KR100432797B1 - Cosmetics for prevention of replicative senescence of human keratinocytes containing retinoic acid as active ingredients - Google Patents
Cosmetics for prevention of replicative senescence of human keratinocytes containing retinoic acid as active ingredients Download PDFInfo
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- KR100432797B1 KR100432797B1 KR10-2001-0046911A KR20010046911A KR100432797B1 KR 100432797 B1 KR100432797 B1 KR 100432797B1 KR 20010046911 A KR20010046911 A KR 20010046911A KR 100432797 B1 KR100432797 B1 KR 100432797B1
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- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 150000002266 vitamin A derivatives Chemical class 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/36—Carboxylic acids; Salts or anhydrides thereof
- A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/203—Retinoic acids ; Salts thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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Abstract
본 발명은 레티노산 화합물(Retinoic Acid)을 유효성분으로 함유하는 인간 정상 피부의 복제 노화 예방용 화장품에 관한 것으로서, 구체적으로는 트랜스 레티노산, 3,4-디디하이드로 레티노산 및 9-시스 레티노산을 포함하는 레티노산 화합물을 유효성분으로 하며, 본 발명의 레티노산 화합물은 인간 점막 상피세포 및 인간 피부 상피세포의 수명을 연장시킬 뿐 아니라 복제노화(replicative senescence)를 억제시켜 노화를 방지하고 상기 상피세포의 노화에 따른 다양한 질환의 치료제 또는 예방제로 사용할 수 있다.The present invention relates to a cosmetic for preventing replication aging of human normal skin containing a retinoic acid as an active ingredient, specifically, trans retinoic acid, 3,4-didihydroretinoic acid and 9-cis retinoic acid A retinoic acid compound comprising an active ingredient, the retinoic acid compound of the present invention not only prolongs the lifespan of human mucosal epithelial cells and human skin epithelial cells, but also inhibits senescence by preventing replication senescence and prevents the aging of the epithelium. It can be used as a therapeutic or prophylactic agent for various diseases according to aging of cells.
Description
본 발명은 레티노산 화합물을 유효성분으로 함유하는 인간 상피세포의 노화방지제에 관한 것으로서, 구체적으로는 트랜스 레티노산, 3,4-디디하이드로레티노산 및 9-시스 레티노산을 포함하는 레티노산 화합물을 유효성분으로 하며, 인간 점막 상피세포 및 인간 피부 상피세포의 수명을 연장시키고 노화를 억제하여 구강 내 창상 및 발치창, 외상성 궤양(traumatic ulcer), 구각증(Angular cheilosis) 및 구강내 화상의 다양한 구강 질환을 치료ㆍ예방하고, 또한 피부 내 창상 및 피부 노화를 방지하고 상기 피부 노화를 방지하는 효과로써 화장품 성분으로 사용할 수 있다.The present invention relates to an anti-aging agent of human epithelial cells containing a retinoic acid compound as an active ingredient, specifically, a retinoic acid compound containing trans retinoic acid, 3,4-didihydroretinoic acid and 9-cis retinoic acid. As an active ingredient, it prolongs the lifespan of human mucosal epithelial cells and human skin epithelial cells and inhibits aging, thereby preventing oral wounds and anterior wounds, traumatic ulcers, Angular cheilosis and various oral burns. It can be used as a cosmetic ingredient with the effect of treating and preventing diseases, and also preventing skin wounds and skin aging and preventing the skin aging.
복제노화(replicative senescence) 현상은 노화(organismic aging)의 근본적인 원인으로서(Wantanabe Y. et al.,Oncogene, 1997,14, 2025-2032), 노화된 세포들이 생체내에 축적되고 이들 세포들로부터 발현되는 표현형이 변경됨으로써 여러 가지 노화와 관련된 병리의 원인이 된다고 추정되고 있다(Dimri G.D. et al.,Proc. Natl. Acad. Sci., 1995,92, 9363-9367).Replicative senescence is a fundamental cause of aging (Wantanabe Y. et al., Oncogene , 1997, 14 , 2025-2032), in which aged cells accumulate in vivo and are expressed from these cells. Changes in the phenotype are presumed to be a cause of various aging-related pathologies (Dimri GD et al., Proc. Natl. Acad. Sci. , 1995, 92 , 9363-9367).
복제노화(replicative senescence)는 세포분열(cell division)이 누적되어 일어나는 것으로, 이러한 사실은 증식(proliferation)이 '세포분열 시계(mitoticclock)'(Bodnor A.G. et al.,Science, 1998,279, 349-352)에 의해 제한된다는 것을 암시하는 것이다. 이러한 현상은 단백질 및 RNA 합성과정에서 발생하는 불규칙적인 손상(damage)이나 변이(mutation)의 축적 또는 유전적으로 프로그램화된 어떠한 과정에 의해 나타나는 것이라는 보고가 있었다(Sugawara O. et al.,Science, 1990,247, 707-710).Replicative senescence is the accumulation of cell division, and this fact suggests that proliferation is a 'mitoticclock' (Bodnor AG et al., Science , 1998, 279 , 349-). 352). This phenomenon has been reported to be caused by the accumulation of irregular damage or mutations in protein and RNA synthesis or by any genetically programmed process (Sugawara O. et al., Science , 1990). , 247 , 707-710).
또한, 세포주기 조절단백질(cell cycle regulatory proteins)과 항암단백질(tumor reppressors)이 상기 복제노화에 영향을 미친다는 보고가 있었는데, 정상(wild-type) p53 및 p21WAF1/CIP1단백질의 발현 수준의 증가가 몇몇 배양세포의 증식능력 상실을 유도하며 이로써 세포 노화의 원인이 됨이 확인된 바 있다(Brown J.P. et al.,Science, 1997, 277, 831-834; Gallimore P.H. et al.,Cell Growth Differ. 1997,8, 763-771; Sugrue M. Mm. et al.,Proc. Natl. Acad. Sci., 1997,94, 9648-9653).In addition, cell cycle regulatory proteins and tumor reppressors have been reported to affect the replication aging, wild-type p53 and p21WAF1 / CIP1Increasing the level of protein expression has been shown to lead to the loss of proliferation of some cultured cells, thereby causing cell aging (Brown J.P. et al.,Science, 1997, 277831-834; Gallimore P.H. et al.,Cell growth differ. 1997,8, 763-771; Sugrue M. Mm. et al.,Proc. Natl. Acad. Sci., 1997,94, 9648-9653).
이외에도, G1 Cdk(G1 cyclin-dependent kinase) 억제자의 하나인 p16INK4A단백질이 다양한 형태의 세포에서 세포의 노화에 관여한다는 것이 보고되었다(Alcorta D.A. et al.,Proc. Notl. Acad. Sci., 1996,93, 13742-13747; Loughran O. et al.,Oncogene, 1996,13, 561-568; Reznikoff C. A. et al.,Cancer Res., 1996,56, 2886-2890; Palmero I. et al.,Oncogene, 1997,15, 495-503; Uhrbom L. et al.,Oncogene, 1997,15, 505-514). 특히, 노화세포에서 p16INK4A는 Cdk4와 Cdk6 모두에 복합체를 형성함이 확인되었으며, 이러한 결과에기초하여 p16INK4A단백질의 발현수준을 증가시키는 조절현상이 세포의 노화과정 중 성장이 정지되는 최종단계에 나타나는 가장 중요한 분자적 변화로 인정되었다. 또한, 노화된 인간 섬유아세포(fibroblast) 내 p16INK4A단백질의 발현수준이 활발히 증식중인 섬유아세포에 비해 증가되었음이 확인되었으며, 불멸화된 세포주(immortal cell line)에서 p16INK4A유전자는 변이되거나 결실되어 있음이 확인되었다(Vojta P. et al.,Biochem. Biophys. Acta., 1995,1242, 29-41).In addition, p16, one of the G1 cyclin-dependent kinase (Gdk) inhibitors,INK4AIt has been reported that proteins are involved in cellular aging in various types of cells (Alcorta D.A. et al.,Proc. Notl. Acad. Sci., 1996,93, 13742-13747; Loughran O. et al.,Oncogene, 1996,13, 561-568; Reznikoff C. A. et al.,Cancer res., 1996,56, 2886-2890; Palmero I. et al.,Oncogene, 1997,15, 495-503; Uhrbom L. et al.,Oncogene, 1997,15, 505-514). Especially in senescent cellsINK4AWas found to form complexes on both Cdk4 and Cdk6, based on these results.INK4ARegulatory phenomena that increase the level of protein expression were recognized as the most important molecular changes in the final stages of cell growth cessation. In addition, p16 in aged human fibroblastsINK4AIt was confirmed that the expression level of the protein was increased compared to fibroblasts which are actively proliferating. P16 in the immortal cell lineINK4AGenes have been identified as mutated or deleted (Vojta P. et al.,Biochem. Biophys. Acta., 1995,1242, 29-41).
종양억제 유전자의 유전자 산물인 pRb 역시 인간 섬유아세포의 노화에 관여하는 것으로 알려져 있으며, pRb에 의해 p16INK4A단백질의 발현수준이 전사단계에서 음성적으로 조절되며, pRb가 결여된 세포내에서 사이클린 D/Cdk4 및 사이클린 D/Cdk6가 형성되지 않으며 노화 세포에서 모든 Cdk가 과발현된 p16INK4A와 복합체를 형성한다는 것이 보고되면서 pRb가 노화에 관여함이 알려져 있다.PRb, a gene product of tumor suppressor genes, is also known to be involved in aging of human fibroblasts. PRb negatively regulates the expression level of p16 INK4A protein in the transcriptional phase and cyclin D / Cdk4 in cells lacking pRb. And it has been reported that cyclin D / Cdk6 is not formed and that all Cdks form complexes with p16 INK4A overexpressed in senescent cells and pRb is involved in aging.
또한, 포스파티딜이노시톨 특이적 포스포리파아제 C(phosphatidylinositol- specific phospholipase C)가 활성화됨으로써 특정 유형의 세포노화에 관여한다는 것이 보고된 바 있다(Choudhury G.G. et al.,FEBS Lett., 1991,293, 211-214).In addition, it has been reported that phosphatidylinositol-specific phospholipase C is involved in the activation of certain types of cell aging by activating phosphatidylinositol-specific phospholipase C (Choudhury GG et al., FEBS Lett. , 1991, 293 , 211-21). 214).
한편, 동물 상피세포는 제한된 증식능력을 갖고 있음이 알려져 있는데(Kang M.K. et al.,Cell Growth Differ., 1998,9, 85-95; Min B.M. et al., Exp. Cell Res, 1999,249, 377-385; Dimri G.D. et al.,Proc. Natl. Acad. Sci., 1995,92,9363-9367), 제한된 횟수의 세포분열을 거친 후에는 더 이상 세포분열을 행하지 아니하는 복제노화 단계로 들어가게 된다고 알려져 있다.Animal epithelial cells are known to have limited proliferative capacity (Kang MK et al., Cell Growth Differ. , 1998, 9 , 85-95; Min BM et al., Exp. Cell Res, 1999, 249 , 377-385; entered into Dimri GD et al, Proc Natl Acad Sci, 1995, 92, 9363-9367), after passing through the cell division of a limited number of steps to replicate the aging do not do any further cell division..... It is known.
