WO2023210401A1

WO2023210401A1 - Method for determining condition of skin on basis of lymph vessels in skin

Info

Publication number: WO2023210401A1
Application number: PCT/JP2023/015121
Authority: WO
Inventors: 菜緒染井; エンフトールガントゥムル; 恭子井上; 健太朗加治屋; 信之三川; 新介秋田
Original assignee: 株式会社資生堂
Priority date: 2022-04-28
Filing date: 2023-04-14
Publication date: 2023-11-02

Abstract

The purpose of the present invention is to elucidate the relationship between lymph vessels in the skin and lymphedema as well as the relationship between lymph vessels in the skin and the skin, and to provide thereby a method for evaluating lymphedema and the condition of the skin. Parameters relating to the structure and/or morphology of lymph vessels in the skin were discovered to be related to lymphedema of the skin and to the condition of the skin, and there is provided a method for evaluating the condition of the skin on the basis of the structure and/or morphology of lymph vessels in the skin.

Description

How to determine skin condition based on skin lymph vessels

The present invention relates to a method for determining skin condition using the relationship between the structure and/or form of skin lymph vessels and skin condition, a method for predicting skin lymph vessel structure, and a method for screening skin condition improving drugs.

A circulatory system consisting of a vascular system and a lymphatic system runs throughout the living body. Blood vessels are responsible for maintaining body temperature and supplying nutrients and oxygen to tissues throughout the body, while lymphatic vessels are responsible for transporting immune cells and collecting excess tissue fluid and foreign substances. The lymphatic system is composed of lymph capillaries present in peripheral areas, collecting lymph vessels formed by aggregation of lymph capillaries, and lymph nodes located throughout the body. The collecting lymph vessels eventually converge into the thoracic duct or right lymph trunk, which connects to the veins. Lymphatic capillaries have closed, blind ends and are composed of a single layer of squamous epithelial cells. Compared to capillaries, lymph capillaries have looser connections between endothelial cells and larger intercellular spaces, and can collect large proteins and foreign substances that cannot be collected in blood vessels. When the function of lymph vessels is inhibited for some reason, tissue fluid accumulates in peripheral tissues, causing swelling and edema.

Lymphedema is a disease in which swelling occurs due to local lymph fluid increase and/or poor collection. Lymph that seeps into the tissues is collected by the lymph vessels, but it is not collected and accumulates in the limbs, causing swelling. Lymphedema has many causes, but it often occurs after cancer surgery or radiation therapy. When lymphedema occurs, it worsens its appearance and impairs motor function. In addition, due to poor lymph flow, inflammation that occurs at the wound site can spread to the entire limb, resulting in cellulitis. It has also been reported that abnormal proliferation of keratinocytes and condensation of dermal collagen occur in affected areas with lymphedema (Non-Patent Document 1: Emerg Infect Dis. 2004 Nov; 10(11): 1938-1946).

Lymphedema that develops after cancer surgery significantly lowers patients' QOL, but there is little detailed physiological information about it, and its treatments include lymphatic drainage through massage, compression with elastic bags, clothing, and exercise. can be mentioned. In addition, it has been reported that lymphatic circulation decreases in postmenopausal women, which is one of the causes of swelling (Non-Patent Document 2: Surg Radiol Anat 13 (3):221-226. doi:10.1007/ BF01627990). It has been reported that administration of estrogen improves or prevents swelling and lymphedema (Patent Document 1: Japanese Patent Application Laid-Open No. 2013-071913).

JP2013-071913A International Publication No. 2019/097958 International Publication No. 2018/030520

The purpose of the present invention is to elucidate the relationship between skin lymph vessels and lymphedema, as well as the relationship between skin lymph vessels and the skin, by clarifying the characteristics of skin lymph vessels that cause lymphedema. , thereby providing a method for evaluating lymphedema and further skin conditions.

