KR20210070064A - MELANIN PRODUCTION INHIBITING COMPOSITION COMPRISING microRNA-2478 - Google Patents
MELANIN PRODUCTION INHIBITING COMPOSITION COMPRISING microRNA-2478 Download PDFInfo
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Abstract
Description
본 발명은 microRNA-2478을 포함하는 멜라닌 생성 억제용 조성물 및 피부 색소 침착 질환 치료용 약학적 조성물에 관한 것이다.The present invention relates to a composition for inhibiting melanin production, including microRNA-2478, and a pharmaceutical composition for treating skin pigmentation diseases.
사람의 피부색을 결정하는 멜라닌(melanin)은 멜라노사이트(melanocyte)에서 생성된다. 구체적으로 멜라닌은 생체 내 존재하는 아미노산인 티로신(tyrosine)을 기질로 하여, 멜라노사이트에 존재하는 티로시나제(tyrosinase) 등의 효소에 의한 중합화 산화 반응으로 형성되는 흑갈색의 색소이다. 이렇게 형성된 멜라닌은 멜라노사이트의 수지상 돌기를 통하여 케라티노사이트(keratinocyte)라는 표피세포로 이동하게 된다. 케라티노사이트로 이동한 멜라닌은 케라티노사이트가 표피에서 떨어져 나갈 때에 피부에서 함께 떨어져 나감으로써 제거될 수 있다. 그러나, 생체 내에 멜라닌을 분해하는 효소가 없기 때문에 한번 형성된 멜라닌은 생체 내에서는 분해되지 않는다. 따라서, 피부를 밝게 하기 위하여는 멜라닌의 생성을 억제하는 것이 관건이라 할 수 있다. Melanin, which determines a person's skin color, is produced in melanocytes. Specifically, melanin is a black-brown pigment formed by polymerization oxidation reaction by enzymes such as tyrosinase present in melanocytes using tyrosine, an amino acid present in a living body, as a substrate. The melanin thus formed moves through the dendrites of the melanocytes to the epidermal cells called keratinocytes. Melanin that has migrated to keratinocytes can be removed by pulling away from the skin as the keratinocytes break away from the epidermis. However, since there is no enzyme decomposing melanin in vivo, melanin once formed is not degraded in vivo. Therefore, in order to brighten the skin, it can be said that suppressing the production of melanin is the key.
최근 정서적으로 흰 피부를 선호하는 동양권의 생활 수준 향상과 더불어 피부 흑화가 자외선에 의한 피부 노화로 인식되면서 미백 및 색소 침착 억제에 관한 연구의 필요성이 점차 증대되고 있다. 그에 따라 아스코르빈산(ascorbic acid), 하이드로퀴논(hydroquinone), 글루타치온(glutathione), 알부틴(arbutin) 등의 티로시나아제 저해 활성을 나타내는 물질들이 화장료나 의약품에 배합되어 사용되어 왔으나, 이들 중 대부분의 것은 효과가 불충분하거나 제형상 불안정한 면이 있어 활용도가 떨어진다. 특히 하이드로퀴논과 같은 화합물은 강한 탈색 작용을 나타내며 그 자체가 피부 감작성을 가지고 있어 피부 알레르기 등을 유발할 수 있고, 정상적인 피부의 기능을 변화시켜 백반증을 유발하는 등의 부작용을 나타내어 피부에 대한 안전성 측면에서 그 사용이 제한되고 있다. Recently, along with the improvement of living standards in the East, who prefer white skin emotionally, skin blackening is recognized as skin aging caused by ultraviolet rays, and the need for research on whitening and pigmentation suppression is gradually increasing. Accordingly, substances exhibiting tyrosinase inhibitory activity, such as ascorbic acid, hydroquinone, glutathione, and arbutin, have been mixed and used in cosmetics and pharmaceuticals, but most of them It has insufficient effectiveness or is unstable in terms of formulation, so its usefulness is low. In particular, compounds such as hydroquinone exhibit a strong bleaching action and have skin sensitization themselves, which can cause skin allergies, etc. its use is limited.
또한, 상기와 같이 티로시나아제의 활성을 저해하는 물질이라 하더라도 이러한 물질이 각질 형성 세포인 케라티노사이트에 작용하여 멜라닌의 생합성을 촉진시키는 인자의 생성을 증가시킨다면, 결과적으로 미백 작용은 약화되거나 오히려 악효과를 거둘 수 있기 때문에 단순히 티로시나아제의 활성 저해제가 좋은 미백제라고 볼 수 없으며, 이에 많은 티로시나아제 저해제가 개발되고 있으나 거의 대부분이 우수한 미백 효과를 나타내지 않는 것으로 알려져 있다.In addition, even if the substance inhibits the activity of tyrosinase as described above, if the substance acts on keratinocytes, which are keratinocytes, and increases the production of factors that promote the biosynthesis of melanin, as a result, the whitening effect is weakened or rather Because it can have a bad effect, it cannot be said that a simple tyrosinase activity inhibitor is a good whitening agent. Therefore, many tyrosinase inhibitors are being developed, but most of them are known to not show an excellent whitening effect.
이에 따라, 미백 또는 색소 침착 억제 효과가 우수한 물질에 대한 연구 및 개발이 요구되고 있다.Accordingly, there is a demand for research and development of a material having an excellent effect of inhibiting whitening or pigmentation.
본 발명의 목적은 microRNA-2478(microRNA-2478)을 포함하는 멜라닌 생성 억제용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for inhibiting melanogenesis comprising microRNA-2478 (microRNA-2478).
본 발명의 다른 목적은 microRNA-2478을 포함하는, 피부 색소 침착 질환의 예방 또는 치료용 약학적 조성물 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin pigmentation diseases, including microRNA-2478.
본 발명의 또 다른 목적은 microRNA-2478을 포함하는 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide a cosmetic composition comprising microRNA-2478.
본 발명의 또 다른 목적은 microRNA-2478을 포함하는 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition comprising microRNA-2478.
상기와 같은 목적을 달성하기 위한 본 발명의 일 측면은 miR-2478 microRNA-2478(microRNA-2478)을 포함하는 을, 멜라닌 생성 억제용 조성물에 관한 것이다.One aspect of the present invention for achieving the above object relates to a composition for inhibiting melanogenesis, comprising miR-2478 microRNA-2478 (microRNA-2478).
본 발명에서 “miRNA(microRNA)”는 21 내지 25 뉴클레오타이드(nt)의 단일 가닥 RNA 분자로서, 진핵생물의 유전자 발현을 제어하는 조절 물질이다. miRNA는 표적 mRNA에 상보적으로 결합하여 전사 후 유전자 억압자(post-transcriptional gene suppressor)로써 작용하며, 번역 억제와 mRNA 불안정화를 유도하는 역할을 하는 것으로 알려져 있다.In the present invention, “miRNA (microRNA)” is a single-stranded RNA molecule of 21 to 25 nucleotides (nt), and is a regulatory substance that controls gene expression in eukaryotes. miRNA is known to function as a post-transcriptional gene suppressor by binding complementarily to the target mRNA, and to induce translational repression and mRNA destabilization.
본 발명에서는 miR-2478(microRNA-2478)가 Rap1a(Ras-related protein Rap-1a)의 유전자 발현을 제어할 수 있음을 규명하였다. 이에 본 발명자들은 상기 miR-2478를 이용하여 Rap1a의 발현을 조절함으로써 멜라닌 생성을 억제할 수 있는 멜라닌 생성 억제용 조성물을 제공하고자 한다.In the present invention, it was identified that miR-2478 (microRNA-2478) can control the gene expression of Rap1a (Ras-related protein Rap-1a). Accordingly, the present inventors intend to provide a composition for inhibiting melanogenesis capable of inhibiting melanogenesis by regulating the expression of Rap1a using the miR-2478.
구체적으로 상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것일 수 있다.Specifically, the miR-2478 may be composed of the nucleic acid sequence of SEQ ID NO: 1.
또한 구체적으로, 상기 miR-2478은 우유 엑소좀으로부터 분리된 것일 수 있다. 보다 구체적으로 소 유래 우유 엑소좀으로부터 분리된 것일 수 있다.Also specifically, the miR-2478 may be isolated from milk exosomes. More specifically, it may be isolated from cow-derived milk exosomes.
본 발명에서, “우유 엑소좀”은 우유 유래의 지질 이중충의 소낭을 말한다. 상기 엑소좀은 우유 유래의 고유의 단백질 및 핵산 등을 포함하고 있으며 30 ~ 200 nm 크기의 생체 나노입자로, 내부에 DNA, RNA, 펩타이드 등으로 이루어진 정보가 담겨있다. 엑소좀은 내부의 물질을 체액 내 분해 효소 등으로부터 안전하게 운반하여 인접 세포 또는 원거리 세포에 정보 전달을 하여 세포 주변의 미세 환경에 영향을 미친다. 특히, 우유 유래의 우유 엑소좀은 식품으로도 접할 수 있을 뿐만 아니라 많은 양의 엑소좀이 추출되며, 긴 시간 동안 안정한 상태로 보관할 수 있다는 장점이 있다.In the present invention, "milk exosome" refers to a vesicle of a lipid bilayer derived from milk. The exosomes contain milk-derived proteins and nucleic acids, and are biological nanoparticles with a size of 30 to 200 nm, and information composed of DNA, RNA, peptides, etc. is contained therein. Exosomes affect the microenvironment around cells by safely transporting internal substances from decomposing enzymes in body fluids, etc., and transmitting information to adjacent or distant cells. In particular, milk-derived milk exosomes can be used as food as well as a large amount of exosomes are extracted and have the advantage that they can be stored in a stable state for a long time.
본 발명의 일 실시예에서 우유 엑소좀 처리에 따라 miR-2478의 발현량을 확인한 결과, 우유 엑소좀의 농도 의존적으로 miR-2478의 발현량이 증가함을 확인하였다(도 1). 상기 결과를 통해, 상기 miR-2478은 우유 엑소좀으로부터 분리될 수 있음을 확인하였다.As a result of confirming the expression level of miR-2478 according to the treatment of milk exosomes in an embodiment of the present invention, it was confirmed that the expression level of miR-2478 increased in a concentration-dependent manner of milk exosomes (FIG. 1). Through the above results, it was confirmed that the miR-2478 can be isolated from milk exosomes.