상피세포의 배양을 통해 관찰한 결과, 상피세포의 복제노화에는 몇가지 뚜렷한 특징이 확인되었다. 노화된 세포들은 더 이상의 성장을 멈추고 생리학적 유사분열물질(mitogen)의 자극이 있더라도 세포주기의 S기(S phage)로 들어가지 않으며, 세포주기의 진행에 필요한 몇몇 유전자들의 발현이 억제된다. 그러나, 노화된 세포들은 여전히 대사활성을 유지하고 있으며, 일정시기 동안은 단백질 또는 RNA 합성능력을 보유하고 있음이 보고되었다(Dimri G.D. et al.,Proc. Natl. Acad. Sci., 1995,92, 9363-9367; Saunders N.A. et al.,Biochem. Biophy. Res. Commun., 1993,197, 46-54).As a result of culturing epithelial cells, some distinctive features were found in the replication aging of epithelial cells. Aged cells stop further growth and enter the S phase of the cell cycle even if they are stimulated by physiological mitogen, and expression of some genes necessary for the progression of the cell cycle is suppressed. However, it has been reported that aged cells still maintain metabolic activity and have protein or RNA synthesis capacity for some time (Dimri GD et al., Proc. Natl. Acad. Sci. , 1995, 92 , 9363-9367; Saunders NA et al., Biochem.Biophy.Res.Commun. , 1993, 197 , 46-54).
최근, 활발히 증식하는 인간 정상 상피세포에서는 텔로머레이즈(telomerase)의 활성이 발견되나, 노화된 세포에서는 그 활성이 현저히 감소됨이 확인되었으며, 이로부터 텔로머레이즈 활성의 크기와 세포의 노화간에 양성적 관계가 있음이 확인되었다(Harle-Bachor C. et al.,Proc. Natl. Acad. Sci., 1996,93, 6476-6481).Recently, telomerase activity was found in actively proliferating human normal epithelial cells, but it was confirmed that its activity is significantly reduced in aged cells. From this, it is positive between the size of telomerase activity and the aging of cells. Relationship was found (Harle-Bachor C. et al., Proc. Natl. Acad. Sci. , 1996, 93 , 6476-6481).
상기의 인간 상피세포는 크게 인간 점막 상피세포 및 인간 피부 상피세포로 나누어지며, 인간 점막 상피세포로 구성된 상피조직 및 인간 피부 상피세포로 구성된 상피조직은 기능상 우리 몸의 외부에서 피부의 표면을 덮어주는 역할을 하는 전형적인 보호상피이나, 형태학적으로는 큰 차이점을 갖고 있다. 즉, 인간 피부 상피조직은 잘 발달된 두꺼운 각화세포층 (cornified cell layer)을 갖고 있으나, 인간 점막 상피조직은 인간 피부 상피조직과 비교시 매우 얇은 각화세포층을 갖거나혹은 각화세포층이 없어 [예, 치은낭 상피 (oral sulcular epithelium) 및 경계 상피 (junctional epithelium)] 두 상피조직은 차이점을 가지고 있다. 형태학적 차이점 이외에도 발현되는 단백질이나 화학적 성질이 달라 두 인간 상피조직은 상당한 차이점을 보이고 있다.The human epithelial cells are largely divided into human mucosal epithelial cells and human skin epithelial cells, epithelial tissue composed of human mucosal epithelial cells and epithelial tissue composed of human skin epithelial cells are functionally covering the surface of the skin from the outside of our body. Typical protective epithelium that plays a role, but with great morphological differences. In other words, human skin epithelial tissue has a well-developed thick cornified cell layer, but human mucosal epithelial tissue has a very thin keratinocyte layer or no keratinocyte layer compared to human skin epithelial tissue [eg, gingiva Oral sulcular epithelium and junctional epithelium] The two epithelial tissues differ. In addition to the morphological differences, there are significant differences between the two human epithelial tissues due to different proteins and chemical properties.
레티노산(retinoic acid)은 비타민 A의 유도체로서, 배발생(embryogenesis), 생식(reproduction), 시각(vision), 세포의 성장 및 수많은 성인조직의 정상적인 분화에 있어 기본적인 역할을 한다고 알려져 있다(Sauret J.H. et al.,Horm. Res., 1995,43, 89-92).Retinoic acid is a derivative of vitamin A and is known to play a fundamental role in embryoogenesis, reproduction, vision, cell growth and normal differentiation of numerous adult tissues (Sauret JH). et al., Horm. Res. , 1995, 43 , 89-92).
레티노산을 인간 피부에 국소적으로 투여하면, 햇빛에 의해 유도된 미세하고 깊은 주름 및 색소 침착 등의 피부 노화를 개선시킬 수 있으며(Varani J. et al.,Am. J. Pathol., 1990,136, 1275-1281), 또한 세포외기질의 파괴와 관련된 많은 메탈로프로테아제(metalloproteinase)의 유도를 억제한다는 것이 확인되었다(Varani J. et al.,J. Invest. Dermatol. Symp. Proc., 1998,3, 57-60). 레티노산은 또한 상피세포의 분화 감소, 글라이코스아미노글라이칸(glycoamino-glycan) 유사 물질의 표피내 침착, 진피내 세포성의 증가, 새로운 세포외기질의 합성 및 섬유아세포의 활성화로 표피 비후에 영향을 준다고 보고된 바 있다(Varani J. et al.,Skin Pharmacol., 1991,4, 254-261).Topical administration of retinoic acid to human skin can improve skin aging, such as fine, deep wrinkles and pigmentation induced by sunlight (Varani J. et al., Am. J. Pathol. , 1990, 136 , 1275-1281), as well as inhibiting the induction of many metalloproteinases associated with the destruction of extracellular matrix (Varani J. et al., J. Invest. Dermatol. Symp. Proc. , 1998, 3 , 57-60). Retinoic acid is also reported to affect epidermal thickening by decreasing epidermal differentiation, intradermal deposition of glycosamino-glycan-like substances, increased intradermal cellularity, synthesis of new extracellular matrix and activation of fibroblasts. (Varani J. et al., Skin Pharmacol ., 1991, 4 , 254-261).
하지만, 레티노산이 인간 상피세포의 증식 및 복제노화를 조절하는 유전자의발현에 영향을 미침으로써 인간 상피세포의 노화방지에 효과를 갖는다는 것에 대해서는 이제까지 보고된 바 없었다.However, it has never been reported that retinoic acid has an effect on anti-aging of human epithelial cells by affecting the expression of genes that regulate the proliferation and replication aging of human epithelial cells.
이에, 본 발명자들은 레티노산이 세포증식 및/또는 복제 노화를 조절하는 유전자의 발현 및 단백질의 활성에 영향을 줌으로써 인간 점막 상피세포 및 인간 피부 상피세포의 수명을 연장시킬 수 있음을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors confirmed that retinoic acid can extend the lifespan of human mucosal epithelial cells and human skin epithelial cells by affecting the expression of genes and the activity of proteins that regulate cell proliferation and / or replication aging. Completed.
본 발명의 목적은 레티노산 화합물을 유효성분으로 함유하는 인간 상피세포의 노화방지제을 제공하는 것이다.An object of the present invention is to provide an anti-aging agent of human epithelial cells containing a retinoic acid compound as an active ingredient.
본 발명의 다른 목적은 레티노산 화합물을 유효성분으로 함유하는 인간 점막 상피세포 및 인간 피부 상피세포의 노화방지제를 제공하는 것이다.Another object of the present invention is to provide an anti-aging agent for human mucosal epithelial cells and human skin epithelial cells containing a retinoic acid compound as an active ingredient.
본 발명의 또 다른 목적은 레티노산 화합물을 유효성분으로 함유하여 구강 내 창상 및 발치창, 외상성 궤양(traumatic ulcer), 구각증(Angular cheilosis) 및 구강내 화상의 다양한 구강 질환을 치료ㆍ예방에 유용하게 사용할 수 있는 용도를 제공한다.Another object of the present invention is to contain a retinoic acid compound as an active ingredient, which is useful for treating and preventing various oral diseases such as oral wounds and ankles, traumatic ulcers, Angular cheilosis, and intraoral burns. Provides a usable use.
또한, 본 발명의 다른 목적은 레티노산 화합물을 유효성분으로 함유하여 피부 내 창상 및 피부 노화를 방지하고, 상기 피부 노화를 방지하는 효과로써 화장품 성분으로 사용할 수 있는 용도를 제공한다.In addition, another object of the present invention is to provide a use that can be used as a cosmetic ingredient as an effect to prevent the skin aging and the skin aging, the skin containing the retinoic acid compound as an active ingredient.
도 1a는 본 발명의 트랜스 레티노산(all-trans retinoic acid)이 인간 정상 구강 상피세포(normal human oral keratinocytes, 이하 'NHOK'라 함)의 증식에 미치는 영향을 나타내는 그래프이고, Figure 1a is a graph showing the effect of the trans-retinoic acid (all-trans retinoic acid) of the present invention on the proliferation of normal human oral keratinocytes (hereinafter referred to as 'NHOK'),
도 1b는 본 발명의 트랜스 레티노산(all-trans retinoic acid)이 인간 정상 피부 상피세포(normal human epidermal keratinocytes, 이하 'NHEK'라 함)의 증식에 미치는 영향을 나타내는 그래프이고, 1B is a graph showing the effect of the all-trans retinoic acid of the present invention on the proliferation of normal human epidermal keratinocytes (hereinafter referred to as 'NHEK'),
도 2a는 본 발명의 트랜스 레티노산의 농도의 변화에 따라, SA-β-gal 염색에 의해 복제노화를 유도하여 노화된 NHOK(PDL 22)의 세포를 위상차현미경으로 촬영한 사진이고, FIG. 2A is a photograph of NHOK (PDL 22) cells aged by inducing replication aging by SA-β-gal staining according to a change in the concentration of trans retinoic acid of the present invention.
a : 대조군 b : 트랜스 레티노산 1 nMa: control group b: trans retinoic acid 1 nM
c : 트랜스 레티노산 10 nM d : 트랜스 레티노산 100 nMc: trans retinoic acid 10 nM d: trans retinoic acid 100 nM
도 2b는 상기도 2a의 결과로서, 전체 세포수에 대하여 복제노화가 일어난 NHOK(PDL 22) 세포의 비율(%)를 나타낸 그래프이고, FIG. 2B is a graph showing the percentage (%) of NHOK (PDL 22) cells in which replication aging has occurred with respect to the total cell number as a result of FIG. 2A ,
도 3a은 본 발명의 트랜스 레티노산이 NHOK의 세포분열 능력에 미치는 영향을 계속적인 계대배양을 통해 세포분열(PDL) 수로서 나타낸 세포 성장곡선이며, Figure 3a is a cell growth curve showing the effect of the trans retinoic acid of the present invention on the cell division capacity of NHOK as cell division (PDL) number through continuous passage,
도 3b는 성장이 빠른 NHOK(PDL 15)에 1 nM의 트랜스 레티노산를 처리하면서 postmitotic stage까지 세포배양하며 SA-β-gal 염색에 의해 복제노화의 정도를 본 것을 위상차 현미경으로 촬영한 사진이고, 3B is a phase contrast microscope photograph showing the degree of replication aging by SA-β-gal staining and culturing the cells to postmitotic stage while treating 1 nM trans retinoic acid in fast-growing NHOK (PDL 15).
a: PDL 18인 NHOK로써 비히클을 처리한 세포a: Cell treated with vehicle with NHOK, PDL 18
b: PDL 20인 NHOK로서 비히클을 처리한 세포b: Cell treated with vehicle as NHOK, PDL 20
c: PDL 22인 NHOK로서 비히클을 처리한 세포c: Cell treated with vehicle as NHOK, PDL 22
d: PDL 18인 NHOK로서 1 nM 레티노산을 처리한 세포d: cells treated with 1 nM retinoic acid as NHOK which is PDL 18
e: PDL 20인 NHOK로서 1 nM 레티노산을 처리한 세포e: cell treated with 1 nM retinoic acid as NHOK which is PDL 20
f: PDL 22인 NHOK로서 1 nM 레티노산을 처리한 세포f: Cell treated with 1 nM retinoic acid as NHOK which is PDL 22
g: PDL 24인 NHOK로서 1 nM 레티노산을 처리한 세포g: Cell treated with 1 nM retinoic acid as NHOK which is PDL 24
h: PDL 26인 NHOK로서 1 nM 레티노산을 처리한 세포 및h: cells treated with 1 nM retinoic acid as NHOK which is PDL 26 and
i: PDL 28인 NHOK로서 1 nM 레티노산을 처리한 세포i: Cell treated with 1 nM retinoic acid as NHOK which is PDL 28
도 3c는 상기도 3b의 결과로서, 전체 세포 수에 대한 노화세포의 비율(%)을 나타낸 그래프이며, 3C is a graph showing the percentage of senescent cells to the total number of cells as a result of FIG. 3B .
