KR102635297B1 - A composition for skin barrier function comprising LINC00302 promoting materials and a method for screening LINC00302 promoting materials - Google Patents
A composition for skin barrier function comprising LINC00302 promoting materials and a method for screening LINC00302 promoting materials Download PDFInfo
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- KR102635297B1 KR102635297B1 KR1020160122319A KR20160122319A KR102635297B1 KR 102635297 B1 KR102635297 B1 KR 102635297B1 KR 1020160122319 A KR1020160122319 A KR 1020160122319A KR 20160122319 A KR20160122319 A KR 20160122319A KR 102635297 B1 KR102635297 B1 KR 102635297B1
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- linc00302
- barrier function
- skin barrier
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
본 명세서에는 LINC00302 발현 또는 활성 촉진 물질을 유효성분으로 포함하는 피부 장벽기능 강화용 조성물 및 LINC00302 발현 또는 활성 촉진 물질의 스크리닝 방법이 개시된다. 상기 LINC00302 발현 또는 활성 촉진 물질을 유효성분으로 포함하는 조성물은 피부 장벽기능을 강화시킬 수 있으며, 피부 보습 또는 항산화 효과를 제공할 수 있다. 또한, 상기 스크리닝 방법에 따르면 LINC00302 유전자의 상대적 발현 촉진 정도를 확인함으로써, 간편하게 피부 장벽기능 강화 물질을 스크리닝할 수 있다.Disclosed herein are a composition for strengthening skin barrier function containing a LINC00302 expression or activity promoting substance as an active ingredient and a screening method for the LINC00302 expression or activity promoting substance. A composition containing the LINC00302 expression or activity promoting substance as an active ingredient can strengthen the skin barrier function and provide skin moisturizing or antioxidant effects. In addition, according to the above screening method, it is possible to easily screen for substances that enhance skin barrier function by confirming the relative expression promotion level of the LINC00302 gene.
Description
본 명세서는 피부 장벽기능 강화용 조성물 및 피부 장벽기능 강화 물질 스크리닝 방법에 관한 것이다.This specification relates to a composition for strengthening the skin barrier function and a method for screening substances for enhancing the skin barrier function.
피부(skin) 중에서 최외각에 위치한 표피(epidermis)의 가장 중요한 기능은, 외부로부터의 다양한 자극, 예컨대 화학 물질, 대기오염물질, 건조한 환경, 자외선 등의 물리, 화학적 자극인자에 대한 방어와 피부를 통한 체내수분의 과도한 발산을 막는 보호기능이며, 이러한 보호기능은 각질 형성세포로 구성된 각질층이 정상적으로 형성되어 있을 때만 유지될 수 있다. 표피 중에서도 가장 바깥에 존재하는 각질층(Stratum corneum, horney layer)은 각질 형성세포로부터 형성되며, 분화가 완결된 각질세포와 그를 둘러싼 지질층으로 구성되어 있다. 각질세포는 표피 최하층에서 지속적으로 증식(proliferation)하는 기저세포(basal cell)가 단계적으로 형태 및 기능상의 변화를 거치며, 피부 표면까지 상승한 특징적인 세포이며, 일정 기간이 경과하면 오래된 각질세포는 피부에서 탈락되고 새로운 각질세포가 그 기능을 대신하게 되는데, 이러한 반복적인 일련의 변화 과정을 표피세포의 분화 혹은 각화(keratinization)라고 부른다. 그런데 이 각화과정이 비정상적으로 과하게 일어나거나, 기저 세포로부터 새로운 세포가 올라와 각질세포가 되는 현상이 반복적으로 일어나지 않으면 피부는 장벽(skin barrier) 기능을 상실하게 되고 그에 따라 피부의 수분 증발량이 증가하면서 건조해지게 된다.The most important function of the epidermis, located in the outermost layer of skin, is to protect the skin from various external stimuli, such as physical and chemical irritants such as chemicals, air pollutants, dry environments, and ultraviolet rays. It is a protective function that prevents excessive dissipation of body moisture through the body, and this protective function can be maintained only when the stratum corneum, composed of keratinocytes, is normally formed. The stratum corneum (horney layer), the outermost layer of the epidermis, is formed from keratinocytes and consists of fully differentiated keratinocytes and a lipid layer surrounding them. Keratinocytes are characteristic cells that rise to the surface of the skin as basal cells that continuously proliferate in the lowest layer of the epidermis undergo gradual changes in form and function. After a certain period of time, old keratinocytes are removed from the skin. They fall off and new keratinocytes take over their function. This repetitive series of changes is called differentiation or keratinization of epidermal cells. However, if this keratinization process occurs abnormally excessively or new cells rise from the basal cells and become keratinocytes repeatedly, the skin loses its skin barrier function, resulting in increased moisture evaporation from the skin and dryness. It becomes ruined.
일 측면에서, 본 발명의 목적은 피부 각질분화를 조절하는 신규한 피부 장벽기능 강화 물질 판별 마커를 제공하는 것이다.In one aspect, the purpose of the present invention is to provide a marker for identifying a novel skin barrier function enhancing material that regulates skin keratin differentiation.
다른 일 측면에서, 본 발명의 목적은 상기 피부 장벽기능 강화 물질 판별 마커를 이용하여 피부 장벽기능 강화 물질을 스크리닝하는 방법을 제공하고자 한다.In another aspect, an object of the present invention is to provide a method for screening a skin barrier function enhancing substance using the skin barrier function enhancing substance discrimination marker.
다른 일 측면에서, 본 발명의 목적은 상기 피부 장벽기능 강화 물질 판별 마커의 발현 또는 활성 촉진 물질을 유효성분으로 포함하는 피부 장벽기능 강화용 조성물을 제공하고자 한다.In another aspect, an object of the present invention is to provide a composition for strengthening the skin barrier function that includes a substance that promotes the expression or activity of the skin barrier function enhancing substance discrimination marker as an active ingredient.
다른 일 측면에서, 본 발명의 목적은 상기 피부 장벽기능 강화 물질 판별 마커를 이용한 피부 장벽기능 관련 피부 상태 진단용 기초정보 제공방법을 제공하고자 한다.In another aspect, the purpose of the present invention is to provide a method of providing basic information for diagnosing skin conditions related to skin barrier function using the skin barrier function enhancing substance discrimination marker.
다른 일 측면에서, 본 발명의 목적은 상기 피부 장벽기능 강화 물질 판별 마커를 이용한 피부 장벽기능 관련 피부 상태 진단용 조성물 또는 키트를 제공하고자 한다.In another aspect, an object of the present invention is to provide a composition or kit for diagnosing skin conditions related to skin barrier function using the skin barrier function enhancing substance discrimination marker.
본 발명의 일 측면은, 피부세포에 시험 물질을 처리하는 단계 및 상기 시험물질을 처리한 피부세포에서 LINC00302의 상대적 발현량을 측정하는 단계를 포함하는 피부 장벽기능 강화 물질 스크리닝 방법을 제공한다.One aspect of the present invention provides a method for screening substances that enhance skin barrier function, comprising treating skin cells with a test substance and measuring the relative expression level of LINC00302 in skin cells treated with the test substance.
본 발명의 일 측면은, LINC00302의 발현 또는 활성 촉진 물질을 유효성분으로 포함하는 피부 장벽기능 강화용 조성물을 제공한다.One aspect of the present invention provides a composition for strengthening the skin barrier function containing a substance that promotes the expression or activity of LINC00302 as an active ingredient.
본 발명의 일 측면은 시험대상으로부터 분리된 피부세포에서 LINC00302의 발현량을 측정하는 단계를 포함하는 피부 장벽기능 관련 피부 상태 진단용 기초 정보 제공 방법을 제공한다.One aspect of the present invention provides a method of providing basic information for diagnosing skin conditions related to skin barrier function, including measuring the expression level of LINC00302 in skin cells isolated from a test subject.
본 발명의 일 측면은, LINC00302의 RNA 검출 시약을 포함하는 피부 장벽기능 관련 피부 상태 진단용 키트를 제공한다.One aspect of the present invention provides a kit for diagnosing skin conditions related to skin barrier function, including the RNA detection reagent of LINC00302.
본 발명의 일 측면은, LINC00302의 RNA 검출 시약을 포함하는 피부 장벽기능 관련 피부 상태 진단용 조성물을 제공한다.One aspect of the present invention provides a composition for diagnosing skin conditions related to skin barrier function, including the RNA detection reagent of LINC00302.