또한 구체적으로, 상기 miR-2478은 Rap1a(Ras-related protein Rap-1a)의 발현을 억제할 수 있다.Also, specifically, the miR-2478 may inhibit the expression of Ras-related protein Rap-1a (Rap1a).
본 발명의 일 실시예에서, 상기 miR-2478을 세포에 도입한 결과 Rap1a의 활성 및 발현량이 현저히 감소함을 확인하였으며(도 12 내지 14), 상기 miR-2478이 Rap1a를 억제시킴으로써 Akt-Gsk3β 경로를 통해 멜라닌 생성을 억제할 수 있음을 확인하였다(도 15). 상기와 같은 결과로부터 본 발명의 miR-2478은 Rap1a의 발현을 조절할 수 있고, 이를 통해 멜라닌 생성을 억제할 수 있음을 규명하였다.In one embodiment of the present invention, as a result of introducing the miR-2478 into cells, it was confirmed that the activity and expression level of Rap1a was significantly reduced ( FIGS. 12 to 14 ), and the miR-2478 inhibited Rap1a, thereby inhibiting the Akt-Gsk3β pathway It was confirmed that melanin production can be inhibited through (FIG. 15). From the above results, it was confirmed that miR-2478 of the present invention can regulate the expression of Rap1a, thereby inhibiting melanin production.
본 발명의 다른 일 측면은 miR-2478(microRNA-2478)을 포함하는, 피부 색소 침착 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating skin pigmentation diseases, including miR-2478 (microRNA-2478).
구체적으로 상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것일 수 있다.Specifically, the miR-2478 may be composed of the nucleic acid sequence of SEQ ID NO: 1.
본 발명에서, “피부 색소 침착 질환”은 멜라닌의 생성으로부터 기인된 피부 색소가 표피에 침착됨으로써 나타나는 모든 질환을 의미한다. 피부 색소 형성은 표피 내 멜라닌의 생성과 분포로부터 기인한다. 포유류 멜라닌 세포에서, 멜라닌은 주 색소 효소인 타이로시나제를 함유한 멜라노좀(melanosome) 내에서 합성된다. In the present invention, "skin pigmentation disease" refers to any disease that is caused by the deposition of skin pigments resulting from the production of melanin on the epidermis. Skin pigmentation results from the production and distribution of melanin in the epidermis. In mammalian melanocytes, melanin is synthesized in melanosomes containing the main pigment enzyme tyrosinase.
상기 피부 색소 침착 질환은 멜라닌 색소의 비정상적 축적과 관련되어 있으며, 자외선에의 과다 노출 또는 대기 오염, 스트레스 등 외부의 환경 변화에 의해 멜라닌이 과다하게 형성된 기미, 주근깨, 흑색점, 모반, 약물에 의한 색소 침착, 염증 후 색소 침착, 피부염에서 발생하는 과색소 침착으로 등의 증상, 나아가 색소 침착에 의한 피부 노화, 피부암 등을 포함할 수 있다.The skin pigmentation disease is related to the abnormal accumulation of melanin pigment, and melanin is excessively formed due to external environmental changes such as excessive exposure to ultraviolet rays or air pollution, stress, freckles, blemishes, nevus, drug-induced It may include symptoms such as pigmentation, post-inflammatory pigmentation, hyperpigmentation occurring in dermatitis, skin aging due to pigmentation, and skin cancer.
구체적으로 상기 피부 색소 침착 질환은 기미, 주근깨, 흑색점, 모반 및 피부암으로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다.Specifically, the skin pigmentation disease may be any one or more selected from the group consisting of melasma, freckles, black spots, nevus, and skin cancer.
본 발명의 일 실시예에서 miR-2478은 세포 독성이 없으며(도 2), 타로시나아제의 활성을 억제하고(도 3), miR-2478을 처리한 경우 티로시나아제의 활성 및 발현이 억제되고, 멜라닌 생합성을 촉진하는 전사인자인 MITF(microphthalmia-associated transcription factor) 의 발현 역시 억제됨을 확인하였다(도 4 및 도 5) 특히, 피부에 색소를 침착시켜 피부를 어둡게 하는 멜라닌의 합성이 감소하는 것을 직접적으로 확인하였다(도 6).In one embodiment of the present invention, miR-2478 has no cytotoxicity (FIG. 2), inhibits the activity of tarosinase (FIG. 3), and when treated with miR-2478, the activity and expression of tyrosinase are inhibited, , it was confirmed that the expression of MITF (microphthalmia-associated transcription factor), a transcription factor that promotes melanin biosynthesis, was also suppressed ( FIGS. 4 and 5 ). In particular, it was confirmed that the synthesis of melanin, which darkens the skin by depositing a pigment on the skin, was reduced. It was directly confirmed (FIG. 6).
상기와 같은 결과로부터 본 발명의 miR-2478을 포함하는 약학적 조성물이 멜라닌 합성을 억제함으로써 과다 색소 침착을 억제할 수 있으며, 멜라닌 합성 및 티로시나아제 활성 등에 기인한 피부 색소 침착 질환에 대해 우수한 치료 효과를 나타낼 수 있음을 확인하였다.From the above results, the pharmaceutical composition containing miR-2478 of the present invention can inhibit hyperpigmentation by inhibiting melanin synthesis, and is an excellent treatment for skin pigmentation diseases caused by melanin synthesis and tyrosinase activity. It was confirmed that the effect can be exhibited.
본 발명의 약학적 조성물은 투여를 위하여, 상기 본 발명 miR-2478 외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.For administration, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the miR-2478 of the present invention. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
또한, 본 발명의 약학적 조성물은 어떠한 제형으로도 적용가능하며, 보다 구체적으로 비경구용 제형일 수 있다. 비경구용 제형으로는 주사용, 도포용, 에어로졸 등의 스프레이 형일 수 있다. 더욱 구체적으로는 주사제 형태일 수 있다.In addition, the pharmaceutical composition of the present invention can be applied in any dosage form, and more specifically, it may be a dosage form for parenteral use. Formulations for parenteral use may be in the form of injections, coatings, sprays, such as aerosols. More specifically, it may be in the form of an injection.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성 용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
본 발명의 또 다른 측면은 miR-2478(microRNA-2478)을 포함하는 약학적 조성물을 치료를 필요로 하는 개체에 약학적으로 유효한 양으로 투여하는 단계를 포함하는 피부 색소 침착 질환의 치료방법에 관한 것이다. '피부 색소 침착 질환'은 상기 설명한 바와 같다.Another aspect of the present invention relates to a method for treating a skin pigmentation disease comprising administering a pharmaceutical composition comprising miR-2478 (microRNA-2478) to an individual in need of treatment in a pharmaceutically effective amount. will be. The 'skin pigmentation disease' is as described above.
본 발명에서 "약학적으로 유효한 양"은 의학적 치료에 적용가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 성병, 연령, 질병의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is a patient's sexually transmitted disease, age, type of disease, severity, drug activity, sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field.
본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있다. 또한 본 발명의 약학적 조성물은 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, the pharmaceutical composition of the present invention may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.
본 발명의 용어 "개체"는 본 발명에 따른 약학적 조성물의 투여에 의해 증상이 호전될 수 있는 피부 색소 침착 질환을 가진 동물 또는 인간을 포함한다. 본 발명에 따른 치료용 조성물을 개체에게 투여함으로써, 피부 색소 침착 질환을 효과적으로 예방 및 치료할 수 있다. The term "subject" of the present invention includes animals or humans having a skin pigmentation disease whose symptoms can be improved by administration of the pharmaceutical composition according to the present invention. By administering the therapeutic composition according to the present invention to an individual, skin pigmentation diseases can be effectively prevented and treated.
본 발명의 용어 "투여"는 어떠한 적절한 방법으로 인간 또는 동물에게 소정의 물질을 도입하는 것을 의미하며, 본 발명에 따른 치료용 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또한, 본 발명에 따른 치료용 조성물은 유효성분이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. The term "administration" of the present invention means introducing a predetermined substance into a human or animal by any suitable method, and the administration route of the therapeutic composition according to the present invention is through any general route as long as it can reach the target tissue. It may be administered orally or parenterally. In addition, the therapeutic composition according to the present invention may be administered by any device capable of moving the active ingredient to the target cell.
본 발명에 따른 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route of administration and the duration, but may be appropriately selected by those skilled in the art.
본 발명의 또 다른 일 측면은 miR-2478(microRNA-2478)을 포함하는 화장료 조성물에 관한 것이다.Another aspect of the present invention relates to a cosmetic composition comprising miR-2478 (microRNA-2478).
구체적으로 상기 화장료 조성물은 피부 미백용 조성물일 수 있다.Specifically, the cosmetic composition may be a composition for skin whitening.
본 발명에서, “미백”은 피부를 하얗게 하는 것을 의미하며 멜라닌의 과도한 합성이나, 티로시나아제 효소의 활성, L-DOPA의 과도한 산화 등으로 인한 기미, 주근깨 등의 다양한 색소 침착을 완화 또는 개선하는 것을 말한다.In the present invention, "whitening" means to whiten the skin, and to relieve or improve various pigmentation such as spots and freckles caused by excessive synthesis of melanin, tyrosinase enzyme activity, excessive oxidation of L-DOPA, etc. say that
본 발명의 일 실시예에서 miR-2478은 세포 독성이 없으며(도 2), 타로시나아제의 활성을 억제하고(도 3), 티로시나아제 및 MITF의 발현을 억제하며(도 4 및 도 5), 멜라닌의 합성을 직접적으로 억제함으로써(도 6) 피부 미백 효과를 유도함을 확인하였다.In an embodiment of the present invention, miR-2478 has no cytotoxicity (FIG. 2), inhibits the activity of tarosinase (FIG. 3), and inhibits the expression of tyrosinase and MITF (FIGS. 4 and 5) , it was confirmed that the skin whitening effect was induced by directly inhibiting the synthesis of melanin (FIG. 6).
상기와 같은 결과로부터 본 발명의 miR-2478을 포함하는 조성물이 우수한 피부 미백 효과를 나타낼 수 있음을 확인하였다.From the above results, it was confirmed that the composition containing miR-2478 of the present invention can exhibit excellent skin whitening effect.