도 4a는 본 발명의 트랜스 레티노산이 NHEK의 세포분열 능력에 미치는 영향을 계속적인 계대배양을 통해 세포분열(PDL) 수로서 나타낸 세포 성장곡선이며, Figure 4a is a cell growth curve showing the effect of the trans retinoic acid of the present invention on the cell division capacity of NHEK as cell division (PDL) number through continuous passage,
도 4b는 성장이 빠른 NHEK(PDL 10.8)에 1 nM의 트랜스 레티노산를 처리하면서 postmitotic stage까지 세포배양하며 SA-β-gal 염색에 의해 복제노화의 정도를본 것을 위상차 현미경으로 촬영한 사진이고, FIG. 4b is a phase contrast microscope photograph showing the degree of replication aging by SA-β-gal staining and culturing cells to postmitotic stage while treating 1 nM trans retinoic acid in fast-growing NHEK (PDL 10.8). FIG.
a: PDL 18.3인 NHEK로써 비히클을 처리한 세포a: Cell treated with vehicle with NHEK, PDL 18.3
b: PDL 20.5인 NHEK로써 vehicle를 처리한 세포b: Cell treated with vehicle with NHEK, PDL 20.5
c: PDL 17.1인 NHEK로서 1 nM retinoic acid를 처리한 세포c: Cell treated with 1 nM retinoic acid as NHEK with PDL 17.1
d: PDL 20.7인 NHEK로서 1 nM retinoic acid를 처리한 세포d: NHEK with PDL 20.7 treated with 1 nM retinoic acid
e: PDL 22.8인 NHEK로서 1 nM retinoic acid를 처리한 세포 및e: NHEK with PDL 22.8, treated with 1 nM retinoic acid, and
f: PDL 25.1인 NHEK로서 1 nM retinoic acid를 처리한 세포f: Cell treated with 1 nM retinoic acid as NHEK, PDL 25.1
도 4c는 상기도 4b의 결과로서, 전체 세포 수에 대한 노화세포의 비율(%)을 나타낸 그래프이고, 4C is a graph showing the percentage of senescent cells to the total number of cells as a result of FIG. 4B ,
도 5a는 1 nM의 트랜스 레티노산에 NHOK를 노출시킨 후, NHOK 내의 텔로머레이즈 활성을 TRAP법으로 분석하여 대조군과 비교한 결과를 나타낸 것이고, Figure 5a shows the results compared to the control by analyzing the telomerase activity in NHOK after exposure to NHOK to 1 nM trans retinoic acid,
도 5b는 1 nM의 트랜스 레티노산에 NHEK를 노출시킨 후, NHEK 내의 텔로머레이즈 활성을 TRAP법으로 분석하여 대조군과 비교한 결과를 나타낸 것이고, Figure 5b shows the results compared to the control by analyzing the telomerase activity in NHEK after exposure of NHEK to 1 nM trans retinoic acid,
도 6a는 1 nM의 트랜스 레티노산에 NHOK을 노출시킨 후, NHOK 내 pRb 단백질의 발현 수준을 웨스턴 블럿 분석으로 측정한 결과이고, Figure 6a is a result of Western blot analysis of the expression level of pRb protein in NHOK after exposure to 1 nM of trans retinoic acid,
도 6b는 1 nM의 트랜스 레티노산에 NHOK을 노출시킨 후, NHOK 내 p16INK4A단백질의 발현 수준을 웨스턴 블럿 분석으로 측정한 결과이며,And Figure 6b is a result of measuring the level of expression after exposure to the NHOK trans retinoic acid of 1 nM, NHOK within p16 INK4A protein by Western blot analysis,
도 6c는 1 nM의 트랜스 레티노산에 NHEK을 노출시킨 후, NHEK 내 p16INK4A단백질의 발현 수준을 웨스턴 블럿 분석으로 측정한 결과를 나타낸 것이다. Figure 6c shows the results of measuring the level of expression after exposure to the NHEK trans retinoic acid of 1 nM, NHEK within p16 INK4A protein by Western blot analysis.
상기 목적을 달성하기 위하여, 본 발명은 레티노산(retinoic acid) 화합물을 유효성분으로 함유하는 인간 상피세포(human keratinocyte)의 노화방지제을 제공한다.In order to achieve the above object, the present invention provides an anti-aging agent of human keratinocytes containing a retinoic acid compound as an active ingredient.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 인간 상피세포는 인간 점막 상피세포 (normal human mucosal keratinocytes) 및 인간 피부 상피세포(normal human epidermal keratinocytes, 이하 'NHEK'라 함)를 포함하며 구체적으로, 상기 인간 점막 상피세포는 인간 구강 상피세포(normal human oral keratinocytes, 이하 'NHOK'라 함)를 특징으로 하며, 본 발명의 레티노산 화합물은 상기 인간 상피세포의 복제노화(replicative senescence)를 억제하여 세포의 수명을 연장시키는 활성을 갖는다.Human epithelial cells of the present invention include normal human mucosal keratinocytes and normal human epidermal keratinocytes (hereinafter referred to as 'NHEK'), specifically, the human mucosal epithelial cells are human oral epithelium Cells (normal human oral keratinocytes, hereinafter referred to as 'NHOK'), the retinoic acid compound of the present invention has the activity of extending the life of the cells by inhibiting the replication senescence of the human epithelial cells (replicative senescence).
또한, 본 발명의 레티노산 화합물은 NHOK 및 NHEK의 복제노화를 억제하여 세포의 수명을 연장시킴으로써 구강 내 창상 및 발치창, 외상성 궤양(traumatic ulcer), 구각증(Angular cheilosis) 및 구강내 화상의 다양한 구강 질환을 치료ㆍ예방하고, 또한 피부 내 창상 및 피부 노화를 방지하고, 상기 피부 노화를 방지하는 효과로써 화장품 성분으로 사용될 수 있다.In addition, the retinoic acid compounds of the present invention inhibit the replication aging of NHOK and NHEK to prolong the life of the cells, thereby causing oral wounds and ankle wounds, traumatic ulcers, Angular cheilosis, and a variety of oral burns. It can be used as a cosmetic ingredient as an effect of treating and preventing oral diseases, preventing intradermal wounds and skin aging, and preventing the skin aging.
상기 레티노산 화합물로는 트랜스 레티노산(all-trans retinoic acid), 3,4-디디하이드로레티노산(3,4-didehydro-retinoic acid) 및 9-시스 레티노산(9-cis retinoic acid)으로 이루어진 군에서 선택되는 것이 바람직하며, 가장 바람직하기로는 트랜스 레티노산를 사용한다.The retinoic acid compound includes all-trans retinoic acid, 3,4-didehydro-retinoic acid, and 9-cis retinoic acid. It is preferably selected from the group, most preferably using trans retinoic acid.
본 발명자들은 증식성이 있는 NHOK 및 NHEK를 레티노산 화합물이 함유된 배양액에서 배양하여 세포의 증식이 보다 촉진됨을 확인하였다(도 1a및도 1b참조).The present inventors cultured the proliferative NHOK and NHEK in a culture solution containing a retinoic acid compound to confirm that the proliferation of the cells is more promoted (see FIGS . 1A and 1B ).
또한, 노화세포 수의 대수적 분석을 통해 트랜스 레티노산의 적용에 의해 노화가 억제됨을 확인하였으며, 이와 함께 세포의 노화에 결정적인 인자로 알려져 있는 pRb 및 p16INK4A단백질의 발현수준과 텔로머레이즈의 활성에 미치는 영향을 확인하였다.Further, were the aging by the application of the trans-retinoic acid through the algebraic analysis of aged cells confirmed suppressed, this in with the activity of pRb and p16 INK4A expression levels and telomer raise of protein, known as the critical factor in the aging of the cells, The impact was confirmed.
빠른 증식성을 갖는 정상 세포가 트랜스 레티노산에 노출된 경우, 텔로머레이즈의 활성이 그대로 유지되거나 다소 증가됨을 확인하였는데(도 5a및도 5b참조), 이러한 결과는 트랜스 레티노산이 거듭되는 증식에 따라 NHOK 및 NHEK 내 텔로머레이즈 활성이 감소되는 것을 방지하여 세포의 복제노화를 억제할 수 있음을 나타낸다.When normal cells with rapid proliferation were exposed to trans retinoic acid, it was confirmed that the activity of telomerase was maintained or increased slightly (see FIGS . 5A and 5B ). It is shown that the telomerase activity in NHOK and NHEK can be prevented from being reduced, thereby inhibiting the aging of cells.
트랜스 레티노산이 처리된 NHOK 및 NHEK 내 p16INK4A단백질의 발현수준은 현저하게 낮게 나타나는데, 이로써 트랜스 레티노산에 의해 세포의 노화가 억제됨을 확인할 수 있다. 즉, 트랜스 레티노산은 세포내 p16INK4A단백질의 발현수준을 저하시켜 NHOK 및 NHEK의 수명을 연장시킬 수 있다. 또한, 낮은 세포분열(Population Doubling Levels, 이하 PDL 이라고 함) 수에서 트랜스 레티노산이 처리된 세포에서의 pRb 단백질의 발현수준은 대등한 세포분열(PDL) 수의 대조군에서의 발현수준에 비해 현저히 낮게 나타났으나, 세포분열(PDL) 수가 증가되면서 대조군에 비해 현저히 증가되는 양상을 나타내었다.The level of expression of the protein within the p16 INK4A-trans retinoic acid treatment NHOK and NHEK appears at remarkably low, and thereby by-trans retinoic acid in the aging cell to determine inhibited. In other words, trans retinoic acid can reduce the expression level of intracellular p16 INK4A protein and extend the life of NHOK and NHEK. In addition, the expression level of pRb protein in trans retinoic acid treated cells at low number of Population Doubling Levels (PDL) is significantly lower than that of control group with comparable cell division (PDL) number. However, as the number of cell division (PDL) was increased, it showed a significant increase compared to the control group.
따라서, 본 발명의 레티노산 화합물은 NHOK 및 NHEK 내 p16INK4A단백질의 발현수준을 억제하고 계속된 증식에도 텔로머레이즈(telomerase) 활성을 지속적으로 유지시킴으로써 NHOK 및 NHEK의 노화를 방지하고, 상기 상피세포의 노화에 따른 다양한 구강질환 및 피부질환의 치료제 또는 예방제로 사용할 수 있다.Accordingly, the retinoic acid compounds of the invention by suppressing the expression level of I p16 INK4A protein NHOK and NHEK even and continued proliferation keep a steady telomerization raised (telomerase) activity and anti-aging of NHOK and NHEK, the epithelium Can be used as a therapeutic or prophylactic agent for various oral diseases and skin diseases according to aging.