본 발명의 일 측면에 따른 신규한 피부 장벽기능 강화 물질 판별 마커인 LINC00302는 각질 형성 세포의 분화에 따라 그 발현량이 증가한다. 따라서, 본 명세서에 개시된 기술을 이용하면 피부에 시험물질을 처리하고 LINC00302 유전자의 상대적 발현 촉진 정도를 확인함으로써, 피부 장벽기능 강화 물질을 간편하고 효율적으로 스크리닝할 수 있다. 또한, 상기 스크리닝된 피부 장벽기능 강화 물질을 통해 피부 장벽기능 강화, 피부 보습 또는 피부 항산화 기능 개선 및 강화에 유용하게 사용할 수 있다. 또한, 본 발명의 일 측면에 따르면, 피부에 포함된 LINC00302 유전자의 검출을 통하여 피부 장벽기능 관련 피부 상태를 빠르고 간단하게 진단할 수 있으며, 이를 통해 피부 장벽기능의 문제를 조기에 예측 또는 판단하여 대응할 수 있다. The expression level of LINC00302, a marker for identifying a novel skin barrier function enhancing material according to one aspect of the present invention, increases with the differentiation of keratinocytes. Therefore, using the technology disclosed in this specification, it is possible to simply and efficiently screen for skin barrier function enhancing substances by treating the skin with a test substance and confirming the relative expression promotion level of the LINC00302 gene. In addition, the screened skin barrier function enhancing substances can be usefully used to strengthen the skin barrier function, moisturize the skin, or improve and strengthen the skin antioxidant function. In addition, according to one aspect of the present invention, skin conditions related to skin barrier function can be quickly and simply diagnosed through detection of the LINC00302 gene contained in the skin, and through this, problems with skin barrier function can be predicted or determined early and responded to. You can.
도 1은 각질형성세포의 분화에 따른 상대적인 LINC00302 RNA 발현량을 나타낸 도이다.
도 2는 LINC00302 유전자의 발현 억제에 따른 각질형성세포에서의 피부 장벽기능 마커인 필라그린(FLG), 인볼루크린(IVL) 및 로리크린(LOR) 감소를 확인한 도이다.
도 3은 각질형성세포에 피부 장벽기능 강화 물질(붉은쌀 추출물)을 적용시 LINC00302 유전자의 상대적 발현량이 증가함을 확인한 도이다. Figure 1 is a diagram showing the relative LINC00302 RNA expression level according to differentiation of keratinocytes.
Figure 2 is a diagram confirming the decrease in skin barrier function markers filaggrin (FLG), involucrin (IVL), and loricrin (LOR) in keratinocytes due to inhibition of expression of the LINC00302 gene.
Figure 3 is a diagram confirming that the relative expression level of the LINC00302 gene increases when a skin barrier function enhancing substance (red rice extract) is applied to keratinocytes.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 명세서에서 "피부"는 생물의 외부를 덮고 있는 기관을 의미하는 것으로서 표피, 진피 및 피하지방층으로 구성되어 있으며 얼굴 또는 몸 전체의 외부를 덮는 조직뿐만 아니라, 두피와 모발을 포함하는 최광의의 개념이다.In this specification, “skin” refers to an organ that covers the outside of a living thing, and is composed of the epidermis, dermis, and subcutaneous fat layer, and is the broadest concept to include the scalp and hair as well as tissues covering the outside of the face or the entire body. am.
본 명세서에서 "유전자의 발현"이라 함은 전사, 번역 및 번역 후 변형 등을 포함하는 최광의의 개념이다.In this specification, “gene expression” is the broadest concept including transcription, translation, and post-translational modification.
본 발명의 일 실시예는 상기 LINC00302를 이용하여 피부 장벽기능 강화 물질을 스크리닝하는 방법을 제공할 수 있다. One embodiment of the present invention can provide a method for screening skin barrier function enhancing substances using LINC00302.
일 실시예로서, 상기 방법은 (a)피부세포에 시험 물질을 처리하는 단계; 및As an example, the method includes (a) treating skin cells with a test substance; and
(b)상기 시험물질을 처리한 피부세포에서 LINC00302(Long intergenic non-protein coding RNA 302)의 상대적 발현량을 측정하는 단계를 포함할 수 있다. (b) It may include measuring the relative expression level of LINC00302 (Long intergenic non-protein coding RNA 302) in skin cells treated with the test substance.
일 실시예로서, 상기 피부 세포는 인간, 마우스, 기니아 피그, 돼지, 조류 등에서 유래된 피부 세포일 수 있다. 상기 피부 세포는 각질형성세포(keratinocyte)를 포함할 수 있다. 다른 일 측면에서, 상기 피부 세포는 사람의 정상 피부 각질 세포를 포함할 수 있다.As an example, the skin cells may be skin cells derived from humans, mice, guinea pigs, pigs, birds, etc. The skin cells may include keratinocytes. In another aspect, the skin cells may include normal human skin keratinocytes.
본 발명의 일 실시예로, 상기 "상대적 발현량"은 시험물질을 처리하기 전의 각질형성세포에서의 LINC00302의 발현량에 대한 시험물질을 처리한 후 각질형성세포에서의 LINC00302의 발현량을 비교하였을 때 발현이 촉진된 정도일 수 있다. 또는, "상대적 발현량"은 시험 물질을 처리한 각질형성세포에서의 LINC00302의 발현량을 시험 물질을 처리하지 않은 각질형성세포에서의 LINC00302의 발현량과 비교하였을 때의 발현이 촉진된 정도일 수 있다. 상기 상대적 발현량은 예컨대, RNA의 상대적 발현량을 포함할 수 있다.In one embodiment of the present invention, the "relative expression level" is a comparison of the expression level of LINC00302 in keratinocytes after treatment with the test substance to the expression level of LINC00302 in keratinocytes before treatment with the test substance. It may be that the expression is promoted. Alternatively, “relative expression level” may be the degree to which expression is promoted when comparing the expression level of LINC00302 in keratinocytes treated with the test substance with the expression level of LINC00302 in keratinocytes not treated with the test substance. The relative expression level may include, for example, the relative expression level of RNA.
상기 관점에서, 일 실시예에 따른 상기 (b)단계는 각질형성세포에 시험물질의 처리하기 전과 후의 LINC00302 발현량을 비교하는 단계를 포함할 수 있다. 또는, 일 실시예로서 상기 (b)단계는 시험물질을 처리한 각질형성세포와 시험물질을 처리하지 않은 각질형성세포의 LINC00302 발현량을 비교하는 단계를 포함할 수 있다.From the above perspective, step (b) according to one embodiment may include comparing the expression level of LINC00302 before and after treatment of the test substance in keratinocytes. Alternatively, as an example, step (b) may include comparing the expression level of LINC00302 in keratinocytes treated with the test substance and keratinocytes not treated with the test substance.
또한, 일 실시예는 상기 (b)단계의 상대적 발현량을 측정한 결과 LINC00302의 발현량이 증가한 경우, 상기 시험물질을 피부 장벽기능 강화물질로 판정하는 단계를 더 포함할 수 있다.In addition, one embodiment may further include the step of determining the test substance as a skin barrier function enhancing substance when the expression level of LINC00302 increases as a result of measuring the relative expression level in step (b).
