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TWI670071B - Composition for promoting hair growth or promoting hair cell growth and use thereof - Google Patents

Composition for promoting hair growth or promoting hair cell growth and use thereof Download PDF

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TWI670071B
TWI670071B TW104139834A TW104139834A TWI670071B TW I670071 B TWI670071 B TW I670071B TW 104139834 A TW104139834 A TW 104139834A TW 104139834 A TW104139834 A TW 104139834A TW I670071 B TWI670071 B TW I670071B
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composition
growth
peptide
adiponectin
cells
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TW104139834A
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TW201717992A (en
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宇廷 席
鄭裕耀
林妍冠
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訊聯生物科技股份有限公司
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Abstract

本發明提供一種促進毛髮增長或促進毛髮細胞生長的組合物,其包含一多胜肽組成物以及脂溶性化合物,其中該脂溶性化合物為脂聯素(adiponectin)或維生素D3;該多胜肽組成物之體積百分比為1%至20%多胜肽組成物,該脂聯素為每毫升0.1奈克(ng/ml)至100 ng/ml,該維生素D3為1奈莫耳濃度(nM)至1000 nM。本發明另提供一種前述之組合物用於製備促進毛髮增長或促進毛髮細胞生長的醫藥品的用途,其中將有效劑量之該醫藥品施予受體以促進受體之毛髮增長或促進毛髮細胞生長,藉以解決現有治療脫毛症藥物效果不彰的問題。The present invention provides a composition for promoting hair growth or promoting hair cell growth, comprising a multi-peptide composition and a fat-soluble compound, wherein the fat-soluble compound is adiponectin or vitamin D3; The volume percentage of the substance is 1% to 20% multipeptide composition, the adiponectin is 0.1 ng/ml to 100 ng/ml per ml, and the vitamin D3 is 1 nanomolar concentration (nM) to 1000 nM. The present invention further provides the use of the aforementioned composition for the preparation of a medicament for promoting hair growth or promoting hair cell growth, wherein an effective amount of the medicament is administered to a recipient to promote hair growth of the recipient or to promote hair cell growth. In order to solve the problem of the ineffectiveness of the existing drugs for treating hair loss.

Description

促進毛髮增長或促進毛髮細胞生長的組合物及其用途Composition for promoting hair growth or promoting hair cell growth and use thereof

本發明係涉及一種促進毛髮增長的組合物,特別是指包含多胜肽組成物以及脂溶性化合物的組合物,其中該脂溶性化合物為脂聯素或維生素D3;本發明另涉及一種前述之組合物用於製備促進毛髮增長的醫藥品的用途;本發明更涉及一種前述之組合物用於製備促進毛髮細胞生長的醫藥品的用途。The present invention relates to a composition for promoting hair growth, and particularly to a composition comprising a multi-peptide composition and a fat-soluble compound, wherein the fat-soluble compound is adiponectin or vitamin D3; the present invention further relates to a combination of the foregoing Use of the medicament for the preparation of a medicament for promoting hair growth; the invention further relates to the use of a composition as described above for the preparation of a medicament for promoting hair cell growth.

脫毛症分為先天性和後天性兩種,先天性見於遺傳因素,後天性多發生於神經性疾病、內分泌疾病、毛囊血流減少、老化等身體因素,再加上壓力增加及生活型態改變都是造成脫毛症的發生原因。Hair loss is divided into two types: congenital and acquired. Congenital conditions are found in genetic factors. Acquired factors occur in neurological diseases, endocrine diseases, hair follicle blood flow reduction, aging and other physical factors, plus increased stress and lifestyle changes. Both cause the cause of hair loss.

已知米諾地爾(minoxidil,MNX)係作用於毛囊真皮乳頭細胞(dermal papilla cell),能而促進細胞生長並使毛囊細胞活化的效果(參照J. Invest.Dermatol., 117, 2001, p.1594-1600)。米諾地爾已由美國食品及藥物管理局核准用於男性以及女性,其中三分之一的男性以及女性雄性禿患者中產生可看見的毛髮生長,另外三分之一的患者中產生細微的毛髮生長,以及剩餘三分之一的患者中沒有產生毛髮生長,因此米諾地爾仍然無法達到令人滿意的效果。Minoxidil (MNX) is known to act on hair follicle dermal papilla cells, which promotes cell growth and activates hair follicle cells (see J. Invest. Dermatol., 117, 2001, p .1594-1600). Minoxidil has been approved by the US Food and Drug Administration for males and females, with one-third of males and female males with baldness producing visible hair growth, and one-third of patients producing subtle Hair growth, and the remaining one-third of the patients did not produce hair growth, so minoxidil still failed to achieve satisfactory results.

為求精進,研究如何使促進毛髮增長或促進毛髮細胞生長更有效率,現有技術實有待改善的必要。In order to improve, it is necessary to study how to promote hair growth or promote hair cell growth, and the prior art needs to be improved.

為了克服現有技術之缺點,本發明的目的在於提供一種能有效達成促進毛髮增長或促進毛髮細胞生長效果的組合物。In order to overcome the disadvantages of the prior art, it is an object of the present invention to provide a composition which can effectively achieve an effect of promoting hair growth or promoting hair cell growth.

為達到上述之發明目的,本發明所採用的技術手段為提供一種促進毛髮增長的組合物,其包含一多胜肽組成物以及脂聯素;其中該多胜肽組成物之體積百分比為1%至20%多胜肽組成物,該脂聯素為每毫升0.1奈克(ng/ml)至100 ng/ml。In order to achieve the above object, the technical means employed by the present invention is to provide a composition for promoting hair growth comprising a multi-peptide composition and adiponectin; wherein the multi-peptide composition has a volume percentage of 1% Up to 20% multipeptide composition, the adiponectin is from 0.1 ng (ng/ml) to 100 ng/ml per ml.