또한 구체적으로, 상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것일 수 있다.Also, specifically, the miR-2478 may consist of the nucleic acid sequence of SEQ ID NO: 1.
본 발명의 화장료 조성물은 용액, 외용 연고, 크림, 폼, 영양 화장수, 유연 화장수, 팩, 유연수, 유액, 메이크업 베이스, 에센스, 비누, 액체 세정료, 입욕제, 선 스크린 크림, 선 오일, 현탁액, 유탁액, 페이스트, 겔, 로션, 파우더, 비누, 계면 활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 패취 및 스프레이로 구성된 군으로부터 선택되는 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention is a solution, external ointment, cream, foam, nourishing lotion, soft lotion, pack, soft water, emulsion, makeup base, essence, soap, liquid detergent, bath agent, sunscreen cream, sun oil, suspension, emulsion Liquid, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch and spray can be prepared in a formulation selected from the group consisting of, but are limited thereto no.
본 발명의 상기 화장료 조성물은 일반 피부 화장료에 배합되는 화장품학적으로 허용 가능한 담체를 1 종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면 유분, 물, 계면 활성제, 보습제, 저급 알코올, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, humectant, lower alcohol, and thickener. , a chelating agent, a pigment, a preservative, a fragrance, etc. may be appropriately mixed, but is not limited thereto.
본 발명의 화장료 조성물에 포함되는 화장품학적으로 허용 가능한 담체는 화장료 조성물의 제형에 따라 다양하다.The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.
본 발명의 제형이 연고, 페이스트, 크림 또는 젤인 경우에는, 담체 성분으로서 동물성 유, 식물성 유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화 아연 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2 종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is an ointment, paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used, but is not limited thereto. These may be used alone or in combination of two or more.
본 발명의 제형이 파우더 또는 스프레이인 경우에는, 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록사이드, 칼슘 실케이트, 폴리아미드 파우더 등이 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로하드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진제를 포함할 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2 종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohard propellants such as, but not limited to, locarbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more.
본 발명의 제형이 용액 또는 유탁액인 경우에는, 담체 성분으로서 용매, 용해화제 또는 유탁화제 등이 이용될 수 있으며, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일 등이 이용될 수 있고, 특히, 목화씨 오일, 땅콩 오일, 옥수수 배종 오일, 올리브 오일, 피마자 오일 및 참깨 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2 종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylglycol oil and the like can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, fatty acid esters of polyethylene glycol or sorbitan may be used, but is not limited thereto. These may be used alone or in combination of two or more.
본 발명의 제형이 현탁액인 경우에는, 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타하이드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있으나, 이에 제한되는 것은 아니다. 이들은 단독으로 사용되거나 2 종 이상 혼합되어 사용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters; Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto. These may be used alone or in combination of two or more.
또한, 본 발명의 조성물은 피부에 직접 도포하거나 살포하는 등의 경피 투여 방법으로 사용될 수 있으며, 본 발명 조성물의 투여 경로는 목적조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. In addition, the composition of the present invention can be used as a transdermal administration method, such as directly applied to the skin or sprayed, and the administration route of the composition of the present invention can be administered through any general route as long as it can reach the target tissue.
본 발명의 조성물의 사용량은 연령, 병변의 정도 등의 개인 차이나 제형에 따라 적절하게 조절될 수 있으며, 1일 1회 내지 수회 적?韜?을 피부에 도포하여 1 주일 내지 수개월 사용될 수 있다. The usage amount of the composition of the present invention may be appropriately adjusted according to individual differences or formulations such as age, degree of lesion, etc., and may be used for one week to several months by applying drops to the skin once to several times a day.
본 발명의 또 다른 일 측면은 miR-2478을 포함하는 식품 조성물일 수 있다.Another aspect of the present invention may be a food composition comprising miR-2478.
구체적으로 상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것일 수 있다.Specifically, the miR-2478 may be composed of the nucleic acid sequence of SEQ ID NO: 1.
또한 구체적으로, 상기 식품 조성물은 미백용일 수 있다.Also specifically, the food composition may be for whitening.
상기 “miR-2478” 및 “미백”에 관한 설명은 전술한 바와 동일하다.The descriptions of “miR-2478” and “whitening” are the same as described above.
상기 식품의 종류에는 특별한 제한은 없다. 본 발명의 miR-2478을 첨가할 수 있는 식품은 소세지, 육류, 빵, 초콜릿류, 스넥류, 캔디류, 과자류, 라면, 피자, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있다. 음료수로 제형화할 경우에 본 발명의 Rap1a의 발현 또는 활성을 억제하는 물질 외에 첨가되는 액체 성분으로는 이에 한정되지는 않으나, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 모노사카라이드(예, 포도당, 과당 등), 디사카라이드(예, 말토오스, 수크로오스 등) 및 폴리사카라이드(예, 덱스트린, 시클로덱스트린 등과 같은 통상적인 당), 및 자일리톨, 소르비톨, 에리스리톨 등의 당 알코올일 수 있다.There is no particular limitation on the type of the food. Foods to which miR-2478 of the present invention can be added include sausage, meat, bread, chocolate, snacks, candy, confectionery, ramen, pizza, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes. When formulated as a beverage, the liquid component added other than the substance that inhibits the expression or activity of Rap1a of the present invention is not limited thereto, but may contain various flavoring agents or natural carbohydrates as additional components as in conventional beverages. can The above-mentioned natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.) and polysaccharides (eg, common sugars such as dextrin, cyclodextrin, etc.), and xylitol, sorbitol , and may be a sugar alcohol such as erythritol.
상기 식품의 종류는 구체적으로 건강기능식품일 수 있다. 상기 건강기능 식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 증진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 유기산, 보호성 콜로이드 점증제, pH 조절제, 안정화제, 보존제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 또한, 본 발명의 건강기능 식품은 과일 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 단독으로 또는 조합으로 사용될 수 있으며, 이러한 첨가제의 비율은 조성물 전체 중량당 0.001 내지 50 중량부의 범위에서 선택되는 것이 일반적이다.The type of food may specifically be a health functional food. The health functional food includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, organic acids, protective colloid thickening Agents, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like may be contained. In addition, the health functional food of the present invention may contain the pulp for the production of fruit and vegetable beverages. These components may be used alone or in combination, and the proportion of these additives is generally selected in the range of 0.001 to 50 parts by weight based on the total weight of the composition.
상기 건강기능식품은 식품의 생체 조절 기능을 강조한 식품으로 물리적, 생화학적, 생물공학적인 방법을 이용하여 특정 목적에 작용 및 발현하도록 부가가치를 부여한 식품이다. 이러한 건강기능 식품의 성분은 생체 방어와 신체 리듬의 조절, 질환의 방지 및 회복에 관계하는 신체 조절 기능을 생체에 대하여 충분히 발휘하도록 설계하여 가공하게 되며, 식품으로 허용 가능한 식품 보조 첨가제, 감미료 또는 기능성 원료를 함유할 수 있다. The health functional food is a food that emphasizes the bioregulatory function of food, and is a food with added value to act and express for a specific purpose using a physical, biochemical, and bioengineering method. The ingredients of these health functional foods are designed and processed to sufficiently exert the body control functions related to the body defense, regulation of body rhythm, prevention and recovery of diseases, and are food supplement additives, sweeteners or functionalities that are acceptable as food. It may contain raw materials.
본 발명의 miR-2478을 건강기능식품(또는 건강기능 음료 첨가물)으로 사용할 경우, 상기 miR-2478을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용하고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 상기 miR-2478의 혼합량은 그의 사용 목적(예방, 건강 또는 개선, 치료적 처치)에 따라 적합하게 결정될 수 있다. When miR-2478 of the present invention is used as a health functional food (or health functional beverage additive), the miR-2478 can be added as it is or used with other food or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the miR-2478 may be appropriately determined depending on the purpose of its use (prevention, health or improvement, therapeutic treatment).
본 발명의 miR-2478을 포함하는 조성물은 독성이 없어 생체 적합성이 우수하며, 티로시나아제 효소 활성 억제 및 멜라닌 합성 억제 효과가 우수하여 피부 미백 및 피부 색소 침착 질환 치료에 널리 활용될 수 있다.The composition comprising miR-2478 of the present invention is non-toxic and has excellent biocompatibility, and has excellent effects of inhibiting tyrosinase enzyme activity and inhibiting melanin synthesis, so it can be widely used for skin whitening and treatment of skin pigmentation diseases.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 우유 엑소좀 처리에 따른 miR-2478의 발현 증가를 확인한 결과를 나타낸 것이다.
도 2는 miR-2478의 독성 여부를 세포 생존율을 통해 확인한 결과를 나타낸 것이다.
도 3은 miR-2478의 티로시나아제 효과 활성 억제 효과를 확인한 결과를 나타낸 것이다.
도 4는 miR-2478의 티로시나아제 및 MITF 의 mRNA 발현 억제 효과를 확인한 결과를 나타낸 것이다(도 4A: 티로시나아제의 mRNA 발현 억제, 도 4B: MITF의 mRNA 발현 억제).
도 5는 miR-2478의 티로시나아제 및 MITF 의 단백질 발현 억제 효과를 확인한 결과를 나타낸 것이다.
도 6은 miR-2478의 멜라닌 합성 억제 효과를 확인한 결과를 나타낸 것이다.
도 7은 miR-2478 억제제 도입시 티로시나아제 활성이 증가한 결과를 나타낸 것이다.
도 8은 miR-2478 억제제 도입시 티로시나아제 및 MITF의 mRNA 발현이 증가한 결과를 나타낸 것이다.
도 9는 miR-2478 억제제 도입시 티로시나아제 및 MITF의 단백질 발현이 증가한 결과를 나타낸 것이다.
도 10은 miR-2478 억제제 도입시 멜라닌 생합성이 증가된 결과를 나타낸 것이다.
도 11은 miR-2478의 Rap1a에 결합하는 부위에 관한 모식도를 나타낸 것이다.
도 12는 miR-2478 도입시 Rap1a에 관한 루시퍼라아제 활성이 감소한 결과를 나타낸 것이다.
도 13은 miR-2478 도입시 Rap1a의 mRNA 발현이 감소된 결과를 나타낸 것이다.