본 발명의 레티노산 화합물은 임상투여시에 경구 또는 비경구로 투여가 가능하며 일반적인 의약품제제의 형태로 사용될 수 있다. 특히 비경구 주사 투여가 바람직하다.Retinoic acid compounds of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations. Parenteral injection administration is particularly preferred.
즉, 본 발명의 레티노산 화합물은 실제 임상투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제 및 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 레티노산 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (Calcium carbonate), 수크로스 (Sucrose) 또는 락토오스 (Lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 및 보존제가 포함될 수 있다.That is, the retinoic acid compound of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used Or using excipients. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, water Prepared with a mixture of sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, liquid solutions, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin.
또한, 본 발명의 약학적 조성물은 비경구로 투여할 수 있으며, 비경구 투여는 피하주사, 정맥주사 또는 근육내 주사에 의한다. 비경구 투여용 제형으로 제제화하기 위해서는 상기 레티노산 화합물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고 이를 앰플 또는 바이알의 단위 투여형으로 제제한다.In addition, the pharmaceutical compositions of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection or intramuscular injection. To formulate into a parenteral formulation, the retinoic acid compound is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.
본 발명의 트랜스 레티노산 화합물의 유효용량은 일반적으로 성인에게 100 ∼ 2000 mg/kg 이고, 바람직하기로는 100 ∼ 1000 mg/kg 이며, 의사 또는 약사의 판단에 따라 일정 시간 간격으로 1 일 수회, 바람직하기로는 하루 1 ∼ 2 회 분할 투여될 수 있다The effective dose of the trans retinoic acid compound of the present invention is generally 100-2000 mg / kg in adults, preferably 100-1000 mg / kg, several times a day at regular time intervals according to the judgment of a doctor or pharmacist. It may be administered in divided doses once or twice a day.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> 트랜스 레티노산에 의한 NHOK의 증식율 측정Example 1 Measurement of Proliferation Rate of NHOK by Trans Retinoic Acid
<단계1> NHOK의 준비<Step 1> Preparation of NHOK
NHOK는 인간 치은조직으로부터 준비하였다(Min B.M. et al.,Int.J.Oncol.,1995,7, 249-256). 조직표본을 18 내지 30 세 사이 연령의 네명의 건강한 인간에게서 얻었다. 상기 조직표본은 칼슘 및 마그네슘이 포함되지 아니한 행크스 균질염용액(calcium- and magnesium-free Hank's balanced salt solution, CMF-HBSS; GIBCO)으로 3 회 세척하였다. 구강 점막(mucosa)으로부터 상피조직을 분리하기 위해 상기 조직에 콜라게네이즈(collagenase type II, 1.0 mg/ml; Sigma Chemical CO.) 및 디스파아제(dispase grade II, 2.4 mg/ml; Boeringer-Mannheim)를 함유한 CMF-HBSS을 첨가하여 95 %의 공기와 5 %의 이산화탄소 조건하에서 37 ℃, 90 분 동안 배양하였다. NHOK를 상기와 같이 분리된 구강 상피조직으로부터 준비하였으며(Min B.M. et al.,Int. J. Oncol., 1995,7, 249-256), 0.15 mM 칼슘과 성장인자 불릿키트(KGM; Clonetics Corp.)를 포함한 상피세포 배양액에서 배양하였다. 세포의 밀도가 대략 70 %가 되었을 때 NHOK를 60 mm 페트리디쉬 당 1 × 105개의 세포수로 분주하였고, 세포 밀도가 대략 70 %가 될때까지 배양한 후, 모든 세포들이 세포증식의 세포분열후기(postmitotic stage)에 이를 때까지 계대배양(subculture)을 시행하였다. 각 계대배양시 마지막에는, 1 × 106및 3 × 106개의 NHOK를 PBS로 1 회 세척하였으며, 세포 침전물(cell pellet)은 분리하여 각각 TRAP 및 웨스턴 블럿 분석을 위해 -70 ℃에서 보관하였다.NHOK was prepared from human gingival tissue (Min BM et al., Int. J. Oncol. , 1995, 7 , 249-256). Tissue samples were obtained from four healthy humans between 18 and 30 years of age. The tissue samples were washed three times with calcium- and magnesium-free Hank's balanced salt solution (CMF-HBSS; GIBCO) that did not contain calcium and magnesium. Collagenase (collagenase type II, 1.0 mg / ml; Sigma Chemical CO.) And dispase grade II (2.4 mg / ml; Boeringer-Mannheim) were used to separate epithelial tissue from oral mucosa. ) And CMF-HBSS were incubated for 90 minutes at 37 ℃ under 95% air and 5% carbon dioxide conditions. NHOK was prepared from isolated oral epithelial tissue (Min BM et al., Int. J. Oncol. , 1995, 7 , 249-256), 0.15 mM calcium and growth factor bullet kit (KGM; Clonetics Corp. Cultured in epithelial cell culture medium. When the cell density reached approximately 70%, NHOK was dispensed at 1 × 10 5 cells per 60 mm Petri dish, and cultured until the cell density reached approximately 70%. Subculture was performed until the postmitotic stage was reached. At the end of each passage, 1 × 10 6 and 3 × 10 6 NHOKs were washed once with PBS, and the cell pellets were separated and stored at −70 ° C. for TRAP and Western blot analysis, respectively.
또한 별도로, 제 16 형 인유두종 바이러스 게놈(papillomavirus-type 16 genome)(Park N.H. et al.,Carcinogenesis, 1991,12, 1627-1631)으로 전이(transfection)시켜 불멸화된(immortal) 인간 구강 상피세포 유래 세포주인HOK-16B 세포(human oral keratinocyte-16B cells)들도 KGM에서 배양하여 하기 실험예 2에서 대조군으로 사용하였다.In addition, an immortal human oral epithelial cell-derived cell line was transfected into a papillomavirus-type 16 genome (Park NH et al., Carcinogenesis , 1991, 12 , 1627-1631). Human oral keratinocyte-16B cells were also cultured in KGM and used as a control in Experimental Example 2 below.
상기와 같이 치은 상피조직 표본으로부터 각각 분리된 NHOK를 60 mm 페트리디쉬에 분주하고 3 일 동안 배양한 후, 원래 분주된 세포의 수를 세포 콜로니 계수에 의해 결정하였다. 또한, 초기 NHOK 배양물의 세포분열(PDL) 수를 결정하기 위해 혈구계를 사용하여 3 개의 페트리디쉬로부터 계수된 세포밀도가 대략 70 %에 이르기까지 세포를 배양한 후, 초기 배양물을 세포증식의 세포분열 후기에 이를 때까지 계대배양하고 각 단계의 마지막에서 하기 수학식 1에 의해 NHOK의 세포분열(PDL) 수를 계산하였다.NHOKs isolated from the gingival epithelial specimens as above were dispensed into 60 mm Petri dishes and incubated for 3 days, and then the number of cells originally dispensed was determined by cell colony count. In addition, after culturing the cells up to approximately 70% cell density counted from three Petri dishes using a hemocytometer to determine the number of cell division (PDL) of the initial NHOK culture, the initial culture was Subcultured until late cell division and at the end of each step, the cell division (PDL) number of NHOK was calculated by the following equation (1).
<수학식 1><Equation 1>
2N= Cf/Ci 2 N = C f / C i
N : PDL 수, Cf: 한 단계의 마지막에서 수확된 전체 세포수,N: number of PDL, C f : total number of cells harvested at the end of one step,
Ci: 분주시 배양 디쉬에 부착된 전체 세포수C i : Total number of cells attached to the culture dish at the time of dispensing
<단계2> 트랜스 레티노산에 의한 NHOK 증식율 측정<Step 2> NHOK growth rate measurement by trans retinoic acid
트랜스 레티노산의 세포증식에 대한 영향을 결정하기 위해, 빠르게 증식하는NHOK(PDL 18)를 24-웰 플레이트에 웰 당 2 ×104cell/ml 개의 세포수로 분주하고 세포밀도가 40 %에 이르기까지(도말 후 24 시간 경과) 배양하였다. 상기 배양된 세포를 1 nM 내지 10 μM의 다양한 농도의 트랜스 레티노산(1 μM, 10 μM, 100 μM, 1 nM, 5 nM 및 10 nM)에 노출시키고 2 또는 4 일간 배양한 후 회수하여 트립판 블루를 배제하는 성질에 의해 살아있는 세포들의 수를 측정하였다. 상기 과정을 4 회 독립적으로 반복 수행하여 세포수의 평균과 표준편차를 측정하였다(도 1a).To determine the effect of trans retinoic acid on cell proliferation, rapidly proliferating NHOK (PDL 18) was dispensed in 24-well plates at 2 x 10 4 cells / ml cell number per well and the cell density reached 40%. Incubated until 24 hours after plating. The cultured cells were exposed to trans retinoic acid (1 μM, 10 μM, 100 μM, 1 nM, 5 nM, and 10 nM) at various concentrations from 1 nM to 10 μM, incubated for 2 or 4 days, and recovered to tryptophan. The number of living cells was measured by the property of excluding blue. The procedure was repeated four times independently to measure the mean and standard deviation of the number of cells ( FIG. 1A ).
도 1a는 본 발명의 트랜스 레티노산(all-transretinoic acid)이 NHOK의 증식에 미치는 영향을 나타내는 그래프이며, 상기 결과로서 1 또는 10 nM의 트랜스 레티노산에 노출된 NHOK 세포들은 대조군에 비해 세포증식이 현저하게 증가하였으며, 최대 세포증식은 1 nM의 레티노산에 노출된 세포에서 관찰되었고, 1 μM 이상의 트랜스 레티노산에 노출된 세포들에서는 투여량 의존적으로 성장이 억제됨이 관찰되었다. Figure 1a is a graph showing the effect of the trans- retinoic acid (all- trans retinoic acid) of the present invention on the growth of NHOK, NHOK cells exposed to 1 or 10 nM trans retinoic acid as a result of cell proliferation compared to the control group This significantly increased, maximal cell proliferation was observed in cells exposed to 1 nM retinoic acid, and dose-dependent growth inhibition was observed in cells exposed to more than 1 μM trans retinoic acid.
<실시예 2> 트랜스 레티노산에 의한 NHEK의 증식율 측정Example 2 Measurement of Growth Rate of NHEK by Trans Retinoic Acid
<단계1> NHEK의 준비<Step 1> Preparation of NHEK
NHEK는 인간 표피(foreskin) 조직으로부터 채취하여 조직표본을 1 내지 3세 사이 연령의 두명의 건강한 인간에게서 얻은 것을 제외하고, 상기 실시예 1의 경우와 동일한 방법으로 처리하였다.NHEK was treated in the same manner as in Example 1, except that tissue samples were taken from human foreskin tissue and tissue specimens were obtained from two healthy humans between 1 and 3 years of age.