較佳的,所述之多胜肽組成物係選自於由血管生成素(angiogenin)、血小板衍生生長因子(platelet-derived growth factor,PDGF)、纖維母細胞生長因子7 (fibroblast growth factor7,FGF7)及其組合所組成之群組。Preferably, the multi-peptide composition is selected from the group consisting of angiogenin, platelet-derived growth factor (PDGF), and fibroblast growth factor 7 (FGF7). ) and a group of its combinations.

較佳的,所述之組合物包含體積百分比為10%的多胜肽組成物以及1 ng/ml的脂聯素。Preferably, the composition comprises 10% by volume of a multi-peptide composition and 1 ng/ml of adiponectin.

較佳的,所述之多胜肽組成物係由以下製備方法所製得:齊備一豬臍帶(umbilical cords)組織;以沖洗液沖洗該豬臍帶組織,使血球細胞或體液完全從豬臍帶組織中移除;由豬臍帶組織分離細胞以進行繼代培養,以獲得細胞激素;將細胞激素及細胞進行凍融循環至少兩次,以獲得一多胜肽混合溶液;以及將該多胜肽混合溶液濃縮去鹽,以獲得一多胜肽組成物,其中該多胜肽組成物之分子量大於3000道爾頓(dalton,Da)。Preferably, the multi-peptide composition is prepared by the following preparation method: preparing a umbilical cords tissue; rinsing the pig umbilical cord tissue with a rinsing liquid to completely separate the blood cells or body fluid from the umbilical cord tissue of the pig Removing; separating cells from pig umbilical cord tissue for subculture to obtain cytokines; freezing and thawing cytokines and cells at least twice to obtain a multi-peptide mixed solution; and mixing the multi-peptide The solution is concentrated to remove salt to obtain a multi-peptide composition wherein the multi-peptide composition has a molecular weight greater than 3000 Daltons (Da).

依據本發明,「沖洗液」如此處所述係指對於豬臍帶組織為等張之溶液;較佳的,所述之沖洗液係90%氯化鈉溶液或磷酸鹽緩衝溶液(phosphate buffered solution,PBS)。According to the present invention, "flushing solution" as used herein refers to a solution which is isotonic to the umbilical cord tissue of a pig; preferably, the rinsing liquid is a 90% sodium chloride solution or a phosphate buffered solution. PBS).

依據本發明,「分離細胞」如此處所述係指將豬臍帶切成約為0.1立方毫米(mm3 )至100 mm3 的碎片至6塊臍帶碎片放置培養皿中,其培養皿中包括10毫升(ml)的培養基[α-最低必需培養基(α-minimum essential medium, α-MEM)以及10%胎牛血清(fetal bovine serum,FBS)]中,並置於恆溫容器培養10天後再去除臍帶碎片,且每週加兩次3毫升(ml)培養基,其中恆溫容器係指二氧化碳(CO2 )培養箱(含有5% CO2 ),以獲得細胞。According to the present invention, "isolated cells" as used herein refers to a piece of pig umbilical cord cut into pieces of about 0.1 cubic millimeters (mm 3 ) to 100 mm 3 into 6 pieces of umbilical cord pieces placed in a petri dish, including 10 in the culture dish. Milliliter (ml) of medium [α-minimum essential medium (α-MEM) and 10% fetal bovine serum (FBS)], and placed in a constant temperature container for 10 days before removing the umbilical cord Fragments, and 3 ml (ml) of medium were added twice a week, wherein the constant temperature container refers to a carbon dioxide (CO 2 ) incubator (containing 5% CO 2 ) to obtain cells.

依據本發明,「繼代培養」如此處所述係指當細胞生長至高密度時,即須收集細胞,經稀釋後分殖至新的培養皿中,以降低細胞密度並維持細胞生長,其稀釋比例依細胞種類而異。According to the present invention, "subculture" as used herein means that when the cells are grown to a high density, the cells are collected, and after dilution, are colonized into a new culture dish to reduce cell density and maintain cell growth. The ratio varies depending on the cell type.

依據本發明,「凍融循環」如此處所述係指將細胞及其細胞激素放置於液態氮中冷凍,然後再將細胞取出至室溫中融化,反覆多次而達到破壞細胞膜之效果。如本案所例示,係將含有細胞之冷凍管直接浸置於液態氮中,再將含有細胞之冷凍管由液態氮中取出並置於37°C水浴解凍,重複以上步驟循環至少兩次,並將破裂的細胞於1000 G離心15分鐘至20分鐘。According to the present invention, the "freeze-thaw cycle" as used herein refers to freezing cells and their cytokines in liquid nitrogen, and then taking the cells out to room temperature for melting, and repeatedly to achieve the effect of destroying the cell membrane. As exemplified in the present case, the cryotube containing the cells is directly immersed in liquid nitrogen, and the cryotube containing the cells is taken out from the liquid nitrogen and thawed in a 37 ° C water bath, and the above steps are repeated at least twice, and The ruptured cells were centrifuged at 1000 G for 15 minutes to 20 minutes.

依據本發明,「濃縮去鹽」如此處所述係指將多胜肽混合溶液放置過濾裝置中,並經由離心及去除上清液,使多胜肽混合溶液達到濃縮並去除鹽類的效果。如本案所例示,係將多胜肽混合溶液置於超濾裝置或離心設備,以減少多胜肽混合溶液的體積、增加濃度以及去除雜質(鹽類),並獲得一組合物,且該組合物之分子量大於3000道爾頓。According to the present invention, "concentrated desalted" as used herein refers to the effect of placing a multi-peptide mixed solution in a filtration device, and centrifuging and removing the supernatant to concentrate and remove salts of the multi-peptide mixed solution. As exemplified in the present invention, the multi-peptide mixed solution is placed in an ultrafiltration device or a centrifugal device to reduce the volume of the multi-peptide mixed solution, increase the concentration, and remove impurities (salts), and obtain a composition, and the combination The molecular weight of the substance is greater than 3000 Daltons.