The present inventors focused on the structure and morphology of lymphatic vessels in the skin of patients with lymphedema, and performed three-dimensional visualization and analysis of the structure of lymphatic vessels in the skin extracted from patients with lymphedema. It has been found that the structure and morphology of lymph vessels in the skin change depending on the severity (types 1 to 4) of skin cancer, and the properties of the lymph vessels change accordingly. Furthermore, when we three-dimensionally visualized and analyzed the lymphatic structure of the skin in healthy people of different ages, we found that the morphology of lymphatic vessels changes with age.
The invention therefore relates to:
[1] A method for determining a skin condition, or a method for determining or providing a cosmetic treatment, based on parameters related to the structure and/or morphology of lymphatic vessels in the skin.
[2] The method according to item 1, wherein the parameter regarding the morphology of lymph vessels in the skin is determined by at least one selected from the group consisting of volume, number of branches, and diameter of lymph vessels.
[3] The method according to item 2, wherein when the parameter related to the morphology of lymph vessels in the skin is the diameter of lymph vessels, it is determined that the skin condition is worsening due to a change in the diameter of lymph vessels.
[4] The method according to item 3, wherein the parameter regarding the morphology of lymph vessels in the skin further takes into consideration the number of branches of lymph vessels, and determines that the skin condition is worsening.
[5] The method according to any one of items 1 to 4, wherein the morphology of the skin lymph vessels is measured by optical coherence tomography or optical ultrasound imaging.
[6] The method according to any one of items 1 to 5, wherein the parameter related to the structure of the skin lymph vessels is determined by the expression level of VE-cadherin in the skin lymph vessels.
[7] The method according to item 6, wherein it is determined that the skin condition is worsening due to an increase in the expression of the VE-cadherin in skin lymph vessels.
[8] A method for screening skin condition improving drugs using parameters related to the morphology of skin lymph vessels as an index.
[9] Adding the candidate drug to the skin,
a step of photographing the morphology of lymph vessels in the skin after addition;
calculating parameters regarding the morphology of skin lymph vessels based on the captured image;
a step of comparing the skin lymphatic vessel morphology in the skin before addition of the candidate drug or in the control drug addition group; and a step of selecting the candidate drug as a skin condition improving drug based on the change in the parameter.
The screening method according to item 8, comprising:
[10] The method according to item 9, wherein the parameter regarding the morphology of the skin lymph vessels is determined by at least one selected from the group consisting of volume, number of branches, and diameter of lymph vessels.
[11] The method according to item 9 or 10, wherein the skin is cultured skin containing lymphatic structure.
[12] A method for screening skin condition improving drugs using the expression level of VE-cadherin in lymphatic endothelial cells as an index.
[13] Adding the candidate drug to the skin,
measuring VE-cadherin expression in skin lymphatic endothelial cells after addition;
A step of comparing the expression of VE-cadherin in the lymphatic endothelial cells of the skin before addition of the candidate drug or in the control drug addition group, and a step of selecting the candidate drug as a skin condition improving drug based on the change in the expression level of VE-cadherin.
The screening method according to item 12, comprising:
[14] The method according to item 13, wherein the skin is cultured skin containing lymphatic structure.
[15] A step of culturing lymphatic endothelial cells in a medium containing a candidate drug; A step of measuring VE-cadherin expression in lymphatic endothelial cells after culture;
Comparing the expression level of VE-cadherin with a control;
The screening method according to item 12, comprising the step of selecting a candidate drug as a skin condition improving drug based on changes in VE-cadherin expression.
[16] A drug for improving skin condition, including an agent for increasing the diameter of skin lymph vessels.

According to the present invention, it is possible to determine the skin condition, especially lymphedema, based on the structure and/or morphology of lymph vessels in the skin.