일 실시예로서, 상기 LINC00302(Long intergenic non-protein coding RNA 302)의 RNA 서열은 ENST00000536536.1 이며 보다 구체적으로 하기의 서열번호 1의 염기 서열로 표시될 수 있다. (http://www.lncipedia.org/db/transcript/LINC00302:1)As an example, the RNA sequence of LINC00302 (Long intergenic non-protein coding RNA 302) is ENST00000536536.1 and can be more specifically represented by the nucleotide sequence of SEQ ID NO: 1 below. (http://www.lncipedia.org/db/transcript/LINC00302:1)
GCCTGCTCCACACCTGGGTCTGACCAGGGTGAGTGGATCTTTTGGAACCTAGACAGAGCACAACTGAAAGGGTTGACTGAACTCCCACAAAGATGTCCTGCACCAGTCTCATGCATCTCGTTCATCAGTGCCTTTCATACCTTCAAGAGCTCTTGCCTCAGAGCATTGCTATGCCTGAGGTGTGTCTCAGAAGCAGCCATTGGCACAGTGGCTTGGGGCTGGGCTCAAAAATTTGCTCTCTTGAAGTTTGGATGTGATTGCTCTGAACAAGCTGCTTTGCCACAATAAATATTTTCAAGTTTGCCTGCTCCACACCTGGGTCTGACCAGGGTGAGTGGATCTTTTGGAACCTAGACAGAGCACAACTGAAAGGGTTGACTGAACTCCCACAAAGATGTCCTGCACCAGTCTCATGCATCTCGTTCATCAGTGCCTTTCATACCTTCAAGAGCTCTTGCCTCAGAGCATTGCTATGCCTGAGGTGTGTCTCAGAAGCAGCCATTGGCACAGTGGCTTGGGGCTGGGCTCAAAAATTTGCTCTCTTGAAGTTTGGATGTGATT GCTCTGAACAAGCTGCTGCTTTGCCACAATAAATATTTTCAAGTTT
본 발명의 일 실시예에 따른 피부 장벽기능 강화 물질 스크리닝 방법은 상기 시험물질을 처리한 피부세포에서 필라그린(Filaggrin), 인볼루크린(Involucrin) 및 로리크린(Loricrin)으로 이루어진 군에서 선택된 하나 이상의 유전자의 상대적 발현량을 측정하는 단계를 더 포함할 수 있다. 또한, 일 실시예로서 상기 필라그린(Filaggrin), 인볼루크린(Involucrin) 및 로리크린(Loricrin)으로 이루어진 군에서 선택된 하나 이상의 유전자의 상대적 발현량이 증가한 경우, 상기 시험 물질을 피부 장벽기능 강화 물질로 판단하는 단계를 더 포함할 수 있다.The method for screening substances for enhancing skin barrier function according to an embodiment of the present invention is to detect at least one selected from the group consisting of Filaggrin, Involucrin, and Loricrin from skin cells treated with the test substance. A step of measuring the relative expression level of genes may be further included. In addition, as an example, when the relative expression level of one or more genes selected from the group consisting of Filaggrin, Involucrin, and Loricrin increases, the test substance is used as a skin barrier function strengthening substance. A judgment step may be further included.
피부 장벽의 최외각을 이루는 각질층은 천연보습인자와 지질 등으로 이루어져 있다. 상기 필라그린(Filaggrin)은 표피 세포에 존재하는 케라틴 섬유에의 부착과 관련된 단백질로서, 표피의 최종 분화를 촉진한다. 전사 후 변형(post-transcriptional modification) 과정을 거쳐 여러 종류의 친수성 아미노산(hydrophilic amino acid)으로 분해되며, 아미노산 풀(pool)이 천연 보습인자(NMF)를 구성하고, 이는 각질층의 수분 유지를 돕는다고 알려져 있다. 또한 필라그린 유전자 돌연변이가 피부 보습 인자들을 감소시키고, 피부 장벽 기능을 손상시키며, 알러젠(allergen)이나 미생물(microbe)에 대한 피부 방어 능력이 감소시키고, T 세포군을 활성화하여 자가면역 메커니즘에 의한 만성 염증(chronic inflammation)을 일으켜 아토피 습진(Atopic eczema)를 유도하는 중요 유전 위험 요소라고도 알려져있다. 상기 인볼루크린(Involucrin)은 각질형성세포의 가교(crosslink)를 이루는 요소 중의 하나로서 세포질에서 발견이 되며, 트랜스글루타미나제 (transglutaminase)에 의해 막 단백질과 연결되어 있다. 상기 로리크린(Loricrin)은 각화외피(cornified envelope)에서 발견되는 주요 단백질의 하나로, 주로 최종적으로 분화된 표피세포에서 발현된다. The stratum corneum, which forms the outermost layer of the skin barrier, is composed of natural moisturizing factors and lipids. Filaggrin is a protein related to attachment to keratin fibers present in epidermal cells and promotes the final differentiation of the epidermis. It is decomposed into various types of hydrophilic amino acids through a post-transcriptional modification process, and the amino acid pool constitutes natural moisturizing factor (NMF), which helps maintain moisture in the stratum corneum. It is known. In addition, filaggrin gene mutation reduces skin moisturizing factors, impairs skin barrier function, reduces skin defense ability against allergens and microbes, and activates T cell population, resulting in chronic inflammation caused by an autoimmune mechanism. It is also known to be an important genetic risk factor that causes chronic inflammation and induces atopic eczema. Involucrin is one of the elements that form the crosslink of keratinocytes and is found in the cytoplasm, and is connected to membrane proteins by transglutaminase. Loricrin is one of the major proteins found in the cornified envelope and is mainly expressed in terminally differentiated epidermal cells.
상기 LINC00302와 피부 장벽기능과의 관계는 아직까지 알려진바 없었으나, 본 발명의 발명자들은 연구를 통하여 피부 각질형성세포(keratinocyte)가 분화함에 따라 상기 LINC00302 유전자의 발현이 증가함을 밝혀냈다. 또한, 일 실시예로서 분화된 각질형성세포에서 LINC00302를 제거(knock-down)시킬 경우, 피부 장벽기능 마커들, 예를 들어 필라그린(Filaggrin), 인볼루크린(Involucrin), 로리크린(Loricrin) 등의 발현량이 감소된다. 이는 상기 LINC00302이 피부 장벽기능 강화물질 판별 마커로서 기능할 수 있음을 의미한다.The relationship between LINC00302 and skin barrier function has not yet been known, but through research, the inventors of the present invention found that the expression of the LINC00302 gene increases as skin keratinocytes differentiate. In addition, as an example, when LINC00302 is knocked down from differentiated keratinocytes, skin barrier function markers such as Filaggrin, Involucrin, and Loricrin The expression level of etc. is reduced. This means that LINC00302 can function as a marker for determining skin barrier function enhancing substances.
본 발명의 일 실시예로서 상기 피부 장벽기능 강화물질로 판정하는 단계는 " 상대적 발현량"이 약 1.1 내지 2배 이상 증가한 경우, 피부 장벽기능 강화물질로 판정하는 것을 포함할 수 있다. 즉, 시험물질을 처리하기 전의 각질형성세포에서의 LINC00302의 발현 정도에 대한 시험물질을 처리한 후 각질형성세포에서의 LINC00302의 발현 정도를 비교하였을 때 발현이 약 1.1 내지 2배 이상 증가한 경우, 피부 장벽기능 강화물질로 판정할 수 있다. 또는, 시험 물질을 처리한 각질형성세포에서의 LINC00302의 발현 정도를 시험 물질을 처리하지 않은 각질형성세포에서의 LINC00302의 발현 정도와 비교하였을 때의 발현이 약 1.1 내지 2배 이상 증가한 경우, 피부 장벽기능 강화물질로 판정할 수 있다. 예를 들어, 상기 LINC00302의 상대적 발현량의 증가정도는 1.1 배 이상, 1.2배 이상, 1.3 배 이상, 1.4배 이상, 1.5 배 이상, 1.6배 이상, 1.7배 이상, 1.8배 이상, 1.9배 이상, 또는 2배 이상일 수 있으나, 이에 제한되는 것은 아니다. 상기 발현 정도는 통계적 유의성을 확보한 상태에서 측정된 결과이다. 통계적 유의성이라는 개념은 생물학적 통계분석법을 통하여 유의적인 차이를 보이는 경우로, 정량적인 경우 p value가 0.05 미만으로 차이가 나는 경우를 포함한다.As an embodiment of the present invention, the step of determining that the substance is a skin barrier function enhancing substance may include determining that the substance is a skin barrier function enhancing substance when the "relative expression level" increases by about 1.1 to 2 times or more. That is, when comparing the expression level of LINC00302 in keratinocytes after treatment with the expression level of LINC00302 in keratinocytes before treatment with the test substance, if the expression increases by about 1.1 to 2 times or more, the skin It can be judged as a barrier function enhancing material. Alternatively, when the expression level of LINC00302 in keratinocytes treated with the test substance is compared with the expression level of LINC00302 in keratinocytes not treated with the test substance, if the expression increases by about 1.1 to 2 times or more, the skin barrier It can be judged as a functional enhancing substance. For example, the degree of increase in the relative expression level of LINC00302 is 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, Or it may be twice or more, but is not limited thereto. The expression level is a result measured with statistical significance secured. The concept of statistical significance refers to cases where a significant difference is shown through biological statistical analysis methods, and in quantitative cases, it includes cases where the p value is less than 0.05.
일 실시예에 따른 상기 (a) 단계에서, 상기 시험물질은 세포의 리포좀(liposome) 또는 백터(vector)에 트렌스펙션시킬 수 있으며, 세포는 각질형성세포, 즉 케라티노사이트(keratinocyte)일 수 있다. 상기 트랜스펙션은 미세 주입법, 칼슘 포스페이트 공동-침전법, 전기 천공법 또는 리포좀 이용법 등을 이용하여 실시할 수 있으나, 이에 한정되는 것은 아니다.In step (a) according to one embodiment, the test substance may be transfected into a liposome or vector of a cell, and the cell may be a keratinocyte, that is, a keratinocyte. . The transfection may be performed using microinjection, calcium phosphate co-precipitation, electroporation, or liposome use, but is not limited thereto.
