TWI392733B - Method, nucleic acid and pharmaceutical composition for inhibiting tyrosinase translation - Google Patents
Method, nucleic acid and pharmaceutical composition for inhibiting tyrosinase translation Download PDFInfo
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Description
本發明係關於一種抑制黑色素(melanin)生成之方法。具體而言，本發明係關於一種藉由抑制酪胺酸酵素之轉譯，以抑制黑色素生成之方法。The present invention relates to a method of inhibiting the production of melanin. In particular, the present invention relates to a method for inhibiting melanin production by inhibiting translation of tyrosinase.
動物之體色深淺與黑色素之生成息息相關，其中尤以麥拉寧黑色素為最。麥拉寧黑色素係由麥拉寧母細胞(melanoblast)所形成，其形成係受到多方刺激影響，如年齡增加、環境污染、遺傳因素等，但最大的因素係來自過多曝曬紫外線。當接受日曬時，動物體表之酪胺酸酵素(tyrosinase)活化會被活化，而代謝酪胺酸(tyrosine)成為左旋多巴對苯二酚(dopaquinone)，並進一步轉換成麥拉寧黑色素。所生成之麥拉寧黑色素藉由麥拉寧母細胞自身之樹突狀結構，將裝滿黑色素小囊(melanosome)之黑色素傳導到角質細胞(keratin)，角質細胞經由角質分化，由肌膚基底層移動到上層形成上層角質細胞，當角質細胞老化萎縮後，細胞核消失，而形成大量含有角質細胞與黑色素的死細胞，使皮膚暗沉並出現斑點。The body color of animals is closely related to the production of melanin, especially melanin melanin. Melanine melanin is formed by melanoblast, and its formation is affected by multiple stimuli, such as age, environmental pollution, genetic factors, etc., but the biggest factor comes from excessive exposure to ultraviolet light. When exposed to sunlight, the activation of tyrosinase on the surface of the animal is activated, while the metabolism of tyrosine becomes levodopa hydroquinone and further converted to melanin melanin. . The resulting melanin melanin transports melanin filled with melanin vesicles to keratin cells by the dendritic structure of the melanin parent cells themselves. The keratinocytes are differentiated by keratinocytes and the basal layer of the skin. Move to the upper layer to form the upper keratinocytes. When the keratinocytes age and shrink, the nucleus disappears, and a large number of dead cells containing keratinocytes and melanin are formed, which makes the skin dull and spots appear.
目前美白之方式分為物理美白及化學換膚美白兩種。物理美白包含淺層磨皮與脈衝光照射兩種方式：(1)淺層磨皮包含微晶或鹽晶磨皮及鑽石微雕，其藉正壓或負壓之真空吸力，利用微細粒子清除臉上老舊角質。因其作用僅在表皮淺層，只能改善粉刺、毛孔粗大、使膚色較白淨，但對於深層斑點及細紋之消除卻無效果；(2)脈衝光照射利用寬頻波以多波長之脈衝光光束照射皮膚，使全臉一致性的色素變淡，脈衝光可作用至真皮層，增強血管收縮刺激纖維組織再生，但作用強度弱，需多次治療方具預期效果，而且，皮膚經脈衝光多次照射後是否有後遺症，尚屬未知。At present, the method of whitening is divided into physical whitening and chemical skinning and whitening. Physical whitening includes two methods: shallow microdermabrasion and pulsed light irradiation: (1) Shallow microdermabrasion contains microcrystalline or salt crystal microdermabrasion and diamond micro-carving, which uses fine particles to remove the face by vacuum suction of positive or negative pressure. Old horny. Because its effect is only in the shallow layer of the epidermis, it can only improve the acne, the pores are coarse, and the skin color is white, but it has no effect on the elimination of deep spots and fine lines; (2) Pulse light irradiation uses multi-wavelength pulsed light with wide frequency wave The light beam illuminates the skin, so that the pigment of the whole face is lightened. The pulsed light can act on the dermis layer to enhance the vasoconstriction and stimulate the regeneration of the fibrous tissue. However, the intensity of the action is weak, and the treatment needs multiple treatments, and the skin is pulsed. Whether there are sequelae after multiple exposures is unknown.
化學換膚美白包含使用去角質成分或漂白劑成分達到美白效果。Chemical skin whitening involves the use of exfoliating ingredients or bleaching ingredients to achieve a whitening effect.
去角質成分包含酸類(如果酸、甘醇酸、水楊酸或A酸)或左旋維他命C，其作用於皮膚角質層，包含淺層換膚與深層換膚。淺層換膚原理為使用具有腐蝕性或會引起焦痂的成分擦塗於皮膚，以去除角質，然而如停留之時間或濃度控制失當，將會造成程度不一之損傷，甚至造成換膚後皮膚抵抗力降低，變得容易感染或引超過敏反應，尤當皮膚發炎時反而引起發炎後色素沉著之反效果；深層換膚則會有燒灼感且更不舒服，使用者通常在換膚前30分鐘到60分鐘服用阿斯匹靈，以阻斷***素，有助於緩和換膚時的燒灼感，深層換膚因滲透較深，會造成結硬痂，如該換膚區域未接受適當處置，則可能感染並進一步導致疤痕之產生。此外，淺層及深層換膚期間仍需注意減少臉部摩擦，故使用者會被要求正躺睡眠、小心淋浴、最少的臉部摩擦、不要流汗、不要剔臉和避免陽光等，造成生活上之不便。漂白劑成份包含對苯二酚(hydroquinone)、鞠酸(kojic acid)及杜鵑花酸(azelaic acid)，其藉由抑制酪胺酸酶之作用阻止酪胺酸轉換成左旋多巴對苯二酚，以降低麥拉寧母細胞製造麥拉寧黑色素的能力。但這些漂白劑皆具有刺激性，易引起刺灼及紅斑等敏感性的現象，特別是臨床顯示長期使用對苯二酚(特別是高劑量)會有永久性黑色素過多，於少數黑人及東方人上亦被報導會發生後天性黃褐症(ochronosis)，故需於使用2至3個月後完全停止使用這些漂白劑。Exfoliating ingredients contain acids (if acid, glycolic acid, salicylic acid or A acid) or L-vitamin C, which acts on the stratum corneum of the skin, including shallow skin resurfacing and deep skin resurfacing. The principle of shallow skin resurfacing is to use a corrosive or irritating component to rub the skin to remove keratin. However, if the time of stay or the concentration control is improper, it will cause different degrees of damage, even after skin resurfacing. The skin's resistance is reduced, it becomes easy to infect or induces more sensitive reactions, especially when the skin is inflamed, but it causes the anti-inflammatory effect after inflammation; deep skin resurfacing will have a burning sensation and is more uncomfortable, the user usually before the skin resurfacing Taking aspirin from 30 minutes to 60 minutes to block prostaglandins helps to ease the burning sensation during skin resurfacing. Deep skin resurfacing can cause hard knots due to deep penetration. If the skin area is not properly accepted Disposal may cause infection and further lead to scarring. In addition, during shallow and deep skin rejuvenation, attention should be paid to reducing facial friction, so users will be required to lie down, take care of showers, minimize facial friction, do not sweat, do not smear face and avoid sunlight, etc. Inconvenience. The bleaching agent comprises hydroquinone, kokuic acid and azelaic acid, which prevents the conversion of tyrosine to levodopa hydroquinone by inhibiting the action of tyrosinase. To reduce the ability of melanin to produce melanin melanin. However, these bleaching agents are irritating and easily cause sensitization such as stinging and erythema. In particular, clinically, long-term use of hydroquinone (especially high dose) has permanent melanin, in a few black and oriental people. It has also been reported that acquired ochronosis, so it is necessary to completely stop using these bleaching agents after 2 to 3 months of use.