<단계2> 트랜스 레티노산에 의한 NHEK 증식율 측정Step 2 Measurement of NHEK Proliferation Rate by Trans Retinoic Acid
트랜스 레티노산의 세포증식에 대한 영향을 결정하기 위해, 빠르게 증식하는 NHEK(PDL 11)를 24-웰 플레이트에 웰 당 2 ×104cell/ml 개의 세포수로 분주하고 세포밀도가 40 %에 이르기까지(도말 후 24 시간 경과) 배양하였다. 상기 배양된 세포를 1 nM 내지 0.5 μM의 다양한 농도의 트랜스 레티노산(0.5 μM, 100 μM, 10 nM 및 1 nM)에 노출시키고 2 또는 4 일간 배양한 후 회수하여 트립판 블루를 배제하는 성질에 의해 살아있는 세포들의 수를 측정하였다. 상기 과정을 4 회 독립적으로 반복 수행하여 세포수의 평균과 표준편차를 측정하였다(도 1b).To determine the effect of trans retinoic acid on cell proliferation, rapidly propagating NHEK (PDL 11) was dispensed in 24-well plates at 2 x 10 4 cells / ml cell number per well and the cell density reached 40%. Incubated until 24 hours after plating. The cultured cells were exposed to various concentrations of trans retinoic acid (0.5 μM, 100 μM, 10 nM, and 1 nM) of 1 nM to 0.5 μM, cultured for 2 or 4 days, and recovered to remove trypan blue. The number of living cells was measured by The procedure was repeated four times independently to measure the mean and standard deviation of the cell numbers ( FIG. 1B ).
도 1b는 NHEK의 증식에 미치는 영향을 나타낸 결과로서, 1 nM의 트랜스 레티노산에 노출된 NHEK 세포들은 대조군에 비해 세포증식이 현저하게 증가하였으며, 최대 세포증식은 1 nM의 레티노산에 노출된 세포에서 관찰되었고, 0.5 μM 이상의 트랜스 레티노산에 노출된 세포들에서는 성장이 억제됨이 관찰되었다. Figure 1b shows the effect on the proliferation of NHEK, NHEK cells exposed to 1 nM trans retinoic acid significantly increased cell proliferation compared to the control group, the maximum cell proliferation is cells exposed to 1 nM retinoic acid The growth was inhibited in cells exposed to trans retinoic acid above 0.5 μM.
<실시예 3> 트랜스 레티노산에 의한 NHOK의 노화 억제율 측정Example 3 Determination of Aging Inhibition Rate of NHOK by Trans Retinoic Acid
노화된 NHOK는 β-갈락토시다아제(β-galactosidase)에 양성적으로 염색되는데(Dimri G.D. et al.,Proc. Natl. Acad. Sci., 1995,92, 9363-9367), 이러한 성질을 이용하여 트랜스 레티노산에 의한 NHOK의 노화 억제율을 조사하였다. 상기 실시예 1과 같이 배양된 NHOK를 35 mm 페트리디쉬에 분주하여 배양하고, PBS로 1회 세척한 후 2 % 포름알데히드 및 0.2 % 글루타르알데히드를 포함한 PBS로 고정한 후 1 mM의 MgCl2가 보충적으로 함유된 PBS로 세척하였다. 상기 세포들을 1 mg/ml의 5-브로모-4-클로로-3-인돌릴 β-D-갈락토시드(5-bromo-4-chloro-3-indolyl β-D-galactoside, X-Gal; Promega Corp.)를 함유한 SA-β-gal 염색용액[조성: 150 mM NaCl, 2 mM MgCl2, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 40 mM 시트르산, 12 mM 인산나트륨(sodium phosphate), pH 6.0]에 37 ℃에서 정치하여 하룻밤동안 염색하였다.Aged NHOKs are positively stained for β-galactosidase (Dimri GD et al., Proc. Natl. Acad. Sci. , 1995, 92 , 9363-9367). The inhibition of aging of NHOK by trans retinoic acid was investigated. NHOK cultured as in Example 1 was dispensed in 35 mm Petri dish, incubated, washed once with PBS, fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde, and supplemented with 1 mM of MgCl 2 . Washed with PBS contained. The cells were treated with 1 mg / ml of 5-bromo-4-chloro-3-indolyl β-D-galactoside (5-bromo-4-chloro-3-indolyl β-D-galactoside, X-Gal; SA-β-gal stain solution containing Promega Corp. [composition: 150 mM NaCl, 2 mM MgCl 2 , 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 40 mM citric acid, 12 mM sodium phosphate, pH 6.0, and left at 37 ℃ to dye overnight.
대표적 계수를 확인하기 위하여, 배양된 각 세포 배지를 4 등분하여 각 구역당 최소 2 구역을 위상차 현미경(Olympus IMT-2 phase-contrast microscope)으로 촬영하여 세포를 계수한 결과 각 구역당 최소 500 개 이상의 세포가 계수되었다.To determine representative counts, each cultured cell medium was divided into 4 sections and at least 2 zones of each zone were photographed with an Olympus IMT-2 phase-contrast microscope. The cells were counted.
노화 NHOK(PDL 22)를 21일 동안 1, 10 또는 100 nM 레티노산에 노출시킨 후 SA-β-gal 염색법을 사용하여 배지내 노화세포의 비율(%)을 결정하였다. 노화세포의 존재 비율(%)은 전체 세포수에 대한 SA-β-gal 양성세포의 비율(%)로 결정하였고, 평균 비율(%)과 표준 편차는 3 회의 독립된 실험으로부터 계산하였다.Aging NHOK (PDL 22) was exposed to 1, 10 or 100 nM retinoic acid for 21 days and then the percentage of senescent cells in medium was determined using SA-β-gal staining. The percentage of senescent cells was determined as the percentage of SA-β-gal positive cells to the total cell number, and the average percentage and standard deviation were calculated from three independent experiments.
도 2a는 트랜스 레티노산의 농도의 변화에 따라, SA-β-gal 염색에 의해 복제노화를 유도하여 노화된 NHOK(PDL 22)의 세포를 위상차현미경으로 촬영한 사진이고, 상기 트랜스 레티노산의 농도는 a: 대조군, b: 1 nM의 트랜스 레티노산, c: 10 nM의 트랜스 레티노산 및 d: 100 nM의 트랜스 레티노산으로 변화를 주어 관찰하였다.도 2b는 상기도 2a의 결과로서, 전체 세포수에 대하여 복제노화가 일어난 NHOK(PDL 22)의 세포의 비율(%)를 나타낸 그래프로서, 저농도인 1 nM의 트랜스 레티노산에 노출된 세포에서 복제노화가 대조군에 비해 현저히 억제되었다. FIG. 2A is a photograph of NHOK (PDL 22) cells aged by induction of replication aging by SA-β-gal staining according to a change in the concentration of trans retinoic acid. Was observed with a: control, b: 1 nM trans retinoic acid, c: 10 nM trans retinoic acid, and d: 100 nM trans retinoic acid. FIG. 2B is a graph showing the percentage (%) of NHOK cells (PDL 22) in which replication aging has occurred with respect to the total number of cells as a result of FIG. 2A , and is replicated in cells exposed to low concentrations of 1 nM trans retinoic acid. Aging was significantly inhibited compared to the control.
상기도 2a및도 2b의 결과로부터, 트랜스 레티노산에 노출된 세포의 노화가 효과적으로 억제됨을 알 수 있으며 특히, 1 nM의 트랜스 레티노산이 적용된 경우 최대로 복제노화가 억제되어 NHOK 세포 증식력이 증가되었다. 2A and 2B , it can be seen that the senescence of cells exposed to trans retinoic acid is effectively inhibited. In particular, when 1 nM of trans retinoic acid is applied, replication aging is inhibited to the maximum, thereby increasing NHOK cell proliferation ability.
<실시예 4> 트랜스 레티노산이 NHOK 및 NHEK의 세포분열 능력에 미치는 영향Example 4 Effect of Trans Retinoic Acid on Cell Division Capacity of NHOK and NHEK
트랜스 레티노산에 노출된 세포의 세포분열 능력(mitotic capacity)을 측정하여 상기 트랜스 레티노산이 NHOK 및 NHEK에 미치는 영향을 하기와 같은 방법으로 조사하였다.The effect of trans retinoic acid on NHOK and NHEK was examined by measuring the mitotic capacity of cells exposed to trans retinoic acid in the following manner.
구체적으로, 빠른 증식성을 나타내는 NHOK(PDL 15)를 60 mm 페트리디쉬 당 2 × 105개의 세포수로 분주하였다. 또한, NHEK(PDL 11)의 세포를 동일한 방법으로 분주하였다. 상기 세포에 0.001 % DMSO를 포함하는 KGM 비히클을 처리한 음성 대조군과 KGM에 용해시킨 트랜스 레티노산 1 nM을 처리한 처리군을 준비하였다. 상기 처리군 및 음성 대조군을 세포밀도가 약 70 %가 될 때까지 계속 배양한 후 세포증식이 세포분열 후기(postmitotic stage)에 이를 때까지 연속적으로 계대배양을 수행하였다. 이때, 트랜스 레티노산은 DMSO에 용해시킨 후 KGM으로 희석하여 DMSO의 최종농도가 0.001 %가 되도록 하여 사용하였다. 각 계대배양의 말기에 누적된세포분열(PDL) 수와 배양된 NHOK 및 NHEK 중 노화된 세포의 수, 즉 SA-β-gal 염색에 의한 양성세포의 비율(%)을 결정하였으며, 1 ×PBS로 1 회 세척한 후 얻은 세포 침전물은 TRAP 및 웨스턴 블럿 분석을 위해 -70 ℃에서 냉동 보관하였다(도 3a, 도 3b, 도 3c, 도 4a, 도 4b및도 4c참조).Specifically, NHOK (PDL 15) showing rapid proliferation was dispensed at 2 × 10 5 cell numbers per 60 mm Petri dish. In addition, cells of NHEK (PDL 11) were dispensed in the same manner. A negative control treated with a KGM vehicle containing 0.001% DMSO and a treatment group treated with 1 nM of trans retinoic acid dissolved in KGM were prepared. The treated group and the negative control group were continuously cultured until the cell density became about 70%, and then passaged continuously until cell proliferation reached the postmitotic stage. At this time, the trans retinoic acid was dissolved in DMSO and diluted with KGM to use the final concentration of DMSO to 0.001%. The cumulative number of cell division (PDL) at the end of each passage and the number of aged cells in cultured NHOK and NHEK, ie the percentage of positive cells by SA-β-gal staining, were determined. Cell precipitates obtained after one wash with were stored frozen at −70 ° C. for TRAP and Western blot analysis (see FIGS. 3A, 3B, 3C, 4A, 4B and 4C ).