또한, 일 실시예로서, 상기 LINC00302의 발현 정도는, 공지의 기술, 예컨대, 역전사 중합효소 연쇄반응(RT-PCR), 엘라이자(ELISA), 웨스턴블럿 또는 이뮨 블럿(immune blot)을 이용하여 확인할 수 있으며, 이에 한정되는 것은 아니다.In addition, as an example, the expression level of LINC00302 can be confirmed using known techniques, such as reverse transcription polymerase chain reaction (RT-PCR), ELISA, Western blot or immune blot. It can be done, but is not limited to this.
일 실시예로서 피부 장벽 기능의 저하는 피부 세포 분화가 잘 이루어지지 않는 것일 수 있으며, 이는 피부 각질층이 정상적인 기능을 할 수 없고, 항산화능이 저하된 것을 의미할 수 있다. 이 경우 피부의 수분 보유력이 떨어질 수 있으며, 피부 염증 및 피부 건조가 나타날 수 있다. 따라서 상기 피부 세포 분화를 촉진하는 물질은 피부 보습, 피부 장벽 기능 강화 및 아토피 증상을 경감 또는 치료하는 효과를 나타낼 수 있다. 즉, 본 발명의 일 실시예에 따른 상기 피부 장벽기능 강화 물질은 피부 보습 물질, 피부 항산화 물질 등을 의미하는 것일 수 있다.As an example, a decrease in skin barrier function may indicate poor skin cell differentiation, which may mean that the stratum corneum of the skin cannot function normally and antioxidant capacity is reduced. In this case, the skin's ability to retain moisture may decrease, and skin inflammation and skin dryness may occur. Therefore, the substance that promotes skin cell differentiation can have the effect of moisturizing the skin, strengthening the skin barrier function, and alleviating or treating atopic symptoms. That is, the skin barrier function enhancing material according to an embodiment of the present invention may mean a skin moisturizing material, a skin antioxidant material, etc.
본 발명의 일 실시예는, LINC00302의 발현 또는 활성 촉진 물질을 유효성분으로 포함하는 피부 장벽기능 강화용 조성물을 제공할 수 있다. One embodiment of the present invention can provide a composition for strengthening the skin barrier function containing a substance that promotes the expression or activity of LINC00302 as an active ingredient.
일 실시예에 있어서, 상기 LINC00302의 발현 또는 활성 촉진 물질은 LINC00302 RNA의 번역을 촉진하는 물질을 포함할 수 있다. 다른 일 실시예에 있어서, 상기 LINC00302의 발현 또는 활성 촉진 물질은 상술된 피부 장벽기능 강화 물질 스크리닝 방법에 따라 스크리닝된 피부 장벽기능 강화 물질을 포함할 수 있다.In one embodiment, the material that promotes the expression or activity of LINC00302 may include a material that promotes translation of LINC00302 RNA. In another embodiment, the substance that promotes the expression or activity of LINC00302 may include a skin barrier function enhancing substance screened according to the skin barrier function enhancing substance screening method described above.
본 발명의 일 실시예에 따른 상기 조성물은 상기 LINC00302 의 발현 또는 활성 촉진 물질을 조성물 총 중량에 대하여 0.00001 내지 30 중량%로 포함할 수 있다.The composition according to an embodiment of the present invention may include 0.00001 to 30% by weight of a substance that promotes the expression or activity of LINC00302 based on the total weight of the composition.
본 명세서에 기재된 상기 일 실시예들에 따른 LINC00302의 발현 또는 활성을 촉진함으로써 각질 형성 세포의 분화를 조절하여 피부의 정상적인 피부 장벽 기능을 회복하도록 할 수 있다. 이에, 일 실시예로서 상기 조성물은 피부 보습 및 항산화 중 하나 이상의 용도를 포함할 수 있다.By promoting the expression or activity of LINC00302 according to the embodiments described herein, the differentiation of keratinocytes can be regulated to restore the skin's normal skin barrier function. Accordingly, as an example, the composition may include one or more of skin moisturizing and antioxidant purposes.
또한, 본 발명의 일 실시예에 따른 상기 조성물은 피부 외용제 조성물일 수 있으며, 보다 구체적으로 화장품 조성물 또는 약학 조성물을 포함할 수 있다.Additionally, the composition according to an embodiment of the present invention may be a composition for external application to the skin, and more specifically, may include a cosmetic composition or a pharmaceutical composition.
일 실시예로서, 상기 화장품 조성물은 국소 적용에 적합한 모든 제형으로 제공될 수 있다. 예를 들면, 용액, 수상에 유상을 분산시켜 얻은 에멀젼, 유상에 수상을 분산시켜 얻은 에멀젼, 현탁액, 고체, 겔, 분말, 페이스트, 포말(foam) 또는 에어로졸 조성물의 제형으로 제공될 수 있다. 이러한 제형의 조성물은 당해 분야의 통상적인 방법에 따라 제조될 수 있다.As an example, the cosmetic composition may be provided in any formulation suitable for topical application. For example, it may be provided in the form of a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, solid, gel, powder, paste, foam, or aerosol composition. Compositions of this dosage form can be prepared according to conventional methods in the art.
일 실시예로서, 상기 화장품 조성물은 상기한 물질 이외에 주 효과를 손상시키지 않는 범위 내에서, 바람직하게는 주 효과에 상승효과를 줄 수 있는 다른 성분들을 함유할 수 있다. 본 발명에 따른 화장품 조성물은 비타민, 고분자 펩티드, 고분자 다당 및 스핑고 지질로 이루어진 군에서 선택된 물질을 포함할 수 있다. 또한 상기 화장품 조성물은 일 실시예로서, 보습제, 에몰리언트제, 계면 활성제, 자외선 흡수제, 방부제, 살균제, 산화 방지제, pH 조정제, 유기 및무기 안료, 향료, 냉감제 또는 제한(制汗)제를 포함할 수 있다. 상기 성분의 배합량은 본 발명의 목적 및 효과를 손상시키지 않는 범위 내에서 당업자가 용이하게 선정 가능하며, 그 배합량은 조성물 총 중량을 기준으로 0.01 내지 5 중량%, 구체적으로 0.01 내지 3 중량%일 수 있다.As an example, the cosmetic composition may contain other ingredients in addition to the above-mentioned substances, preferably within a range that does not impair the main effect, and can have a synergistic effect on the main effect. The cosmetic composition according to the present invention may contain a substance selected from the group consisting of vitamins, high molecular weight peptides, high molecular weight polysaccharides, and sphingolipids. In addition, the cosmetic composition may include, as an example, a moisturizer, an emollient, a surfactant, an ultraviolet absorber, a preservative, a disinfectant, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, a coolant, or an antiperspirant. You can. The mixing amount of the above components can be easily selected by a person skilled in the art within the range that does not impair the purpose and effect of the present invention, and the mixing amount may be 0.01 to 5% by weight, specifically 0.01 to 3% by weight, based on the total weight of the composition. there is.
일 실시예로서 상기 약학적 조성물은 피부 장벽 기능 약화에 의한 질병의 예방 또는 치료용일 수 있으며, 예를 들면 건조 피부, 아토피 등의 예방 또는 치료용일 수 있다.As an example, the pharmaceutical composition may be used for the prevention or treatment of diseases caused by weakened skin barrier function, for example, for the prevention or treatment of dry skin, atopy, etc.
또한, 일 실시예로서 상기 약학 조성물은 국소 적용에 적합한 모든 제형으로 제공될 수 있다. 예를 들면, 경구, 경피(trandermally), 정맥, 근육, 피하주사에 의해 투여될 수 있다. 일 실시예로서 상기 약학 조성물은 주사제, 피부 외용 용액제, 현탁제, 유액제, 겔, 패취 또는 분무제일 수 있으나, 이에 제한되는 것은 아니다. 상기 제형은 당해 분야의 통상적인 방법에 따라 용이하게 제조될 수 있으며, 계면 활성제, 부형제, 수화제, 유화 촉진제, 현탁제, 삼투압 조절을 위한 염 또는 완충제, 착색제, 향신료, 안정화제, 방부제, 보존제 또는 기타 상용하는 보조제를 적당히 사용할 수 있다.Additionally, as an example, the pharmaceutical composition may be provided in any formulation suitable for topical application. For example, it can be administered orally, transdermally, intravenously, intramuscularly, or by subcutaneous injection. As an example, the pharmaceutical composition may be an injection, a solution for external use on the skin, a suspension, an emulsion, a gel, a patch, or a spray, but is not limited thereto. The formulation can be easily prepared according to conventional methods in the field, and contains surfactants, excipients, wetting agents, emulsification accelerators, suspending agents, salts or buffers for adjusting osmotic pressure, colorants, flavorings, stabilizers, preservatives, preservatives or Other commonly used supplements can be used appropriately.