基於上述理由，業界仍須一種兼具便利、有效且安全之抑制黑色素形成之方法。For the above reasons, the industry still needs a method that combines convenience, effectiveness, and safety to inhibit melanin formation.
發明概述Summary of invention
本發明之一目的在於提供一種抑制酪胺酸酵素轉譯之方法，其包含抑制細胞中由酪胺酸酵素第一序列區TIF-1(序列辨識編號1)或酪胺酸酵素第二序列區TIF-2(序列辨識編號2)所編碼片段之轉譯。It is an object of the present invention to provide a method for inhibiting translation of tyrosinase comprising inhibiting a first sequence region of tyrosinase from a cell, TIF-1 (SEQ ID NO: 1) or a second sequence region of tyrosinase, TIF -2 (Sequence Identification Number 2) translation of the encoded fragment.
本發明之另一目的在於提供一種抑制麥拉寧黑色素生成之方法，其包含抑制細胞中由酪胺酸酵素第一序列區TIF-1(序列辨識編號1)或酪胺酸酵素第二序列區TIF-2(序列辨識編號2)所編碼片段之轉譯。Another object of the present invention is to provide a method for inhibiting melanin melanin production comprising inhibiting a first sequence region of tyrosinase in a cell, TIF-1 (SEQ ID NO: 1) or a second sequence region of tyrosidase Translation of the fragment encoded by TIF-2 (Sequence Identification Number 2).
本發明之再一目的在於提供一種核酸分子，其可抑制細胞中由酪胺酸酵素第一序列區TIF-1(序列辨識編號1)或酪胺酸酵素第二序列區TIF-2(序列辨識編號2)所編碼片段之轉譯。A further object of the present invention is to provide a nucleic acid molecule which inhibits TIF-1 (SEQ ID NO: 1) or tyrosinase second sequence region TIF-2 in a cell from tyrosinase (sequence identification) No. 2) Translation of the encoded segment.
本發明之又一目的在於提供一種醫藥組合物，其可用以抑制細胞中酪胺酸酵素之轉譯，其包含治療有效量之前述核酸分子。It is a further object of the present invention to provide a pharmaceutical composition useful for inhibiting translation of tyrosidase in a cell comprising a therapeutically effective amount of the aforementioned nucleic acid molecule.
本發明之另一目的在於提供一種醫藥組合物，其可用以抑制細胞中麥拉寧黑色素之生成，其包含治療有效量之前述核酸分子。Another object of the present invention is to provide a pharmaceutical composition for inhibiting the production of melanin melanin in a cell comprising a therapeutically effective amount of the aforementioned nucleic acid molecule.
發明詳細說明Detailed description of the invention
本發明係利用專一性抑制酪胺酸酵素轉譯之方法，以抑制酪胺酸酵素之作用，並進而抑制麥拉寧黑色素之生成，具有作用快速、有效、操作簡便、安全而無副作用、無毒、可重覆操作之優點，以達到防止或減少麥拉寧黑色素生成、堆積，進而達到美白之作用。The invention utilizes the method of specifically inhibiting the translation of tyrosinase to inhibit the action of tyrosinase and further inhibit the formation of melanin melanin, and has the functions of rapid, effective, simple operation, safety, no side effects, no toxicity, The advantages of repeated operation can be achieved to prevent or reduce the formation and accumulation of melanin melanin, thereby achieving the effect of whitening.
本發明提供一種抑制酪胺酸酵素轉譯之方法，其包含抑制細胞中由酪胺酸酵素第一序列區TIF-1(序列辨識編號1)或酪胺酸酵素第二序列區TIF-2(序列辨識編號2)所編碼片段之轉譯。The present invention provides a method for inhibiting translation of tyrosinase, which comprises inhibiting a first sequence region of tyrosinase from a cell, TIF-1 (SEQ ID NO: 1) or a second sequence region of tyrosinase, TIF-2 (sequence) Identification number 2) Translation of the encoded segment.
根據本發明，當特異性抑制酪胺酸酵素第一序列區或酪胺酸酵素第二序列區所編碼片段之轉譯時，可有效抑制酪胺酸酵素之生產，並進一步抑制麥拉寧黑色素之生成。當施用於皮膚時，可使膚色變淺，達到美白之效果。本發明中酪胺酸酵素第一序列區係相對於酪胺酸酵素基因位置之第79至99個核苷酸；酪胺酸酵素第二序列區係相對於酪胺酸酵素基因位置之第179至199個核苷酸。According to the present invention, when the translation of the fragment encoding the first sequence region of tyrosinase or the second sequence region of tyrosidase is specifically inhibited, the production of tyrosidase can be effectively inhibited, and the melanin melanin is further inhibited. generate. When applied to the skin, the skin tone can be lightened to achieve a whitening effect. In the present invention, the first sequence of tyrosinase is relative to the 79th to 99th nucleotides of the tyrosinase gene position; the second sequence of tyrosidase is relative to the position of the tyrosinase gene. Up to 199 nucleotides.