FIG. 1A shows three-dimensional images of lymph vessels in normal skin (normal) and skin of lymphedema patients (types 1 to 4). FIG. 1B shows the results of measuring the volume of lymph vessels from a three-dimensional image of lymph vessels. FIG. 1C shows the results of measuring the diameter of a lymphatic vessel from a three-dimensional image of the lymphatic vessel. FIG. 1D shows the results of measuring the number of branches of lymph vessels from a three-dimensional image of lymph vessels. FIG. 2 shows the results of fluorescence staining and photographing of VE-cadherin in normal skin (normal) and skin of lymphedema patients (type 2, type 4). FIG. 3A shows three-dimensional images of lymph vessels in the cheek skin of a person in their 20s and the cheek skin of a person in their 60s. FIG. 3B shows the results of measuring the volume of lymph vessels from three-dimensional images of lymph vessels in young people (26 to 45 years old) and middle-aged people (46 to 67 years old). FIG. 3C shows the results of measuring the diameter of lymph vessels from three-dimensional images of lymph vessels in young people (26 to 45 years old) and middle-aged people (46 to 67 years old). Figure 3D shows the results of measuring the number of lymphatic vessel branches from three-dimensional images of lymphatic vessels in young people (26 to 45 years old) and middle-aged people (46 to 67 years old). Figure 4 shows the intensity of R-CHP measured in stained photographs obtained by R-CHP/Hoechst staining on normal skin (normal) and skin of lymphedema patients (type 1 + type 2, type 3 + type 4). The results are shown below.

The present invention relates to a method for determining skin condition based on parameters related to the structure and/or morphology of skin lymph vessels.

Skin lymph vessels can be observed by performing known observation techniques on the skin. Techniques for observing lymph vessels include scintigraphy, indocyanine green fluorescence angiography (ICG), redundant skin staining, optical coherence tomography, and optical ultrasound imaging. Microscopic lymph vessels that exist in the dermis of the skin are particularly called lymph capillaries, and can be observed using these techniques.

Although scintigraphy is an established method for diagnosing lymphedema and is capable of visualizing lymph fluid and its flow in living organisms, the use of isotopes as contrast media places a heavy burden on patients. The ICG contrast method has also been established as a method for diagnosing lymphedema, and lymph fluid and its flow can be visualized by subcutaneously injecting indocyanine green. Although these methods for labeling lymph fluid can visualize relatively large collecting lymph vessels and determine the transport capacity of lymph vessels, the lymph fluid exudates into the tissue, making it possible to visualize relatively large collecting lymph vessels. There is a problem in that the background of lymphatic capillaries in the peripheral region becomes high. On the other hand, by staining excess skin and observing it using a fluorescence microscope, it became possible to construct a three-dimensional structure of the smaller lymph capillaries present in the skin. Staining of the skin can target proteins specifically expressed in lymphatic vessels. Such proteins include podoplanin and the like. As an example, a skin sample can be fluorescently labeled specifically in lymph vessels by using an anti-podoplanin antibody as a primary antibody and a fluorescently labeled secondary antibody. By making it transparent using a transparentizing reagent, observation using a light sheet microscope becomes possible (Patent Document 3: International Publication No. 2018/030520).

Optical coherence tomography (OCT) is a method that uses coherent near-infrared broadband light to image a vertical cross section of the measurement target as a backscattered intensity distribution, and obtains a three-dimensional image of the microstructure of the skin. (Non-patent document 3: Sci Rep. 2016 Feb 19;6:21122. doi: 10.1038/srep21122). Photo-ultrasonic imaging uses an ultrasonic sensor to receive ultrasonic waves emitted by tissues that have absorbed pulsed light emitted from a laser, and can obtain three-dimensional images in real time by performing parallel signal processing on the received signals. (Non-patent document 4: F1000Res. 2018; 7: 1813. doi: 10.12688/f1000research.16743.2)

Lymph vessels in the dermis, especially lymph capillaries, visualized and three-dimensionally constructed using lymph vessel observation techniques can be analyzed in terms of volume, diameter, and number of branches. The volume of lymph vessels relates to the proportion of lymph vessels in the dermis to a unit volume. The diameter of a lymphatic vessel can be expressed as the average diameter calculated by calculating the diameter over the entire length of a lymphatic vessel in the dermis. The number of branches of a lymphatic vessel can be expressed as the number of branches included in a unit volume. The volume, diameter, and number of branches can be appropriately determined for a three-dimensionally constructed lymphatic vessel by computer processing. As an example, Imaris3D/4D image analysis software (Zeiss) can be used to determine the volume, diameter, and number of branches of a three-dimensionally constructed lymphatic vessel.