도 14는 miR-2478 도입시 Rap1a의 단백질 발현이 감소된 결과를 나타낸 것이다.
도 15는 phospho-Gsk3β, Gsk3β, pAkt 및 Akt의 단백질 발현을 측정하여 우유 엑소좀의 멜라닌 생성 조절 경로를 확인한 결과를 나타낸 것이다(A: 우유 엑소좀 처리, B: miR-2478 도입, C: 우유 엑소좀 처리 후 miR-2478 억제제 도입). 1 shows the results of confirming the increase in the expression of miR-2478 according to the milk exosome treatment.
Figure 2 shows the results of confirming the toxicity of miR-2478 through cell viability.
3 shows the results of confirming the inhibitory effect of miR-2478 tyrosinase effect activity.
4 shows the results of confirming the inhibitory effect on mRNA expression of tyrosinase and MITF of miR-2478 (FIG. 4A: inhibition of mRNA expression of tyrosinase, FIG. 4B: inhibition of mRNA expression of MITF).
5 shows the results of confirming the inhibitory effect on protein expression of tyrosinase and MITF of miR-2478.
6 shows the results of confirming the melanin synthesis inhibitory effect of miR-2478.
7 shows the result of increased tyrosinase activity upon introduction of the miR-2478 inhibitor.
8 shows the results of increased mRNA expression of tyrosinase and MITF upon introduction of the miR-2478 inhibitor.
9 shows the results of increased protein expression of tyrosinase and MITF upon introduction of the miR-2478 inhibitor.
10 shows the result of increased melanin biosynthesis upon introduction of the miR-2478 inhibitor.
11 is a schematic diagram of the miR-2478 binding site to Rap1a.
12 shows the result of reduced luciferase activity with respect to Rap1a upon introduction of miR-2478.
13 shows the result of reduced Rap1a mRNA expression upon introduction of miR-2478.
14 shows the result of reduced Rap1a protein expression upon introduction of miR-2478.
15 shows the results of confirming the melanogenesis regulation pathway in milk exosomes by measuring the protein expression of phospho-Gsk3β, Gsk3β, pAkt and Akt (A: milk exosome treatment, B: miR-2478 introduction, C: milk introduction of miR-2478 inhibitor after exosome treatment).
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.
실시예 1. 우유 엑소좀(milk exosome) 제조방법Example 1. Method for preparing milk exosomes
원심분리기를 이용하여 소 유래 우유에서 엑소좀을 추출하였다. 보다 구체적으로는, 우유를 튜브에 분주하여, 2000 g와 10,000 g에서 각각 10분 동안 원심분리하여 상층액을 수거하였다. 수거한 상층액을 0.45 μm, 0.2 μm 필터로 여과한 다음, phosphate buffered saline (PBS)를 혼합하였다. 이후, Exoquick exosome precipitation solution (System Biosciences)을 PBS와 혼합하여 넣어 준 후, 30 분 동안 휴지시키고, 1500 g에서 30 분간 다시 원심분리하였다. 상층액을 제거하고 엑소좀 펠렛을 PBS에 용해시켜 이후 실험에 이용하였다.Exosomes were extracted from cow-derived milk using a centrifuge. More specifically, milk was dispensed into tubes, and the supernatant was collected by centrifugation at 2000 g and 10,000 g for 10 minutes, respectively. The collected supernatant was filtered through 0.45 μm and 0.2 μm filters, and then phosphate buffered saline (PBS) was mixed. Thereafter, the Exoquick exosome precipitation solution (System Biosciences) was mixed with PBS and put, rested for 30 minutes, and centrifuged again at 1500 g for 30 minutes. The supernatant was removed and the exosome pellet was dissolved in PBS and used for subsequent experiments.
실험예 1. 우유 엑소좀 내의 miRNA(microRNA) 확인Experimental Example 1. Identification of miRNA (microRNA) in milk exosomes
피부 색소 침착에 관여하는 우유 엑소좀 내의 microRNA을 확인하였다. 소에 서 유래한 우유 엑소좀을 이용하여 microRNA 프로파일링을 조사한 결과, miR-2478이 가장 많이 존재한다는 연구 결과가 보고된 바 있음에 따라(Izumi, H., Tsuda, M., Sato, Y., Kosaka, N., Ochiya, T., Iwamoto, H., Namba, K., andTakeda, Y. (2015). Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages. J. Dairy Sci. 98, 2920-2933.), 본 발명에서는 우유 엑소좀을 처리함에 따라 miR-2478이 증가하는지 확인하였다.MicroRNAs in milk exosomes involved in skin pigmentation were identified. As a result of investigating microRNA profiling using cow-derived milk exosomes, it was reported that miR-2478 was the most abundant (Izumi, H., Tsuda, M., Sato, Y. , Kosaka, N., Ochiya, T., Iwamoto, H., Namba, K., and Takeda, Y. (2015). Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages. J. Dairy Sci. 98 , 2920-2933.), in the present invention, it was confirmed whether miR-2478 was increased by treatment with milk exosomes.
B16F10 세포에 우유 엑소좀을 20 μg/ml, 50 μg/ml 농도로 처리한 후 miR-2478의 발현을 확인하였다. 상기 miR-2478의 서열은 아래와 같다.B16F10 cells were treated with milk exosomes at concentrations of 20 μg/ml and 50 μg/ml, and then the expression of miR-2478 was confirmed. The sequence of miR-2478 is as follows.
6웰 플레이트 상에 B16F10 세포를 2x105 cells/well 의 농도로 분주하고, 우유 엑소좀을 농도 별로 24 시간 동안 처리하였다. 그 후, 세포를 수거하고, 1 ml의 TRI 시약 (Bioline)을 이용하여 추출한 총 RNA 100 ng에 miScript Reverse Transcription kit (Qiagen)을 활용하여 cDNA를 합성하였다. 합성된 생산물 2 μl를 miScript SYBR Green kit (Qiagen)를 사용하여 PCR 반응을 실시하였다. 다음과 같은 PCR 조건 하에서 유전자를 증폭시켰다. 즉, Roto gene Q PCR 기기 (Qiagen)을 이용하여 94°C 에서15 분, 그 후 94°C에서 15 초, 55°C에서 30 초 및 70°C에서 20 초의 40 주기로 증폭시켰다. 유전자 발현은 SNORD61 발현으로 보정하였으며, miR-2478 검출용 miRNA 특이적 프라이머는 QIAGEN(miScript primer assay)으로 부터 구매하였다.B16F10 cells were dispensed on a 6-well plate at a concentration of 2x10 5 cells/well, and milk exosomes were treated for 24 hours at each concentration. Thereafter, cells were harvested, and cDNA was synthesized using the miScript Reverse Transcription kit (Qiagen) from 100 ng of total RNA extracted using 1 ml of TRI reagent (Bioline). 2 μl of the synthesized product was subjected to PCR reaction using miScript SYBR Green kit (Qiagen). The gene was amplified under the following PCR conditions. That is, using a Roto gene Q PCR machine (Qiagen), amplification was performed at 40 cycles of 15 min at 94 °C, 15 sec at 94 °C, 30 sec at 55 °C and 20 sec at 70 °C. Gene expression was corrected for SNORD61 expression, and miRNA-specific primers for miR-2478 detection were purchased from QIAGEN (miScript primer assay).
그 결과, 도 1에 나타난 바와 같이 우유 엑소좀을 처리한 경우, miR-2478의 발현이 증가되는 것을 확인하였으며, 처리된 우유 엑소좀의 농도가 증가할수록 miR-2478의 발현이 증가함을 확인하였다. As a result, as shown in FIG. 1 , it was confirmed that the expression of miR-2478 was increased when the milk exosomes were treated, and it was confirmed that the expression of miR-2478 increased as the concentration of the treated milk exosomes increased. .
이를 통해 miR-2478은 우유 엑소좀으로부터 분리될 수 있으며, 우유 엑소좀 농도 의존적으로 증가하는 것을 알 수 있었다.Through this, miR-2478 could be separated from milk exosomes, and it was found that milk exosomes increased in a concentration-dependent manner.
실험예 2. miR-2478의 세포 독성 확인Experimental Example 2. Confirmation of cytotoxicity of miR-2478
세포주에 주입된 miR-2478의 세포 독성 여부를 확인하였다. miRNA mimmic을 도입한 군을 대조군(negative control)으로 하였다(NC).It was confirmed whether miR-2478 injected into the cell line was cytotoxic. A group into which miRNA mimmic was introduced was used as a negative control (NC).
구체적으로, B16F10 세포를 96 웰 플레이트에 5x104 cells/ml이 되도록 분주하고 37℃, 5% CO2 조건의 배양기에서 24 시간 배양하였다. 상기 배양된 세포에서 miR-2478 mimic(Genpharma)의 형질주입은 리포펙타민(lipofectamine 2000, Invitrogen)을 이용하여 수행하였다. microRNA mimic과 대조군은 최종적으로 50 nM 농도를 사용하였다. 대조군은 Genepharma에서 구입한 negative control(NC) miRNA mimmic을 형질 주입하였다. 상기 세포를 24 시간 동안 배양한 후, 각 웰에는 10 μl의 EZ-Cytox 세포생존율 분석 시약(Daeil Lab Service)을 처리하여 37℃에서 1 시간 배양하였다. 플레이트 리더기로 450 nm에서 흡광도를 측정하여 대조군의 흡광도와 비교하여 B16F10 세포에 대한 독성 정도를 조사하였다.Specifically, B16F10 cells were aliquoted to a volume of 5x10 4 cells/ml in a 96-well plate and cultured for 24 hours in an incubator at 37°C and 5% CO 2 conditions. Transfection of miR-2478 mimic (Genpharma) in the cultured cells was performed using lipofectamine (lipofectamine 2000, Invitrogen). For microRNA mimic and control, 50 nM concentration was finally used. The control group was transfected with a negative control (NC) miRNA mimmic purchased from Genepharma. After culturing the cells for 24 hours, each well was treated with 10 μl of EZ-Cytox cell viability assay reagent (Daeil Lab Service) and incubated at 37° C. for 1 hour. By measuring the absorbance at 450 nm with a plate reader, the degree of toxicity to B16F10 cells was investigated compared with the absorbance of the control group.
