JP3863462B2 - Test method for skin diseases and sensitive skin, and kit used for the method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a simple skin disease and sensitive skin testing method and a kit for carrying out the method.
[0002]
[Prior art and problems to be solved by the invention]
In recent years, skin diseases such as sensitive skin and atopic dermatitis have been increasing due to changes in living environment, a decrease in the ability to respond on the living body, modulation of immunity, and the like, and are becoming a social problem. In particular, atopic dermatitis (which may be abbreviated as “AD” in the present specification) is a disease in which the total number of patients or the number of potential patients is one in 10 and two in Japan. However, the cause of this has not been fully elucidated, and the examination is performed mainly by clinical or medical examinations based on clinical symptoms such as the nature and distribution of the rash, and often relies on a gross or empirical diagnosis by a doctor.
[0003]
More quantitative examination methods for diseases include blood biochemical examination methods and pathological examinations. In the blood biochemical examination method, blood is collected invasively, blood components, various examination markers, cells and the like are examined, and in the pathological examination, a biopsy of skin lesion tissue and the like are examined for the presence or absence of abnormalities in the cell level. For example, in the diagnosis of atopic dermatitis, a blood biochemical test method such as measurement of serum IgE level is used. These methods are simple methods because doctors, nurses, and clinical technologists need to collect blood with a syringe or surgically remove a living tissue (affected area) (biopsy). Furthermore, even from the patient's side, blood collection and biopsy use a needle, syringe, and sharp scalpel, which imposes a mental and physical burden.
[0004]
There are scratch tests, prick tests, tape stripping tests, etc. as simple clinical tests for skin diseases, and for the specific diagnosis of atopic dermatitis, adsorbing secretory immunoglobulin A (IgA) secretion secreted from the skin A patch for examining atopic dermatitis has been disclosed (Japanese Patent Laid-Open No. 6-032733). By affixing such a patch to the skin and measuring secretory IgA absorbed after a predetermined time, atopic dermatitis can be specifically diagnosed noninvasively and easily.
[0005]
By the way, C-fibers, which are unmyelinated sensory nerves, exist in the skin, and their terminals transmit the noxious stimulation of the skin to the center as free nerve endings, while releasing neuropeptides through nerve branching at the periphery. It is known to cause vasodilation and the like. In addition, there are reports that expression of various neurotrophic factors in the epidermis is increased in psoriasis and atopic dermatitis which are inflammatory skin diseases. Among various neurotrophic factors, nerve growth factor (sometimes abbreviated as “NGF” in this specification) is known to act on part of sympathetic nerves and sensory nerves, and on their development and differentiation. ing. In addition, neurotrophic factor 4 (sometimes abbreviated as “NT-4” in the present specification) is considered to act on the development and differentiation of sensory neurons. According to M. Grewe et al. (J. Invest. Delmatol. 114: 1108-1112, 2000), immune samples show that NT-4 expression is increased in samples surgically collected from AD patients compared to normal epidermis. Is described scientifically.
[0006]
However, the relationship between such neurotrophic factors and skin diseases such as psoriasis and atopic dermatitis has not been elucidated yet. In addition, detection of neurotrophic factors in skin, particularly human skin, is usually performed by invasive techniques such as immunohistochemical treatment of surgically removed skin biopsies, and non-invasive measures of neurotrophic factors in the skin It was thought that there was no way to measure easily.
[0007]
[Means for Solving the Problems]
As a result of intensive studies on the relationship between neurotrophic factors and psoriasis and atopic dermatitis skin, the present inventors have surprisingly found that NGF in a stratum corneum sample obtained by a noninvasive method such as tape stripping and the like. It was found that the abundance of NT-4 was statistically significantly higher in lesion sites of certain skin diseases than in healthy sites. In addition, since neurotrophic factors such as NGF and NT-4 were not considered to exist near the surface of the epidermis, in a stratum corneum sample obtained by a simple method of collecting only the surface layer of the epidermis such as tape stripping It is also surprising that those factors can be detected. Thus, it has been found that skin diseases can be examined by a non-invasive and simple method that does not rely on invasive means.