도 3a은 본 발명의 트랜스 레티노산이 NHOK의 세포분열 능력에 미치는 영향을 계속적인 계대배양을 통해 세포분열(PDL) 수로서 나타낸 세포 성장곡선이고,도 3b는 성장이 빠른 NHOK(PDL 15)에 1 nM의 트랜스 레티노산를 처리하면서 postmitotic stage까지 세포배양하며 SA-β-gal 염색에 의해 복제노화의 정도를 본 것으로 도 내의 a: PDL 18인 NHOK로써 비히클을 처리한 세포, b: PDL 20인 NHOK로서 비히클을 처리한 세포, c: PDL 22인 NHOK로서 비히클을 처리한 세포, d: PDL 18인 NHOK로서 1 nM 레티노산을 처리한 세포, e: PDL 20인 NHOK로서 1 nM 레티노산을 처리한 세포, f: PDL 22인 NHOK로서 1 nM 레티노산을 처리한 세포, g: PDL 24인 NHOK로서 1 nM 레티노산을 처리한 세포, h: PDL 26인 NHOK로서 1 nM 레티노산을 처리한 세포 및 i: PDL 28인 NHOK로서 1 nM 레티노산을 처리한 세포를 위상차 현미경으로 촬영한 사진이고,도 3c는 상기도 3b의 결과로서, 전체 세포 수에 대한 노화세포의 비율(%)을 나타낸 것이다. Figure 3a is a cell growth curve showing the effect of the trans retinoic acid of the present invention on the cell division ability of NHOK as a cell division (PDL) number through continuous passage, Figure 3b is a fast growth NHOK (PDL 15) 1 Cell culture to the postmitotic stage with treatment of nM trans retinoic acid and the degree of replication aging by SA-β-gal staining.The cells treated with vehicle with NHOK of a: PDL 18 in the figure, b: NHOK of PDL 20 Cell treated with vehicle, c: cell treated with NHOK as PDL 22, d: cell treated with 1 nM retinoic acid as NHOK as PDL 18, cell treated with 1 nM retinoic acid as NHOK as PDL 20 f: cells treated with 1 nM retinoic acid with NHOK as PDL 22, g cells treated with 1 nM retinoic acid as NHOK as PDL 24, cells treated with 1 nM retinoic acid as NHOK as PDL 26 and i : Phase-contrast microscopy of cells treated with 1 nM retinoic acid as NHOK, PDL 28 A photograph, Figure 3c shows the percentage of senescent cells for as a result of the Figure 3b, the total number of cells.
상기 NHOK의 세포분열을 알 수 있는도 3a, 도 3b및도 3c의 결과를 보다 상세하게 살펴보면, 음성 대조군으로 비히클을 처리한 NHOK(PDL 15)는세포분열(PDL) 수가 20까지 양호하게 증식함이 확인되었으며 이는 세포 성장곡선의 대수성장기(log phase)에 해당하는 것이었다. 일부 NHOK 세포들은 세포분열(PDL) 20에 이르러 복제노화의 양상이 나타냈으나, 세포분열(PDL) 22에 이르러서는 대부분의 세포들에서 복제노화의 특징인 SA-β-gal 염색에 대해 양성반응을 나타내었고 성장이 정지되었다. 그러나 1 nM의 트랜스 레티노산에 노출된 NHOK 배양산물에서는 세포증식의 대수성장기가 세포분열(PDL) 0으로부터 세포분열(PDL) 26까지에서 관찰되었으며, 이들 세포들은 세포분열(PDL) 28까지 증식한 후 세포증식이 중지되었다. Referring to the results of FIGS . 3A, 3B, and 3C , which show cell division of NHOK, in detail, NHOK (PDL 15) treated with a vehicle as a negative control was well grown to 20 cell division (PDL) numbers. This has been confirmed and corresponds to the log phase of the cell growth curve. Some NHOK cells showed signs of replication aging by reaching cell division (PDL) 20, but positive responses to SA-β-gal staining, a characteristic of replication aging, in most cells by cell division (PDL) 22. And growth was stopped. However, in NHOK cultures exposed to 1 nM of trans retinoic acid, logarithmic growth phase of cell proliferation was observed from cell division (PDL) 0 to cell division (PDL) 26, and these cells proliferated to cell division (PDL) 28. Cell proliferation was stopped afterwards.
또한,도 4a는 트랜스 레티노산이 NHEK의 세포분열 능력에 미치는 영향을 계속적인 계대배양을 통해 PDL(세포분열) 수로서 나타낸 세포 성장곡선을 나타낸 것으로서,도 4b는 성장이 빠른 NHEK(PDL 10.8)에 1 nM의 트랜스 레티노산를 처리하면서 postmitotic stage까지 세포배양하며 SA-β-gal 염색에 의해 복제노화의 정도를 본 것을 도 내의 a: PDL 18.3인 NHEK로써 비히클을 처리한 세포, b: PDL 20.5인 NHEK로써 비히클을 처리한 세포, c: PDL 17.1인 NHEK로서 1 nM 레티노산을 처리한 세포, d: PDL 20.7인 NHEK로서 1 nM 레티노산을 처리한 세포 및 e: PDL 22.8인 NHEK로서 1 nM 레티노산을 처리한 세포를 위상차 현미경으로 촬영한 사진이고,도 4b는 전체 세포 수에 대한 노화세포의 비율(%)을 도시한 것이다.In addition, Figure 4a shows the cell growth curve showing the effect of trans retinoic acid on the cell division ability of NHEK as PDL (cell division) number through continuous passage, Figure 4b is a fast growth NHEK (PDL 10.8) Cell culture to postmitotic stage with 1 nM trans retinoic acid and the degree of replication aging by SA-β-gal staining were treated with vehicle with NHEK of a: PDL 18.3 in the figure, b: NHEK with PDL 20.5 Cells treated with vehicle, c: cells treated with 1 nM retinoic acid as NHEK with PDL 17.1, d: cells treated with 1 nM retinoic acid with NHEK with PDL 20.7, and 1 nM retinoic acid with NHEK with PDL 22.8 Is a photo taken with a phase contrast microscope of the cells treated, Figure 4b shows the ratio (%) of senescent cells to the total number of cells.
상기 상기 NHEK의 세포분열을 알 수 있는도 4a, 도 4b및도 4c의 결과를보다 구체적으로 살펴보면, 음성 대조군으로 비히클을 처리한 NHEK(PDL 11)는 세포분열(PDL) 수가 18까지 양호하게 증식함이 확인되었으며 이는 세포 성장곡선의 대수성장기(log phase)에 해당하는 것이었다. 일부 NHEK 세포들은 PDL 20에 이르러 대부분 세포들에게서 복제노화의 양상이 나타냈으나, PDL 20.5에 이르러서는 대부분의 세포들에서 복제노화의 특징인 SA-β-gal 염색에 대해 양성반응을 나타내었고 성장이 정지되었다. 그러나, 1 nM의 트랜스 레티노산에 노출된 NHEK 배양산물에서는 세포증식의 대수성장기가 PDL 0으로부터 PDL 23까지에서 관찰되었으며, 이들 세포들은 PDL 25까지 증식한 후 세포증식이 중지되었다. 따라서, 1 nM의 트랜스 레티노산이 처리된 세포가 노화의 억제 효과가 우수하고 따라서 세포분열의 수가 증가하였다. 결론적으로, 노화 세포 수의 대수적인 분석을 통해서 트랜스 레티노산의 작용으로 노화가 억제됨을 알 수 있다.Looking at the results of Figure 4a, 4b and 4c that can be seen the cell division of the NHEK in more detail, NHEK (PDL 11) treated with a vehicle as a negative control, the number of cell division (PDL) is proliferated well up to 18 This corresponds to the log phase of the cell growth curve. Some NHEK cells reached PDL 20 and showed signs of replication aging in most cells, but by PDL 20.5 most cells showed positive response to SA-β-gal staining, a characteristic of replication aging. Was stopped. However, in NHEK cultures exposed to 1 nM of trans retinoic acid, logarithmic growth phase of cell proliferation was observed from PDL 0 to PDL 23, and these cells proliferated to PDL 25 and ceased cell proliferation. Therefore, cells treated with 1 nM of trans retinoic acid have an excellent inhibitory effect on aging and thus increase the number of cell divisions. In conclusion, through the algebraic analysis of the number of senescent cells, it can be seen that senescence is inhibited by the action of trans retinoic acid.
<실시예 5> 트랜스 레티노산이 NHOK 및 NHEK의 텔로머레이즈 활성에 미치는 영향Example 5 Effect of Trans Retinoic Acid on the Telomerase Activity of NHOK and NHEK
트랜스 레티노산에 노출된 NHOK 및 NHEK의 텔로머레이즈의 활성 변화를 조사하기 위하여, 텔로머레이즈의 활성을 TRAP-eze 텔로머레이즈 검출키트(Oncor Inc.)를 이용한 TRAP 분석방법에 의해 측정하였다.In order to investigate the activity change of telomerase of NHOK and NHEK exposed to trans retinoic acid, the activity of telomerase was measured by TRAP assay using TRAP-eze telomerase detection kit (Oncor Inc.).
구체적으로, 상기 실시예 4에서 준비하여 냉동 보관시킨 세포 침전물(cell pellet)을 사용하여 이를 200 ㎕의 1 ×CHAPS 완충용액(조성: pH 7.5의 10 mM Tris-HCl, 1 mM MgCl2, 1 mM EDTA, 0.5 % 3-[(3-cholamidopropyl)-dimethylammoniumSpecifically, using a cell pellet prepared in Example 4 and frozen and stored in 200 μl of 1 × CHAPS buffer solution (composition: 10 mM Tris-HCl, 1 mM MgCl 2 , 1 mM pH 7.5). EDTA, 0.5% 3-[(3-cholamidopropyl) -dimethylammonium
]-1-propanesulfonate)에 용해시키고, 얼음상에서 30 분간 배양한 후, 4 ℃,12,000 rpm에서 30 분간 원심분리하였다. 이로부터 분리한 상층액을 TRAP 분석을 위해 드라이아이스(dry ice) 상에서 냉각시킨 후, 상층액의 단백질 농도를 바이오-라드(Bio-rad)사의 단백질 분석용 시약을 사용하여 측정하였다. 이때, 텔로머레이즈 활성을 가지는 HOK-16B 세포 파쇄물을 양성 대조군으로 사용하였고, 200 ㎍/㎖의 RNase A(Sigma Chemical Co.)로 처리된 HOK-16B 세포 파쇄물을 음성 대조군으로 사용하였다.] -1-propanesulfonate), incubated for 30 minutes on ice, and centrifuged for 30 minutes at 4 ℃, 12,000 rpm. The supernatant separated therefrom was cooled on dry ice for TRAP analysis, and the protein concentration of the supernatant was measured using a reagent for protein analysis of Bio-rad. At this time, HOK-16B cell lysate having telomerase activity was used as a positive control, and HOK-16B cell lysate treated with 200 μg / ml of RNase A (Sigma Chemical Co.) was used as a negative control.
TRAP 분석을 위하여, 1.0 ㎍의 세포성 단백질을 포함하는 세포 용해물(cell lysate) 2 ㎕를 1 ×TRAP 반응완충액[조성: 20 mM Tris-HCl(pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005 % Tween 20, 1 mM EGTA], 4 가지 데옥시뉴클레오티드 삼인산(deoxynucleotide triphosphate) 각각 50 μM, 3,000 Ci/mmol의 (γ-32p)ATP (Amersham Corp.), 20 μCi로 말단 표지된 TS 프라이머 0.05 μg, 프라이머 혼합액 1 ㎕ 및 Taq DNA 중합효소(Perkin-Elmer) 0.4 unit를 포함하는 48 ㎕의 용액에 첨가하여 준비한 반응 혼합용액을 30 ℃에서 30 분간 배양한 후, 상기 텔로머레이즈 반응산물을 DNA 증폭기(DNA thermal cycler, Perkin-Elmer)를 사용하여 PCR 반응을 수행하였다. PCR 반응은 94 ℃에서 30 초 및 55 ℃에서 30 초의 조건으로 30 회 반복수행한 후, 최종적으로 72 ℃에서 10 분 동안 1 회 연장시켰다. 상기 PCR 산물을 12.5 %의 비변성 폴리아크릴아마이드 겔(non-denaterued polyacrylamide gel)에서 60 W로 90 분간 전기 영동을 수행하였으며, 이때 1 ×Tris-붕산염(borate) EDTA 완충용액을 전개용매로 사용하였다. 전기영동이 끝난 겔을 건조시킨 후, 방사성 표지를 오토래디오그래피(autoradiography)로 확인하였다.For TRAP analysis, 2 μl of cell lysate containing 1.0 μg of cellular protein was added to 1 × TRAP reaction buffer [composition: 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA], 4 deoxynucleotide triphosphates 50 μM each, 3,000 Ci / mmol of (γ-32p) ATP (Amersham Corp.), TS primers terminated with 20 μCi 0.05 After incubating the reaction mixture solution prepared by adding to 48 μl solution containing μg, 1 μl of primer mixture and 0.4 unit of Taq DNA polymerase (Perkin-Elmer), the telomerase reaction product was incubated at 30 ° C. for 30 minutes. PCR reactions were performed using an amplifier (DNA thermal cycler, Perkin-Elmer). The PCR reaction was repeated 30 times at 30 [deg.] C. at 30 [deg.] C. and 30 seconds at 55 [deg.] C. and finally extended once at 72 [deg.] C. for 10 minutes. The PCR product was subjected to electrophoresis for 90 minutes at 60 W in a 12.5% non-denaterued polyacrylamide gel, wherein 1 × Tris-borate EDTA buffer was used as the developing solvent. . After drying the electrophoresis gel, radiolabeling was confirmed by autoradiography.