그 결과, 도 2에 나타난 바와 같이 대조군과 miR-2478을 도입한 세포 모두에서 생존율 변화가 나타나지 않은 바, 본 발명의 miR-2478이 독성이 없음을 확인하였다.As a result, as shown in FIG. 2 , there was no change in viability in both the control group and the cells introduced with miR-2478, confirming that miR-2478 of the present invention was not toxic.
실험예 3. miR-2478의 미백 효과 확인Experimental Example 3. Confirmation of whitening effect of miR-2478
3-1. 티로시나아제(Tyrosinase, TYR) 효소 활성 억제3-1. Inhibition of tyrosinase (TYR) enzyme activity
B16F10 세포에 miR-2478을 과발현시킨 후, 티로시나아제 효소 억제 활성을 확인하였다. After overexpressing miR-2478 in B16F10 cells, tyrosinase enzyme inhibitory activity was confirmed.
구체적으로, B16F10세포에서 miR-2478 mimic의 형질주입은 lipofectamine 2000 (Invitrogen)을 이용하여 실시하였다. microRNA mimic과 대조군(Genpharma)은 최종적으로 50 nM을 사용하였다. 상기 세포를 24 시간 배양한 후 배지를 제거하고 PBS로 2 회 세척하였다. 1% Triton X-100, 5 mM EDTA, 0.1% 0.1 M PMSF(phenylmethyl sulfonyl fluoride)를 혼합한 0.1 M 포스페이트 완충용액으로 상기 세포를 수거한 후 얼음에서 30 분간 용해시켰다. 그 후, 4℃, 16,500 g 조건으로 30 분간 원심분리해서 상층액을 티로시나아제 활성 측정 용액으로 사용하였다. 상기 상층액 40 μl에 80 μl의 0.1 M 포스페이트 완충용액과 40 μl 티로시나아제를 첨가하였다. 기질로서 40 μl의 10 mM DOPA를 첨가한 후 37℃에서 30 분간 반응시킨 다음, 플레이트 리더기를 이용하여 파장 475 nm에서 흡광도를 측정하였다. Specifically, transfection of miR-2478 mimic in B16F10 cells was performed using lipofectamine 2000 (Invitrogen). 50 nM was finally used for microRNA mimic and control (Genpharma). After culturing the cells for 24 hours, the medium was removed and washed twice with PBS. The cells were harvested with 0.1 M phosphate buffer mixed with 1% Triton X-100, 5 mM EDTA, and 0.1% 0.1 M PMSF (phenylmethyl sulfonyl fluoride), and then lysed on ice for 30 minutes. Thereafter, the solution was centrifuged for 30 minutes at 4°C and 16,500 g, and the supernatant was used as a solution for measuring tyrosinase activity. 80 μl of 0.1 M phosphate buffer and 40 μl of tyrosinase were added to 40 μl of the supernatant. After adding 40 μl of 10 mM DOPA as a substrate and reacting at 37° C. for 30 minutes, absorbance was measured at a wavelength of 475 nm using a plate reader.
그 결과, 도 3에 나타난 바와 같이 miR-2478을 과발현시킨 경우 대조군에 비해 티로시나아제 활성이 48% 감소하였는 바, miR-2478이 티로시나아제의 활성을 억제함으로써 미백 효과를 나타낼 수 있음을 확인하였다.As a result, as shown in FIG. 3, when miR-2478 was overexpressed, the tyrosinase activity was reduced by 48% compared to the control. It was confirmed that miR-2478 can exhibit a whitening effect by inhibiting the activity of tyrosinase. did.
3-2. 티로시나아제 및 MITF의 mRNA 발현 억제 확인3-2. Confirmation of mRNA expression inhibition of tyrosinase and MITF
B16F10 세포에 miR-2478을 과발현시킨 후, 티로시나아제 및 전사인자인 MITF(microphthalmia-associated transcription factor) 의 mRNA 발현을 확인하였다. miR-2478 과발현 방법 및 조건은 상기 3-1과 동일하다.After overexpressing miR-2478 in B16F10 cells, mRNA expression of tyrosinase and a transcription factor MITF (microphthalmia-associated transcription factor) was confirmed. The miR-2478 overexpression method and conditions are the same as in 3-1 above.
B16F10세포에서 우유 엑소좀을 처리 하고 24 시간 후에 세포를 수거하였다. 상기 수거한 세포에 Tri 시약(Bioline)을 반응시켜 total RNA를 분리하였다. 역전사 반응을 실시하기 위하여 1 μg RNA에 dNTP, M-MLV reverse-transcriptase (Promega) 등을 첨가한 다음 37℃에서 1 시간 반응시켜 cDNA를 합성하였다. TYR과 MITF mRNA 발현을 측정하기 위해 SYBR Green PCR Master mix(Bioline)를 이용하여 실시간 중합효소 연쇄반응을 수행하였다. 특정 유전자를 증폭하기 위한 PCR 조건은 다음과 같이 시행하였다. 95℃에서 10 분간 반응시킨 다음, 15 초 95℃, 15 초 60℃, 72℃ 15 초를 한 주기로 하여 40 cycles 동안 증폭하였다. 표적 유전자 mRNA 발현량은 actin 발현량에 대한 상대적인 발현량으로 보정하였다. 사용된 프라이머는 하기 표 2에 나타난 바와 같다. B16F10 cells were treated with milk exosomes and the cells were harvested 24 hours later. Total RNA was isolated by reacting the collected cells with Tri reagent (Bioline). In order to perform the reverse transcription reaction, dNTP, M-MLV reverse-transcriptase (Promega), etc. were added to 1 μg RNA, and then reacted at 37° C. for 1 hour to synthesize cDNA. Real-time polymerase chain reaction was performed using SYBR Green PCR Master mix (Bioline) to measure TYR and MITF mRNA expression. PCR conditions for amplifying a specific gene were performed as follows. After reacting at 95°C for 10 minutes, amplification was carried out for 40 cycles with a cycle of 15 seconds at 95°C, 15 seconds at 60°C, and 72°C for 15 seconds. The target gene mRNA expression level was corrected for the expression level relative to the actin expression level. The primers used are as shown in Table 2 below.
그 결과, 도 4A에 나타난 바와 같이, miR-2478을 과발현시킨 경우, 티로시나아제의 mRNA 발현이 억제됨을 확인하였다. 이로부터 miR-2478이 티로시나아제의 발현을 억제함으로써 미백 효과를 나타낼 수 있음을 확인하였다.As a result, as shown in FIG. 4A , it was confirmed that the mRNA expression of tyrosinase was suppressed when miR-2478 was overexpressed. From this, it was confirmed that miR-2478 can exhibit a whitening effect by inhibiting the expression of tyrosinase.
또한, 도 4B에 나타난 바와 같이 miR-2478을 과발현시킨 경우, MITF의 mRNA 발현이 억제됨을 확인하였다. 이로부터 miR-2478이 MITF의 발현을 억제함으로써 미백 효과를 나타낼 수 있음을 확인하였다.In addition, as shown in FIG. 4B , when miR-2478 was overexpressed, it was confirmed that the mRNA expression of MITF was suppressed. From this, it was confirmed that miR-2478 can exhibit a whitening effect by suppressing the expression of MITF.
3-3. 티로시나아제 및 MITF의 단백질 발현 억제 확인3-3. Confirmation of inhibition of protein expression of tyrosinase and MITF
B16F10 세포에 miR-2478을 과발현시킨 후, 티로시나아제 및 전사인자인 MITF의 단백질 발현을 확인하였다. miR-2478 과발현 방법 및 조건은 상기 3-1과 동일하다.After overexpressing miR-2478 in B16F10 cells, protein expression of tyrosinase and MITF, a transcription factor, was confirmed. The miR-2478 overexpression method and conditions are the same as in 3-1 above.
구체적으로, B16F10세포주를 2×105 cells/well로 6 웰 플레이트에 부착시킨 다음, miR-2478을 처리하고 24 시간 동안 배양하였다. 수거한 세포를 16,500 g에서 15 분간 원심 분리하여 상등액을 취한 후, 단백질 농도를 BSA 키트(Bio-rad)로 정량하였다. Specifically, the B16F10 cell line was attached to a 6-well plate at 2×10 5 cells/well, treated with miR-2478, and cultured for 24 hours. The collected cells were centrifuged at 16,500 g for 15 minutes to obtain a supernatant, and then the protein concentration was quantified with a BSA kit (Bio-rad).
추출한 단백질은 10% SDS 폴리아크릴마이드 젤을 이용하여 단백질을 분리하였다. 상기 분리한 단백질은 니트로셀룰로오스막(GE Health care)으로 옮긴 후, 항체의 비특이적 결합을 방지하기 위하여 1 시간 동안 5% 탈지분유에 반응하였다. TYR (abcam), MITF (abcam), Actin (Sigma)의 1차 항체를 1:1000의 비율로 희석하여 4℃에서 12 - 18 시간 니트로셀룰로오스막과 반응시켰다. 이후, PBS로 세척한 다음 HRP-tagged anti-rabbit 항체를 첨가하여 상온에서 30분 동안 반응시켰다. ECL 키트 (Santa Cruz Inc.)를 사용하여 단백질 밴드를 관찰하였다.The extracted protein was separated using a 10% SDS polyacrylamide gel. The separated protein was transferred to a nitrocellulose membrane (GE Health care), and then reacted with 5% skim milk for 1 hour to prevent non-specific binding of the antibody. Primary antibodies of TYR (abcam), MITF (abcam), and Actin (Sigma) were diluted at a ratio of 1:1000 and reacted with a nitrocellulose membrane at 4°C for 12 - 18 hours. After washing with PBS, HRP-tagged anti-rabbit antibody was added and reacted at room temperature for 30 minutes. Protein bands were observed using an ECL kit (Santa Cruz Inc.).