根據本發明，抑制由酪胺酸酵素第一序列區TIF-1或酪胺酸酵素第二序列區TIF-2所編碼片段轉譯之方法，可依習知抑制一特定片段轉譯之方法進行，較佳地，係使用一可抑制由酪胺酸酵素第一序列區TIF-1或酪胺酸酵素第二序列區TIF-2所編碼片段之核酸分子，該核酸分子係使用RNA干擾(RNA interference；RNAi)、核酸酵素(ribozyme)或反股(antisense)機制抑制由酪胺酸酵素第一序列區TIF-1或酪胺酸酵素第二序列區TIF-2所編碼片段之轉譯；更佳地，其係使用RNA干擾機制。According to the present invention, the method for inhibiting translation of a fragment encoded by the first sequence region of tyrosinase TIF-1 or the second sequence region of tyrosidase TIF-2 can be carried out by conventional methods for inhibiting translation of a specific fragment. Preferably, a nucleic acid molecule which inhibits a fragment encoded by the first sequence region of tyrosinase TIF-1 or the second sequence region of tyrosidase TIF-2, which uses RNA interference (RNA interference; RNAi), ribozyme or antisense mechanism inhibits translation of a fragment encoded by the first sequence region of tyrosinase TIF-1 or the second sequence region of tyrosidase TIF-2; more preferably, It uses an RNA interference mechanism.
RNA干擾機制係指經由雙股RNA(double-strand RNA；dsRNA)誘導轉譯後同源mRNA降解的過程。RNAi係先於細胞內形成雙股RNA，接著該雙股RNA會經雙股切割酶(dicer)作用切成小片段干擾RNA，即siRNAs(short interfering RNA；small interference RNA)，siRNAs再和RNA誘導沉默複合體(RISC，RNA-induced silencing complex)結合，進而降解同源的mRNAs，而具有基因沉默(gene silencing)的特性。於本發明之一具體實施例中，該核酸分子係包含一第一反股RNA分子或一第二反股RNA分子；其中該第一反股RNA分子具有如序列辨識編號4所示之序列；該第二反股RNA分子具有如序列辨識編號6所示之序列；其中第一反股分子之GC量為42.9%，第二反股分子之GC量則為57.1%。The RNA interference mechanism refers to the process of inducing homologous mRNA degradation after translation by double-strand RNA (dsRNA). RNAi forms double-stranded RNA in the cell, and then the double-stranded RNA is cleaved into small interfering RNAs by short-stranding enzymes, ie, short interfering RNA (small interference RNA), siRNAs and RNA induction. The RISC (RNA-induced silencing complex) binds, thereby degrading homologous mRNAs, and has the property of gene silencing. In a specific embodiment of the present invention, the nucleic acid molecule comprises a first anti-strand RNA molecule or a second anti-strand RNA molecule; wherein the first anti-strand RNA molecule has a sequence as shown in sequence identification number 4; The second anti-strand RNA molecule has a sequence as shown in SEQ ID NO: 6; wherein the GC amount of the first anti-strand molecule is 42.9%, and the GC amount of the second anti-strand molecule is 57.1%.
視需要，該核酸分子另包含一第一正股RNA分子或一第二正股RNA分子；其中該第一正股RNA分子具有如序列辨識編號3所示之序列；該第二正股RNA分子具有如序列辨識編號5所示之序列。該等正股RNA分子配合該等反股分子可提早於基因複製階段即分別與DNA之正股及反股結合，以抑制酪胺酸酵素之轉錄。Optionally, the nucleic acid molecule further comprises a first positive stranded RNA molecule or a second positive stranded RNA molecule; wherein the first positive stranded RNA molecule has a sequence as shown in SEQ ID NO: 3; the second positive stranded RNA molecule There is a sequence as shown in sequence identification number 5. The positive-stranded RNA molecules can be combined with the anti-strand molecules of the DNA to bind to the positive and anti-strand of the DNA, respectively, to inhibit the transcription of tyrosinase.
本文中所言之「正股」乙詞係指可轉錄出一由目標基因編碼之產物，通常係指目標基因之編碼序列，於本發明中即為酪胺酸酵素第一序列區TIF-1或酪胺酸酵素第二序列區TIF-2所編碼之片段。本文中所言之「反股」乙詞係指其轉錄之RNA於正常生理條件下可與正股轉錄之RNA進行鹼基配對，進而形成一雙股RNA之核酸；較佳地，正股與反股於正常生理條件下所形成之雙股RNA之鹼基係完全配對。正股與反股之設計係為本發明所屬技術領域中具一般知識者所熟知。As used herein, the term "positive stock" refers to a product that is transcribed from a gene encoded by a target gene, and generally refers to the coding sequence of the target gene. In the present invention, the first sequence region of tyrosinase is TIF-1. Or a fragment encoded by the second sequence region of tyrosinase, TIF-2. As used herein, the term "anti-share" refers to the nucleic acid of a transcribed RNA that can be base-paired with normal-stranded RNA under normal physiological conditions to form a double-stranded RNA; preferably, a positive strand The bases of the double-stranded RNA formed under the normal physiological conditions are completely matched. The design of the positive and negative shares is well known to those of ordinary skill in the art to which the invention pertains.
一般而言，為使RNA分子可充分發揮其RNA干擾之效果，通常於各股之3'端包含一突出段。較佳地，根據本發明之該第一反股RNA分子、該第二反股RNA、該第一正股RNA分子、該第二正股RNA於3'端包含一突出段；更佳地，該突出段係為個數少於五之胸腺嘧啶(thiamine)；尤佳地；該突出段係為二個之胸腺嘧啶。In general, in order for RNA molecules to fully exert their RNA interference effect, a protruding segment is usually included at the 3' end of each strand. Preferably, the first anti-strand RNA molecule, the second anti-strand RNA, the first positive-stranded RNA molecule, and the second positive-stranded RNA according to the present invention comprise a protruding segment at the 3' end; more preferably, The protruding segment is less than five thiamine; more preferably; the protruding segment is two thymines.