較佳的,所述之由豬臍帶組織分離細胞以進行繼代培養並獲得細胞激素之步驟中,係將分離所得之細胞置於不含血清之培養基培養細胞3天至18天,以獲得細胞激素。Preferably, in the step of separating cells from pig umbilical cord tissue for subculture and obtaining cytokines, the isolated cells are cultured in serum-free medium for 3 days to 18 days to obtain cells. hormone.

更佳的,所述之由豬臍帶組織分離細胞以進行繼代培養並獲得細胞激素之步驟中,係將分離所得之細胞置於不含血清之培養基培養細胞12天,以獲得細胞激素。More preferably, in the step of separating cells from pig umbilical cord tissue for subculture and obtaining cytokines, the isolated cells are cultured in serum-free medium for 12 days to obtain cytokines.

較佳的,所述之豬臍帶組織係來自無特定病原(specific pathogen free,SPF)豬。Preferably, the pig umbilical cord tissue is derived from a specific pathogen free (SPF) pig.

本發明另提供一種促進毛髮增長的醫藥組合物,其包含有效劑量之前述之多胜肽組成物與脂聯素,以及藥學上可接受之載劑(pharmaceutically acceptable vehicle)。The present invention further provides a pharmaceutical composition for promoting hair growth comprising an effective amount of the aforementioned multi-peptide composition and adiponectin, and a pharmaceutically acceptable vehicle.

本發明又提供一種前述之組合物用於製備促進毛髮增長的醫藥品的用途,其中將有效劑量之該醫藥品施予受體以促進受體之毛髮增長。The invention further provides the use of a composition as described above for the manufacture of a medicament for promoting hair growth, wherein an effective amount of the medicament is administered to a recipient to promote hair growth of the recipient.

本發明再提供一種前述之組合物用於製備促進毛髮細胞生長的醫藥品的用途,其中將有效劑量之該醫藥品施予受體以促進受體之毛髮細胞生長。The invention further provides the use of a composition as described above for the manufacture of a medicament for promoting growth of hair cells, wherein an effective amount of the medicament is administered to a recipient to promote hair cell growth of the recipient.

較佳的,所述之組合物更包含0.1微莫耳濃度(mM)至100 mM米諾地爾(minoxidil,MNX)、1奈莫耳濃度(nM)至1000 nM維生素D3或其組合。Preferably, the composition further comprises 0.1 micromolar concentration (mM) to 100 mM minoxidil (MNX), 1 nanomolar concentration (nM) to 1000 nM vitamin D3 or a combination thereof.

更佳的,所述之米諾地爾為1 mM,該維生素D3為10 nM。More preferably, the minoxidil is 1 mM and the vitamin D3 is 10 nM.

較佳的,所述之組合物更包含1 nM至1000 nM的維生素D3。Preferably, the composition further comprises from 1 nM to 1000 nM of vitamin D3.

更佳的,所述之維生素D3為10 nM。More preferably, the vitamin D3 is 10 nM.

本發明再提供一種促進毛髮增長的組合物,其包含一多胜肽組成物以及維生素D3 (vitamin D3),其中該多胜肽組成物之體積百分比為1%至20%多胜肽組成物,該維生素D3為1 nM至1000 nM。The present invention further provides a composition for promoting hair growth comprising a multi-peptide composition and vitamin D3 (vitamin D3), wherein the volume fraction of the multi-peptide composition is from 1% to 20% of the peptide composition, The vitamin D3 is from 1 nM to 1000 nM.

較佳的,所述之多胜肽組成物之體積百分比為10%多胜肽組成物,該維生素D3為10 nM。Preferably, the multi-peptide composition has a volume percentage of 10% polypeptide composition and the vitamin D3 is 10 nM.

本發明另提供一種促進毛髮增長的醫藥組合物,其包含有效劑量之前述之多胜肽組成物與維生素D3,以及藥學上可接受之載劑。The invention further provides a pharmaceutical composition for promoting hair growth comprising an effective amount of the aforementioned multi-peptide composition and vitamin D3, and a pharmaceutically acceptable carrier.

本發明又提供一種如前述之組合物用於製備促進毛髮增長的醫藥品的用途,其中將有效劑量之該醫藥品施予受體可促進受體之毛髮增長。The invention further provides the use of a composition as hereinbefore described for the manufacture of a medicament for promoting hair growth, wherein administration of an effective amount of the medicament to a recipient promotes hair growth of the recipient.

較佳的,所述之醫藥品是局部或全身性施予受體受脫毛症影響的皮膚之區域。Preferably, the pharmaceutical product is a region of the skin which is locally or systemically administered to the recipient to be affected by the hair loss.

更佳的,所述之受體受脫毛症影響的皮膚之區域為頭皮。More preferably, the area of the skin affected by the hair loss is scalp.

較佳的,所述之受體是人類或動物。Preferably, the receptor is a human or an animal.

本發明的組合物係可利用熟習此技藝者所詳知的技術,將上述的多胜肽組成物與脂聯素與一藥學上可接受之載劑、或多胜肽組成物與維生素D3與一藥學上可接受之載劑,製備成一適用本發明組合物之劑型。其中本發明所述之「醫藥上可接受之載劑」包含,但不限於溶劑(solvent)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、黏結劑(binding agent)、賦形劑(excipient)、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防腐劑(preservative)、潤滑劑(lubricant)、表面活性劑(surfactant),及其他類似或適用本發明之載劑。The compositions of the present invention may be a combination of the above-described multi-peptide composition with adiponectin and a pharmaceutically acceptable carrier, or a multi-peptide composition and vitamin D3, using techniques well known to those skilled in the art. A pharmaceutically acceptable carrier is prepared in a dosage form suitable for use in the compositions of the present invention. The "pharmaceutically acceptable carrier" as used in the present invention includes, but is not limited to, a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent. , excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, surface activity Surfactant, and other carriers similar or suitable for use in the present invention.

