Patent Application
20130252247
The present invention relates to a kit for screening skin-activating substances and a method for screening skin-activating substances using the same, and more particularly to a kit for screening skin-activating substances and a method for screening skin-activating substances using the same, in which skin-activating substances such as anti-aging substances and whitening substances are screened by isolating and purifying substances, which induce the expression of Klotho gene in skin cells, using the kit comprising Klotho gene known as an anti-aging gene.
Skin aging naturally occurs with time or is mainly caused by sun exposure. As the skin ages, the time for the horny layer of the epidermis to be replaced is increased so that the horny layer becomes thicker, and the content of elastic components and moisturizing components in the dermis decreases or the arrangement thereof changes so that the elasticity and water content of the dermis decrease, resulting in wrinkle formation. Photoaging caused by UV radiation is a cause of skin aging, and methods capable of preventing the change in the skin caused by UV radiation have been actively studied. Further, methods for preventing age-related aging, that is, genetically programmed aging, have also been studied. Such efforts have been focused on finding mechanisms that control aging.
Such efforts include studies on the Klotho gene. Klotho-knockout mice show arteriosclerosis, vascular calcification, soft tissue calcification, emphysema, activity reduction, gonadal dysgenesis, sterility, skin atrophy, ataxia, hypoglycemia and hyperphosphatemia, which are related to an increase in the concentration of 1,25(OH)2D3 (Mutation of the mouse klotho gene leads to a syndrome resembling ageing, Nature 1997; 390. 45-51). On the other hand, an increase in the expression of the Klotho protein indicates increases in life expectancy, insulin resistance and IGF-1 resistance (Kurosu et al., 2005). It was reported that single nucleotide polymorphisms in the human Klotho protein are associated with a reduction in life expectancy, osteoporosis, stroke and coronary artery disease (Arking et al., 2002. Kawano et al., 2002; Mull in et al., 2005. Ogata et al., 2002; Yamada et al., 2005).
Previous studies show that Klotho is expressed mainly in human kidneys and is also expressed in the placenta, the prostate and the small intestines. The recognition of importance of Klotho has increased, but the expression of Klotho in skin cells, including normal human keratinocytes, fibroblasts or melanocytes, has not yet been reported, and a study on a substance capable of increasing the expression of Klotho has also been reported.
Accordingly, the present inventors have constructed a kit capable of increasing the expression of Klotho protein in skin cells and have identified the expression of Klotho protein in skin cells, including keratinocytes, fibroblasts and melanocytes, using the constructed kit, suggesting that skin-activating substances, including anti-aging substances and skin-whitening substances, can be screened using the kit, thereby completing the present invention.
Therefore, it is an object of the present invention to provide a kit for screening skin-activating substances by determining whether the Klotho gene is expressed in skin cells, and a method for screening skin-activating substances using the kit.
In order to accomplish the above object, the present invention provides a kit for screening skin-activating substances by screening substances which increases the expression of Klotho protein in skin cells, the kit comprising Klotho protein, Klotho protein amplification primers of SEQ ID NOS: 1 and 2, a probe of SEQ ID NO: 3, and skin cells.
The present invention also provides a method for screening a skin-activating substance using the kit.
The inventive kit for screening skin-activating substances can quickly and conveniently screen skin-activating substances, such as anti-aging substances, whitening substances and moisturizing substances, by determining whether Klotho gene is expressed in skin cells.
a shows the results of RT-PCR amplification performed to determine whether Klotho protein is expressed in human embryonic kidney cells (HEK293), keratinocytes (NHK) and fibroblasts (NHF), and
The present invention relates to a method capable of identifying the expression of Klotho protein in skin cells. According to the method of the present invention, skin-activating substances capable of inducing the expression of the anti-aging gene Klotho in skin cells can be screened. Examples of the skin-activating substances include anti-aging substances, which reduce skin wrinkles or improve skin elasticity or make the skin soft, whitening substances, which brighten skin tone or reduce skin pigmentations, such as discolorations, freckles, blemish, and skin tanning by sunlight, and skin-moisturizing substances.
The inventive screening kit capable of identifying the expression of Klotho protein in skin cells comprises Klotho protein, Klotho protein amplification primers of SEQ ID NOS: 1 and 2, a probe of SEQ ID NO: 3 (see Table 1), and skin cells, and can identify anti-aging substances by screening substances which increase the expression level of Klotho protein in the skin cells.