使用RNA干擾轉譯因子在細胞質發生並不進入細胞核，故不具嵌入染色體進而產生不可逆之基因突變的危險，同時核酸本體能在細胞中自然分解與代謝，亦無累積殘留的問題，其使用量較無限制，劑量多寡除了效果之外，僅有成本的考量並無其它副作用。The use of RNA interference translation factors does not enter the nucleus in the cytoplasm, so there is no risk of embedding the chromosomes to produce irreversible gene mutations. At the same time, the nucleic acid body can be naturally decomposed and metabolized in the cells, and there is no cumulative residue problem. Limitations, in addition to the effect, there are no other side effects from the cost considerations.
核酸酵素係指具有催化能力的RNA分子，可以經由互補的序列而結合至其目標RNA序列，並進而切割其目標RNA，本發明所屬技術領域中具一般知識之人士可依本說明書之揭示而設計合宜之核酸酵素以抑制酪胺酸酵素之轉譯。A nucleic acid enzyme refers to an RNA molecule having catalytic ability to bind to its target RNA sequence via a complementary sequence, and thereby cleave its target RNA, and a person having ordinary knowledge in the technical field of the present invention can design according to the disclosure of the present specification. A suitable nucleic acid enzyme to inhibit the translation of tyrosinase.
以反股抑制轉譯之方法係指藉由反義核酸分子結合至目標基因之互補序列，而干擾目標基因的早期或晚期的複製過程，於本發明則為設計可結合至酪胺酸酵素第一序列區TIF-1或酪胺酸酵素第二序列區TIF-2互補序列之反義分子，本發明所屬技術領域中具一般知識之人士可依本說明書之揭示而設計合宜之反股以抑制酪胺酸酵素之轉譯。The method for inhibiting translation by anti-strand refers to the early or late replication process of the target gene by binding the antisense nucleic acid molecule to the complementary sequence of the target gene, and in the present invention is designed to bind to the first tyrosinase. An antisense molecule of the sequence region TIF-1 or the tyrosinase second sequence region TIF-2 complementary sequence, and those having ordinary knowledge in the art to which the present invention pertains can design a suitable anti-strand to suppress cheese according to the disclosure of the present specification. Translation of aminase.
本發明之方法中運送核酸至細胞之方法可為習知之核酸分子運送方法。其中較佳地，該核酸係以電子穿孔(electroporation)、超音波、離子導入、自然吸收、或以脂質體(liposome)、γ-聚麩胺酸(γ-polyglutamic acid，γ-PGA)、磷酸鈣、病毒或二乙基胺基乙基-類糊精(diethylaminoethyl-dextran)輔助方式運送至該細胞中；更佳地，該等核酸分子係以電子穿孔方式運送，電子穿孔係於直流電場作用的瞬間，使細胞膜表面產生約105至200 μm之疏水或親水的微小通道，這種通道可維持幾毫秒到幾秒，然後自行恢復，於此期間生物大分子如核酸可通過這種微小的通道進入細胞中。當直接施於動物體之體表皮膚時，電子穿孔方式具有非侵入性、無痛、可控制深度、不破壞皮膚結構，及進入皮膚速度快效率高等優點。The method of transporting nucleic acids to cells in the method of the present invention may be a conventional nucleic acid molecule delivery method. Preferably, the nucleic acid is subjected to electron perforation, ultrasonication, iontophoresis, natural absorption, or liposome, γ-polyglutamic acid (γ-PGA), phosphoric acid. Calcium, virus or diethylaminoethyl-dextran is transported to the cells in an auxiliary manner; more preferably, the nucleic acid molecules are transported by electron perforation, and electron perforation is applied to a DC electric field. Instantly, the surface of the cell membrane produces a hydrophobic or hydrophilic microchannel of about 105 to 200 μm. This channel can be maintained for a few milliseconds to a few seconds and then recovers itself, during which biological macromolecules such as nucleic acids can pass through this tiny channel. Enter the cell. When applied directly to the skin surface of an animal body, the electronic perforation method has the advantages of non-invasive, painless, controllable depth, no damage to the skin structure, and high speed and efficiency in entering the skin.
根據本發明之方法，該細胞係為哺乳動物細胞；較佳為人類細胞；更佳為人類皮膚細胞；最佳為麥拉寧母細胞。According to the method of the present invention, the cell line is a mammalian cell; preferably a human cell; more preferably a human skin cell; preferably a melanin parent cell.
根據本發明使用核酸抑制之方法具有下列優點：(1)可以阻斷酪胺酸基因複製與表現蛋白產物；(2)有效直接減少麥拉寧黑色素；(3)選取之序列能專一性抑制酪胺酸酵素基因，故阻斷酪胺酸酵素效果佳；(4)持續抑制酪胺酸酵素活性效果較長；(5)核酸並非刺激物質，與習用之對苯二酚或鞠酸相較，不具有過敏或其他不適應等問題；(6)使用此方法屬於深層美白，與習用之淺層磨皮或脈衝光等物理美白方法僅具淺層淡斑效果相較，功效更佳且不傷及表皮細胞；與作用在皮膚角質層之果酸、甘醇酸、水楊酸或A酸等酸類或左旋維他命C之化學換膚美白方法相較，本發明之方法不會造成皮膚損傷，更無換膚後皮膚抵抗力降低、易受感染或過敏反應等副作用。The method of using nucleic acid inhibition according to the present invention has the following advantages: (1) can block tyrosine gene replication and express protein product; (2) directly reduce melanin melanin; (3) the selected sequence can specifically inhibit casein Amino acid enzyme gene, so blocking tyrosinase effect; (4) sustained inhibition of tyrosinase activity is longer; (5) nucleic acid is not a stimulating substance, compared with conventional hydroquinone or tannic acid, Does not have allergies or other incompatibility problems; (6) the use of this method is deep whitening, and the physical whitening method such as shallow microdermabrasion or pulsed light is only better than the shallow blemish effect, the effect is better and does not hurt And epidermal cells; compared with the chemical skin whitening method of acid, glycolic acid, salicylic acid or acid acid or levorotatory vitamin C which acts on the stratum corneum of the skin, the method of the invention does not cause skin damage, and Side effects such as reduced skin resistance, vulnerability to infection or allergic reactions without skin rejuvenation.