本發明所述之「醫藥組合物」可以多種形式存在,該等形式包含,但不限於液體、半固體及固體藥劑形式,諸如溶液(solution)、乳劑(emulsion)、懸浮液(suspension)、粉末(powder)、錠劑(tablet)、丸劑(pill)、口含錠(lozenge)、片劑(troche)、口嚼膠(chewing gum)、膠囊(slurry)、脂質體以及其他類似或適用本發明之劑型。The "pharmaceutical composition" of the present invention may exist in various forms including, but not limited to, liquid, semi-solid, and solid pharmaceutical forms such as solutions, emulsions, suspensions, powders. (powder), tablet, pill, lozenge, troche, chewing gum, slurry, liposome, and the like or other suitable for use in the present invention Formulation.

本發明所述之「有效劑量」係指在劑量上及對於所需要之時間而言對達成促進毛髮增長結果有效之量;依據本發明,係指透過施予特定範圍量之多胜肽組成物與脂聯素、或多胜肽組成物與維生素D3,能夠達成使得毛髮增長之功效;更佳的是能夠達成加乘效果(synergistic effect)的功效。"Effective dose" as used herein means an amount effective to achieve a hair growth promoting effect at a dose and for a desired period of time; in accordance with the present invention, a multi-peptide composition is administered by administering a specific range of amounts. With the adiponectin, or the multi-peptide composition and the vitamin D3, the effect of hair growth can be achieved; more preferably, the effect of achieving a synergistic effect can be achieved.

本發明再提供一種前述之組合物用於製備治療脫毛症的醫藥品的用途,其中將有效劑量之該醫藥品施予受體以治療受體之脫毛症。The invention further provides the use of a composition as described above for the manufacture of a medicament for the treatment of alopecia, wherein an effective amount of the medicament is administered to a recipient for the treatment of receptor depilation.

本發明所述之「脫毛症」,又稱禿毛症、無毛症、或稀毛症,是指局部或全身被毛脫落的總稱,以脫毛為主要特徵。禿頭則為於頭皮的毛髮脫落所形成的症狀。The "hair loss" described in the present invention, also known as alopecia, hairlessness, or thin hair, is a general term for local or whole body hair loss, and is mainly characterized by hair removal. Baldness is a symptom of hair loss from the scalp.

依據本發明,脂聯素或維生素D3皆屬於「脂溶性化合物」的一種。According to the present invention, adiponectin or vitamin D3 is a kind of "fat-soluble compound".

本發明的優點在於本發明包含多胜肽組成物以及脂聯素的組合物,可有效促進毛髮增長與促進細胞生長,且多胜肽組成物以及脂聯素的組合對於促進毛髮增長與促進細胞生長的效果皆具有加乘效果。本發明另一包含多胜肽組成物以及維生素D3的組合物可有效促進毛髮增長,且多胜肽組成物以及維生素D3的組合對於促進毛髮增長亦具有加乘效果。An advantage of the present invention is that the present invention comprises a composition of a multi-peptide composition and adiponectin, which is effective for promoting hair growth and promoting cell growth, and a combination of a multi-peptide composition and adiponectin for promoting hair growth and promoting cells The effects of growth have a multiplier effect. Another composition comprising the multi-peptide composition and vitamin D3 of the present invention is effective for promoting hair growth, and the combination of the multi-peptide composition and vitamin D3 also has a multiplying effect on promoting hair growth.

以下配合圖式及本發明之較佳實施例,進一步闡述本發明為達成目的所採取的技術手段。The technical means adopted by the present invention for achieving the object are further explained below in conjunction with the drawings and the preferred embodiments of the present invention.

多胜肽組成物的製備方法,係由中華民國發明專利申請案第103135440號「促進真皮乳頭細胞生長之組成物及其醫藥組合物與其製備方法」中所製備而得,詳細製備方法如下製備例1至製備例6所述:The preparation method of the multi-peptide composition is prepared by the Chinese Patent Application No. 103135440 "Composition for promoting the growth of dermal papilla cells and a pharmaceutical composition thereof and a preparation method thereof", and the detailed preparation method is as follows. 1 to Preparation Example 6:

製備例1 由豬臍帶分離細胞Preparation Example 1 Separation of cells from pig umbilical cord

齊備一豬臍帶,將豬臍帶以75%乙醇清洗20秒至30秒後,再以PBS清洗後,將臍帶切成3至4等份並至於以滅菌之培養盤中。以手術刀或鑷子沿臍帶靜脈將臍帶打開,並將臍帶以兩鑷子將臍帶拉開,小心將動脈移除以避免任何血液潑濺至華通氏膠(Wharton’s jelly,臍帶內膠狀結締組織),同時亦將靜脈移除。將華通氏膠塊從線羊膜(cord amnion)移除並置於新鮮且含有α-MEM培養皿中以保持其潤濕。接著以外科剪刀臍帶切成約為0.1 mm3 至100 mm3 以形成臍帶碎片,並將4塊至6塊臍帶碎片放置培養盤中,其培養盤中包括10 ml的培養基(α-MEM以及10% FBS)中,並置於CO2 培養箱(含有5% CO2 )。每週加兩次3 ml培養基。10天後將臍帶碎片移除,並以PBS洗滌後再加入新鮮培養基,以使細胞培養至80%至90%滿,並進行繼代培養以增殖細胞。A pig umbilical cord is prepared, and the pig umbilical cord is washed with 75% ethanol for 20 seconds to 30 seconds, and then washed with PBS, and then the umbilical cord is cut into 3 to 4 aliquots and sterilized in a culture dish. Open the umbilical cord with a scalpel or forceps along the umbilical cord vein, and pull the umbilical cord apart with the umbilical cord. Carefully remove the artery to avoid any blood spilling into the Wharton's jelly. The vein is also removed. The Huatong's block was removed from the cord amnion and placed in a fresh and containing alpha-MEM dish to keep it moist. The umbilical cord of the surgical scissors is then cut into approximately 0.1 mm 3 to 100 mm 3 to form umbilical cord fragments, and 4 to 6 umbilical cord fragments are placed in a culture dish containing 10 ml of medium (α-MEM and 10). In % FBS), placed in a CO 2 incubator (containing 5% CO 2 ). Add 3 ml of medium twice a week. After 10 days, the umbilical cord fragments were removed, and washed with PBS and then fresh medium was added to culture the cells to 80% to 90% full, and subcultured to proliferate the cells.