본 발명의 일 실시예에 따른 약학 조성물의 유효 성분은 대상의 연령, 성별, 체중, 병리 상태 및 그 심각도, 투여 경로 또는 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 적용량 결정은 당업자의 수준 내에 있으며, 이의 1일 사용 용량은 예를 들어 0.1mg/kg/일 내지 5000mg/kg/일, 보다 구체적으로는 50 mg/kg/일 내지 500 mg/kg/일이 될 수 있으나, 이에 제한되는 것은 아니다.The active ingredient of the pharmaceutical composition according to an embodiment of the present invention will vary depending on the subject's age, gender, weight, pathological condition and severity, route of administration, or the judgment of the prescriber. Determination of application dosage based on these factors is within the level of one skilled in the art, the daily usage dosage being, for example, 0.1 mg/kg/day to 5000 mg/kg/day, more specifically 50 mg/kg/day to 500 mg/kg. /It may be work, but is not limited to this.
본 발명의 일 실시예는 상기 LINC00302를 피부 장벽기능 관련 피부 상태 정도를 진단할 수 있는 바이오 마커로서 제공할 수 있으므로, 본 발명의 다른 일 실시예는 이를 이용하여 피부 장벽기능 관련 피부 상태 진단용 조성물 또는 피부 장벽기능 관련 피부 상태 진단용 키트를 제공할 수 있다. 또한, 본 발명의 다른 일 실시예는 이를 이용한 피부 장벽기능 관련 피부 상태 진단용 기초 정보 제공 방법 또는 피부 장벽기능 관련 피부 상태 진단방법을 제공할 수 있다.Since one embodiment of the present invention can provide LINC00302 as a biomarker capable of diagnosing the degree of skin condition related to skin barrier function, another embodiment of the present invention uses it to prepare a composition for diagnosing skin condition related to skin barrier function or A kit for diagnosing skin conditions related to skin barrier function can be provided. In addition, another embodiment of the present invention can provide a method of providing basic information for diagnosing skin conditions related to skin barrier function or a method of diagnosing skin conditions related to skin barrier function using the same.
구체적으로, 본 발명의 일 실시예는 시험대상으로부터 분리된 피부세포에서 LINC00302의 발현량을 측정하는 단계를 포함하고, 상기 LINC00302 발현량은 LINC00302의 RNA 발현량인 피부 장벽 기능관련 피부 상태 진단용 기초 정보 제공 방법 또는 피부 장벽 기능관련 피부 상태 진단방법을 제공할 수 있다.Specifically, one embodiment of the present invention includes measuring the expression level of LINC00302 in skin cells isolated from a test subject, and the expression level of LINC00302 is the RNA expression level of LINC00302, which is basic information for diagnosing skin conditions related to skin barrier function. A method of providing or a method of diagnosing skin conditions related to skin barrier function can be provided.
일 실시예로서 상기 방법들은 상기 시험대상으로부터 분리된 피부세포에서의 LINC00302 발현량을 정상대조군 피부세포에서의 LINC00302 발현량과 비교하는 단계를 더 포함할 수 있다. 이때, 일 실시예로서 상기 시험대상으로부터 분리된 피부세포 및 정상대조군 피부세포는 각질형성세포(keratinocyte)일 수 있다.As an example, the methods may further include comparing the expression level of LINC00302 in skin cells isolated from the test subject with the expression level of LINC00302 in normal control skin cells. At this time, as an example, the skin cells isolated from the test subject and the normal control skin cells may be keratinocytes.
또한, 일 실시예로서 상기 방법들은 상기 시험대상으로부터 분리된 피부 세포에서의 LINC00302 발현량이 정상대조군 피부세포에서의 LINC00302 발현량보다 낮을 경우 피부 장벽 기능관련 피부 상태에 이상이 있는 것으로 정보를 제공하거나, 진단하는 단계를 더 포함할 수 있다. 예를 들어, 상기 시험대상으로부터 분리된 피부세포에서의 LINC00302 발현량이 정상대조군 피부세포에서의 LINC00302 발현량보다 약 1.1 내지 2배 이상 낮은 경우, 피부 장벽기능 관련 피부 상태에 이상이 있는 것으로 판단할 수 있다. 보다 구체적으로, 예를 들어, 상기 시험대상으로부터 분리된 피부세포에서의 LINC00302의 발현량이 정상대조군 피부세포에 비하여, 1.1 배 이상, 1.2배 이상, 1.3 배 이상, 1.4배 이상, 1.5 배 이상, 1.6배 이상, 1.7배 이상, 1.8배 이상, 1.9배 이상, 또는 2배 이상 낮은 경우, 피부 장벽기능 관련 피부 상태에 이상이 있는 것으로 판단할 수 있다. 상기 발현량은 통계적 유의성을 확보한 상태에서 측정된 결과이다. 통계적 유의성이라는 개념은 생물학적 통계분석법을 통하여 유의적인 차이를 보이는 경우로, 정량적인 경우 p value가 0.05 미만으로 차이가 나는 경우를 포함한다. In addition, as an example, the methods provide information that there is an abnormality in the skin condition related to skin barrier function when the expression level of LINC00302 in skin cells isolated from the test subject is lower than the expression level of LINC00302 in normal control skin cells, or A diagnosis step may be further included. For example, if the expression level of LINC00302 in skin cells isolated from the test subject is about 1.1 to 2 times lower than the expression level of LINC00302 in normal control skin cells, it can be determined that there is an abnormality in the skin condition related to skin barrier function. there is. More specifically, for example, the expression level of LINC00302 in skin cells isolated from the test subject is 1.1 times more, 1.2 times more, 1.3 times more, 1.4 times more, 1.5 times more, and 1.6 times more than normal control skin cells. If it is two or more times, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more lower, it can be judged that there is a problem with the skin condition related to the skin barrier function. The expression level is a result measured with statistical significance secured. The concept of statistical significance refers to cases where a significant difference is shown through biological statistical analysis methods, and in quantitative cases, it includes cases where the p value is less than 0.05.
본 발명의 일 실시예는 상기 LINC00302의 RNA 검출 시약을 포함하는 피부 장벽기능 관련 피부 상태 진단용 조성물을 제공할 수 있다. One embodiment of the present invention can provide a composition for diagnosing skin conditions related to skin barrier function, including the RNA detection reagent of LINC00302.
또다른 본 발명의 일 실시예는 상기 LINC00302의 RNA 검출 시약을 포함하는 피부 장벽기능 관련 피부 상태 진단용 키트를 제공할 수 있으며, 상기 키트는 상술된 피부 장벽기능 관련 피부 상태 진단용 기초 정보 제공 방법 또는 피부 장벽기능 관련 피부 상태 진단방법이 기재되어 있는 지시서를 더 포함할 수 있다.Another embodiment of the present invention may provide a kit for diagnosing skin conditions related to skin barrier function including the RNA detection reagent of LINC00302, and the kit may be used as a method of providing basic information for diagnosing skin conditions related to skin barrier function as described above or the skin condition. It may further include instructions that describe methods for diagnosing skin conditions related to barrier function.
일 실시예로서, 상기 피부 장벽기능 관련 피부 상태 진단용 조성물 또는 피부 장벽기능 관련 피부 상태 진단용 키트에 포함되는 LINC00302 RNA 검출 시약은 LINC00302에 특이적으로 결합하는 프라이머 및 프로브 중 하나 이상을 포함할 수 있다. As an example, the LINC00302 RNA detection reagent included in the composition for diagnosing skin conditions related to skin barrier function or the kit for diagnosing skin conditions related to skin barrier function may include one or more of a primer and a probe that specifically binds to LINC00302.
본 명세서에서 "프로브(probe)"란 유전자의 표적 부위와 상보적으로 결합할 수 있는 서열의 염기를 갖는 폴리뉴클레오티드, 그 변이체, 또는 폴리뉴클레오티드와 이에 결합된 표지 물질을 포함하는 것을 의미한다. As used herein, “probe” means a polynucleotide having a base sequence capable of complementary binding to the target site of a gene, a variant thereof, or a polynucleotide and a labeling substance bound thereto.