本發明茲另提供一種抑制麥拉寧黑色素生成之方法，其包含抑制細胞中由酪胺酸酵素第一序列區TIF-1或酪胺酸酵素第二序列區TIF-2所編碼片段之轉譯。The present invention further provides a method of inhibiting melanin melanin production comprising inhibiting translation of a fragment encoded by a first sequence region of tyrosidase TIF-1 or a second sequence region of tyrosinase TIF-2 in a cell.
本發明茲再提供一種核酸分子，其係抑制細胞中由酪胺酸酵素第一序列區TIF-1或酪胺酸酵素第二序列區TIF-2所編碼片段之轉譯。The present invention further provides a nucleic acid molecule which inhibits translation of a fragment encoded by the first sequence region of tyrosidase TIF-1 or the second sequence region of tyrosidase TIF-2 in a cell.
本發明茲又提供一種用以抑制細胞中酪胺酸酵素之轉譯之醫藥組合物，其包含治療有效量之前述核酸分子。The invention further provides a pharmaceutical composition for inhibiting translation of tyrosidase in a cell comprising a therapeutically effective amount of the aforementioned nucleic acid molecule.
本發明另提供一種用以抑制細胞中麥拉寧黑色素之生成之醫藥組合物，其包含治療有效量之前述核酸分子。The invention further provides a pharmaceutical composition for inhibiting the production of melanin melanin in a cell comprising a therapeutically effective amount of the aforementioned nucleic acid molecule.
根據本發明之醫藥組合物可為習用任何可包含核酸分子之劑型，較佳地，其係呈粉狀、液狀、水膠狀、乳液狀或霜狀。The pharmaceutical composition according to the present invention may be any dosage form which may comprise a nucleic acid molecule, preferably in the form of a powder, a liquid, a hydrogel, an emulsion or a cream.
視需要，根據本發明之組合物另包含施用所需之試劑，例如，以電子穿孔、超音波、離子導入、自然吸收、或以脂質體、γ-聚麩胺酸、磷酸鈣、病毒或二乙基胺基乙基-類糊精輔助方式運送所需之試劑。較佳地，當以電子穿孔方式遞送時，該組合物另包含電子穿孔方式所需之試劑，可增加導電度及穿透力，例如21 mM之HEPES、137 mM之NaCl2 ,、5 mM之KCl2 ,、0.7 mM之Na2 HPO4 ,、6 mM之葡萄糖；pH 7.5。The composition according to the invention further comprises, if necessary, a reagent required for administration, for example, by electron perforation, ultrasonication, iontophoresis, natural absorption, or by liposome, γ-polyglutamic acid, calcium phosphate, virus or two The ethylaminoethyl-dextrin assists in the delivery of the required reagents. Preferably, when delivered by electronic perforation, the composition further comprises an agent required for electron perforation to increase conductivity and penetration, such as 21 mM HEPES, 137 mM NaCl 2 , 5 mM KCl 2 , 0.7 mM Na 2 HPO 4 , 6 mM glucose; pH 7.5.
另一方面，根據本發明之組合物視其抑制機制之不同，另包含緩衝液，於本發明之一較佳具體實施例中，當使用RNA干擾機制時，可另包含siRNA緩衝溶液以穩定核酸避免核酸快速分解，例如100.0 mM之KCl、30.0 mM之HEPES-pH 7.5、1.0 mM之MgCl。In another aspect, the composition according to the present invention further comprises a buffer depending on its inhibition mechanism. In a preferred embodiment of the present invention, when an RNA interference mechanism is used, an siRNA buffer solution may be further included to stabilize the nucleic acid. Avoid rapid breakdown of nucleic acids, such as 100.0 mM KCl, 30.0 mM HEPES-pH 7.5, 1.0 mM MgCl.
於本發明之一較佳具體實施例中，該緩衝液包含20 mM之KCl、0.15 mM之CaCl2 、10 mM之K2 HPO4 /KH2 PO4 、25 mM之HEPES、2 mM之EGTA、5 mM之MgCl2 、50 mM之麩胺基硫(glutathione)及2 mM之ATP，pH 7.6，可同時進行RNA干擾且合於電子穿孔之需要。In a preferred embodiment of the present invention, the buffer comprises 20 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 , 25 mM HEPES, 2 mM EGTA, 5 mM MgCl 2 , 50 mM glutathione and 2 mM ATP, pH 7.6, can simultaneously perform RNA interference and meet the needs of electron perforation.
本發明之核酸分子可與其他的美白成分組合，以達到更佳之美白效果，於本發明之一較佳具體實施例中，其另包含左旋維他命C、鞠酸(kojic acid)、杜鵑花酸(azelaic acid)、對苯二酚或皮質類固醇(corticosteroids)。The nucleic acid molecule of the present invention can be combined with other whitening ingredients to achieve a better whitening effect. In a preferred embodiment of the present invention, it further comprises L-vitamin C, kojic acid, and azalea ( Azelaic acid), hydroquinone or corticosteroids.
茲以下列實例予以詳細說明本發明，唯並不意味本發明僅侷限於此等實例所揭示之內容。The invention is illustrated by the following examples, which are not intended to limit the invention.