[0008]
Even more surprisingly, the abundance of NGF and NT-4 in the stratum corneum samples of those who have no pruritic site is the NGF and NGF in the stratum corneum samples from healthy sites of patients with skin disease lesion sites. It was also found that it was statistically significantly less than the abundance of NT-4, specifically below the detection limit. From this, it may be possible to examine whether an individual is susceptible to skin diseases, such as atopic or sensitive skin, by measuring the abundance of NGF and / or NT-4 in the stratum corneum. .
[0009]
Accordingly, the present invention provides a method for noninvasively examining skin diseases and sensitive skin of an individual characterized by measuring the abundance of NGF and / or NT-4 in a stratum corneum sample.
[0010]
Preferably, the method is
(A) NGF and / or NT-4 in the stratum corneum sample from the site (a) suspected to be affected or sensitive skin of the individual suffering from skin disease or having sensitive skin Stratum corneum sample derived from the site (b) corresponding to the site of another individual who does not suffer from skin disease or does not have sensitive skin Measuring the amount of NGF and / or NT-4 present in the medium, and
(B) Compare the abundance of NGF and / or NT-4 in the stratum corneum sample from site (a) with the abundance of NGF and / or NT-4 in the stratum corneum sample from site (b), respectively. Process,
Comprising.
[0011]
Preferably, the stratum corneum sample is taken by a tape stripping method.
[0012]
In a further embodiment, the present invention provides a method for assaying an individual's skin disease susceptibility or sensitive skin characterized by measuring the abundance of NGF and / or NT-4 in a stratum corneum sample To do.
Preferably, the method comprises
(A) measuring the abundance of NGF and / or NT-4 in the stratum corneum sample of the individual,
(B) The stratum corneum sample derived from the site corresponding to the site of one or more individuals not affected by skin disease or having sensitive skin as a control, using the value obtained in (A) Comparing the abundance of NGF and / or NT-4 in the medium,
Comprising.
[0013]
Preferably, the stratum corneum sample is obtained by non-invasive means.
Preferably, the stratum corneum sample is taken by tape stripping.
[0014]
The present invention further provides a kit for measuring the abundance of NGF and / or NT-4 in stratum corneum samples,
Means for applying to the skin and then peeling to obtain a stratum corneum sample; and
Means for measuring NGF and / or means for measuring NT-4;
Comprising.
[0015]
Preferably, the means for taking such a stratum corneum sample is an adhesive tape.
[0016]
Such a kit implements a method for measuring the abundance of NGF and / or NT-4 in a stratum corneum sample to detect the above-mentioned skin diseases or non-invasively test skin disease susceptibility. Available for.
[0017]
According to the present invention, the abundance value of NGF or NT-4 in the lesion site of skin disease shows a significant difference from that in the healthy site, so the degree of sensory stimulation such as “itch” in that site or sensitive skin No, whether or not the test subject suffers from a skin disease or the degree of the disease can be objectively diagnosed. Specifically, the abundance of NGF or NT-4 is below the detection limit in healthy sites.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
The skin diseases that can be examined and tested according to the present invention include any diseases as long as the abundance of NGF and NT-4 can be significantly distinguished from the relevant values in healthy sites. However, examples of diseases that can be suitably diagnosed according to the present application include AD, psoriasis vulgaris, senile xeroderma, and so-called sensitive skin although it is not a disease. The “individual” in the present invention means any mammal including humans.
[0019]
Factors that make skin condition sensitive include skin barrier function decline, skin irritation threshold decline, skin dryness, contact dermatitis-causing substances, physicochemical stimulation, stress, physical condition, seasonal changes, ultraviolet rays, physiology, etc. Is mentioned. Furthermore, it may be possible to make the skin sensitive by mistaken skin care, or to become sensitive only by the assumption of the person. For these reasons, it is thought that the category generally called “sensitive skin” has increased in recent years.
Such “sensitive skin” is currently defined as:
(1) “Skins that normally react to many substances that are not common to many humans, such as pharmaceutical external preparations, cosmetics, plants, UV rays, metals, etc., and are prone to skin problems. Skin that is physically sensitive to substances (pollen, fragrance, etc.) and irritating substances (alcohol, etc.) “Specifically, it is considered to be atopic, allergic, or hypersensitive, and it is easy to dry the skin. It is a human who is prone to skin irritation and / or is susceptible to rash due to reduced skin barrier function. Includes skin that is susceptible to sweat, alcohol, fragrance, physical irritation, and acne / pimples; and (2) “Skin inherent due to lack of sleep, overwork, menstruation, seasonal change, mental stress, etc. When skin resistance or skin physiology weakens, the skin may be temporarily prone to skin problems against irritants.