본 명세서에서 "프라이머(primer)"란 유전자의 표적 부위에 해당하는 특정 영역을 PCR을 이용하여 증폭하기 위하여 사용하는 유전자 특정 영역의 말단에 상보적으로 결합할 수 있는 서열의 염기를 갖는 폴리뉴클레오티드 또는 그 변이체를 의미한다. 상기 프라이머는 특정 영역 말단과 완전히 상보적일 것을 요구하지 않으며, 상기 말단에 혼성화되어 이중사슬 구조를 형성할 정도로 상보적이라면 사용될 수 있다.As used herein, “primer” refers to a polynucleotide having a base sequence that can bind complementary to the end of a specific region of a gene used to amplify a specific region corresponding to the target region of a gene using PCR, or It means that variant. The primer is not required to be completely complementary to the end of a specific region, and can be used as long as it is complementary enough to hybridize to the end to form a double chain structure.
본 명세서에서 "혼성화(hybridization)"란 2개의 단일 가닥 핵산이 상보적인 염기 서열들의 페어링(pairing)에 의하여 이합체 구조(duplex structure)를 형성하는 것을 의미한다. 혼성화는 단일 가닥 핵산 서열 간의 상보성이 완전할 경우(perfect match) 뿐 아니라 일부 미스매치(mismatch) 염기가 존재하여도 일어날 수 있다.As used herein, “hybridization” means forming a duplex structure by pairing complementary base sequences of two single-stranded nucleic acids. Hybridization can occur not only when the complementarity between single-stranded nucleic acid sequences is perfect (perfect match), but also when some mismatched bases exist.
본 명세서에서 "폴리뉴클레오티드(polynucleotide)"란 복수개의 뉴클레오티드의 중합체를 의미하는 것으로, 통상적인 의미의 수십개의 뉴클레오티드의 중합체인 올리고뉴클레오티드를 포함하는 광의의 폴리뉴클레오티드를 의미한다.As used herein, “polynucleotide” refers to a polymer of a plurality of nucleotides, and refers to a polynucleotide in the broad sense including oligonucleotides, which are polymers of dozens of nucleotides in the conventional sense.
이하, 실시예를 통하여, 본 발명의 구성 및 효과를 보다 구체적으로 설명한다. 그러나 아래 실시예는 본 발명에 대한 이해를 돕기 위해 예시의 목적으로만 제공된 것일 뿐 본 발명의 범주 및 범위가 그에 의해 제한되는 것은 아니다. Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. However, the examples below are provided only for illustrative purposes to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.
[실험예 1][Experimental Example 1]
피부 각질형성세포의 분화에 따른 LINC00302 의 RNA 발현 양상을 확인하기 위하여, 하기의 실험을 실시하였다.In order to confirm the RNA expression pattern of LINC00302 according to the differentiation of skin keratinocytes, the following experiment was performed.
먼저, 인간 1차 각질형성세포(Human primary keratinocyte)를 론자사(Lonza, Inc. Walkersville, MD, USA)에서 구입하여 60파이 디쉬에 분주한 뒤, 각질형성배지(Keratinocyte Growth Medium, KGM-Gold™, http://www.lonza.com/products-services/bio-research/primary-cells/human-cells-and-media/keratinocytes-and-media/kgm-gold-keratinocyte-growth-medium.aspx)를 사용하여 분화시켰다. 이때, 컨플루언시(Confluency)가 100%가 되기 까지는 세포증식(proliferating)이 일어나는 단계이므로, 100%가 될 때를 ‘0 day’로 하였다. ‘0 day’가 되었을 때 1.2 mM CaCl2가 포함된 KGM-Gold™ 각질형성배지로 배양하기 시작하고 이때부터 분화를 유도하였다. First, human primary keratinocytes were purchased from Lonza, Inc. Walkersville, MD, USA, distributed in 60 pi dishes, and then cultured in keratinocyte growth medium (KGM-Gold™). , http://www.lonza.com/products-services/bio-research/primary-cells/human-cells-and-media/keratinocytes-and-media/kgm-gold-keratinocyte-growth-medium.aspx) Differentiation was used. At this time, cell proliferating occurs until confluency reaches 100%, so the time when confluency reaches 100% is considered '0 day'. At 'day 0', culture began with KGM-Gold™ keratinogenesis medium containing 1.2mM CaCl 2 and differentiation was induced from then on.
분화를 시작한 후 0day, 4day, 7 day째 되는 날(0D, 4D, 7D), 상기 각질형성세포에서 RNA를 추출하였다. 트리졸(Trizol, Invitrogen, Carlsbad, CA, USA) 1ml 을 이용하여 상기 세포를 녹인 뒤, 원심분리기를 이용하여 상층액을 분리하였으며, 이소프로판올(isopropanol) 0.35ml를 넣어 핵산의 펠렛을 형성시키고, 70% EtOH로 세척한 후 펠렛을 물에 녹여 RNA를 분리하였다. 약 1μg의 RNA를 올리고 디티(oligo dT) 및 역전사 효소(reverse transcripase)를 이용하여 cDNA로 만든 뒤, LINC00302 RNA에 특이적인 프라이머(assay ID: Hs03665776_g1)를 이용하여 Applied biosystems사의 taqman 방식의 RT-qPCR을 실시하여 정량분석하였다. RNA was extracted from the keratinocytes on the 0th, 4th, and 7th day after differentiation began (0D, 4D, 7D). After dissolving the cells using 1 ml of Trizol (Invitrogen, Carlsbad, CA, USA), the supernatant was separated using a centrifuge, and 0.35 ml of isopropanol was added to form a nucleic acid pellet. After washing with % EtOH, the pellet was dissolved in water to isolate RNA. Approximately 1 μg of RNA was made into cDNA using oligo dT and reverse transcripase, and then RT-qPCR using Applied biosystems' taqman method using a primer specific for LINC00302 RNA (assay ID: Hs03665776_g1). A quantitative analysis was performed.
상기 PCR은 하기의 온도사이클에 따라 총 45사이클로 진행하였다.The PCR was carried out for a total of 45 cycles according to the temperature cycle below.
- 전변성(Pre-denaturation): 95℃, 10min, - Pre-denaturation: 95℃, 10min,
- 변성(Denaturation): 95℃, 10sec, - Denaturation: 95℃, 10sec,
- 어닐링(Annealing) 및 신장(Extension): 60℃, 1min- Annealing and Extension: 60℃, 1min
LINC00302 RNA 발현량은 하우스키핑(housekeeping) 유전자인 RPLP0의 RNA(Genebank Accession No.: NM_001002.3) 발현량(assay ID:Hs99999902_m1)으로 정규화(normalization) 하였으며, 이를 도 1에 나타내었다. The LINC00302 RNA expression level was normalized to the RNA (Genebank Accession No.: NM_001002.3) expression level (assay ID: Hs99999902_m1) of the housekeeping gene RPLP0, which is shown in Figure 1.
그 결과, 도 1에 나타난 바와 같이 각질형성세포가 분화함에 따라 LINC00302 RNA의 발현도 증가함을 확인할 수 있다.As a result, as shown in Figure 1, it can be confirmed that the expression of LINC00302 RNA increases as keratinocytes differentiate.
[실험예 2][Experimental Example 2]
각질형성세포에서 LINC00302의 발현 억제시 피부 장벽마커가 감소함를 확인하기 위하여, 하기의 실험을 실시하였다.In order to confirm that skin barrier markers decrease when the expression of LINC00302 is suppressed in keratinocytes, the following experiment was performed.
먼저, 상기 [실험예 1]에 기재된 방법과 동일한 방법으로 각질형성세포를 배양한 다음, 상기 각질형성세포에서 LINC00302 의 발현을 억제(knock down)시키기 위해서 하기 두 종류의 siRNA를 각각 도입하였다.First, keratinocytes were cultured in the same manner as described in [Experimental Example 1], and then the following two types of siRNA were introduced to knock down the expression of LINC00302 in the keratinocytes.
