JP4235037B2 - Evaluation method of skin aging degree - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method and an evaluation kit for evaluating the degree of skin aging based on biological aging of the skin.
[0002]
[Prior art and problems to be solved by the invention]
Skin age-related changes are recognized by the appearance of wrinkles, sagging, and the like, and when touched by elasticity, elasticity, and the like. Therefore, estimation of apparent skin aging, so-called skin age, can be done relatively easily by judgment from appearance, and more numerical objective can be achieved by using instrumental measurement that assumes contact with the skin surface. Skin age can be estimated from various data. Actually, as a method for determining the skin age, a method of estimating the actual age based on the external appearance of wrinkles and sagging (see, for example, Patent Document 1 and Patent Document 2), and a method in which a vibrating body is brought into contact with the skin. A method for determining skin characteristics and skin age based on vibration changes (for example, see Patent Document 3) and a method for estimating age based on the dermal brightness of an ultrasonic skin image (for example, see Patent Document 4) have been reported.
[0003]
However, it is considered that the aging change of the skin begins with biological aging changes in many skins such as cell aging and matrix aging before the appearance and feel. Therefore, the degree of skin aging is preferably evaluated using such biological aging changes in the skin as an index, but such an evaluation method is hardly known. For example, an age estimation method using an average area of keratinocytes in the epidermis as an index (see, for example, Patent Document 5) is known. It was hard to say that it was a change in age.
[0004]
An object of this invention is to provide the method of evaluating the aging degree of skin based on the biological aging change of skin.
[0005]
[Patent Document 1]
JP 2002-330943 A [Patent Document 2]
JP 2002-360544 A [Patent Document 3]
Japanese Patent Laid-Open No. 2001-212087 [Patent Document 4]
JP 2000-83954 A [Patent Document 5]
JP-A-11-299792 [0006]
[Means for Solving the Problems]
The present inventor examined the relationship between aging force and non-muscle cell generation, and surprisingly found that the dermal fibroblast generation force decreases with aging and stimulates dermal fibroblasts. It was found that the degree of skin aging can be evaluated by measuring the change in force generated in the cells when a substance is added.
[0007]
That is, the present invention provides a method for evaluating the degree of skin aging of a subject, comprising adding a stimulating substance to a collagen gel in which skin fibroblasts derived from the subject are embedded, and measuring a change in force generated in the cells. Is to provide.
[0008]
The present invention also provides a skin aging evaluation kit comprising a collagen gel in which skin fibroblasts are embedded, a stimulating substance, and a cell generation tension measuring device.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
In the evaluation method of the present invention, skin fibroblasts collected from a subject are used as samples. Skin fibroblasts can be collected by punch biopsy or the like. The collection site includes the face, the upper arm inner part, the upper arm outer part, the thigh inner part, the thigh outer part, the abdomen, the skin of the back part, etc., and may depend on the method of using the evaluation results, An abdominal part, an upper arm inner part, an inner thigh part, etc., which are non-exposed parts and are not noticeable of wounds at the time of collection, are preferable.
[0010]
The collected skin fibroblasts are embedded in a collagen gel and used for measuring the force generated by the cells. Fibroblast embedding in collagen gel is a known method (Kolodney MS, Wysolmerski RB, Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. J Cell Biol., 117, 73-82 (1992), Alternatively, Kolodney MS, Elson EL, Correlation of myosin light chain phosphorylation with isometric contraction of fobroblasts. J Biol Chem, 268, 23850-55 (1993)) can be prepared, for example, as follows.
[0011]
The collected skin fibroblasts are cultured, for example, in Dulbecco's modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS), and cultured fibroblasts are isolated by a conventional method. FBS-containing DMEM is added to the obtained cells so as to be 3-4 million cells / mL and suspended again. On the other hand, a collagen gel preparation solution is prepared by a conventional method using a collagen solution (Nitta gelatin, type IA). To this collagen gel preparation solution, the previously prepared cell suspension solution is added so that the final cell density becomes 3 × 10 ⁵ cells / mL to 5 × 10 ⁵ cells / mL, and the fibroblast-embedded collagen gel preparation solution is added. Can be produced.