Parameters related to the morphology of lymphatic vessels in the skin can be determined by at least one selected from the group consisting of volume, diameter, and number of branches of lymphatic vessels. The volume, diameter, and number of branches may each be used as parameters related to the morphology. Furthermore, a new parameter may be derived by combining at least two parameters among volume, number of branches, and diameter.

As lymphedema worsens, the diameter and volume of skin lymph vessels decrease (Figures 1A, B, C). Therefore, the diameter of lymphatic vessels in the skin can be correlated with the severity of lymphedema. Further, in early lymphedema (types 1 and 2), the number of branches increases, and then in advanced lymphedema (types 3 and 4), the lymph vessels degenerate and the number of branches decreases (FIGS. 1A, D). Therefore, parameters related to the morphology of lymphatic vessels in the skin, such as changes in volume, diameter or number of branches, can be associated with the severity of lymphedema, and even thresholds can be established for parameters related to the morphology of lymphatic vessels associated with the severity of lymphedema. You can also set As an example, the diameter threshold can be set to, for example, 40 μm or 35 μm, and lymphedema can be determined when the diameter is lower than the threshold. As an example, the volume threshold can be set to, for example, 2.5% or 2.0%, and lymphedema can be determined when the volume is lower than the threshold. Furthermore, lymphedema can also be determined by further taking into account the number of branches of lymphatic vessels in the skin. As an example, if the diameter and/or volume of cutaneous lymph vessels decreases while the number of branches of cutaneous lymph vessels increases, this indicates that the cutaneous lymph vessels have become thinner and have more branches. This shows that lymphedema can be determined in its early stages. On the other hand, a decrease in the diameter and/or volume of the skin lymph vessels, as well as a decrease in the number of branches of the skin lymph vessels, indicates that the lymph vessels have degenerated, leading to lymphedema and further skin conditions. Deterioration, ie, advanced lymphedema, can be determined.

The relationship between lymphedema and skin conditions is also known, and it is known that lymphedema increases the thickness of the epidermis, including the stratum corneum (Non-Patent Document 1). Further, as lymphedema worsens, collagen production increases and degeneration occurs in the dermis (Figure 4). Then, worsening of lymphedema is also associated with worsening of skin condition. Thereby, the skin condition can also be determined by determining the severity of lymphedema. Therefore, the skin condition can be determined based on parameters related to the structure and/or morphology of skin lymph vessels. More specifically, changes in the volume, diameter, or number of branches of skin lymph vessels can be associated with deterioration of skin condition. It can be determined that the skin condition is worsening if the volume and/or diameter decreases. Furthermore, the skin condition can also be determined by further taking into account the number of branches of lymphatic vessels in the skin.