Specific skin states of “sensitive skin” defined in (1) or (2) include dry skin, acne, hypersensitive skin, easily irritated skin, and ultraviolet sensitive skin. appear.
[0020]
In the present invention, the “site suspected of suffering from a skin disease” means not only the appearance of an abnormality in the skin but also a site where the patient feels something abnormal on the skin, for example, itching. It includes the part which is. The “part close to the relevant part of the individual” means a part in the vicinity of the part where the abnormality is observed in the patient and where the abnormality is not found. “A site corresponding to the relevant site of another individual who does not suffer from a skin disease” refers to a healthy body that does not have a skin abnormality corresponding to the body of a patient with a skin abnormality. It means a human part. For example, if the “site suspected of having a skin disease” is the inside of the upper arm / forearm of the patient, the “site corresponding to the site of another individual not suffering from a skin disease” It is preferable that it is the site | part inside the upper arm and forearm of another healthy person, or its vicinity. Then, the amount of NGF or NT-4 in the “site suspected of suffering from skin disease” as a control “site near the relevant site of the individual” or “another subject not suffering from skin disease” If it is statistically significantly higher than the NGF or NT-4 amount in the “part corresponding to the relevant part of the individual”, it can be evaluated as “affected by skin disease”. The presence or absence of a statistically significant difference can be determined by a conventional statistical process such as t-test. The evaluation of the part having sensitive skin could be performed in the same manner.
[0021]
In the present invention, the “susceptibility to skin disease” means whether or not the skin disease is likely to occur or recur in the future, including whether the skin disease is present or not. A person having a constitution or sensitive skin may fall under an individual who is susceptible to skin diseases. Such an assessment of “susceptibility to skin disease” can be made, for example, by determining the amount of NGF and / or NT-4 in a stratum corneum sample of an individual to be measured of one or more individuals not suffering from skin disease. This can be done by comparing the amount of NGF and / or NT-4 in the stratum corneum sample derived from the site corresponding to the site as a control value and testing whether there is a statistically significant difference. When the amount of NGF and / or NT-4 of a plurality of individuals is used as the control value, the average value of the amount of NGF and / or NT-4 of the plurality of individuals is used as the control value. In addition, if the amount of NGF and NT-4 in the stratum corneum sample of the individual to be measured is below the detection limit of the corresponding measurement method, it may be possible to determine that “it is difficult to suffer from a skin disease”. Sensitive skin testing could be done in the same way.
[0022]
According to the present invention, skin disease can be examined by comparing, for example, the value of NGF in any one of the above abnormalities with the value of NGF at the site to be diagnosed. However, it is possible to know the progress of the disease by comparing the NGF value or NT-4 value with both healthy sites and the sites to be diagnosed.
[0023]
In the present invention, the sample collected by a non-invasive method for measuring the abundance of NGF and NT-4 is not the whole epidermis obtained by surgical removal by M. Grewe et al. Samples obtained by means usually used for sampling the stratum corneum in the field, such as tape stripping, are used. The present invention is based on the premise that it has been found that the abundance of NGF and NT-4 in the stratum corneum sample, not the whole epidermis sample, has a clear relationship with skin diseases. Furthermore, the sample that can be used in the present invention includes a horny piece produced by rubbing or the like, scales, dandruff, dirt, nails, desquamation produced by scratching behavior, and the like.
[0024]
Tape stripping means is preferred as means for collecting the stratum corneum sample. The tape stripping means may be a support (adhesive tape) having an adhesive spread on its surface, and may be a conventional adhesive cellophane tape. The support of the adhesive tape includes paper, suf, cotton, cloth, non-woven fabric, polyester resin, urethane resin, polyethylene resin, ethylene-vinyl acetate copolymer, soft polyvinyl chloride film, aluminum foil Etc., or a complex thereof. Examples of the adhesive include natural rubber, polyisobutylene rubber, polybutadiene rubber, silicone rubber, polyisoprene rubber, styrene-isoprene-styrene block copolymer rubber and other synthetic rubber, polypropylene resin, polystyrene resin, and polyacrylate ester. Examples include synthetic resins such as polymer resins. Further, rosin, hydrogenated rosin and its ester, polyterpene resin, petroleum resin and the like can be used as a tackifier. Such an adhesive tape has high adhesiveness with the skin, and the adhesive patch is reliably applied to the skin.