[0012]
On the other hand, a container for producing a collagen gel was produced using silicon rubber, a silicone tube, a cloth tape, a culture dish, and the like. The above fibroblast-embedded collagen gel preparation solution is poured into a groove made of silicon rubber, incubated at 37 ° C. for gelation, and after 24 hours, further incubated in serum-free medium for 1 to 2 days. A blast-embedded collagen gel can be prepared.
[0013]
The collagen used here may be an animal-derived collagen, but in particular, mammals such as humans, mice, rats, rabbits, guinea pigs and cows are preferred. These collagens may be collected and used from tissues by a known method, or commercially available products (for example, type I-A manufactured by Nitta Gelatin Co., Ltd.) may be purchased and used.
[0014]
The force generated in skin fibroblasts embedded in collagen gel is the force generated when actin-myosin polymerizes to form stress fibers, which can be called myofibrils, and contracts. The measurement is performed according to a known method (Kolodney MS, Wysolmerski RB, J Cell Biol., 117, 73-82 (1992), Kolodney MS, Elson EL, J Biol Chem, 268, 23850-23855 (1993)). be able to.
[0015]
For example, one side of the fibroblast-embedded collagen gel prepared above is fixed in a serum-free medium kept at about 37 ° C. in a beaker, and the other is isotonic transducer (for example, Isotonic Transducer, 8 gf, T-7-8-240, ORIENTEC, JAPAN, or PAT-08, Physio Tech.JAPAN, etc.), after applying a load equivalent to 50-300 mg, stabilize for about 30 minutes to 1 hour, Add about 1 mL of stimulating substance prepared in serum-free DMEM medium to a concentration of about 50 times the final concentration, measure the change in force generated with an isotonic transducer, and amplify (for example, Physio Tech., Signal Conditioner MAC -2) After recording (for example, MP100A-CE, AcqKnowledge 3.5, both Biopack system Inc. Santabarbara).
[0016]
The stimulating substance may be any substance that generates a force (tension) by stimulating the actin-myosin system of fibroblasts, such as thrombin, lysophosphatidic acid (LPA), etc. And fetal bovine serum (FBS), monopalmitoyl phosphatidic acid (MLPA), lysophosphatidylcholine palmitoyl (LPCP), endothelin (ET-1), Platelet-derived growth factor (PDGF) can also be used.
[0017]
Using human abdomen-derived dermal fibroblasts (passage number 2-3), subcultured to prepare cells with passage number 5-7 and passage numbers 16-19 (in vitro senescent fibroblasts), When the force generated by the fibroblasts when embedded in collagen gel and stimulated with thrombin and LPA was measured, the senescent fibroblasts showed 4.8 +/− 3.3 dyne and 2.5 +/− 3, respectively. .1 dyne (mean value +/− SD), and significantly reduced cell generation compared to young fibroblasts (32.3 +/− 25.3 dyne, 11.3 +/− 6.8 dyne, respectively) (Example 1). Moreover, when the force which generate | occur | produces the cell at the time of thrombin stimulation was measured about the fibroblast derived from the 62-79-year-old elderly person and the fibroblast derived from 1-4 years old infants, the cell derived from an elderly person is 13.3+. This was /−9.0 dyne, which was significantly lower than cells derived from infants (24.5 +/− 2.0 dyne) (Example 2). That is, the force generated by cells of dermal fibroblasts decreases with age.
Therefore, by measuring the force generated by the skin fibroblasts collected from the subject, the degree of aging of the subject's cells and the degree of aging of the skin can be evaluated.
[0018]
The skin aging evaluation kit of the present invention is a kit for carrying out the above-described method for evaluating the skin aging of a subject, and includes a collagen gel in which the above-described skin fibroblasts are embedded, a stimulating substance, and a cell generation tension measurement. A device (for example, an isotonic transducer or the like) is provided.
[0019]
The evaluation method and evaluation kit of the present invention are used for, for example, a surgical policy in estimating wound healing rate in surgery, a transplant skin creation and selection policy in regenerative medicine, and a guideline for selecting a cosmetic according to skin age. It can be used.