The morphology of skin lymph vessels also changes depending on age (Figure 3A). There is a tendency for the volume of lymph vessels to decrease with age (Figure 3B). Furthermore, in young people, the diameter of lymph vessels in the skin is large and the number of branches is small, while in middle-aged and elderly people, the diameter of lymph vessels is small and the number of branches is large (FIGS. 3C and D). Since skin conditions worsen with age, lymphatic vessel morphology has been shown to be related to skin conditions even in normal skin. Therefore, the morphology of lymph vessels in the skin is not only an indicator of the degree of deterioration of the pathological condition of lymphedema, but also an indicator of the deterioration of normal skin. As an example, the diameter threshold can be set to, for example, 35 μm or 30 m, and if the diameter is lower than the threshold, it can be determined that the skin condition is worsening. The volume threshold can be set as appropriate, and for example, 2.5% or 2.0% can be used. If it is lower than such a threshold, it can be determined that the skin condition is deteriorating. Furthermore, the skin condition can also be determined by further taking into account the number of branches of lymphatic vessels in the skin. As an example, if the diameter and/or volume of cutaneous lymph vessels decreases while the number of branches of cutaneous lymph vessels increases, this indicates that the cutaneous lymph vessels have become thinner and have more branches. Shows and allows you to determine your skin condition. Determination of skin condition is done for cosmetic purposes. Cosmetic treatments can be provided to maintain the health of the lymphatic vessels when it is determined that the lymphatic vessels have become narrower, have decreased volume, and/or have an increased number of branches. Such cosmetic treatments may include skin massage and presentation of appropriate cosmetics such as skin condition improving agents. Accordingly, another aspect of the invention also relates to a method for determining or providing a cosmetic treatment based on parameters relating to the structure and/or morphology of lymphatic vessels in the skin. The skin condition improving agent provided is preferably a skin condition improving agent screened by the screening method of the present invention.

The expression of VE-cadherin in the cutaneous lymph vessels can be mentioned as a parameter related to the structure of the cutaneous lymph vessels. As lymphedema worsens, the expression level of VE-cadherin in skin lymph vessels increases (Figure 2). Therefore, the expression level of VE-cadherin in the lymphatic vessels of the skin can be correlated with the severity of lymphedema. The expression level of VE-cadherin can be observed by staining a skin sample and using a fluorescence microscope using an anti-VE-cadherin antibody and a fluorescently labeled secondary antibody. VE-cadherin is present in both lymph vessels and blood vessels, so in order to distinguish between lymph vessels and blood vessels, co-staining with podoplanin, which is specifically expressed in lymph vessels, may be performed, or it may be possible to distinguish between lymph vessels and blood vessels based on the structure of the vessels. It may also be distinguished from lymph vessels. In yet another embodiment, the measurement may be performed by isolating lymph vessels from a skin sample and measuring the protein amount or mRNA amount of the isolated lymph vessels. The amount of protein or mRNA can be measured based on conventional methods, such as Western blotting or quantitative PCR. It has been shown that as lymphedema worsens, the expression level of VE-cadherin in skin lymph vessels increases. This makes it possible to correlate the expression level of VE-cadherin in skin lymph vessels with the severity of lymphedema, and the greater the expression level of VE-cadherin, the worse the lymphedema becomes. Since a relationship between lymphedema and the structure of lymph vessels has been shown, parameters related to the structure of lymph vessels, like parameters related to the morphology of lymph vessels, are not only indicators of the degree of deterioration of the pathological condition of lymphedema. This is an indicator of deterioration of normal skin.

VE-cadherin is a protein with a molecular weight of about 140 kDa, also called cadherin-5 or CD144, and belongs to the cadherin superfamily. VE-cadherin is specific to endothelial cells, is expressed in lymphatic endothelial cells and vascular endothelial cells, and is present in cell membranes (Patent Document 2: International Publication No. 2019/097958). VE-cadherin molecules are known to be involved in the permeability of vascular and lymphatic endothelium. Without intending to be limited by theory, increased expression of VE-cadherin may increase adhesion between lymphatic endothelial cells and inhibit lymph fluid collection.

Another aspect of the present invention may relate to a method of predicting the lymphatic structure of the skin from the skin condition based on the relationship between parameters based on the structure and/or morphology of the lymphatic vessels of the skin and the skin condition. By determining the skin condition based on the epidermal condition and/or the degeneration of dermal collagen, the lymphatic structure of the skin can be predicted, and thereby the progression of swelling and edema can be predicted.