도 5a는 1 nM의 트랜스 레티노산에 NHOK를 노출시킨 후, NHOK 내의 텔로머레이즈 활성을 TRAP법으로 분석하여 대조군과 비교한 결과를 나타낸 것이고, 상기 결과로는 세포분열(PDL) 17, 19 및 22인 비히클 처리된 NHOK 음성 대조군으로부터 얻어진 세포 추출물(도 5a의 레인 4, 6 및 8) 및 세포분열(PDL) 17, 19, 22, 25 및 26인 트랜스 레티노산이 처리된 NHOK 처리군으로부터 얻어진 세포 추출물(도 5a의 레인 5, 7, 9, 11 및 12)에서 현저하기 증가된 텔로머레이즈 활성이 확인되었다. 상기 NHOK 음성 대조군에서는 세포분열(PDL) 수의 변화에 따라 텔로머레이즈의 활성이 거의 유사하였으나, 트랜스 레티노산 처리된 NHOK 처리군에서는 세포분열(PDL) 수가 증가함에 따라 텔로머레이즈 활성이 유지되거나 약간 상승함이 확인되었다. 또한, 복제노화의 특징을 나타내는 비히클 처리된 NHOK(PDL 23) 및 트랜스 레티노산 처리된 세포들(PDL 27 및 28)에서는 텔로머레이즈 활성이 일정하게 유지되어 빠른 증식성을 나타내는 NHOK로부터 얻어진 세포 추출물에 비해 텔로머레이즈 활성이 거의 검출되지 아니하였다(도 5a의 레인 10, 13 및 14). Figure 5a shows the results compared to the control by analyzing the telomerase activity in NHOK after exposure of NHOK to 1 nM trans retinoic acid, TRAP method, the cell division (PDL) 17, 19 and Cells obtained from NHOK treated group treated with transretinoic acid with cell extract (lanes 4, 6 and 8 of FIG. 5A) obtained from vehicle treated NHOK negative control group 22 and cell division (PDL) 17, 19, 22, 25 and 26 Significantly increased telomerase activity was observed in the extract (lanes 5, 7, 9, 11 and 12 of FIG. 5A ). In the NHOK negative control, the activity of telomerase was almost similar to the change in the number of cell division (PDL), but in the transretinoic acid treated NHOK group, the telomerase activity was maintained as the number of cell division (PDL) increased. A slight rise was confirmed. In addition, cell extracts obtained from NHOK exhibiting rapid proliferative properties in vehicle treated NHOK (PDL 23) and trans retinoic acid treated cells (PDL 27 and 28), which exhibited characteristics of replication aging, with constant telomerase activity. Almost no telomerase activity was detected (lanes 10, 13 and 14 of FIG. 5A ).
도 5b는 동일한 목적으로 1 nM의 트랜스 레티노산에 NHEK를 노출시킨 후, NHEK 내의 텔로머레이즈 활성을 TRAP법으로 분석하여 대조군과 비교한 결과를 나타낸 것이다. Figure 5b shows the results compared to the control by analyzing the telomerase activity in NHEK by exposure to NHEK to 1 nM trans retinoic acid for the same purpose.
그 결과, PDL 13.3, 16.5 및 19.8인 비히클 처리된 NHEK 음성 대조군으로부터 얻어진 세포 추출물(도 5b의 레인 4, 6 및 8) 및 세포분열(PDL) 13.6, 17.1,20.7 및 22.6인 트랜스 레티노산이 처리된 NHEK 처리군으로부터 얻어진 세포 추출물(도 5b의 레인 5, 7, 9 및 11)에서 현저하기 증가된 텔로머레이즈 활성이 확인되었다. NHEK 음성 대조군에서는 세포분열(PDL) 수의 변화에 따라 텔로머레이즈의 활성이 거의 유사하였으나, 트랜스 레티노산 처리된 NHEK 처리군에서는 세포분열(PDL) 수가 증가함에 따라 텔로머레이즈 활성이 유지되거나 약간 상승함이 확인되었다. 복제노화의 특징을 나타내는 비히클 처리된 NHEK(PDL 20.5) 및 레티노산 처리된 세포들(PDL 24.3 및 PDL 25.1)에서는 텔로머레이즈 활성이 일정하게 유지되어 빠른 증식성을 나타내는 NHEK로부터 얻어진 세포 추출물에 비해 텔로머레이즈 활성이 거의 검출되지 아니하였다(도 5b의 레인 10, 12 및 13).As a result, cell extracts (lanes 4, 6 and 8 of FIG. 5B) obtained from vehicle treated NHEK negative controls with PDL 13.3, 16.5 and 19.8 and trans retinoic acid with cell division (PDL) 13.6, 17.1, 20.7 and 22.6 were treated. Significantly increased telomerase activity was observed in cell extracts obtained from NHEK treated groups (lanes 5, 7, 9 and 11 of Figure 5b ). In the NHEK negative control, the activity of telomerase was almost similar with the change of the number of cell division (PDL), but in the trans retinoic acid treated NHEK group, the telomerase activity was maintained or slightly increased as the number of cell division (PDL) increased. The rise was confirmed. Vehicle treated NHEKs (PDL 20.5) and retinoic acid treated cells (PDL 24.3 and PDL 25.1), which are characterized by replication aging, maintain constant telomerase activity compared to cell extracts obtained from NHEKs showing rapid proliferation. Little telomerase activity was detected (lanes 10, 12 and 13 in FIG. 5B ).
따라서, 빠른 증식성을 갖는 NHOK 및 NHEK가 트랜스 레티노산에 노출된 경우, 텔로머레이즈의 활성이 그대로 유지되거나 다소 증가됨을 상기도 5a및도 5b에서 알 수 있으며, 상기 트랜스 레티노산이 거듭되는 증식에 따라 NHOK 및 NHEK 내의 텔로머레이즈 활성이 감소되는 것을 방지하여 세포의 복제노화를 억제할 수 있음을 나타낸다.Therefore, when NHOK and NHEK having fast proliferative properties are exposed to trans retinoic acid, it can be seen in FIGS . 5A and 5B that the activity of telomerase remains or is slightly increased, and the trans retinoic acid is repeatedly reproduced. Thus, it is possible to prevent the reduction of telomerase activity in NHOK and NHEK, thereby inhibiting the aging of cells.
<실시예 6> 트랜스 레티노산이 NHOK 및 NHEK 내 p16Example 6 Trans Retinoic Acid in p16 in NHOK and NHEK INK4AINK4A 단백질의 수준에 미치는 영향Effect on the level of protein
트랜스 레티노산에 노출된 NHOK 및 NHEK 세포내 p16INK4A단백질의 발현수준을조사하기 위해, 웨스턴 블럿 분석(Min B.M. et al.,Int. J. Oncol., 1995,7, 249-256)을 항 인간 p53 모노클로날 항체(PAb1801; Oncogene Sciences), 항 인간 p21WAF1/CIP1(187) 모노클로날 항체, 항 쥐 Rb(IF8) 모노클로날 항체 및 항 인간 p16(C-20) 폴리클로날 항체(Santa Cruz Biotechnology)를 사용하여 수행하였다.In order to investigate the NHOK and level of expression of the protein within the p16 INK4A NHEK cells exposed to trans retinoic acid, Western blot analysis (Min BM et al., Int . J. Oncol., 1995, 7, 249-256) an anti-human p53 monoclonal antibody (PAb1801; Oncogene Sciences), anti human p21 WAF1 / CIP1 (187) monoclonal antibody, anti murine Rb (IF8) monoclonal antibody and anti human p16 (C-20) polyclonal antibody ( Santa Cruz Biotechnology).
각 항체들을 사용하여 웨스턴 블럿 분석을 수행한 후, 상기 막을 1 ×Ponceau S 염색용액으로 10 분 동안 염색하여 레인(lane) 당 첨가된 전체 단백질을 정량하였다.After performing Western blot analysis using each antibody, the membrane was stained with 1 × Ponceau S staining solution for 10 minutes to quantify the total protein added per lane.
도 6a는 1 nM의 트랜스 레티노산에 NHOK을 노출시킨 후, NHOK 내 pRb 단백질의 발현 수준을 웨스턴 블럿 분석으로 측정한 결과로서, 비히클 대조군 내 서로 다른 세포분열(PDL) 수를 갖는 NHOK에서는 서로 pRb 단백질의 양이 유사한 정도로 나타났으나, 트랜스 레티노산이 처리된 NHOK에서는 초기 배양(PDL 18)시에는 매우 낮았으며, 세포분열(PDL) 수가 증가함에 따라 점진적으로 증가하였다. 낮은 세포분열(PDL) 수에서 트랜스 레티노산이 처리된 세포에서의 pRb 단백질의 발현수준은 대등한 세포분열(PDL) 수의 대조군에서의 발현수준에 비해 현저히 낮게 나타났으나, 세포분열(PDL) 수가 증가되면서 대조군에 비해 현저히 증가되는 양상을 나타내었다. FIG. 6A shows Western blot analysis of expression levels of pRb protein in NHOK after exposure to 1 nM of trans retinoic acid. In NHOKs with different cell division (PDL) numbers in the vehicle control group, pRb The amount of protein was similar, but it was very low in the initial culture (PDL 18) in NHOK treated with trans retinoic acid, and gradually increased as the number of cell division (PDL) increased. The expression level of pRb protein in cells treated with trans retinoic acid at low cell division (PDL) number was significantly lower than that in the control group of comparable cell division (PDL) number, but the number of cell division (PDL) The increase was significantly increased compared to the control.
도 6b는 1 nM의 트랜스 레티노산에 NHOK을 노출시킨 후, NHOK 내 p16INK4A단백질의 발현 수준을 웨스턴 블럿 분석으로 측정한 결과로서, 트랜스 레티노산이 처리된 구강 상피세포 내 p16INK4A단백질의 발현수준은 현저하게 낮게 나타나는데, 이로써 트랜스 레티노산에 의해 세포의 노화가 억제됨을 확인할 수 있다. 즉, 트랜스 레티노산은 세포내 p16INK4A단백질의 발현수준을 저하시켜 NHOK의 수명을 연장시킬 수 있다. Figure 6b of 1 nM After exposing the NHOK trans retinoic acid, NHOK within p16 INK4A the level of expression of the protein as a result of measurement by Western blot analysis, the level of expression of I p16 INK4A protein trans retinoic acid is the oral epithelium treatment Remarkably low, it can be seen that the inhibition of aging of the cell by trans retinoic acid. In other words, trans retinoic acid can reduce the expression level of the intracellular p16 INK4A protein and extend the life of NHOK.