製備例2 繼代培養細胞Preparation 2 Subcultured cells

藉由真空抽吸器吸除培養基,並以5 ml PBS清洗培養盤中之細胞。再次移除PBS,並將3 ml且濃度為0.25%胰蛋白酶(trypsin)-乙二胺四乙酸(EDTA)加到各培養盤中;將培養盤放置在37°C、5分鐘後。將分離的細胞收集於15 ml離心管中,以400 G離心5分鐘後,去除上清液並加入2 ml培養基。藉由吸量管將細胞重新懸浮,並吸取10微升(μl)細胞並與10 ul剛果藍(trypan blue)混合並以自動細胞計數器進行細胞計數。The medium was aspirated by a vacuum aspirator and the cells in the plate were washed with 5 ml of PBS. The PBS was again removed, and 3 ml of a concentration of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) was added to each plate; the plate was placed at 37 ° C for 5 minutes. The separated cells were collected in a 15 ml centrifuge tube, centrifuged at 400 G for 5 minutes, and the supernatant was removed and 2 ml of the medium was added. The cells were resuspended by a pipette and 10 microliters (μl) of cells were aspirated and mixed with 10 ul of trypan blue and counted in an automated cell counter.

製備例3 製備細胞激素(cytokines)Preparation Example 3 Preparation of cytokines (cytokines)

為產生細胞激素,首先將製備例2所得之細胞接種到細胞培養皿中,且每培養皿生長至3x105 至7x105 個細胞的密度。將細胞培養在37°C且CO2 培養箱中,培養基每週更換兩次(約每3天至4天),直到細胞生長至90%滿。To produce cytokines, the cells obtained in Preparation Example 2 were first inoculated into a cell culture dish, and grown to a density of 3 x 10 5 to 7 x 10 5 cells per dish. The cells were cultured in a CO 2 incubator at 37 ° C and the medium was changed twice a week (approximately every 3 to 4 days) until the cells were grown to 90% full.

當細胞長至90%滿時,以5 ml PBS洗滌細胞兩次,再將8 ml不含血清之培養基加到每個培養皿,並於37°C且CO2 培養箱中培養12天,以獲得細胞激素。When the cells were 90% full, wash the cells twice with 5 ml PBS, add 8 ml of serum-free medium to each dish, and incubate for 12 days at 37 ° C in a CO 2 incubator. Obtain cytokines.

製備例4 收集多胜肽混合溶液Preparation Example 4 Collection of multi-peptide mixed solution

部分細胞激素會釋放至培養基中,而另一些細胞激素則駐留在細胞內。收集培養基,並將附著的細胞暴露於3 ml且濃度為0.2%胰蛋白酶-EDTA在37°C處理5分鐘,收集細胞並以40 G離心5分鐘並去除上清液,再以10 ml的PBS洗滌已離心沉澱的細胞1次。再次離心並去除上清液,然後再將細胞重新懸浮在3 ml PBS並置於冷凍管中,再將含有細胞之冷凍管直接浸置於液態氮中。將含有細胞之冷凍管置於37°C水浴解凍,重複以上凍融循環兩次以打破細胞。將破裂的細胞於1000 G離心15分鐘至20分鐘獲得上清液和細胞裂解物。混合上清液和細胞裂解物得多胜肽混合溶液,並藉由免疫酵素測定法(enzyme-linked immunosorbent assay,ELISA)檢測上清液或多胜肽混合溶液[例如重組鹼性成纖維細胞生長因子(basic fibroblast growth factor, bFGF)或血小板衍生生長因子(PDGF)等]的濃度。Some cytokines are released into the medium, while others lie in the cells. The medium was collected, and the attached cells were exposed to 3 ml and treated with 0.2% trypsin-EDTA for 5 minutes at 37 ° C. The cells were collected and centrifuged at 40 G for 5 minutes and the supernatant was removed, followed by 10 ml of PBS. The precipitated cells were washed once for 1 time. Centrifuge again and remove the supernatant, then resuspend the cells in 3 ml PBS and place in a cryotube, then immerse the cryotube containing the cells directly in liquid nitrogen. The cryotube containing the cells was thawed in a 37 ° C water bath, and the above freeze-thaw cycles were repeated twice to break the cells. The ruptured cells were centrifuged at 1000 G for 15 minutes to 20 minutes to obtain supernatant and cell lysate. Mixing the supernatant and cell lysate with a peptide mixture solution, and detecting the supernatant or multi-peptide mixed solution by enzyme-linked immunosorbent assay (ELISA) [eg, recombinant basic fibroblast growth) The concentration of a factor (basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF), etc.].