實例一：於魚體中專一性抑制酪胺酸酵素之活性Example 1: Specific inhibition of tyrosinase activity in fish
電子穿孔：Electroporation:
本實例使用BTXElectro Cell Manipulator(ECM)600系統進行電子穿孔。將50個斑馬魚之新生受精卵置於電極距離為2 mm之小管中。所使用之核酸分子為各為100 μg/mL之第一反股RNA分子(序列辨識編號4)、第二反股RNA分子(序列辨識編號6)、第一正股RNA分子(序列辨識編號3)及第二正股RNA分子(序列辨識編號5)，且該等RNA分子於3'端具有二個胸腺嘧啶分子，並溶於一包含20 mM之KCl、0.15 mM之CaCl2 、10 mM之K2 HPO4 /KH2 PO4 、25 mM之HEPES、2 mM之EGTA、5 mM之MgCl2 、50 mM之麩胺基硫(glutathione)及2 mM之ATP，pH 7.6之緩衝液中。所使用之電子穿孔條件如下：250 V、375 V與500 V/cm；脈衝間距為1至2秒；脈衝長度為0.32至0.4毫秒；脈衝數為3。將已處理之魚卵以Yamamoto沙林(saline)溶液潤洗數次，並於Yamamoto沙林溶液中培養5至6日後，移置乾淨水中。This example uses BTX The Electro Cell Manipulator (ECM) 600 system performs electron perforation. The 50 zebrafish newborn fertilized eggs were placed in small tubes with an electrode distance of 2 mm. The nucleic acid molecule used is a first anti-strand RNA molecule of 100 μg/mL each (SEQ ID NO: 4), a second anti-strand RNA molecule (SEQ ID NO: 6), and a first positive strand RNA molecule (SEQ ID NO: 3) And a second positive stranded RNA molecule (SEQ ID NO: 5), and the RNA molecules have two thymine molecules at the 3' end and are dissolved in a 20 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 , 25 mM HEPES, 2 mM EGTA, 5 mM MgCl 2 , 50 mM glutathione and 2 mM ATP, pH 7.6 buffer. The electron perforation conditions used were as follows: 250 V, 375 V and 500 V/cm; pulse spacing was 1 to 2 seconds; pulse length was 0.32 to 0.4 milliseconds; pulse number was 3. The treated fish eggs were rinsed several times with a Yamamoto saline solution and cultured for 5 to 6 days in a Yamamoto sarin solution, and then placed in clean water.
反轉錄聚合酶連鎖反應Reverse transcription polymerase chain reaction
取5 μg之全RNA於200 μL微量離心管，利用ImProm-IITM 反轉錄酶(Promega，USA)進行合成cDNA。以合成好的cDNA作為模板，加入酪胺酸酵素基因序列專一性引子(序列辨識編號7及8)進行聚合酶連鎖反應擴增分析。Take 5 μg of total RNA in a 200 μL microcentrifuge tube and use ImProm-II TM reverse transcriptase (Promega) , USA) for the synthesis of cDNA. Using the synthesized cDNA as a template, a tyrosinase gene sequence specific primer (SEQ ID NO: 7 and 8) was added for polymerase chain reaction amplification analysis.
聚合酶連鎖反應之反應液為10 μL之10mM Tris-HCl，pH 8.3、90 mM KCl、1 mM MnCl2 、0.2 mM dNTPs、1 mM寡(dT)6、5 mM亂數六員引子(random hexamer primer)及1.25單位之rTh DNA聚合酶(Perkin-Elmer，CA)。將樣品於100℃加熱一分鐘，再於70℃靜置15分鐘，繼以42℃5分鐘供反轉錄反應。於反轉錄反應後，40 μL之鉗合混合液(5%甘油、10 mM Tris-HCl，pH 8.3、100 mM KCl、0.75 mM EGTA、0.05% Tween 20、1.5 mM MgCl2 及0.25 mM之引子)進行60循環之聚合酶連鎖反應(94℃進行1分鐘，62℃進行2分鐘、72℃進行3分鐘)。The polymerase chain reaction reaction solution was 10 μL of 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl 2 , 0.2 mM dNTPs, 1 mM oligo (dT) 6, 5 mM random six-member primer (random hexamer) Primer ) and 1.25 units of rTh DNA polymerase (Perkin-Elmer) , CA). The sample was heated at 100 ° C for one minute and then allowed to stand at 70 ° C for 15 minutes, followed by a reverse transcription reaction at 42 ° C for 5 minutes. After the reverse transcription reaction, 40 μL of the clamped mixture (5% glycerol, 10 mM Tris-HCl, pH 8.3, 100 mM KCl, 0.75 mM EGTA, 0.05% Tween 20, 1.5 mM MgCl 2 and 0.25 mM primer) A 60-cycle polymerase chain reaction was carried out (94 ° C for 1 minute, 62 ° C for 2 minutes, and 72 ° C for 3 minutes).
南方墨點分析Southern ink dot analysis
將聚合酶連鎖反應產物以1.2%瓊脂洋菜膠分析，並轉移至耐龍(nylon)膜上，並以UV交聯。再於55℃由雜交反應液及以DIG-11-dUTP標示之cDNA探針進行雜交反應。所得墨點以5 mL之清洗緩衝液I於室溫清洗一次，再以10 mL之清洗緩衝液II於65℃清洗兩次，接著以10 mL之阻斷溶液阻斷。將稀釋之抗-DIG-AP接合至75 mU/mL阻斷溶液，並於10 mL之抗-DIG-AP溶液中培育該膜30分鐘。再以清洗溶液清洗該膜2次15分鐘；並於10 mL之檢測緩衝液中平衡(equilibrate)2至5分鐘；將該膜進一步移至10 mL新鮮配置之呈色-基質溶液，於避光下反應，並以加入50 mL之水終止呈色反應。The polymerase chain reaction product was analyzed as 1.2% agarwood gel and transferred to a nylon membrane and cross-linked by UV. The hybridization reaction was carried out at 55 ° C from the hybridization reaction solution and the cDNA probe indicated by DIG-11-dUTP. The resulting dots were washed once with 5 mL of Wash Buffer I at room temperature, and then washed twice with 10 mL of Wash Buffer II at 65 ° C, followed by blocking with 10 mL of blocking solution. The diluted anti-DIG-AP was conjugated to a 75 mU/mL blocking solution and the membrane was incubated in 10 mL of anti-DIG-AP solution for 30 minutes. The membrane was washed twice with a washing solution for 15 minutes; equilibrated in 10 mL of assay buffer for 2 to 5 minutes; the membrane was further moved to 10 mL of freshly formulated color-matrix solution, protected from light. The reaction was carried out and the color reaction was terminated by the addition of 50 mL of water.
其結果示於圖1。可知使用根據本發明之RNAi核酸分子專一性抑制酪胺酵素之轉錄。The result is shown in Fig. 1. It is known that the RNAi nucleic acid molecule according to the present invention specifically inhibits the transcription of tyrase.
實例二：於麥拉寧母細胞中專一性抑制酪胺酸酵素之活性Example 2: Specific inhibition of tyrosinase activity in melanin parent cells
本實例之實施方法於實例一類似，惟其材料改使用小鼠之麥拉寧母細胞，其結果示於圖2，亦顯示使用根據本發明之RNAi核酸分子專一性抑制酪胺酵素之轉錄。The method of the present example is similar to that of Example 1, except that the material is changed to the mouse melanin parent cell, and the results are shown in Fig. 2, which also shows that the transcription of tyrase is specifically inhibited by using the RNAi nucleic acid molecule according to the present invention.