그 결과, 도 5에 나타난 바와 같이 miR-2478을 과발현시킨 경우, mRNA의 발현이 감소된 것과 마찬가지로, 티로시나아제 및 MITF의 단백질 발현이 모두 감소된 것을 확인하였다. 상기와 같은 결과는 본 발명의 우유 엑소좀에 포함된 miR-2478이 티로시나아제 및 MITF의 발현을 억제함으로써 미백 효과를 유도함을 나타내는 것이다.As a result, as shown in FIG. 5 , when miR-2478 was overexpressed, it was confirmed that both the protein expression of tyrosinase and MITF was reduced, similarly to the decrease in mRNA expression. The above results indicate that miR-2478 contained in the milk exosome of the present invention induces a whitening effect by inhibiting the expression of tyrosinase and MITF.
3-4. 멜라닌 합성 억제효과 확인3-4. Confirmation of melanin synthesis inhibitory effect
B16F10 세포에 miR-2478을 과발현시킨 후, 멜라닌 함량을 측정하였다. miR-2478 과발현 방법 및 조건은 상기 3-1과 동일하다.After overexpressing miR-2478 in B16F10 cells, the melanin content was measured. The miR-2478 overexpression method and conditions are the same as in 3-1 above.
구체적으로, B16F10 세포주에 miR-2478을 주입한 다음, 24시간 동안 배양하고 세포를 수거하였다. 회수한 세포에 10% DMSO를 포함하는 1N NaOH를 첨가한 다음 90℃에서 1 시간 반응시켜 멜라닌이 충분히 용출될 수 있도록 한 후 플레이트 리더기를 이용하여 파장 405nm에서 흡광도를 측정하였다.Specifically, miR-2478 was injected into the B16F10 cell line, then cultured for 24 hours and cells were harvested. 1N NaOH containing 10% DMSO was added to the recovered cells, followed by reaction at 90° C. for 1 hour so that melanin could be sufficiently eluted, and absorbance was measured at a wavelength of 405 nm using a plate reader.
그 결과, 도 6에 나타난 바와 같이, miR-2478을 과발현시킨 경우, 멜라닌 합성이 저해되는 것을 확인하였으며, 이로부터 직접적으로 멜라닌 합성을 억제하는 효과가 우수함을 확인하였다.As a result, as shown in FIG. 6, when miR-2478 was overexpressed, it was confirmed that melanin synthesis was inhibited, and from this, it was confirmed that the effect of directly inhibiting melanin synthesis was excellent.
실험예 4. miR-2478의 멜라닌 생합성 과정에의 영향 확인Experimental Example 4. Confirmation of the effect of miR-2478 on the melanin biosynthesis process
우유 엑소좀에 들어있는 miR-2478이 직접적으로 멜라닌 생합성 과정에 관여하는지 알아보기 위해 우유 엑소좀을 B16F10 세포에 50 μg/ml 농도로 처리한 후, NC억제제 및 miR-2478 억제제을 각각 도입하여 미백 효과를 조사하였다. To determine whether miR-2478 contained in milk exosomes is directly involved in the melanin biosynthesis process, milk exosomes were treated with B16F10 cells at a concentration of 50 μg/ml, and then NC inhibitors and miR-2478 inhibitors were introduced respectively for whitening effect. was investigated.
구체적으로, NC억제제 및 miR-2478 억제제는 Genpharma 에서 구입하여 사용하였다. 각각 리포펙타민(lipofectamine)을 이용하여 50 nM 처리 농도로 형질 도입(transfection)하였으며, 24 시간 후 세포를 수집한 후 활성 및 발현 정도를 관찰하였다.Specifically, NC inhibitors and miR-2478 inhibitors were purchased from Genpharma and used. Each of lipofectamine (lipofectamine) was transfected at a treatment concentration of 50 nM, and the cells were collected 24 hours later, and the activity and expression level were observed.
4-1. 티로시나아제 효소 활성 억제4-1. Inhibition of tyrosinase enzyme activity
상기 실험예 3-1과 동일한 방법을 이용하여 티로시나아제 효소 억제 활성을 확인하였다.Tyrosinase enzyme inhibitory activity was confirmed using the same method as in Experimental Example 3-1.
그 결과, 도 7에 나타난 바와 같이, 우유 엑소좀을 B16F10 세포에 50 μg/ml 농도로 처리한 후, miR-2478 억제제를 도입한 B16F10 세포에서 대조군 및 NC억제제를 도입한 경우에 비해 티로시나아제 활성이 유의하게 증가함을 확인하였다. 이로부터 miR-2478에 의해 티로시나아제의 활성이 억제됨을 다시 확인하였다.As a result, as shown in FIG. 7, after the milk exosomes were treated at a concentration of 50 μg/ml in B16F10 cells, the miR-2478 inhibitor was introduced into the B16F10 cells compared to the case of introducing the control and NC inhibitor tyrosinase. It was confirmed that the activity significantly increased. From this, it was confirmed again that the activity of tyrosinase was inhibited by miR-2478.
4-2. 티로시나아제 및 MITF의 mRNA 발현 억제 확인4-2. Confirmation of mRNA expression inhibition of tyrosinase and MITF
상기 실험예 3-2와 동일한 방법을 이용하여 티로시나아제의 mRNA 발현을 확인하였다.The mRNA expression of tyrosinase was confirmed using the same method as in Experimental Example 3-2.
그 결과, 도 8에 나타난 바와 같이, miR-2478 억제제를 도입한 B16F10 세포에서 대조군 및 NC억제제를 도입한 경우에 비해 티로시나아제 및 MITF의 mRNA 발현이 유의하게 증가함을 확인하였다. 구체적으로, MITF의 mRNA 발현은 miR-2478을 억제한 세포에서 약 2.5배 증가하였으며, 티로시나아제의 mRNA 발현도 약 2배 증가하였다. 이로부터 miR-2478에 의해 티로시나아제 및 MITF의 mRNA 발현이 억제됨을 다시 확인하였다.As a result, as shown in FIG. 8 , it was confirmed that mRNA expression of tyrosinase and MITF was significantly increased in B16F10 cells into which the miR-2478 inhibitor was introduced, compared to the case where the control and NC inhibitors were introduced. Specifically, the mRNA expression of MITF was increased about 2.5-fold in miR-2478-inhibited cells, and the mRNA expression of tyrosinase was also increased about 2-fold. From this, it was confirmed again that the mRNA expression of tyrosinase and MITF was suppressed by miR-2478.
4-3. 티로시나아제 및 MITF의 단백질 발현 억제 확인4-3. Confirmation of inhibition of protein expression of tyrosinase and MITF
상기 실험예 3-3와 동일한 방법을 이용하여 티로시나아제 및 전사인자인 MITF의 단백질 발현을 확인하였다.Protein expression of tyrosinase and MITF, a transcription factor, was confirmed using the same method as in Experimental Example 3-3.
그 결과, 도 9에 나타난 바와 같이, miR-2478 억제제를 도입한 B16F10 세포에서 대조군 및 NC억제제를 도입한 경우에 비해 티로시나아제 및 MITF의 단백질 발현이 모두 증가된 것을 확인하였다. 또한 티로시나아제의 mRNA 발현이 유의하게 증가함을 확인하였다. 이로부터 miR-2478에 의해 티로시나아제의 발현이 억제됨을 다시 확인하였으며, miR-2478이 직접적으로 멜라닌 생합성과정에 관여한다는 것을 확인하였다.As a result, as shown in FIG. 9 , it was confirmed that both the protein expression of tyrosinase and MITF was increased in B16F10 cells into which the miR-2478 inhibitor was introduced, compared to the case where the control and NC inhibitor were introduced. In addition, it was confirmed that the mRNA expression of tyrosinase was significantly increased. From this, it was confirmed again that the expression of tyrosinase was suppressed by miR-2478, and it was confirmed that miR-2478 was directly involved in the melanin biosynthesis process.
4-4. 멜라닌 합성 억제4-4. Inhibition of melanin synthesis
상기 실험예 3-4와 동일한 방법을 이용하여 멜라닌 함량을 측정하였다. The melanin content was measured using the same method as in Experimental Example 3-4.
그 결과, 도 10에 나타난 바와 같이 miR-2478 억제제를 도입한 B16F10 세포에서 대조군 및 NC억제제를 도입한 경우에 비해 멜라닌 함량이 유의하게 증가함을 확인하였다. 이로부터 miR-2478에 의해 멜라닌 생합성이 억제됨을 다시 확인하였다.As a result, as shown in FIG. 10 , it was confirmed that the melanin content was significantly increased in the B16F10 cells into which the miR-2478 inhibitor was introduced, compared to the case where the control and the NC inhibitor were introduced. From this, it was confirmed again that melanin biosynthesis was inhibited by miR-2478.
실험예 5. miR-2478의 표적 유전자 확인Experimental Example 5. Identification of the target gene of miR-2478
5-1. miR-2478의 표적 유전자인 Rap1a 결합 부위 확인5-1. Confirmation of Rap1a binding site, a target gene of miR-2478
miR-2478에 의해 영향을 받는 세포 내의 유전자를 조사하였다. 구체적으로, microRNA의 표적 유전자와 결합 부위를 예측할 수 있는 프로그램인 타겟 스캔(Target Scan)을 이용하여 miR-2478에 결합하는 쥐와 인간에 존재하는 유전자를 조사하였으며, 그 결과 Rap1a(Ras-related protein Rap-1a)를 선발하였다.Genes in cells affected by miR-2478 were investigated. Specifically, the genes present in mice and humans that bind to miR-2478 were investigated using Target Scan, a program that can predict the target gene and binding site of microRNA. As a result, Rap1a (Ras-related protein) Rap-1a) was selected.
miR-2478이 Rap1a 유전자를 직접적으로 조절하는지 알아보기 위하여 먼저 miR-2478이 Rap1a에 결합하는 부위를 조사하였으며, Rap1a의 잠재적인 miR-2478 결합 부위를 돌연변이 시킨 플라스미드를 제작하였다. 구체적으로, miR-2478이 결합하는 부위를 함유한 Rap1a 단편을 pGL3-control 벡터에 삽입하여 재조합 벡터(pGL3-Rap1a-wt)를 구축하였다. QuickChange site-directed mutagenesis kit (Stratagene)을 활용하여 miR-2478에 결합하는 Rap1a 부위를 돌연변이 시켰다(pGL3-Rap1a-mut).To find out whether miR-2478 directly regulates the Rap1a gene, the site where miR-2478 binds to Rap1a was first investigated, and a plasmid in which the potential miR-2478 binding site of Rap1a was mutated was constructed. Specifically, a recombinant vector (pGL3-Rap1a-wt) was constructed by inserting the Rap1a fragment containing the miR-2478 binding site into the pGL3-control vector. Using the QuickChange site-directed mutagenesis kit (Stratagene), the Rap1a site binding to miR-2478 was mutated (pGL3-Rap1a-mut).
miR-2478의 Rap1a에 결합하는 부위에 관한 모식도는 도 11에 나타난 바와 같다.A schematic diagram of the miR-2478 binding site to Rap1a is shown in FIG. 11 .