In another aspect of the present invention, the present invention also relates to a method for screening skin condition improving drugs using parameters related to the morphology of skin lymph vessels as an index. More specifically, the screening method of the present invention includes the following steps:
adding a candidate drug to the skin;
a step of photographing the morphology of lymph vessels in the skin after addition;
calculating parameters regarding the morphology of lymph vessels based on the captured images;
a step of comparing parameters regarding the morphology of lymph vessels in the skin before addition of the candidate drug or in a control drug addition group; and a step of selecting the candidate drug as a skin condition improving drug based on the change in the parameter.
including. By performing this method, candidate drugs can be screened as skin condition improving agents. The volume, number of branches, and/or diameter of lymph vessels can be used as parameters regarding the morphology of lymph vessels in the skin. The candidate agent may be repeatedly added to the skin over a period of time during which changes in parameters related to lymphatic vessel morphology occur. The control drug added group may differ only in that no candidate drug is added. The skin used in the screening method of the present invention may be cultured skin containing lymphatic structure, cultured tissue of surplus skin, animal skin, or skin of a living human body.

In yet another aspect of the invention, the invention also relates to a method for screening skin condition improving drugs based on VE-cadherin expression in skin lymphatic cells. More specifically, the screening method of the present invention is as follows:
adding a candidate drug to the skin;
measuring VE-cadherin expression in skin lymphatic cells after addition;
a step of comparing the expression of VE-cadherin in the skin lymph vessels before addition of the candidate drug or a control drug addition group; and a step of selecting the candidate drug as a skin condition improving drug based on the change in VE-cadherin expression.
including. By performing this method, candidate drugs capable of suppressing VE-cadherin expression in skin lymph vessels can be screened as skin condition improving agents. The control drug added group may differ only in that no candidate drug is added. The skin used in the screening method of the present invention may be cultured skin containing lymphatic structure, cultured tissue of surplus skin, or animal skin.

In yet another embodiment, the screening method may be performed on cultures of lymphatic cells.
Specifically:
culturing lymphatic vessel cells in a medium containing a candidate drug;
measuring VE-cadherin expression in lymphatic cells after culture;
The method includes a step of comparing the expression level of VE-cadherin with a control, and a step of selecting a candidate drug as a skin condition improving drug based on the change in VE-cadherin expression. The lymphatic cells may be cultured alone or in combination with other cells, and the control may be a group that differs only in the absence of the candidate agent or the candidate agent. It may be a group before drug addition. For example, it may be cultured as a skin culture. Candidate drugs capable of suppressing VE-cadherin expression in lymphatic cells can be screened as skin condition improving agents. Lymphatic cells are especially lymphatic endothelial cells.

As the candidate drug used in the screening method, any drug included in any library can be used. As an example, a known cosmetic material library or pharmaceutical library may be used. These libraries may be compound libraries or extract libraries.

In the present invention, the skin condition improving agent refers to a drug that can improve the general properties of the skin, particularly the skin conditions related to lymphedema or swelling. The skin condition improving agent may be incorporated into cosmetics, pharmaceuticals, or quasi-drugs, or may be incorporated into foods, such as nutritional supplements such as supplements. Without intending to be limited by theory, improving the expression of VE-cadherin and the function of lymphatic vessels in the skin can improve skin conditions, particularly swelling or lymphedema. Therefore, the skin condition improving agent can also be referred to as an agent for treating, preventing or improving swelling or lymphedema, and can also be referred to as an agent for increasing the diameter and/or volume of lymph vessels. The skin condition improving agent can be administered by any route such as transdermally, mucosally, orally, subcutaneously, or intramuscularly, but transdermal administration is preferred from the viewpoint of acting on the lymphatic vessels directly under the skin.

All documents mentioned herein are incorporated by reference in their entirety.

The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Changes in the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, may be made without departing from the spirit of the present invention.