本實例中亦使用西方墨點測試酪胺酸酵素之表現，其係將麥拉寧母細胞之全蛋白質進行SDS-聚丙烯醯胺凝膠電泳，並將電泳結束後之膠片以移轉緩衝液浸泡平衡5分鐘，另將PVDF膜以100%甲醇浸漬數秒，同樣以移轉緩衝液浸泡平衡5分鐘，在正極導板上依序放上3 mm紙、膠片、PVDF膜與3mm 47紙，再將負極導板蓋上以26伏特電壓轉印90分鐘轉印結束後取出PVDF膜，浸在TBS中漂洗5分鐘，於阻斷溶液中緩慢震盪2小時，進行阻斷。結束後以TBS漂洗5分鐘，加入10mL之一次抗體溶液(anti tyrosinase polyclone antibody，1：3000)，於室溫下緩慢震盪雜交1-2小時。倒掉一次抗體溶液，以TBS溶液清洗3次，每次5分鐘，再加入10 mL二次抗體溶液(Goat Anti-mouse IgG-HRP conjugate，1：5000)，於室溫下緩慢震盪雜交1-2小時。倒掉二次抗體溶液，以TBS溶液清洗3次，每次5分鐘，加入呈色劑呈色，最後浸泡於蒸餾水中終止呈色反應，呈色結束後將PVDF膜自然風乾，並照相記錄實驗結果。In this example, the western blot is also used to test the performance of tyrosinase, which is to carry out SDS-polyacrylamide gel electrophoresis on the whole protein of the melanin parent cell, and the film after the electrophoresis is transferred to the buffer buffer. Soak and equilibrate for 5 minutes, and immerse the PVDF membrane in 100% methanol for a few seconds. Also equilibrate for 5 minutes with transfer buffer. Place 3 mm paper, film, PVDF membrane and 3mm 47 paper on the positive guide. The negative electrode guide was covered with a voltage of 26 volts for 90 minutes. After the transfer was completed, the PVDF film was taken out, immersed in TBS for 5 minutes, and slowly shaken in the blocking solution for 2 hours to block. After the end, the cells were rinsed with TBS for 5 minutes, and 10 mL of an antibody solution (anti tyrosinase polyclone antibody, 1:3000) was added thereto, and hybridization was slowly shaken at room temperature for 1-2 hours. Pour off the antibody solution once, wash it with TBS solution 3 times for 5 minutes, then add 10 mL of secondary antibody solution (Goat Anti-mouse IgG-HRP conjugate, 1:5000), and shake slowly at room temperature. 2 hours. The secondary antibody solution was poured out, washed with TBS solution for 3 times, each time for 5 minutes, the coloring agent was added for coloring, and finally immersed in distilled water to terminate the color reaction. After the coloring, the PVDF film was naturally air-dried, and the photo recording experiment was performed. result.
其結果示於圖3。其顯示使用根據本發明之RNAi核酸分子專一性抑制酪胺酵素之表現。The result is shown in Fig. 3. It shows that the use of the RNAi nucleic acid molecule according to the invention specifically inhibits the performance of tyrase.
實例三：於魚體內改變體色(一)Example 3: Changing body color in fish (1)
本實例使用BTXElectro Cell Manipulator(ECM)600系統進行電子穿孔。將50個菊池氏細鯽(medaka)之新生受精卵置於電極距離為2 mm之小管中。所使用之核酸分子為各為100 μg/mL之第一反股RNA分子(序列辨識編號4)、第二反股RNA分子(序列辨識編號6)、第一正股RNA分子(序列辨識編號3)及第二正股RNA分子(序列辨識編號5)，且該等RNA分子於3'端具有二個胸腺嘧啶分子，並溶於一包含20 mM之KCl、0.15 mM之CaCl2 、10 mM之K2 HPO4 /KH2 PO4 、25 mM之HEPES、2 mM之EGTA、5 mM之MgCl2 、50 mM之麩胺基硫(glutathione)及2 mM之ATP，pH 7.6之緩衝液中。所使用之電子穿孔條件如下：250 V、375V與500 V/cm；脈衝間距為1至2秒；脈衝長度為0.32至0.4毫秒；脈衝數為3。將已處理之魚卵以Yamamoto沙林(saline)溶液潤洗數次，並於Yamamoto沙林溶液中培養5至6日後，移置乾淨水中。This example uses BTX The Electro Cell Manipulator (ECM) 600 system performs electron perforation. Fifty newborn medaka fertilized eggs were placed in small tubes with an electrode distance of 2 mm. The nucleic acid molecule used is a first anti-strand RNA molecule of 100 μg/mL each (SEQ ID NO: 4), a second anti-strand RNA molecule (SEQ ID NO: 6), and a first positive strand RNA molecule (SEQ ID NO: 3) And a second positive stranded RNA molecule (SEQ ID NO: 5), and the RNA molecules have two thymine molecules at the 3' end and are dissolved in a 20 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 , 25 mM HEPES, 2 mM EGTA, 5 mM MgCl 2 , 50 mM glutathione and 2 mM ATP, pH 7.6 buffer. The electron perforation conditions used were as follows: 250 V, 375 V, and 500 V/cm; the pulse pitch was 1 to 2 seconds; the pulse length was 0.32 to 0.4 msec; and the number of pulses was 3. The treated fish eggs were rinsed several times with a Yamamoto saline solution and cultured for 5 to 6 days in a Yamamoto sarin solution, and then placed in clean water.
實驗結果示於圖4及5，其可知施予反股RNA分子(A、B、C)之魚體色明顯淡於控制組(a、b、c)尤以脊柱之體色更為明顯。The experimental results are shown in Figures 4 and 5. It can be seen that the body color of the fish administered with the anti-strand RNA molecules (A, B, C) is significantly lighter than that of the control group (a, b, c), especially the body color of the spine.
實例四：於魚體內改變體色(二)Example 4: Changing body color in fish (2)
本實例之方法與實例三相同，除改以斑馬魚(zebrafish)為實驗動物。The method of this example is the same as in Example 3 except that zebrafish is used as an experimental animal.