- si-LINC00302 #1-si-LINC00302 #1
(sense) 5’- ACUGAACUCCCACAAAGAU - 3’(서열번호 2) (sense) 5’- ACUGAACUCCCACAAAGAU - 3’ (SEQ ID NO. 2)
(antisense) 5’- A UCU UUG UGG GAG UUC AGU - 3’(서열번호 3) (antisense) 5’- A UCU UUG UGG GAG UUC AGU - 3’ (SEQ ID NO: 3)
- si-LINC00302 #2-si-LINC00302 #2
(sense) 5’- GAGCACAACUGAAAGGGUU - 3’(서열번호 4)(sense) 5’- GAGCACAACUGAAAGGGUU - 3’ (SEQ ID NO: 4)
(antisense) 5’- AACCCUUUCAGUUGUGCUC - 3’(서열번호 5)(antisense) 5’- AACCCUUUCAGUUGUGCUC - 3’ (SEQ ID NO: 5)
그 다음, 상기 각질형성세포를 37℃ 5% CO2 인큐베이터에서 7일간 배양한 뒤, 상기 [실험예 1]에 기재된 방법과 동일한 방법으로 RNA를 추출하고 cDNA를 합성하였다.Next, the keratinocytes were cultured in an incubator at 37°C and 5% CO 2 for 7 days, and then RNA was extracted and cDNA was synthesized in the same manner as described in [Experimental Example 1].
LINC00302 RNA에 특이적인 프라이머(assay ID: Hs03665776_g1), 피부 장벽기능 마커 유전자인 필라그린(FLG) mRNA(Genebank Accession No.: NM_002016)에 특이적인 프라이머 (assay ID: Hs00856927_g1), 인볼루크린(IVL) mRNA(Genebank Accession No.: NM_005547)에 특이적인 프라이머 (assay ID: Hs00846307_s1), 로리크린(LOR) mRNA(Genebank Accession No.: NM_000427)에 특이적인 프라이머 (assay ID:Hs01894962_s1)를 이용하여 어플라이드 바이오시스템사(Applied biosystems)사의 택맨 유전자발현시스템(TaqMan geneexpressionassaykit, AppliedBiosystems, FosterCity, CA)을 이용하여 RT-qPCR을 상기 [실험예 1]과 동일 조건에서 실시하고 정량분석하였다.LINC00302 Primer specific to RNA (assay ID: Hs03665776_g1), primer specific to filaggrin (FLG) mRNA (Genebank Accession No.: NM_002016), a skin barrier function marker gene (assay ID: Hs00856927_g1), involucrin (IVL) Applied Biosystems using primers specific for mRNA (Genebank Accession No.: NM_005547) (assay ID: Hs00846307_s1) and primers specific for loricrin (LOR) mRNA (Genebank Accession No.: NM_000427) (assay ID: Hs01894962_s1). RT-qPCR was performed under the same conditions as [Experimental Example 1] using the TaqMan gene expression assay kit (Applied Biosystems, FosterCity, CA) from Applied Biosystems and quantitative analysis was performed.
LINC00302 RNA 발현량은 하우스키핑(housekeeping) 유전자인 RPLP0의 mRNA(Genebank Accession No.: NM_001002.3) 발현량(assay ID:Hs99999902_m1)으로 정규화(normalization) 하고, 이를 도 2에 나타내었다.The LINC00302 RNA expression level was normalized to the mRNA (Genebank Accession No.: NM_001002.3) expression level (assay ID: Hs99999902_m1) of the housekeeping gene RPLP0, and this is shown in Figure 2.
그 결과, 도 2에 나타난 바와 같이 각질형성세포에서 LINC00302을 제거한 경우(si-LINC00302 #1, si-LINC00302 #2) 피부 장벽기능 마커 유전자인 필라그린, 인볼루크린 및 로리크린의 유전자 발현량도 감소하였음을 확인할 수 있다.As a result, as shown in Figure 2, when LINC00302 was removed from keratinocytes (si-LINC00302 #1, si-LINC00302 #2), the gene expression levels of filaggrin, involucrin, and loricrin, which are skin barrier function marker genes, were also increased. It can be confirmed that it has decreased.
[실험예 3][Experimental Example 3]
본 발명의 일 실시예에 따른 스크리닝 방법에 따라 시험물질의 LINC00302 상대적 발현량을 측정하였다.The relative expression level of LINC00302 of the test substance was measured according to the screening method according to an embodiment of the present invention.
먼저, 상기 [실험예 1]에 기재된 방법과 동일한 방법으로 각질형성세포를 배양한 다음, 상기 세포에 시험물질로서 피부 장벽강화 물질로 알려진 붉은쌀 추출물을 300 ppm 처리하였다. 3일 후 상기 [실험예 1]에 기재된 방법과 동일한 방법으로 RNA를 추출하고 cDNA를 만든 뒤 LINC00302 특이적인 프라이머로 RT-qPCR하였다. First, keratinocytes were cultured in the same manner as described in [Experimental Example 1], and then the cells were treated with 300 ppm of red rice extract, known as a skin barrier strengthening substance, as a test substance. After 3 days, RNA was extracted using the same method as described in [Experimental Example 1], cDNA was created, and RT-qPCR was performed using LINC00302-specific primers.
상기 붉은쌀 추출물은 대한민국 공개특허공보 제10-2015-0139298호에 개시된 방법에 따라 제조하였으며, 상기 문헌은 그 전체가 본 명세서에 참조로서 포함된다.The red rice extract was prepared according to the method disclosed in Korean Patent Publication No. 10-2015-0139298, which is incorporated herein by reference in its entirety.
그 결과, 도 3에 나타난 바와 같이 붉은쌀 추출물을 처리한 세포에서의 LINC00302 상대적 발현량이 상기 붉은쌀 추출물을 처리하지 않은 대조군(control)과 대비하여 약 1.5배 높게 나타났으므로, 상기 붉은쌀 추출물은 피부 장벽강화 물질임을 확인할 수 있었다.As a result, as shown in Figure 3, the relative expression level of LINC00302 in cells treated with the red rice extract was approximately 1.5 times higher compared to the control group that was not treated with the red rice extract, so the red rice extract It was confirmed that it was a skin barrier strengthening substance.
본 발명의 일 실시예에 따른 조성물의 제형예를 아래에서 설명하나, 다른 여러 가지 제형으로도 응용 가능하며, 이는 본 발명을 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.A formulation example of a composition according to an embodiment of the present invention will be described below, but it can be applied to various other formulations, and this is not intended to limit the present invention, but is only intended to provide a detailed explanation.
[제형예 1] 영양화장수[Formulation example 1] Nutritional lotion
하기 표 1에 기재된 조성에 따라 통상적인 방법으로 영양화장수를 제조할 수 있다.Nutritional lotion can be prepared by a conventional method according to the composition shown in Table 1 below.
[제형예 2] 영양로션[Formulation example 2] Nutritional lotion
하기 표 2에 기재된 조성에 따라 통상적인 방법으로 영양로션을 제조할 수 있다.Nutritional lotion can be prepared by conventional methods according to the composition shown in Table 2 below.
[제형예 3] 영양크림[Formulation example 3] Nutrition cream
하기 표 3에 기재된 조성에 따라 통상적인 방법으로 영양크림을 제조할 수 있다.Nutritional cream can be prepared by a conventional method according to the composition shown in Table 3 below.
[제형예 4] 주사제[Formulation Example 4] Injectable
하기 표 4에 기재된 조성에 따라 통상적인 방법으로 주사제를 제조할 수 있다.Injections can be prepared by conventional methods according to the composition shown in Table 4 below.
[제형예 5] 연고[Formulation Example 5] Ointment
하기 표 5에 기재된 조성에 따라 통상적인 방법으로 연고를 제조할 수 있다.The ointment can be prepared by a conventional method according to the composition shown in Table 5 below.