[0025]
Further, in the case of the method according to the present invention for testing the susceptibility to skin diseases, the sample to be measured for the abundance of NGF and NT-4 is not limited to a sample collected by a non-invasive method. It may be the entire epidermis obtained by selective extraction.
[0026]
The amount of NGF and / or NT-4 present in each sample prepared in this way is soluble in each sample via a biochemical method known per se, such as a solvent extraction method, preferably freeze-thaw method, ultrasonic crushing method, etc. It is desirable to measure after extracting NGF or NT-4 by an extraction method for preparing a fraction. The solvent used for extraction may be any conventional physiological buffer, such as Dulbecco's phosphate buffered saline, but is not limited thereto.
[0027]
For the measurement of NGF, methods such as enzyme immunoassay, radioimmunoassay, and Western blotting based on the reactivity with an antibody against NGF can be used. NT-4 can be measured by methods such as enzyme immunoassay, radioimmunoassay, and Western blotting. In either case, NGF measurement and NT-4 measurement are possible, but from the viewpoint of ease of operation, sensitivity, equipment, etc., a method by an enzyme immunoassay using the above antibody is desirable. An enzyme immunoassay (ELISA) kit for measuring NGF and NT-4 is commercially available from, for example, PROMEGA.
[0028]
NGF and NT-4 measured as described above are each compared with the value of a healthy site as a control and the site to be examined (that is, the site suspected of having a skin disease).
[0029]
In each of the above methods, it is preferable to use an antibody against NGF and an antibody against NT-4 as means for measuring NGF and NT-4, respectively. Each of such antibodies can be obtained by a known production method, but it is convenient to use a commercially available product in accordance with the mode recommended by the vendor.
[0030]
Thus, according to the present invention, there is also provided a kit configured so that antibodies for measuring NGF and NT-4 can be used in combination. Use in combination means that NGF and NT-4 can be measured on the spot or at different times or locations, but the results are in a state of final comparison. To do. This kit can include reagents (for example, a buffering agent) and fixtures (microtiter plate, pipette, etc.) that facilitate the above-described measurement.
[0031]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0032]
Preparation of stratum corneum sample and preparation of measurement sample
Lesions of AD inflammatory skin disease in patients diagnosed with AD (upper arm / forearm inner, waist, legs, wrists, throat, etc.) and corresponding healthy sites in the vicinity of the patient, as well as AD First, an adhesive cellophane tape was applied to a healthy site corresponding to a healthy person who did not have a lesion site, and immediately peeled off. Phosphate buffered physiological saline was added to the stratum corneum attached to the tape, freeze-thawed and sonicated, and then a soluble protein fraction containing NGF and NT-4 was obtained by centrifugation.
[0033]
The operations for each of the above samples were performed by a common method specifically described.
[0034]
The stratum corneum was collected by slightly improving the method described in I. Horii et al. (J. Dermatol., 121: 587-592 (1989)). First, a piece of adhesive tape (Nichiban cello tape (registered trademark)) was affixed to the sample collection part, and the tape piece was peeled off. The tape piece (24 mm × 45 mm) was chopped, immersed in Dulbecco's phosphate buffered saline, and stored at −20 ° C. until extraction. The extraction was performed by treating the immersion liquid for 2 minutes on ice using an ultrasonic crusher (Astrason (registered trademark) Heat Systems-Ultrasonics) equipped with a probe. Insolubles were removed by centrifugation at 14000 g for 10 minutes at 4 ° C. The supernatant thus obtained was subjected to the following experiment.
[0035]
Measurement example of NGF and NT-4 NGF concentration and NT-4 concentration in the measurement sample (supernatant) prepared by the above-mentioned method are measured by NGF and NT-4 enzyme immunoassay (ELISA) kit (PROMEGA), respectively. Made by the company). At that time, each sample was diluted 2-fold with the diluent provided in the kit.