製備例5 多胜肽混合溶液之濃度及去鹽(desalting)Preparation Example 5 Concentration and desalting of a multi-peptide mixed solution

將製備例4所獲得之多胜肽混合溶液放入超濾裝置(型號為Amicon® stir cell)或離心設備(Amicon® filter centrifugation device)以減少多胜肽混合溶液的體積以及增加濃度,以獲得經濃縮之多胜肽混合溶液。經濃縮之多胜肽混合溶液由超濾裝置或離心設備中放入收集管中。取2 ml經濃縮之多胜肽混合溶液以評估多胜肽混合溶液的濃度。Preparation Example 4 The peptide obtained was poured as much as ultrafiltration device (Model Amicon ® stir cell) or centrifugal device (Amicon ® filter centrifugation device) to reduce the volume of the multi-peptide and increasing concentrations of the mixed solution, to obtain Concentrated multi-peptide mixed solution. The concentrated multi-peptide mixed solution is placed in a collection tube by an ultrafiltration device or a centrifugal device. 2 ml of the concentrated multi-peptide mixed solution was taken to evaluate the concentration of the multi-peptide mixed solution.

製備例6 冷凍乾燥(lyophilization)多胜肽混合溶液Preparation Example 6 Lyophilization Polypeptide Mixture Solution

將製備例5所獲得之50 ml經濃縮之多胜肽混合溶液置於夾鏈袋並於-80°C過夜(12小時至16小時)。將含有經濃縮之多胜肽混合的袋子一同放入冷凍乾燥機,以冷凍乾燥多胜肽混合溶液(4天至5天),以獲得凍乾的多胜肽混合粉末。將凍乾的多胜肽混合粉末以無菌水再懸浮,並以0.22 μm過濾膜過濾後獲得一多胜肽組成物,並將該多胜肽組成物儲存於-80°C,其中該多胜肽組成物包含:每毫升20奈克(ng/ml)至200 ng/ml血管生成素,較佳為55 ng/ml;2 ng/ml至20 ng/ml,較佳為6 ng/ml PDGF;以及2 ng/ml至20 ng/ml,較佳為4 ng/ml纖維母細胞生長因子7 (Fibroblast Growth Factor 7, FGF7)。50 ml of the concentrated multi-peptide mixed solution obtained in Preparation Example 5 was placed in a zipper bag and left at -80 ° C overnight (12 hours to 16 hours). The bag containing the concentrated multi-peptide was mixed together in a freeze dryer to freeze-dry the mixture of the peptides (4 days to 5 days) to obtain a lyophilized multi-peptide mixed powder. The lyophilized multi-peptide mixed powder was resuspended in sterile water and filtered through a 0.22 μm filter membrane to obtain a multi-peptide composition, and the multi-peptide composition was stored at -80 ° C, wherein the multi-win The peptide composition comprises: 20 ng/ml to 200 ng/ml angiogenin per ml, preferably 55 ng/ml; 2 ng/ml to 20 ng/ml, preferably 6 ng/ml PDGF And 2 ng/ml to 20 ng/ml, preferably 4 ng/ml Fibroblast Growth Factor 7, FGF7.

製備例7 分離毛囊(hair follicle)之體外試驗Preparation Example 7 In vitro test for isolating hair follicles

使用BL6小鼠並犧牲後,將其含鬍鬚部分的皮膚用剪刀及手術刀取下並放入PBS{含1%PSA [含青霉素(penicillin)、鏈黴素(streptomycin)及兩性黴素B (amphotericin B)的抗生素-抗真菌劑(antibiotic-antimycotic, 100X)]及0.5%兩性黴素B (fungizone)}中保持濕潤,接著使用PBS (含1% PSA及0.5% fungizone)清洗3次。先將乾淨培養皿放入少許PBS (含1% PSA及0.5% fungizone),再將含鬍鬚部分的皮膚放入。將含鬍鬚部分的皮膚翻面可以見到毛囊,利用已滅菌鑷子及乾淨手術刀小心分離毛囊,將分離的毛囊放入含PBS (含1% PSA及0.5% fungizone)的乾淨培養皿中等待使用。After using BL6 mice and sacrificed, the skin containing the beard parts was removed with scissors and a scalpel and placed in PBS {containing 1% PSA [containing penicillin, streptomycin and amphotericin B ( The amphotericin B) anti-fungal antibiotic (antibiotic-antimycotic, 100X) and 0.5% amphotericin B (fungizone) were kept moist, followed by 3 washes with PBS (containing 1% PSA and 0.5% fungizone). Put the clean petri dish into a little PBS (containing 1% PSA and 0.5% fungizone), then put the skin with the beard part. The hair follicle can be seen by turning the skin containing the beard part. The hair follicles are carefully separated by using a sterile scorpion and a clean scalpel. The separated hair follicles are placed in a clean Petri dish containing PBS (containing 1% PSA and 0.5% fungizone). .

製備例8 毛囊真皮乳頭細胞的分離與培養Preparation 8 Isolation and culture of hair follicle dermal papilla cells

將小鼠BL6犧牲後取其含鬍鬚的皮膚,並隔夜置於PBS (含1% PSA及0.5% fungizone)。分離整個毛囊後將毛囊的末端小球(end-bulb)切出,並培養於毛囊真皮乳頭細胞培養基(購買自promocell公司)中,更換培養基直到細胞生長,之後每2天至3天更換一次培養基。The mouse BL6 was sacrificed and its beard-bearing skin was taken and placed overnight in PBS (containing 1% PSA and 0.5% fungizone). After separating the whole hair follicle, the end-bulb of the hair follicle was excised and cultured in the hair follicle dermal papilla cell culture medium (purchased from Promocell), the medium was changed until the cells were grown, and then the medium was changed every 2 to 3 days. .