In one embodiment of the present invention, the skin cells may be normal human keratinocytes (NHK), normal human fibroblasts (NHF) or normal human melanocytes (NHM), but are not limited thereto.
The present invention also relates to a method for screening skin-activating substances using the above kit, the method comprising the steps of: treating skin cells with candidates and determining whether Klotho protein is expressed in the skin cells treated with the candidates; and isolating and purifying substances, which increase the expression of the Klotho protein, among the candidates.
In one Example of the present invention, it was found using the above kit that retinoic acid and retinol, known as anti-aging substances, increased the expression of Klotho protein, suggesting that the screening method according to the present invention is suitable for screening anti-aging substances and skin-activating substances, which have not yet been known.
Hereinafter, the present invention will be described in further detail with reference examples and test examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, and various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
In order to examine whether Klotho protein is expressed in skin cells, primers of SEQ ID NOS: 1 and 2, which can amplify Klotho gene (NCBI accession No. NM-004795), were constructed. Then, RT-PCR was performed using the primer set and a probe of SEQ ID NO: 3 (TaqMan™ fluorogenic probe) in order to detect Klotho mRNA in the cDNA of keratinocytes (NHK), fibroblasts (NHF), NHM-promoblack (Normal Human Epidermal Melanocytes (NHEM) juvenile foreskin, Promocell: melanocytes derived from dark-skinned persons), NHM-DP (NHM-dark pigment, Invitrogen: melanocytes derived from dark-skinned persons) and NHM-MP (NHM-moderate pigment, Invitrogen: melanocytes derived from Asian). The PCR reaction was performed for 35 cycles, each consisting of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, and extension at 72° C. for 60 sec. As a positive control, the cDNA of human embryonic kidney cells (HEK293) known to express Klotho gene was used. The results of the RT-PCR amplification are shown in
In addition, the expression level of Klotho mRNA in each type of cells was analyzed by quantitative real-time PCR, and the results of the analysis are shown in
In order to examine the effects of retinoic acid and retinol (known as anti-aging substances) on the expression of Klotho in keratinocytes, quantitative real time PCR was performed under the same conditions as described in Test Example 1, and the expression levels of Klotho protein were examined at 2 days and 8 days after treatment of the cells with each of 0.1 μM and 1 μM of retinoic acid and 1 μM and 10 μM of retinol. The results of the examination are shown in
As can be seen from the results in
Human neonatal epidermal keratinocytes (HEK; Lonza, NHEK-Neo-Neonatal Normal Human Epidermal Keratinocytes, Pooled) were dispensed in medium (KBM-gold, Lonza) in a 6-well plate at a density of 1×105 cells per well and cultured at 37° C. for 24 hours, after which the medium was replaced with OPTI-MEM medium (GIBCO). Meanwhile, human neonatal epidermal keratinocytes were cultured in OPTI-MEM medium (GIBCO) containing 100 nM of siRNA (small interfering RNA; Dharmacon, ON-TARGETplus) having a sequence complementary to Klotho gene and 10 μl of transfection reagent (RNAiMAX, Invitrogen) at 37° C. for 20 minutes, after which these cells were added to the above cultured cells and incubated at 37° C. for 6 hours. Then, the cells were incubated in DMEM medium at 37° C. for 4 days. As control groups, HEK cells treated with siRNA (sc in
To examine whether the Klotho gene was knockdown, quantitative real-time PCR was performed under the same conditions as described in Test Example 1, and the results of the PCR are shown in
As can be seen from the results in
In order examine the influence of Klotho protein on MMP-9 known to degrade collagen to cause skin aging, the change in the expression level of MMP-9 in Klotho-knockdown human neonatal epidermal keratinocytes (HEK) was analyzed by RT-PCR.