5-2. Rap1a에 대한 miR-2478의 활성 조절 확인5-2. Confirmation of activity regulation of miR-2478 on Rap1a
miR-2478에 의한 Rap1a 조절 여부를 조사하기 위해 루시퍼라제 리포터 분석을 실시하였다.To investigate whether Rap1a is regulated by miR-2478, a luciferase reporter assay was performed.
구체적으로, Cos7 세포주를 3x104 cells/well로 분주하여 24 웰 플레이트에서 24시간 배양하였다. 구축한 pGL3-Rap1a (50ng)와 miR-2478 mimic (50nM) (Genepharm)를 lipofectamine 2000 (Invitrogen)을 사용하여 세포에 주입하여 형질전환을 유도하였다 각 시료의 형질전환 효율을 보정하기 위하여 pRL-TK 벡터를 함께 첨가하였다. 형질전환 세포는 2일간 배양한 후 TD-20/20 Luminometer (Turner Biosystems)를 사용하여 dual luciferase assay kit(promega)로 루시퍼라제 활성을 측정하였다. Specifically, the Cos7 cell line was seeded at 3x10 4 cells/well and cultured in a 24-well plate for 24 hours. Transformation was induced by injecting the constructed pGL3-Rap1a (50ng) and miR-2478 mimic (50nM) (Genepharm) into cells using lipofectamine 2000 (Invitrogen). In order to correct the transformation efficiency of each sample, pRL-TK Vector was added together. Transformed cells were cultured for 2 days and then luciferase activity was measured with a dual luciferase assay kit (promega) using a TD-20/20 Luminometer (Turner Biosystems).
그 결과, 도 12에 나타난 바와 같이 miR-2478을 도입한 세포에서 Rap1a가 약 75%로 유의하게 루시퍼라아제 활성이 감소하였고, miR-2478에 반응하는 Rap1a을 돌연변이시킨 플라스미드에서는 대조군과 차이가 없는 것을 확인하였다. As a result, as shown in FIG. 12, Rap1a was significantly reduced in luciferase activity by about 75% in miR-2478-introduced cells, and in the plasmid mutated with Rap1a responding to miR-2478, there was no difference from the control confirmed that.
5-3. Rap1a에 대한 miR-2478의 유전자 발현 조절 확인5-3. Confirmation of gene expression regulation of miR-2478 for Rap1a
miR-2478을 B16F10 세포 내에 도입 후, 상기 실험예 3-2와 동일한 방법을 이용하여 Rap1a의 mRNA 발현을 확인하였다.After miR-2478 was introduced into B16F10 cells, mRNA expression of Rap1a was confirmed using the same method as in Experimental Example 3-2.
구체적으로, B16F10세포에서 miR-2478 mimic의 형질주입(transfection)은 lipofectamine 2000 (Invitrogen)을 이용하여 수행하였다. microRNA mimic과 대조군(Genpharma)은 최종적으로 50 nM을 사용하였다. 상기 세포를 24시간 배양한 후, 수거하여Tri 시약(Bioline)을 반응시켜 total RNA를 분리하였다. 역전사 반응을 실시하기 위하여 1 μg RNA에 dNTP, M-MLV reverse-transcriptase (Promega) 등을 첨가한 다음 37℃에서 1시간 반응시켜 cDNA를 합성하였다. Rap1a mRNA 발현을 측정하기 위해 SYBR Green PCR Master mix(Bioline)를 이용하여 실시간 중합효소 연쇄반응을 수행하였다. 특정 유전자를 증폭하기 위한 PCR 조건은 다음과 같이 시행하였다. 95℃에서 10분간 반응시킨 다음, 15초 95℃, 15초 60℃, 72℃ 15초를 한 주기로 하여 40 cycles 동안 증폭하였다. 표적 유전자 mRNA 발현량은 actin 발현량에 대한 상대적인 발현량으로 보정하였다. 사용된 프라이머는 하기 표 3에 나타난 바와 같다. Specifically, transfection of miR-2478 mimic in B16F10 cells was performed using lipofectamine 2000 (Invitrogen). 50 nM was finally used for microRNA mimic and control (Genpharma). After culturing the cells for 24 hours, they were collected and reacted with Tri reagent (Bioline) to isolate total RNA. In order to perform the reverse transcription reaction, dNTP, M-MLV reverse-transcriptase (Promega), etc. were added to 1 μg RNA, and then reacted at 37° C. for 1 hour to synthesize cDNA. Real-time polymerase chain reaction was performed using SYBR Green PCR Master mix (Bioline) to measure Rap1a mRNA expression. PCR conditions for amplifying a specific gene were performed as follows. After reacting at 95°C for 10 minutes, amplification was carried out for 40 cycles with 15 seconds 95°C, 15
그 결과, 도 13에 나타난 바와 같이, miR-2478을 도입한 세포에서 Rap1a의 mRNA 발현이 감소함을 확인하였다. 이는 본 발명이 우유 엑소좀 내의 miR-2478이 Rap1a의 유전자 발현 감소에 영향을 미침을 나타내는 것이다.As a result, as shown in FIG. 13 , it was confirmed that the mRNA expression of Rap1a was decreased in the cells into which miR-2478 was introduced. This indicates that the present invention affects miR-2478 in milk exosomes on the reduction of Rap1a gene expression.
5-4. Rap1a에 대한 miR-2478의 단벡질 발현 조절 확인5-4. Confirmation of protein expression regulation of miR-2478 for Rap1a
miR-2478을 B16F10 세포 내에 도입 후, 상기 실험예 3-3과 동일한 방법을 이용하여 Rap1a의 단백질 발현을 확인하였다. 또한, 아무것도 도입하지 않은 대조군과 함께 Rap1a에 반응하지 않는 let-7i를 도입한 세포를 miR-2478을 도입한 세포와 비교하였다. 도입한 let-7i 서열은 다음과 같다.After miR-2478 was introduced into B16F10 cells, Rap1a protein expression was confirmed using the same method as in Experimental Example 3-3. In addition, cells introduced with let-7i that did not respond to Rap1a together with a control group to which nothing was introduced were compared with cells introduced with miR-2478. The introduced let-7i sequence is as follows.
B16F10 세포주를 6 웰 플레이트에 2×105 cells/well로 부착시킨 후, 50 nM 의 miR-2478 mimic(Genpharma)을 lipofectamine 2000(Invitrogen)이용하여 형질 주입하였다. 24시간 후, 상기 세포를 수거하였으며, 수거한 세포를 16,500 g에서 15분간 원심 분리하여 상층액을 취하였다. 단백질 농도는 BSA kit (Bio-rad)를 이용하여 정량하였다. 추출한 단백질들은 1% SDS 폴리아크릴마이드 젤을 이용하여 분리하였다. 상기 분리한 단백질은 니트로셀룰로오스막(GE Health care)으로 옮긴 후, 항체 비특이적 결합을 방지하기 위하여 1시간 동안 5% 탈지분유에 반응시켰다. Rap1a (Novus)의 1차 항체를 1:1000의 비율로 희석하여 4℃에서 12 - 18시간 니크로셀룰로오스막에 결합시켰다. 이후, HRP-tagged anti-rabbit 항체와 결합시켜 상온에서 30분 동안 반응시켰다. ECL kit(Santa Cruz Inc)를 사용하여 단백질 밴드를 확인하였다. After attaching the B16F10 cell line to a 6-well plate at 2×10 5 cells/well, 50 nM miR-2478 mimic (Genpharma) was transfected using lipofectamine 2000 (Invitrogen). After 24 hours, the cells were harvested, and the collected cells were centrifuged at 16,500 g for 15 minutes to obtain a supernatant. Protein concentration was quantified using a BSA kit (Bio-rad). The extracted proteins were separated using 1% SDS polyacrylamide gel. The separated protein was transferred to a nitrocellulose membrane (GE Health care), and then reacted with 5% skim milk for 1 hour to prevent antibody non-specific binding. The primary antibody of Rap1a (Novus) was diluted at a ratio of 1:1000 and bound to a nitrocellulose membrane at 4°C for 12 to 18 hours. Then, it was combined with an HRP-tagged anti-rabbit antibody and reacted at room temperature for 30 minutes. Protein bands were identified using an ECL kit (Santa Cruz Inc).
그 결과, 도 14에 나타난 바와 같이 대조군과 Rap1a에 반응하지 않는 let-7i를 도입한 세포에서는 Rap1a 단백질 발현 차이가 없었지만, miR-2478을 도입한 세포에서는 Rap1a 단백질 발현이 감소하였다. 상기와 같은 결과는 miR-2478이 Rap1a 유전자 발현을 직접적으로 억제함을 나타내는 것이다.As a result, as shown in FIG. 14 , there was no difference in Rap1a protein expression between the control group and the cells introduced with let-7i that did not respond to Rap1a, but Rap1a protein expression was decreased in the cells introduced with miR-2478. The above results indicate that miR-2478 directly inhibits Rap1a gene expression.
실험예 6. miR-2478의 멜라닌 생성 조절 경로 확인Experimental Example 6. Confirmation of the melanogenesis regulation pathway of miR-2478
본 발명의 우유 엑소좀이 어떤 경로를 통해 멜라닌 생성을 조절하는지 확인하였다. It was confirmed through which pathway the milk exosomes of the present invention regulate melanin production.
구체적으로, B16F10 세포에 Rap1a siRNA를 도입 후 웨스턴블롯을 이용하여 phospho-Gsk3β, Gsk3β, pAkt 및 Akt의 단백질 발현을 측정하였다. Specifically, protein expression of phospho-Gsk3β, Gsk3β, pAkt and Akt was measured using western blot after Rap1a siRNA was introduced into B16F10 cells.