[0036]
As a result of ELISA, absorbance was measured with a MICROPLATE READER Model 450 (manufactured by BIO-RAD) at a wavelength of 450 nm.
[0037]
Since the amount of stratum corneum collected varies depending on the target site and skin disease, the amount of protein in the measurement sample is measured using Micro BCA (trademark) Protein Assay Reagent Kit (PIERCE), and NGF per sample protein Alternatively, NT-4 was calculated and used as the NGF value and NT-4 value in the stratum corneum.
[0038]
Results and discussion
The results of comparing NGF values and NT-4 values in the stratum corneum of AD and healthy individuals are shown in FIGS.
From the results of FIGS. 1 and 2, the NGF and NT-4 values in the horny layer sample derived from the lesion site of the AD inflammatory skin disease site of the patient diagnosed with AD are the horny layer sample derived from the healthy site of the patient. And it was statistically significantly higher than any NGF and NT-4 values in healthy sites of another healthy person who was not diagnosed with AD. Therefore, it was shown that skin diseases can be diagnosed using NGF values and / or NT-4 values in stratum corneum samples collected by tape stripping as indices.
[0039]
Surprisingly, the NGF and NT-4 values of stratum corneum samples from healthy sites of patients diagnosed with AD are those of healthy individuals who have not been diagnosed with AD despite being healthy sites. It was significantly higher than the NGF and NT-4 values in the strata. Therefore, it was revealed that the stratum corneum of individuals suffering from AD has significantly higher NGF values and NT-4 values even at sites that are not affected sites, although not as much as the affected sites. In addition, the NGF and NT-4 values in the stratum corneum of healthy individuals who have not been diagnosed with AD were below the detection limit. By utilizing this fact, it is possible to test whether an individual has a predisposition to suffer from a skin disease using the NGF value and / or NT-4 value in the stratum corneum sample as an index.
[Brief description of the drawings]
FIG. 1 is a result of comparing NGF values in the stratum corneum of AD patients and healthy individuals.
FIG. 2 shows the result of comparing NT-4 values in the stratum corneum of AD patients and healthy individuals.

Claims (10)

A method for noninvasively examining a skin disease or sensitive skin of an individual characterized by measuring the abundance of NGF and / or NT-4 in a stratum corneum sample. (A) NGF and / or NT- in the stratum corneum sample from the site (a) suspected to be affected or sensitive skin of said individual having skin disease or having sensitive skin The stratum corneum derived from the abundance of 4 and the site corresponding to the site of the same individual as a control or the site corresponding to the site of another individual who does not suffer from skin disease or does not have sensitive skin (b) Measuring NGF and / or NT-4 abundance in the sample, and
(B) Compare the abundance of NGF and / or NT-4 in the stratum corneum sample from site (a) with the abundance of NGF and / or NT-4 in the stratum corneum sample from site (b), respectively. Process,
The method of claim 1 comprising: The method according to claim 1 or 2, wherein the stratum corneum sample is obtained by tape stripping. A method for assaying susceptibility or sensitive skin of an individual's skin disease, characterized by measuring the abundance of NGF and / or NT-4 in a stratum corneum sample. (A) measuring the abundance of NGF and / or NT-4 in the stratum corneum sample of the individual,
(B) The stratum corneum sample derived from the site corresponding to the site of one or more individuals not affected by skin disease or having sensitive skin as a control, using the value obtained in (A) Comparing the abundance of NGF and / or NT-4 in the medium,
The method of claim 4 comprising: 6. The method according to claim 4 or 5, wherein the stratum corneum sample is obtained by non-invasive means. The method according to any one of claims 4 to 6, wherein the stratum corneum sample is obtained by tape stripping. A kit for measuring the abundance of NGF and / or NT-4 in a stratum corneum sample,
Means for applying to the skin and then peeling to obtain a stratum corneum sample; and
Means for measuring NGF and / or means for measuring NT-4;
A kit comprising: The kit according to claim 8, wherein the means for collecting the stratum corneum sample is an adhesive tape. The kit according to claim 8 or 9, for carrying out the method according to any one of claims 1 to 5.

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