製備例9 角質細胞(keratinocyte)的培養Preparation 9 Culture of keratinocytes

取人類的皮膚組織,並以PBS (含1% PSA及0.5% fungizone)清洗3次。將皮膚組織置於培養皿後,以手術剪成小片段約為0.1 mm3 至100 mm3 並移除脂肪與結締組織。將小片段皮膚組織製於含有每毫升2.4單位(U/ml)的分散酶(Dispase® II)的培養皿於4°C隔夜培養,隔天將小片段皮膚組織浸泡於PBS,並輕輕地以彎鉗從小片段皮膚組織的表皮(呈微白且半透明狀)將真皮(呈粉紅、不透明且粘稠狀)分離至新的含有4 ml PBS的培養皿。添加1 ml 0.25%胰蛋白酶於含有真皮的培養皿中(最終濃度為0.05%胰蛋白酶),再將真皮切成細小片段並重複以微量吸管多次吸排直到變混濁後,添加0.5 ml胎牛血清以停止胰蛋白酶的分解作用。將經胰蛋白酶分解的真皮經100 μm的細胞過濾裝置分離出呈單顆狀的細胞溶液,並置於15 ml離心管以200G離心5分鐘後,移除上清液後加入角質細胞培養基(購買自Thermo Fisher公司)回溶下層細胞並種植於培養皿,隔天更換培養基,之後每三天更換培養基,被培養的細胞即為角質細胞。Human skin tissue was taken and washed 3 times with PBS (containing 1% PSA and 0.5% fungizone). After the skin tissue is placed in a petri dish, it is surgically cut into small pieces of about 0.1 mm 3 to 100 mm 3 and the fat and connective tissue are removed. A small piece of skin tissue was prepared in a Petri dish containing 2.4 units (U/ml) of dispase (Dispase ® II) per ml overnight at 4 ° C, and the small pieces of skin tissue were soaked in PBS every other day, and gently The dermis (pale, opaque, and viscous) was separated from the epidermis (white, translucent) of the small segment of the skin tissue by a curved forceps into a new Petri dish containing 4 ml PBS. Add 1 ml of 0.25% trypsin to the dermis containing the dermis (final concentration of 0.05% trypsin), then cut the dermis into small pieces and repeat the pipetting multiple times with a micropipette until turbidity, add 0.5 ml fetal bovine serum To stop the decomposition of trypsin. The trypsin-decomposed dermis was separated into a single cell solution by a 100 μm cell filtration device, and centrifuged at 200 G for 5 minutes in a 15 ml centrifuge tube. After removing the supernatant, the keratinocyte medium was added (purchased from Thermo Fisher Company) reconstituted the lower layer cells and planted them in a culture dish. The medium was changed every other day, and then the medium was changed every three days. The cultured cells were keratinocytes.

實施例1 毛髮增長實驗 取製備例7所製得含鬍鬚的毛囊做為實驗,實驗分為控制組[威廉斯培養基E (Williams medium E)並添加10 ng/ml皮質醇(hydrocortisone)及10 μg/ml胰島素]、1mM MNX、10 nM維生素D3、體積百分比為0.001%咖啡因(caffeine)以及1 ng/ml脂聯素;前述五組中,各組再分為施予及未施予體積百分比為10%多胜肽組成物,該10%多胜肽組成物係由製備例6所製得之多胜肽組成物添加至各組中達10%用量。各組分別施予完成後,進行25天在37°C、5% CO2 的培養與觀察鬍鬚增長的情形,其中有施予MNX、維生素D3、咖啡因、脂聯素或多胜肽組成物的各組每3至4天再施予一次。 表1、各組施予與未施予10%多胜肽組成物於第5至25天的鬍鬚長度變化。 -:表示未施予;+:表示施予。Example 1 Hair Growth Experiment The hair follicles prepared in Preparation Example 7 were taken as an experiment. The experiment was divided into control group [Williams medium E and 10 ng/ml corcorolone and 10 μg were added. /ml insulin], 1 mM MNX, 10 nM vitamin D3, 0.001% caffeine and 1 ng/ml adiponectin; in the above five groups, each group was subdivided into administered and unadministered volume percentage As a 10% multi-peptide composition, the multi-peptide composition prepared in Preparation Example 6 was added to each group in an amount of 10%. After each group was administered separately, 25 days of culture at 37 ° C, 5% CO 2 and observation of whisker growth, including administration of MNX, vitamin D3, caffeine, adiponectin or polypeptide composition Each group was administered once every 3 to 4 days. Table 1. Changes in beard length on days 5 to 25 of the 10% multi-peptide composition administered and not administered to each group. -: indicates no administration; +: indicates administration.

請參閱圖1及表1所示,就控制組而言,添加10%多胜肽組成物無論是在第5天或是至第25天,鬍鬚增長情況與控制組添加10%多胜肽組成物無顯著差異;就1 mM MNX組而言,在第5天至第25天合併添加10%多胜肽組成物鬍鬚有增長趨勢,未添加10%多胜肽組成物鬍鬚則沒有顯著增長,與控制組未添加10%多胜肽組成物無顯著差異;1 ng/ml脂聯素組而言,添加10%多胜肽組成物無論是在第5天或是至第25天,鬍鬚增加的相對長度皆顯著大於單獨施予1 ng/ml脂聯素以及10%多胜肽組成物的總和,因此脂聯素與多胜肽組成物的組合具有加乘效果,得到了無法預期的效果。此外,就10 nM維生素D3組而言,添加10%多胜肽組成物在第20天,鬍鬚增加的相對長度(43.35%)顯著大於單獨施予1 ng/ml脂聯素(19.68%)以及10%多胜肽組成物(20.21%)的總和,因此維生素D3與多胜肽組成物的組合具有加乘效果,亦得到了無法預期的效果。 表2、各組施予與未施予10%多胜肽組成物於第5至10天的鬍鬚長度變化。 -:表示未施予;+:表示施予。Please refer to Figure 1 and Table 1. For the control group, add 10% multi-peptide composition, whether on day 5 or to day 25, beard growth and control group added 10% peptide composition There was no significant difference in the material; in the 1 mM MNX group, the addition of 10% polypeptide composition had a tendency to grow on the 5th to 25th day, and there was no significant increase in the whisker without the addition of 10% polypeptide composition. There was no significant difference between the control group and the control group without adding 10% peptide composition; in the 1 ng/ml adiponectin group, the addition of 10% peptide composition, whether on day 5 or to day 25, increased beard The relative lengths were significantly greater than the sum of 1 ng/ml adiponectin and 10% multi-peptide composition alone, so the combination of adiponectin and multi-peptide composition had additive effects, which had unpredictable effects. . In addition, for the 10 nM vitamin D3 group, the addition of 10% polypeptide composition on day 20, the relative length of whisker increase (43.35%) was significantly greater than the administration of 1 ng / ml adiponectin (19.68%) and The sum of the 10% multi-peptide composition (20.21%), so the combination of vitamin D3 and the multi-peptide composition has an additive effect, and an unexpected effect is obtained. Table 2. Changes in beard length on days 5 to 10 for each group administered with and without the 10% polypeptide composition. -: indicates no administration; +: indicates administration.

