibody treatment was performed at room temperature for 30 minutes.
After the biotinylated secondary antibody treatment, a biotinylated chromogenic enzyme / avidin complex treatment is performed at room temperature for 30 minutes (ABC method).
Was done.

After washing with PBS, diaminobenzidine treatment (Dojindo 349-00903, DAB reaction)
Then, the color was developed. When the color was sufficiently developed, the product was washed with water and dried. Positive rate of split-layer dermal wound exudate was measured for type I collagen 6
/ 7 (6 of 7 cases), type III collagen 3/7 (3 of 7 cases), TGF-β 0/7 (0 of 7 cases), and fibronectin 1/7 (1 of 7 cases) there were. Generally, it is said that type III collagen is produced and accumulated in the wound healing process, and is eventually replaced by type I collagen. The above results can be said to reflect this process. The pH of the wound surface was 8.13 ± 0.16.

Example 2 Exudate from the wound surface of 12 cases of pressure ulcer and intractable leg ulcer was adsorbed on a nitrocellulose membrane, and an anti-collagen antibody (I
(Type, type III), anti-TGF-β antibody and anti-fibronectin antibody as primary antibodies, and immunohistochemical examination using a biotinylated anti-mouse antibody as the secondary antibody was performed in the same manner as in Example 1.

The positive rate of pressure ulcer and intractable leg ulcer exudate in 12 cases was 10/12 (1 out of 12 cases) of type I collagen, respectively.
0), type III collagen 8/12 (8 out of 12),
TGF-β 1/12 (1 of 12 cases) and fibronectin 2/12 (2 of 12 cases).

It is said that during the wound healing process, type III collagen is produced and accumulated and is eventually replaced by type I collagen. In the examples of the present invention, however, the same tendency was observed because type III collagen was detected in a larger amount. Was done. In addition,
The pH of the wound surface was 7.90 ± 0.20. It has been found that the method of the present invention for detecting components of wound exudate by immunoblot method can be one means of knowing the condition of the wound surface and the degree of the treatment process.

Example 3 The procedure of Example 1 was repeated, except that the nitrocellulose membrane was applied to the wound surface and the exudate was collected and adsorbed, and the same results as in Example 1 were obtained. .

Example 4 The procedure of Example 2 was repeated, except that the nitrocellulose membrane was applied to the wound surface and the exudate was collected and adsorbed, and the same results as in Example 2 were obtained. .

In the case of intractable ulcers such as leg ulcers and pressure ulcers, the wound healing process is prolonged. When attention is paid to collagen during the wound healing process, the collagen confirmed in the wound shifts from type III to type I as the healing progresses. Next, the present invention will be described in detail with reference to Reference Examples, but the present invention is not limited to only these cases.

REFERENCE EXAMPLE Type III collagen-positive cases are found in the early stage of granulation of intractable ulcers such as leg ulcers and pressure ulcers. That is, the reference photograph shown in FIG. H. This is a case of his sacral decubitus, which is an intractable ulcer but shows granulation. The reference photograph shown in FIG.
9 years old S. His case of greater trochanteric pressure ulcer is a refractory ulcer, but is an early finding of granulation. Further, the reference photograph shown in FIG. M. This is a case of his sacral pressure ulcer, in which necrotic tissue remains.
The reference photograph shown in FIG.
U. This is a case of his greater trochanteric pressure ulcer, with necrotic tissue remaining.

Cases of type I collagen positive and type III collagen negative are wound surfaces such as mature granulation tissue and shallow skin defect wounds that are expected to be relatively epidermal. That is, the reference photograph shown in FIG. N. It is a case of his sacral decubitus and a mature granulation surface.
In addition, the reference photograph shown in FIG. S.
It is his second degree burn wound and his lower leg findings 2 weeks after the injury. The reference photograph shown in FIG.
K. I. It is his second degree burn wound and his lower leg findings one week after the injury. The reference photograph shown in FIG. 8 is the same case as the above reference photograph G, and is an example of a skin wound for skin transplantation to a burn wound.