그 결과, 세포분열(PDL) 수가 증가함에 따라 트랜스 레티노산 처리된 NHOK 또는 비히클 처리된 NHOK 모두에서 세포내 p16INK4A단백질의 발현수준이 점차적으로 현저하게 증가하였다. 상기 트랜스 레티노산 처리된 NHOK군의 노화세포(PDL 28)에서 확인된 p16INK4A단백질의 발현수준은 비히클 처리된 대조군에서와 유사하였으나, 트랜스 레티노산이 처리된 그외의 세포분열(PDL) 수의 NHOK에서는 비히클이 처리된 NHOK에서보다 현저히 낮았다. 이러한 결과는 트랜스 레티노산이 NHOK 내의 텔로머레이즈 활성은 지속적으로 유지시키는 반면, 세포 내 p16INK4A단백질의 발현수준은 감소시켜 노화를 억제함을 나타낸다.As a result, cell division (PDL) according to the number increases, the level of expression of the protein within the cell in both p16 INK4A-trans retinoic acid-treated or vehicle treated NHOK NHOK is increased gradually as significantly. The expression level of p16 INK4A protein in the senescent cells (PDL 28) of the trans-retinoic acid-treated NHOK group was similar to that of the vehicle-treated control group, but in other cell division (PDL) -treated NHOKs treated with trans-retinoic acid. Vehicle was significantly lower than in treated NHOKs. These results indicate that trans retinoic acid maintains telomerase activity in NHOK while inhibiting aging by decreasing the expression level of p16 INK4A protein in cells.
도 6c는 1 nM의 트랜스 레티노산에 NHEK을 노출시킨 후, NHEK 내 p16INK4A단백질의 발현 수준을 웨스턴 블럿 분석으로 측정한 결과이며, 세포분열(PDL) 수가 증가함에 따라 트랜스 레티노산 처리된 NHEK 또는 비히클 처리된 NHEK 모두에서 세포내 p16INK4A단백질의 발현수준이 점차적으로 현저히 증가하였다. 상기 트랜스 레티노산 처리된 NHEK군의 노화세포(PDL 24.3 ∼ 25.1)에서 확인된 p16INK4A단백질의 발현수준은 비히클 처리된 대조군에서와 유사하였으나, 트랜스 레티노산이 처리된 그외의 세포분열(PDL) 수의 NHEK에서는 비히클이 처리된 NHOK에서보다 현저히 낮았다. Figure 6c after exposing the NHEK trans retinoic acid of 1 nM, NHEK within a measurement of the expression level to the analysis Western blot results of the p16 INK4A protein, cell division (PDL) number increases, according to a trans retinoic acid treatment NHEK or the expression level of p16 INK4A within proteins both in the vehicle treated NHEK were gradually significantly increased. The expression level of p16 INK4A protein in senescent cells (PDL 24.3-25.1) of the trans-retinoic acid-treated NHEK group was similar to that of the vehicle-treated control group, but the number of other cell divisions (PDL) treated with trans-retinoic acid was In NHEK the vehicle was significantly lower than in the treated NHOK.
상기 결과로부터, 트랜스 레티노산이 NHEK 내의 텔로머레이즈 활성은 지속적으로 유지시키는 반면, 세포 내 p16INK4A단백질의 발현수준은 감소시켜 노화를 억제함을 나타낸다.These results indicate that trans retinoic acid maintains telomerase activity in NHEKs while inhibiting aging by decreasing the expression level of p16 INK4A protein in cells.
따라서, 본 발명의 트랜스 레티노산 화합물은 NHOK 및 NHEK 내의 p16INK4A단백질의 발현수준을 억제하고 계속된 증식에도 텔로머레이즈(telomerase) 활성을 지속적으로 유지시킴으로써 NHOK 및 NHEK의 노화를 방지하여 상기 상피세포의 노화에 따른 다양한 질환의 치료제 및 예방제로 사용할 수 있다.Accordingly, trans-retinoic acid compounds of the invention by suppressing the expression level of p16 INK4A protein in NHOK and NHEK and in the continued growth keep a steady telomerization raised (telomerase) activity to prevent the aging of NHOK and NHEK the epithelial cells Can be used as a therapeutic and prophylactic agent for various diseases caused by aging.
한편, 본 발명의 레티노산 화합물의 급성 독성을 알아보기 위하여 하기와 같은 실험을 수행하였다.On the other hand, in order to determine the acute toxicity of the retinoic acid compound of the present invention was carried out the following experiment.
<실험예 1> 랫트에 대한 경구투여 급성 독성실험Experimental Example 1 Oral Acute Toxicity in Rats
6주령의 특정병원부재(SPF) SD계 랫트를 사용하여 급성독성실험을 실시하였다. 군당 2 마리씩의 동물에 트랜스 레티노산을 0.5 % 메틸셀룰로즈 용액에 현탁하여 1 g/kg/15 ml의 용량으로 단회 경구투여하였다. 시험물질 투여후 동물의 폐사여부, 임상증상, 체중변화를 관찰하고 혈액학적 검사와 혈액생화학적검사를 실시하였으며, 부검하여 육안으로 복강장기와 흉강장기의 이상여부를 관찰하였다. 시험결과, 시험물질을 투여한 모든 동물에서 특기할 만한 임상증상이나 폐사된 동물은 없었으며, 체중변화, 혈액검사, 혈액생화학 검사, 부검소견 등에서도 독성변화는 관찰되지 않았다. 이상의 결과 실험된 화합물은 모두 랫트에서 2 g/kg까지 독성변화를 나타내지 않으며 경구 투여 최소치사량 (LD50)은 2 g/kg이상인 안전한 물질로 판단되었다.Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally in trans doses of 1 g / kg / 15 ml in suspension of trans retinoic acid in a 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all of the tested compounds did not show toxic changes up to 2 g / kg in rats, and the minimum oral dose (LD50) of 2 g / kg or more was determined to be a safe substance.
<제제예 1> 시럽제의 제조방법Preparation Example 1 Manufacturing Method of Syrup
본 발명의 레티노산 화합물을 유효성분 2%(중량/부피)로 함유하는 시럽은 다음과 같은 방법으로 제조한다.A syrup containing the retinoic acid compound of the present invention in an active ingredient of 2% (weight / volume) is prepared by the following method.
레티노산 화합물의 산부가염, 사카린, 당을 온수 80 g에 용해시켰다. 이 용액을 냉각시킨 후, 여기에 글리세린, 사카린, 향미료, 에탄올, 소르브산 및 증류수로 이루어진 용액을 제조하여 혼합하였다. 이 혼합물에 물을 첨가하여 100 ㎖가 되게 하였다. 상기 부가염은 실시예에 의한 다른 염으로 대치시킬 수 있다.Acid addition salts, saccharin and sugar of the retinoic acid compound were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.
상기 시럽제의 구성성분은 다음과 같다.The components of the syrup are as follows.
레티노산 화합물·염산염 ·············· 2 gRetinoic acid compound, hydrochloride, 2 g
사카린 ······················· 0.8 gSaccharin 0.8 g
당 ························ 25.4 g25.4 g of sugar
글리세린······················ 8.0 gGlycerin ... 8.0 g
향미료 ······················ 0.04 gSpices ··················· 0.04 g
에탄올 ·······················4.0 gEthanol 4.0 g
소르브산 ······················0.4 g0.4 g of sorbic acid
증류수 ·······················정량Distilled water ·····················
<제제예 2> 정제의 제조방법Preparation Example 2 Preparation of Tablet
유효성분 15 mg이 함유된 정제는 다음과 같은 방법으로 제조한다.A tablet containing 15 mg of active ingredient is prepared by the following method.
레티노산 화합물·염산염 250 g을 락토오스 175.9 g, 감자전분 180 g 및 콜로이드성 규산 32 g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160 g, 활석 50 g 및 스테아린산 마그네슘 5 g을 첨가해서 얻은 혼합물을 정제로 만들었다.250 g of retinoic acid compound and hydrochloride were mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.
상기 정제의 구성성분은 다음과 같다.The components of the tablet are as follows.
레티노산 화합물·염산염············ 250 g250 g of retinoic acid compounds, hydrochloride
락토오스 ···················175.9 gLactose ········ 175.9 g
감자전분 ····················180 gPotato starch ········· 180 g
콜로이드성 규산 ················ 32 gColloidal silicic acid 32 g
10 % 젤라틴 용액10% gelatin solution
감자전분 ····················160 gPotato starch · 160 g
활석 ······················ 50 gTalc · 50 g
스테아르산 마그네슘 ··············· 5 gMagnesium stearate 5 g
<제제예 3> 주사액제의 제조방법Preparation Example 3 Manufacturing Method of Injection Solution
유효성분 10 mg을 함유하는 주사액제는 다음과 같은 방법으로 제조하였다.Injection solution containing 10 mg of the active ingredient was prepared by the following method.
레티노산 화합물·염산염 1 g, 염화나트륨 0.6 g 및 아스코르브산 0.1 g을 증류수에 용해시켜서 100 ㎖을 만들었다. 이 용액을 병에 넣고 20℃에서 30 분간 가열하여 멸균시켰다.1 g of retinoic acid compound, hydrochloride, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.
상기 주사액제의 구성성분은 다음과 같다.The components of the injection solution are as follows.
레티노산 화합물·염산염 ·············1 gRetinoic acid compound, hydrochloride, 1 g
염화나트륨···················0.6 gSodium Chloride ・ ・ ・ ・ 0.6 g
아스코르브산··················0.1 g0.1 g of ascorbic acid
증류수········· ············정량Distilled water ···················
상기에서 살펴본 바와 같이, 본 발명의 레티노산 화합물은 인간 구강 점막 상피세포 및 인간 피부 상피세포를 포함하는 인간 상피세포의 수명을 연장시킬 뿐 아니라 복제노화를 억제시켜 노화를 방지하고 상기 인간 상피세포의 노화에 따른다양한 질환인 구강 내 창상 및 발치창, 외상성 궤양(traumatic ulcer), 구각증(Angular cheilosis) 및 구강내 화상의 다양한 구강 질환을 치료ㆍ예방하고, 피부 내 창상 및 피부 노화를 방지하고, 상기 피부 노화를 방지하는 효과로써 화장품 성분으로 사용할 수 있다.As described above, the retinoic acid compound of the present invention not only prolongs the lifespan of human epithelial cells including human oral mucosal epithelial cells and human skin epithelial cells, but also inhibits aging by preventing aging and preventing the aging of the human epithelial cells. It treats and prevents various oral diseases such as oral wounds and ankle wounds, traumatic ulcers, angular cheilosis and intraoral burns, which are various diseases associated with aging, and prevents skin wounds and skin aging, It can be used as a cosmetic ingredient as an effect of preventing the skin aging.
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| WO1998013020A1 (en) * | 1996-09-27 | 1998-04-02 | Unilever Plc | Skin care compositions containing combinations of compounds for mimicking the effect on skin of retinoic acid |
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| US4877805A (en) * | 1985-07-26 | 1989-10-31 | Kligman Albert M | Methods for treatment of sundamaged human skin with retinoids |
| WO1998013020A1 (en) * | 1996-09-27 | 1998-04-02 | Unilever Plc | Skin care compositions containing combinations of compounds for mimicking the effect on skin of retinoic acid |
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| Biochem Biophys Res Commun,Vol 268(2),pp268-274,2000 * |
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