Moreover, the value of the force generated by skin fibroblasts in each age group is prepared as background data, and by comparing this with the data obtained from the subject, the skin age of the subject can be estimated.
[0020]
【Example】
Example 1 Reduction of fibroblast development tension due to in vitro aging (1) Cell human skin fibroblasts (age; 62, sex; male, abdominal origin, passage number 2-3, PDL (Population doubling time) = 6- 8) was purchased from CELL SYSTEMS-Dainippon Pharmaceutical Co., Ltd., and the number of passages at the time of purchase was 3, and the cells were subcultured with 5% FBS-containing DMEM at a split ratio of 1: 4 when confluent. Passage numbers 5-7 were compared as young (Young) and passages 16-19 as old age groups (Old) aged in vitro.
[0021]
(2) Measurement of cell generation force In the collagen gel culture system, the force was measured according to the method of Kolodeny et al. (Kolodney MS, Wysolmerski RB, Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. J Cell Biol., 117, 73-82 (1992), Kolodney MS, Elson EL, Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts. J Biol Chem, 268, 23850-23855 (1993)). Briefly, after fixing and stabilizing fibroblast-embedded collagen gel (1.5 × 10 ⁶ cells, 1.5 mg / mL collagen, Nitta Gelatin Type I-A) in a serum-free medium kept at 37 ° C. Add 1 mL of substance adjusted to a concentration about 50 times the final concentration with serum-free DMEM (final concentration: thrombin = 0.2 U / mL, LPA (lysophosphatidic acid) = 10 μM), and the change in force generated is Measured with Isotonic Transducer (8gf, T-7-8-240, ORIENTEC, JAPAN), amplified (Physio Tech., Signal Conditioner MAC-2) and recorded (MP100A-CE, AcqKnowledge 3.5, both in Biopack system Inc. Santabarbara ).
[0022]
As shown in FIG. 1, it was confirmed that the senescent fibroblasts aged in vitro significantly reduced the power generated by the cells by stimulation with thrombin and LPA as compared with young fibroblasts. Since the number of cells is the same, it is considered that the power generated by individual cells decreased with aging.
[0023]
Example 2 Reduction of fibroblast development tension due to in vivo aging (1) Cells
In vivo senescent cells are DF014 (age; 62, sex; male), DF151 (age; 79, sex; male), DF181 (age; 64, sex; unknown), DF146 (age; 67, sex; male) , DF149 (age; 75, sex; male), 62-79 years old cells (OLD, n = 5), and DF374 (age; 1, sex; male), DF628 (age; 4, sex ; male), DF775 (age; 3, sex; male), (age; 1, sex; male), DF287 (age; 1, sex; female), DF486 (age; 2, sex; male), 1- Cells derived from 4-year-old infants (YNG, n = 5) were used.
[0024]
(2) Measurement of force generated by cells The same method as in Example 1 was used. 1 mL of thrombin solution adjusted to a concentration about 50 times the final concentration with serum-free DMEM (final concentration 0.2 U / mL) was added, and changes in the generated force were observed.
[0025]
As shown in FIG. 2, it was confirmed that the cell-derived force was significantly reduced by thrombin stimulation in the cells derived from the elderly compared with the cells derived from the 4-year-old infant. Since the number of cells is the same, it is considered that the power generated by individual cells decreased with aging.
[0026]
【The invention's effect】
According to the present invention, the skin aging degree of a subject can be evaluated based on the biological aging change of the skin.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing a decrease in force generation by thrombin and LPA stimulation of in vitro senescent fibroblasts.
FIG. 2 is a diagram showing a decrease in force generation by thrombin stimulation of in vivo senescent fibroblasts.

Claims (2)

  A method for evaluating a change in age of skin fibroblasts of a subject, comprising adding a stimulating substance to a collagen gel in which skin fibroblasts derived from the subject are embedded, and measuring a change in force generated in the cells. .   The evaluation method according to claim 1, wherein the stimulating substance is thrombin and / or lysophosphatidic acid.

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