實驗結果示於圖6，其可知施予反股RNA分子(A)之魚體色明顯淡於野生種(a)，其斑紋並不明顯，有白化現象。The experimental results are shown in Fig. 6. It can be seen that the fish body color of the anti-strand RNA molecule (A) is significantly lighter than that of the wild species (a), and the streaks are not obvious, and there is whitening.
實例五：於麥拉寧母細胞中關閉酪胺酸酵素Example 5: Closing tyrosinase in melanin parent cells
本實例之方法與實例三相同，除改以小鼠麥拉寧母細胞(melanosoma cell)為實驗細胞。The method of this example was the same as that of Example 3 except that mouse melanoma cells were used as experimental cells.
實驗結果示於圖7，其可知施予反股RNA分子(1)之細胞失去了色素，而由黑棕色轉為幾乎為白色。The results of the experiment are shown in Fig. 7, which shows that the cells administered to the anti-strand RNA molecule (1) lose the pigment and turn from dark brown to almost white.
上述實施例僅為說明本發明之原理及其功效，而非限制本發明。習於此技術之人士對上述實施例所做之修改及變化仍不違背本發明之精神。本發明之權利範圍應如後述之申請專利範圍所列。The above-described embodiments are merely illustrative of the principles and effects of the invention, and are not intended to limit the invention. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.
圖1為於魚體中使用RNAi分子改變酪胺酸酵素生成之反轉錄聚合酶連鎖反應之南方墨點結果圖，第1行為控制組；第2行為實驗組。Figure 1 is a graph showing the results of Southern blotting of the reverse transcription polymerase chain reaction of tyrosinase production using RNAi molecules in fish, the first behavior control group; the second behavioral experimental group.
圖2為於麥拉寧母細胞中使用RNAi分子改變酪胺酸酵素生成之反轉錄聚合酶連鎖反應之南方墨點結果圖，第1行為麥拉寧母細胞；第2行為正常皮膚細胞；第3行為經RNAi分子處理之麥拉寧母細胞。Figure 2 is a graph showing the results of the southern blot of the reverse transcription polymerase chain reaction of tyrosinase production using RNAi molecules in the melanin parent cells, the first behavior of melanin mother cells; the second behavior of normal skin cells; 3 behavioral melanin parent cells treated with RNAi molecules.
圖3為於麥拉寧母細胞中使用RNAi分子改變酪胺酸酵素生成之反轉錄聚合酶連鎖反應之西方墨點結果圖，第1行為控制組；第2行為實驗組。Figure 3 is a graph showing the results of western blotting of the reverse transcription polymerase chain reaction of tyrosinase production using RNAi molecules in the melanin parent cells, the first behavioral control group; the second behavioral experimental group.
圖4為使用RNAi分子改變菊池氏細鯽體色之結果圖，(A)、(B)及(C)為以RNAi分子處理之魚體；(a)、(b)及(c)為控制組。Figure 4 is a graph showing the results of using the RNAi molecule to change the body color of Kikuchi. (A), (B) and (C) are fish bodies treated with RNAi molecules; (a), (b) and (c) are controlled. group.
圖5為使用RNAi分子改變菊池氏細鯽體色之結果圖，(A)及(B)為以RNAi分子處理之魚體；(a)及(b)為控制組。Figure 5 is a graph showing the results of using the RNAi molecule to change the body color of Kikuchi. (A) and (B) are fish bodies treated with RNAi molecules; (a) and (b) are control groups.
圖6為使用RNAi分子改變斑馬魚體色之結果圖，(A)為以RNAi分子處理之魚體；(a)為野生斑馬魚。Figure 6 is a graph showing the results of using the RNAi molecule to change the body color of zebrafish, (A) is a fish body treated with RNAi molecules; (a) is a wild zebrafish.
圖7為使用RNAi分子關閉麥拉寧母細胞中之酪胺酸酵素結果圖，1為以RNAi分子處理之細胞；2為控制組。Figure 7 is a graph showing the results of shutting down tyrosinase in melanin parent cells using RNAi molecules, 1 is a cell treated with RNAi molecules; 2 is a control group.
<110> 溫士頓生技股份有限公司 <120> 抑制酪胺酸酵素轉譯之方法、核酸分子及醫藥組合物 <130> 無 <140> <141> <150> <151> <160> 8 <170> PatentIn version 3.2 <210> 1 <211> 21 <212> DNA <213> 人工序列 <400> 1<210> 2 <211> 21 <212> DNA <213> 人工序列 <400> 2<210> 3 <211> 21 <212> RNA <213> 人工序列 <400> 3<210> 4 <211> 19 <212> RNA <213> 人工序列 <400> 4<210> 5 <211> 19 <212> RNA <213> 人工序列 <400> 5<210> 6 <211> 19 <212> RNA <213> 人工序列 <400> 6<210> 7 <211> 20 <212> DNA <213> 人工序列 <400> 7<210> 8 <211> 20 <212> DNA <213> 人工序列 <400> 8 <110> Winston Biotechnology Co., Ltd. <120> Methods for inhibiting the translation of tyrosinase, nucleic acid molecules and pharmaceutical compositions <130> None <140><141><150><151><160> 8 <170> PatentIn version 3.2 <210> 1 <211> 21 <212> DNA <213> Artificial sequence <400> 1 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <400> 2 <210> 3 <211> 21 <212> RNA <213> Artificial sequence <400> 3 <210> 4 <211> 19 <212> RNA <213> Artificial sequence <400> 4 <210> 5 <211> 19 <212> RNA <213> Artificial sequence <400> 5 <210> 6 <211> 19 <212> RNA <213> Artificial sequence <400> 6 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <400> 8
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| Boonanuntanasarn, S et al., "Molecular cloning, gene expression in albino mutants and gene knockdown studies of tyrosinase mRNA in rainbow trout", PIGMENT CELL RESEARCH, Volume: 17, Issue: 4, Pages: 413-421, 2004/08 * |
| Pickart, MA et al., " Functional genomics tools for the analysis of zebrafish pigment", PIGMENT CELL RESEARCH, Volume: 17, Issue: 5, Pages: 461-470, 2004/10 * |
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