Human neonatal epidermal keratinocytes (HEK; Lonza, NHEK-Neo-Neonatal Normal Human Epidermal Keratinocytes, Pooled) were dispensed in medium (KBM-gold, Lonza) in a 6-well plate at a density of 1×105 cells per well and cultured at 37° C. for 24 hours, after which the medium was replaced with OPTI-MEM medium (GIBCO). Meanwhile, human neonatal epidermal keratinocytes were cultured in OPTI-MEM medium (GIBCO) containing 100 nM of siRNA (small interfering RNA; Dharmacon, ON-TARGETplus) having a sequence complementary to Klotho gene and 10 μl of transfection reagent (RNAiMAX, Invitrogen) at 37° C. for 20 minutes, after which these cells were added to the above cultured cells and incubated at 37° C. for 6 hours. Then, the cells were incubated in DMEM medium at 37° C. for 4 days. As control groups, HEK cells treated with siRNA (HEKn sc in
RNA was extracted from the cultured HEK cells using a RNeasy mini kit (Qiagen) and subjected to RT-PCR using a Superscript III kit (Invitrogen) to synthesize cDNA. The synthesized cDNA was subjected to quantitative real-time PCR using a probe (TaqMan® Gene Expression Assays) for MMP-9, thereby analyzing the expression pattern of MMP-9 mRNA. The results of the analysis are shown in
Human neonatal epidermal keratinocytes (HEK; Lonza, NHEK-Neo-Neonatal Normal Human Epidermal Keratinocytes, Pooled) were dispensed in medium (KBM-gold) in a 6-well plate at a density of 5×104 cells per well and cultured at 37° C. for 24 hours, after which the cells were treated with 1 μg/ml of a recombinant Klotho protein (R&D systems, Recombinant Human Klotho) and incubated for 8 days. As a control group, HEK cells not treated with Klotho protein were used.
The HEK culture was loaded on gelatin gel and analyzed by zymography, and the results of the analysis are shown in
As can be seen from the results in
In order to examine the influence of Klotho protein on the expression of hyaluronic acid synthase 2 (HAS2) known as a skin-moisturizing factor, human neonatal epidermal keratinocytes (Lonza, NHEK-Neo-Neonatal Normal Human Epidermal Keratinocytes, Pooled) were treated with a recombinant Klotho protein, and the expression pattern of HAS mRNA in the cells was analyzed.
Human neonatal epidermal keratinocytes (HEK) (Lonza, NHEK-Neo-Neonatal Normal Human Epidermal Keratinocytes, Pooled) were dispersed in medium (KBM-gold) in a 6-well plate at densities of 2×105 cells per each well of 3 wells and 5×104 cells per each well of 3 wells and cultured at 37° C. for 24 hours, after which the cells were treated with a recombinant Klotho protein at concentrations of 1 and 0.5 μg/ml as shown in Table 2 below and were incubated at 37° C. for 2 days and 8 days.
RNA was extracted from the cultured HEK cells using a RNeasy mini kit (Qiagen) and subjected to RT-PCR using a Superscript III kit (Invitrogen) to synthesize cDNA. The synthesized cDNA was subjected to quantitative real-time PCR using a probe (TaqMan® Gene Expression Assays) for HAS2, thereby analyzing the expression pattern of HAS2 mRNA. The results of the analysis are shown in
As can be seen from the results in
In order to examine the influence of Klotho protein on p21 whose expression is known to increase with aging, the change in the expression level of p21 in Klotho-knockdown human neonatal dermal fibroblasts (HDF) was observed by RT-PCR.
Human neonatal dermal keratinocytes (Lonza, NHEK-Neo-Neonatal Human Dermal Keratinocytes, Pooled) were dispensed in a 60-mm dish with DMEM medium at a density of 2×105 cells and cultured at 37° C. for 24 hours, after which the medium was replaced with OPTI-MEM medium (GIBCO). Meanwhile, human neonatal epidermal keratinocytes were cultured in OPTI-MEM medium (GIBCO) containing 100 nM of siRNA (small interfering RNA; Dharmacon, ON-TARGETplus) having a sequence complementary to Klotho gene and 10 μl of transfection reagent (RNAiMAX, Invitrogen) at 37° C. for 20 minutes, after which these cells were added to the above cultured cells and incubated at 37° C. for 6 hours. Then, the cells were incubated in DMEM medium at 37° C. for 4 days. As control groups, HDF cells treated with siRNA (HEKn sc in
RNA was extracted from the cultured HDF cells using a RNeasy mini kit (Qiagen) and subjected to RT-PCR using a Superscript III kit (Invitrogen) to synthesize cDNA. The synthesized cDNA was subjected to quantitative real-time PCR using a probe (TaqMan® Gene Expression Assays) for the senescence marker p21, thereby analyzing the expression pattern of p21 mRNA. The results of the analysis are shown in
As can be seen from the results in
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR11/08132 | 10/28/2011 | WO | 00 | 4/24/2013 |