<110> AMOREPACIFIC CORPORATION <120> A composition for skin barrier function comprising LINC00302 promoting materials and a method for screening LINC00302 promoting materials <130> 16P556IND <160> 5 <170> KopatentIn 2.0 <210> 1 <211> 302 <212> RNA <213> Artificial Sequence <220> <223> RNA of LINC00302 <400> 1 gcctgctcca cacctgggtc tgaccagggt gagtggatct tttggaacct agacagagca 60 caactgaaag ggttgactga actcccacaa agatgtcctg caccagtctc atgcatctcg 120 ttcatcagtg cctttcatac cttcaagagc tcttgcctca gagcattgct atgcctgagg 180 tgtgtctcag aagcagccat tggcacagtg gcttggggct gggctcaaaa atttgctctc 240 ttgaagtttg gatgtgattg ctctgaacaa gctgctttgc cacaataaat attttcaagt 300 tt 302 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense siRNA of LINC00302 #1 <400> 2 acugaacucc cacaaagau 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense siRNA of LINC00302 #1 <400> 3 aucuuugugg gaguucagu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense siRNA of LINC00302 #2 <400> 4 gagcacaacu gaaaggguu 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense siRNA of LINC00302 #2 <400> 5 aacccuuuca guugugcuc 19 <110> AMOREPACIFIC CORPORATION <120> A composition for skin barrier function comprising LINC00302 promoting materials and a method for screening LINC00302 promoting materials <130> 16P556IND <160> 5 <170>CopatentIn 2.0 <210> 1 <211> 302 <212> RNA <213> Artificial Sequence <220> <223> RNA of LINC00302 <400> 1 gcctgctcca cacctgggtc tgaccagggt gagtggatct tttggaacct agacagagca 60 caactgaaag ggttgactga actcccacaa agatgtcctg caccagtctc atgcatctcg 120 ttcatcagtg cctttcatac cttcaagagc tcttgcctca gagcattgct atgcctgagg 180 tgtgtctcag aagcagccat tggcacagtg gcttggggct gggctcaaaa atttgctctc 240 ttgaagtttg gatgtgattg ctctgaacaa gctgctttgc cacaataaat attttcaagt 300 tt 302 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense siRNA of LINC00302 #1 <400> 2 acugaacucc cacaaagau 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense siRNA of LINC00302 #1 <400> 3 aucuuuggg gaguucagu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense siRNA of LINC00302 #2 <400> 4 gagcacaacu gaaaggguu 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense siRNA of LINC00302 #2 <400> 5 aacccuuuca gugugcuc 19
Claims (24)
(a)피부세포에 시험 물질을 처리하는 단계;
(b)상기 시험물질을 처리한 피부세포에서 LINC00302(Long intergenic non-protein coding RNA 302)의 발현량을 측정하는 단계; 및
(c)상기 LINC00302의 상대적 발현량이 1.5배 이상 증가한 경우 상기 시험물질을 피부 장벽기능 강화물질로 판정하는 단계를 포함하는 피부 장벽기능 강화 물질 스크리닝 방법.As a method for screening substances that enhance skin barrier function,
(a) treating skin cells with a test substance;
(b) measuring the expression level of LINC00302 (Long intergenic non-protein coding RNA 302) in skin cells treated with the test substance; and
(c) A screening method for skin barrier function enhancing substances, including the step of determining the test substance as a skin barrier function enhancing substance when the relative expression level of LINC00302 increases by more than 1.5 times.
상기 (b)단계는 상기 시험물질을 처리한 피부세포에서 LINC00302 RNA의 상대적 발현량을 측정하는 것을 포함하는, 피부 장벽기능 강화 물질 스크리닝 방법.According to paragraph 1,
Step (b) includes measuring the relative expression level of LINC00302 RNA in skin cells treated with the test substance.
상기 시험물질을 처리한 피부세포에서 필라그린(Filaggrin), 인볼루크린(Involucrin) 및 로리크린(Loricrin)으로 이루어진 군에서 선택된 하나 이상의 유전자의 상대적 발현량을 측정하는 단계를 더 포함하는 피부 장벽기능 강화 물질 스크리닝 방법.According to paragraph 1,
Skin barrier function further comprising measuring the relative expression level of one or more genes selected from the group consisting of Filaggrin, Involucrin, and Loricrin in skin cells treated with the test substance. Reinforcement material screening method.
필라그린(Filaggrin), 인볼루크린(Involucrin) 및 로리크린(Loricrin)으로 이루어진 군에서 선택된 하나 이상의 유전자의 상대적 발현량이 증가한 경우, 상기 시험 물질을 피부 장벽기능 강화 물질로 판단하는 단계를 더 포함하는 피부 장벽기능 강화 물질 스크리닝 방법.In clause 7,
When the relative expression level of one or more genes selected from the group consisting of Filaggrin, Involucrin, and Loricrin increases, the test substance is judged to be a skin barrier function enhancing substance. Screening method for substances that enhance skin barrier function.
상기 LINC00302의 발현 또는 활성 촉진 물질은 제1항 내지 제5항, 제7항 및 제8항 중 어느 한 항에 따른 방법에 따라 스크리닝된 피부 장벽기능 강화 물질을 포함하는, 피부 장벽기능 강화용 조성물.According to clause 10,
The expression or activity promoting material of LINC00302 is a composition for strengthening the skin barrier function, comprising a skin barrier function enhancing material screened according to the method according to any one of claims 1 to 5, 7, and 8. .
상기 조성물은 LINC00302 발현 또는 활성 촉진 물질을 조성물 총 중량을 기준으로, 0.00001 내지 30 중량%로 포함하는, 피부 장벽기능 강화용 조성물.According to clause 10,
The composition is a composition for strengthening the skin barrier function, comprising 0.00001 to 30% by weight of LINC00302 expression or activity promoting material, based on the total weight of the composition.
상기 조성물은 피부 화장료 조성물인, 피부 장벽기능 강화용 조성물.According to clause 10,
The composition is a skin cosmetic composition, a composition for strengthening the skin barrier function.
상기 조성물은 약학적 조성물인, 피부 장벽기능 강화용 조성물.According to clause 10,
The composition is a pharmaceutical composition, a composition for strengthening the skin barrier function.
상기 약학적 조성물은 피부 장벽 기능 약화에 의한 질병의 예방 또는 치료용인, 피부 장벽기능 강화용 조성물.According to clause 15,
The pharmaceutical composition is a composition for strengthening the skin barrier function, which is used to prevent or treat diseases caused by weakening the skin barrier function.
(a)시험대상으로부터 분리된 피부세포에서 LINC00302(Long intergenic non-protein coding RNA 302)의 발현량을 측정하는 단계; 및
(b)상기 LINC00302의 발현량을 측정한 결과 상기 시험대상으로부터 분리된 피부 세포에서의 LINC00302 발현량이 정상대조군 피부세포에서의 LINC00302 발현량보다 1.5배 이상 낮을 경우 피부 장벽 기능관련 피부 상태에 이상이 있는 것으로 정보를 제공하는 단계를 포함하는 피부 장벽 기능관련 피부 상태 진단용 기초 정보 제공 방법.As a method of providing basic information for diagnosing skin conditions related to skin barrier function,
(a) Measuring the expression level of LINC00302 (Long intergenic non-protein coding RNA 302) in skin cells isolated from the test subject; and
(b) As a result of measuring the expression level of LINC00302, if the expression level of LINC00302 in skin cells isolated from the test subject is more than 1.5 times lower than the expression level of LINC00302 in normal control skin cells, there is an abnormality in skin condition related to skin barrier function. A method of providing basic information for diagnosing skin conditions related to skin barrier function, including the step of providing information.
상기 시험대상으로부터 분리된 피부세포에서의 LINC00302 발현량을 정상대조군 피부세포에서의 LINC00302 발현량과 비교하는 단계를 더 포함하는, 피부 장벽 기능관련 피부 상태 진단용 기초 정보 제공 방법.According to clause 17,
A method of providing basic information for diagnosing skin conditions related to skin barrier function, further comprising comparing the expression level of LINC00302 in skin cells isolated from the test subject with the expression level of LINC00302 in normal control skin cells.
상기 LINC00302의 RNA 검출 시약은 LINC00302 RNA에 특이적으로 결합하는 프라이머 및 프로브 중 하나 이상을 포함하는 피부 장벽 기능관련 피부 상태 진단용 키트.According to clause 20,
The LINC00302 RNA detection reagent is a kit for diagnosing skin conditions related to skin barrier function, including at least one of a primer and a probe that specifically binds to LINC00302 RNA.
상기 LINC00302의 RNA 검출 시약은 LINC00302 RNA에 특이적으로 결합하는 프라이머 및 프로브 중 하나 이상을 포함하는 피부 장벽 기능관련 피부 상태 진단용 조성물.According to clause 23,
The RNA detection reagent of LINC00302 is a composition for diagnosing skin conditions related to skin barrier function, comprising at least one of a primer and a probe that specifically binds to LINC00302 RNA.
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| KR1020160122319A KR102635297B1 (en) | 2016-09-23 | 2016-09-23 | A composition for skin barrier function comprising LINC00302 promoting materials and a method for screening LINC00302 promoting materials |
| PCT/KR2017/008901 WO2018056580A1 (en) | 2016-09-23 | 2017-08-16 | Composition for reinforcing skin barrier function comprising linc00302 expression-promoting material, and method for screening linc00302 promoting material |
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| KR1020160122319A KR102635297B1 (en) | 2016-09-23 | 2016-09-23 | A composition for skin barrier function comprising LINC00302 promoting materials and a method for screening LINC00302 promoting materials |
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| KR102083438B1 (en) * | 2011-11-04 | 2020-03-03 | (주)아모레퍼시픽 | Method for screening substances improving skin barrier function |
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