According to the method of the present invention, when type III collagen is continued for a long period of time or type I collagen is not expressed, prostaglandin which is suspected of blood flow and nutritional disorders at the wound site and therefore promotes blood flow Ointment which administers a preparation (manufactured by Ono Pharmaceutical Co., Ltd.) or a dibutyl cyclic AMP preparation (manufactured by Daiichi Pharmaceutical Co., Ltd.) which promotes angiogenesis, or an ointment which positively promotes granulation, such as orsenone ointment (manufactured by Nippon Redley Co., Ltd.), reflap ointment (Made by Eisai Co., Ltd.) is preferably used. It is also possible to detect locally deficient growth factors by applying the method of the present invention, and to carry out replacement therapy therefor.
In addition, in the case of type I collagen positive and type III collagen negative, it can be cured by general ointment therapy.

[0033]

As described above, the method of the present invention for quickly and easily measuring the exudate from a wound portion enables the measurement and inspection of the degree of wound development on a wound surface, and a kit for the measurement and inspection Is provided. In addition, the method of the present invention enables a method for measuring and examining the degree of wound healing and an examination method for determining a method for treating a wound surface.

[Brief description of the drawings]

FIG. 1 is a reference photograph of a male case of sacral decubitus, which is an intractable ulcer but in which granulation is observed.

FIG. 2 is a reference photograph showing a case of a greater trochanteric pressure ulcer in a woman, which is an intractable ulcer but is in the early stage of granulation.

FIG. 3 is a reference photograph of a male case of sacral decubitus, in which necrotic tissue remains.

FIG. 4 is a reference photograph of a male case of greater trochanteric pressure sore, in which necrotic tissue remains.

FIG. 5 is a reference photograph of a case of a sacral decubitus ulcer in a male, who has an entirely mature granulation surface.

FIG. 6 is a reference photograph showing a second-degree burn wound of a woman and findings of the lower leg two weeks after the injury.

FIG. 7 is a reference photograph showing a second-degree burn wound of a woman, showing findings of the lower leg one week after the injury.

FIG. 8 is a reference photograph showing a female II degree burn wound and an example of a skin wound for skin transplantation into the burn wound.

Claims (11)

[Claims] 1. A method for measuring and estimating the degree of wound development on a wound surface, wherein the exudate from the wound surface is adsorbed on an adsorption film, and components in the exudate are detected by an immunochemical method. 2. The method according to claim 1, wherein the adsorption film is a hydrophobic adsorption film. 3. The method according to claim 2, wherein the hydrophobic adsorption film is a nitrocellulose film, a cellulose acetate film, a vinylidene fluoride film or a nylon film. 4. The method according to claim 1, wherein the immunochemical method is a dot blot method, a Western blot method or a Northern blot method. 5. The method according to claim 1, wherein the component in the wound exudate is collagen, cytokine, fibrin, fibronectin or laminin. 6. The method according to claim 1, wherein the cytokine is TGF-β or PDG.
The method for measuring and inspecting a wound surface according to claim 5, which is F. 7. A method for measuring and estimating the degree of wound development on a wound surface, comprising the steps of: adsorbing an exudate from the wound surface to a nitrocellulose membrane, blocking with a blocking protein, and treating the primary antibody with a specific antibody. Performing a color-enzyme-labeled anti-primary antibody (secondary antibody) treatment to perform color development. 8. The method according to claim 1, wherein the method for measuring the degree of wound progress is a method for measuring and inspecting the degree of wound healing. 9. The measurement / inspection method according to claim 1, wherein the measurement / inspection method of the degree of wound development is a method for determining a wound treatment method. 10. A kit for measuring and inspecting the degree of wound development on a wound surface having at least the following composition. -Hydrophobic membrane-blocking protein-primary antibody-secondary antibody-coloring composition 11. A nitrocellulose membrane as a hydrophobic membrane, a rabbit anti-human type I collagen antibody, a rabbit anti-human type III collagen antibody, an anti-TGF-β as a blocking protein.
The measurement test according to claim 10, wherein the antibody and / or the anti-PDGF antibody, the secondary antibody is a biotinylated anti-mouse / anti-rabbit IgG antibody, and the coloring composition is a streptavidin-biotinylated peroxidase complex and diaminobenzidine. Kit.