Example 1: Analysis of lymph vessels in skin samples from lymphedema patients Surgical surplus skin and normal lower branches of lower limb lymphedema patients whose severity of lymphedema (types 1 to 4) was determined using lymphoscintigraphy Skin samples were provided by Chiba University Hospital. Informed consent was obtained from each patient for the use of excess skin in experiments.

The excess skin tissue of the lower limbs was heat treated (60°C, 1 minute) to peel off the epidermis and fixed overnight with 4% paraformaldehyde (PFA)/physiological saline (PBS). PFA-fixed skin tissue was permeabilized overnight with 2% Triton-X100/PBS, and then blocked overnight with 5% donkey serum/PBS. Thereafter, it was incubated for 3 days with anti-podoplanin antibody (Dako Cytomation, #M3619)/5% donkey serum/PBS as the primary antibody, washed with PBS, and Alexa647-labeled anti-mouse (H+L) antibody (Invitrogen)/as the secondary antibody. Incubated overnight with 5% donkey serum/PBS. The immunostained skin tissue was immersed in a clearing reagent: Rapiclear 1.52 (SunJin Lab Co.) to make it transparent. The sample thus obtained was observed under a fluorescence microscope using LSM880 (ZEISS), and a three-dimensional image of the skin lymph vessels was obtained (FIG. 1A). From the three-dimensional images, the volume, diameter, and number of branches of lymph vessels were determined using Imaris3D/4D image analysis software (Zeiss) (FIGS. 1B to 1D). As lymphedema worsened, the diameter and volume of cutaneous lymph vessels decreased (Fig. 1A, B, C). The number of branches increased in early lymphedema (types 1 and 2), followed by a decrease in the number of branches in advanced lymphedema (types 3 and 4).

Heat treatment (60°C, 1 minute) was applied to the excess skin tissue of the lower limbs to peel off the epidermis, which was fixed overnight in 4% paraformaldehyde (PFA)/physiological saline (PBS). The PFA-fixed skin tissue was defatted using CUBIC-L (Wako) at 37°C for one week, and then blocked overnight with 5% donkey serum/PBS. Thereafter, it was incubated for 3 days with anti-VE-cadherin antibody (Cell signaling, #2500)/5% donkey serum/PBS as the primary antibody, washed, and Alexa568-labeled anti-rabbit (H+L) antibody (Invitrogen) as the secondary antibody. )/5% donkey serum/PBS overnight. The immunostained skin tissue was cleared by immersion in a clearing reagent. CUBIC-R+ (WAKO) was used as a clarifying reagent. The sample thus obtained was observed using a fluorescence microscope using LSM880 (ZEISS) (FIG. 2). As lymphedema worsened, the expression level of VE-cadherin in skin lymph vessels increased.

Example 2: Age-related changes in lymph vessels in healthy subjects Excess cheek skin of women in their 20s to 60s was treated with Obio, LLC. Obtained from.
The excess skin tissue on the cheek was heat treated (60° C., 1 minute) to peel off the epidermis and fixed overnight with 4% paraformaldehyde (PFA)/physiological saline (PBS). PFA-fixed skin tissue was permeabilized overnight with 2% Triton-X100/PBS, and then blocked overnight with 5% donkey serum/PBS. Thereafter, the cells were incubated for 3 days with anti-podoplanin antibody (Dako Cytomation, #M3619)/5% donkey serum/PBS as the primary antibody, washed with PBS, and Alexa647-labeled anti-mouse (H+L) antibody (Invitrogen)/as the secondary antibody. Incubated overnight with 5% donkey serum/PBS. The immunostained skin tissue was immersed in a clearing reagent: Rapiclear 1.52 (SunJin Lab Co.) to make it transparent. The sample thus obtained was observed under a fluorescence microscope using LSM880 (ZEISS), and a three-dimensional image of the skin lymph vessels was obtained (FIG. 3A). From the three-dimensional images, the diameter and number of branches of lymph vessels were determined using Imaris3D/4D image analysis software (ZEISS) (FIGS. 3B and 3C). The morphology of lymphatic vessels in the skin also changed with age (Fig. 3A). There was a tendency for the volume of lymph vessels to decrease with age (Fig. 3B). In addition, in young people, the diameter of lymph vessels was large and the number of branches was small, while in middle-aged and elderly people, the diameter of lymph vessels was small and the number of branches was large (Fig. 3C and D).