B16F10세포주를 6 웰 플레이트에 2×105 cells/well개의 세포로 부착시킨 후, 50 nM 의 Rap1a-siRNA(Genpharma)을 lipofectamine 2000(Invitrogen)이용하여 형질 주입하였다. 24시간 후, 상기 세포를 수거하였으며, 수거한 세포는 15,600 g에서 15분간 원심 분리하여 그 상층액을 취한 후, BSA kit (Bio-rad)를 이용하여 단백질을 정량하였다. 추출한 단백질은10% SDS 폴리아크릴마이드 젤을 이용하여 분리하였다. 상기 분리한 단백질은 니트로셀룰로오스막(GE Health care)으로 옮긴 후, 항체의 비특이적 결합을 방지하기 위하여 1시간 동안 5% 탈지분유에 반응하였다. GSK3β(cell signaling), phospho-GSK3β (cell signaling) Akt(abcam), phosphor-Akt (abcam)의 1차 항체를 1:1000의 비율로 희석하여 4 ℃에서 12 - 18시간 니트로셀룰로오스막에 결합시켰다. 이후, HRP-tagged anti-rabbit 항체와 결합시켜 상온에서 30분 동안 반응시켰다. ECL kit(Santa Cruz Inc)를 사용하여 단백질 밴드를 확인하였다. After attaching the B16F10 cell line to a 6-well plate at 2×10 5 cells/well, 50 nM of Rap1a-siRNA (Genpharma) was transfected with lipofectamine 2000 (Invitrogen). After 24 hours, the cells were harvested, and the collected cells were centrifuged at 15,600 g for 15 minutes to obtain a supernatant, and then protein was quantified using a BSA kit (Bio-rad). The extracted protein was separated using 10% SDS polyacrylamide gel. The separated protein was transferred to a nitrocellulose membrane (GE Health care), and then reacted with 5% skim milk powder for 1 hour to prevent non-specific binding of the antibody. Primary antibodies of GSK3β (cell signaling), phospho-GSK3β (cell signaling) Akt (abcam) and phosphor-Akt (abcam) were diluted at a ratio of 1:1000 and bound to a nitrocellulose membrane at 4°C for 12 to 18 hours. . Then, it was combined with an HRP-tagged anti-rabbit antibody and reacted at room temperature for 30 minutes. Protein bands were identified using an ECL kit (Santa Cruz Inc).
이후, B16F10 세포에 우유 엑소좀을 각각 20 μg/ml 및50 μg/ml을 24시간 동안 처리한 후 웨스턴블롯을 이용하여 phospho-Gsk3β, Gsk3β, pAkt 및 Akt의 단백질 발현을 측정하였다.Thereafter, B16F10 cells were treated with 20 μg/ml and 50 μg/ml of milk exosomes, respectively, for 24 hours, and then protein expression of phospho-Gsk3β, Gsk3β, pAkt and Akt was measured using Western blot.
그 결과, 도 15A에 나타난 바와 같이 우유 엑소좀을 20 μg/ml, 50 μg/ml 각각 처리한 세포에서도 농도 의존적으로 Akt가 활성화됨에 따라 Gsk3β가 불활성화되는 것을 확인하였다. As a result, as shown in FIG. 15A , it was confirmed that Gsk3β was inactivated as Akt was activated in a concentration-dependent manner even in cells treated with milk exosomes at 20 μg/ml and 50 μg/ml, respectively.
또한, B16F10 세포에 miR-2478을 도입한 후, 웨스턴블롯을 이용하여 phospho-Gsk3βGsk3β및 Akt의 단백질 발현을 측정하였다. 웨스턴블롯 방법은 상기와 같다. In addition, after miR-2478 was introduced into B16F10 cells, protein expression of phospho-Gsk3βGsk3β and Akt was measured using Western blot. The Western blot method is as described above.
그 결과, 도 15B에 나타난 바와 같이 miR-2478을 처리하였을 때에도 마찬가지로 Akt가 활성화됨에 따라 Gsk3β가 불활성화되는 것을 확인하였다.As a result, as shown in FIG. 15B , it was confirmed that Gsk3β was inactivated as Akt was also activated when miR-2478 was treated.
나아가, Akt-Gsk3β경로가 miR-2478에 의해 멜라닌 생성을 조절하는 것인지 확인하기 위해 본 발명의 우유 엑소좀 처리 후 miR-2478 억제제를 도입하여 Akt-Gsk3β경로에 변화가 있는지 조사하였다. 웨스턴블롯을 이용하여 phospho-Gsk3βGsk3β및 Akt의 단백질 발현을 측정하였으며, 웨스턴블롯 방법은 상기 기재된 바와 같다. Furthermore, in order to confirm whether the Akt-Gsk3β pathway regulates melanogenesis by miR-2478, a miR-2478 inhibitor was introduced after treatment with milk exosomes of the present invention to investigate whether there is a change in the Akt-Gsk3β pathway. The protein expression of phospho-Gsk3βGsk3β and Akt was measured using western blot, and the western blot method was as described above.
그 결과, 도 15C에 나타난 바와 같이 miR-2478 억제제를 도입한 세포에서 대조군에 비해 Akt 인산화가 감소하였으며, phospho-Gsk3β (ser9)이 감소하였다.As a result, as shown in FIG. 15C, Akt phosphorylation was decreased and phospho-Gsk3β (ser9) was decreased in the cells introduced with the miR-2478 inhibitor compared to the control group.
상기와 같은 결과로부터 본 발명의 우유 엑소좀 내의 miR-2478이 Rap1a를 억제시킴으로써 Akt-Gsk3β경로를 통해 멜라닌 생성을 억제하여 미백 효과를 나타냄을 확인하였다. 이에 따라, 본 발명의 우유 엑소좀에 함유된 성분인 miR-2478은 피부 미백용으로 널리 활용될 수 있다. From the above results, it was confirmed that miR-2478 in the milk exosome of the present invention inhibits Rap1a, thereby inhibiting melanin production through the Akt-Gsk3β pathway, thereby exhibiting a whitening effect. Accordingly, miR-2478, a component contained in the milk exosome of the present invention, can be widely used for skin whitening.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다. The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.
<110> University-Industry Cooperation Group of Kyung Hee University <120> MELANIN PRODUCTION INHIBITING COMPOSITION COMPRISING microRNA-2478 <130> 19PP31045 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> miR-2478 <400> 1 guaucccacu ucugacacca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TYR Forward <400> 2 cgagcctgtg cctcctctaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TYR Reverse <400> 3 ccaggactca cggtcatcca 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MITF Forward <400> 4 caaatggcaa atacgttacc cg 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MITF Reverse <400> 5 caatgctctt gcttcagact ct 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Rap1a Forward <400> 6 caggaaccga gcaatttaca gc 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Rap1a Reverse <400> 7 tgttctttgc caactacccg t 21 <210> 8 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7i miRNA <400> 8 ugagguagua guuugugcug uu 22 <110> University-Industry Cooperation Group of Kyung Hee University <120> MELANIN PRODUCTION INHIBITING COMPOSITION COMPRISING microRNA-2478 <130> 19PP31045 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> miR-2478 <400> 1 guaucccacu ucugacacca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TYR Forward <400> 2 cgagcctgtg cctcctctaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TYR Reverse <400> 3 ccaggactca cggtcatcca 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MITF Forward <400> 4 caaatggcaa atacgttacc cg 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MITF Reverse <400> 5 caatgctctt gcttcagact ct 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Rap1a Forward <400> 6 caggaaccga gcaatttaca gc 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Rap1a Reverse <400> 7 tgttctttgc caactacccg t 21 <210> 8 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7i miRNA <400> 8 ugagguagua guuugugcug uu 22
Claims (13)
상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것인, 멜라닌 생성 억제용 조성물.According to claim 1,
The miR-2478 is composed of the nucleic acid sequence of SEQ ID NO: 1, a composition for inhibiting melanin production.
상기 miR-2478은 우유 엑소좀으로부터 분리된 것인, 멜라닌 생성 억제용 조성물.According to claim 1,
The miR-2478 is a composition for inhibiting melanin production, which is isolated from milk exosomes.
상기 miR-2478은 Rap1a(Ras-related protein Rap-1a)의 발현을 억제하는 것인, 멜라닌 생성 억제용 조성물.According to claim 1,
The miR-2478 is to suppress the expression of Rap1a (Ras-related protein Rap-1a), a composition for inhibiting melanin production.
상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것인, 피부 색소 침착 질환의 예방 또는 치료용 약학적 조성물.6. The method of claim 5,
The miR-2478 is a pharmaceutical composition for the prevention or treatment of skin pigmentation diseases, which consists of the nucleic acid sequence of SEQ ID NO: 1.
상기 피부 색소 침착 질환은 기미, 주근깨, 흑색점, 모반 및 피부암으로 이루어진 군에서 선택되는 어느 하나 이상인 것인, 피부 색소 침착 질환의 예방 또는 치료용 약학적 조성물.6. The method of claim 5,
The skin pigmentation disease is any one or more selected from the group consisting of melasma, freckles, black spots, nevus and skin cancer, a pharmaceutical composition for preventing or treating skin pigmentation disease.
상기 화장료 조성물은 피부 미백용인 것인, 화장료 조성물.9. The method of claim 8,
The cosmetic composition is for skin whitening, the cosmetic composition.
상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것인, 화장료 조성물.9. The method of claim 8,
The miR-2478 is a cosmetic composition consisting of the nucleic acid sequence of SEQ ID NO: 1.
상기 miR-2478은 서열번호 1의 핵산서열로 이루어진 것인, 식품 조성물.12. The method of claim 11,
The miR-2478 is a food composition consisting of the nucleic acid sequence of SEQ ID NO: 1.
상기 식품 조성물은 미백용인 것인, 식품 조성물.12. The method of claim 11,
The food composition is for whitening, the food composition.
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| KR101665753B1 (en) | 2013-07-12 | 2016-10-12 | 동국대학교 산학협력단 | Composition comprising miRNA 675 for suppressing production of melanogenesis |
| US20180343882A1 (en) * | 2015-11-29 | 2018-12-06 | Hadasit Medical Research Services And Development Ltd. | Supplementation of milk formulas with microvesicles isolated from milk |
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