Example 3: Degeneration of collagen in skin samples derived from lymphedema patients Surgical surplus skin of lower limb lymphedema patients whose lymphedema severity (types 1 to 4) was determined using lymphoscintigraphy, and as a control. Normal lower limb skin samples were provided by Chiba University Hospital. Informed consent was obtained from each patient for the use of excess skin in experiments.

The skin samples were frozen and frozen sections with a thickness of 50 μm were obtained. The frozen sections were stained for denatured collagen using R-CHP (3Helix Inc.) according to the protocol. Denatured collagen was quantified using the image analysis software Fiji on the stained images (FIG. 4). Exacerbation of lymphedema increased denatured collagen production in the dermis.

Claims

A method for determining skin condition based on parameters related to the structure and/or morphology of skin lymph vessels.
The method according to claim 1, wherein the parameter regarding the morphology of the skin lymph vessels is determined by at least one selected from the group consisting of volume, number of branches, and diameter of lymph vessels.
The method according to claim 2, wherein when the parameter related to the morphology of lymph vessels in the skin is the diameter of lymph vessels, it is determined that the skin condition is worsening due to a change in the diameter of lymph vessels.
4. The method according to claim 3, wherein the parameter regarding the morphology of lymph vessels in the skin further takes into account the number of branches of lymph vessels, and determines that the skin condition is worsening.
The method according to any one of claims 1 to 4, wherein the morphology of the skin lymph vessels is measured by optical coherence tomography or optical ultrasound imaging.
The method according to claim 1, wherein the parameter related to the structure of the skin lymph vessels is determined by the expression level of VE-cadherin in the skin lymph vessels.
7. The method of claim 6, wherein it is determined that the skin condition is worsening due to increased expression of the VE-cadherin in lymphatic vessels of the skin.
A screening method for skin condition improving drugs using parameters related to the morphology of lymph vessels in the skin as indicators.
adding a candidate drug to the skin;
a step of photographing the morphology of lymph vessels in the skin after addition;
calculating parameters regarding the morphology of skin lymph vessels based on the captured image;
a step of comparing the skin lymphatic vessel morphology in the skin before addition of the candidate drug or in the control drug addition group; and a step of selecting the candidate drug as a skin condition improving drug based on the change in the parameter.
The screening method according to claim 8, comprising:
The method according to claim 9, wherein the parameter regarding the morphology of the skin lymph vessels is determined by at least one selected from the group consisting of the number of branches and the diameter of the lymph vessels.
The method according to claim 9 or 10, wherein the skin is cultured skin containing lymphatic structure.
A screening method for skin condition improving drugs using the expression level of VE-cadherin in lymphatic endothelial cells as an index.
adding a candidate drug to the skin;
measuring VE-cadherin expression in skin lymphatic endothelial cells after addition;
A step of comparing the expression of VE-cadherin in the lymphatic endothelial cells of the skin before addition of the candidate drug or in the control drug addition group, and a step of selecting the candidate drug as a skin condition improving drug based on the change in the expression level of VE-cadherin.
The screening method according to claim 12, comprising:
14. The method of claim 13, wherein the skin is cultured skin containing lymphatic structures.
a step of culturing lymphatic endothelial cells in a medium containing a candidate drug; a step of measuring VE-cadherin expression in lymphatic endothelial cells after culturing;
Comparing the expression level of VE-cadherin with a control;
13. The screening method according to claim 12, comprising the step of selecting a candidate drug as a skin condition improving drug based on changes in VE-cadherin expression.
A drug for improving skin conditions, including an agent that increases the diameter of skin lymph vessels.