請參閱圖2及表2所示,就1 ng/ml脂聯素組而言,合併添加10%多胜肽組成物無論是在第5天或是第10天,鬍鬚增加的相對長度(23.05%、50.12%)皆顯著大於單獨施予1 ng/ml脂聯素(3.08%、5.30%)以及10%多胜肽組成物(11.06%、16.25%)的總和,因此脂聯素與多胜肽組成物的組合具有加乘效果,得到了無法預期的效果。值得留意的是,觀察0.001%咖啡因組中,添加10%多胜肽組成物無論是在第5天或是第10天,相較於單獨添加0.001%咖啡因而言,反而產生抑制鬍鬚生長效果的反向教示(teaching away)。Please refer to Figure 2 and Table 2. For the 1 ng/ml adiponectin group, add 10% multi-peptide composition, whether on day 5 or day 10, the relative length of beard increase (23.05) %, 50.12%) were significantly greater than the sum of 1 ng/ml adiponectin (3.08%, 5.30%) and 10% multi-peptide composition (11.06%, 16.25%), so adiponectin and multi-win The combination of peptide compositions has an additive effect and an unexpected effect is obtained. It is worth noting that in the 0.001% caffeine group, the addition of 10% polypeptide composition, whether on day 5 or day 10, resulted in inhibition of whisker growth compared to 0.001% caffeine alone. The opposite direction (teaching away).

實施例2 真皮乳頭細胞增生實驗Example 2 Dermal papilla cell proliferation experiment

將製備例8的真皮乳頭細胞以每孔3x103 的數量種植於96孔盤,每孔添加0.1 ml的毛囊真皮乳頭細胞培養基。隔天移除培養基後添加新的毛囊真皮乳頭細胞培養基,實驗分為控制組(僅毛囊真皮乳頭細胞培養基)、體積百分比1%至10%多胜肽組成物、0.1 mM至100mM MNX、1 nM至1000 nM維生素D3、體積百分比0.0002%至0.025%咖啡因以及0.1 ng/ml至100 ng/ml脂聯素。經過72小時後,細胞生長的情形以細胞活性分析(MTS試劑係購自於promega公司之CellTiter 96® AQueous One Solution Cell Proliferation Assay)進行量化分析。將20 μl的AQueous One試劑(CellTiter 96® AQueous one solution reagent)添加於每孔皆含有100 μl培養基且為不同組別處理的96孔盤中,並於37°C、5% CO2 培養箱中靜置2小時,再以分光光度計於490 nm測量吸光值,以檢測存活細胞的增生情形。 表3、不同濃度的各組於第3天真皮乳頭細胞存活狀態。 The dermal papilla cells of Preparation Example 8 were seeded in a 96-well plate at a rate of 3 x 10 3 per well, and 0.1 ml of hair follicle dermal papilla cell culture medium was added to each well. After the medium was removed every other day, a new hair follicle dermal papilla cell culture medium was added. The experiment was divided into control group (only hair follicle dermal papilla cell culture medium), volume percentage 1% to 10% peptide composition, 0.1 mM to 100 mM MNX, 1 nM. Up to 1000 nM vitamin D3, volume percentage 0.0002% to 0.025% caffeine, and 0.1 ng/ml to 100 ng/ml adiponectin. After 72 hours, the cell growth was quantified by cell activity assay (MTS reagent was purchased from Promega's CellTiter 96 ® AQueous One Solution Cell Proliferation Assay). Add 20 μl of AQueous One reagent (CellTiter 96 ® AQueous one solution reagent) to a 96-well plate containing 100 μl of medium per well and treated in different groups at 37 ° C in a 5% CO 2 incubator After standing for 2 hours, the absorbance was measured at 490 nm with a spectrophotometer to detect the proliferation of viable cells. Table 3. The survival status of dermal papilla cells on day 3 of each group at different concentrations.

請參閱圖3及表3所示,藉由各組不同濃度單獨試驗,找出各組對於真皮乳頭細胞可承受的劑量範圍,並從中挑選欲進行以下實驗的適合劑量。Referring to Figure 3 and Table 3, the individual doses of different concentrations were tested to find out the range of doses that each group can withstand for dermal papilla cells, and the appropriate dose for the following experiment was selected.

以下同樣將製備例8的真皮乳頭細胞以每孔3x103 的數量種植於96孔盤,每孔添加0.1 ml的毛囊真皮乳頭細胞培養基。隔天移除培養基後添加新的毛囊真皮乳頭細胞培養基培養基,實驗分為控制組(僅毛囊真皮乳頭細胞培養基),1 ng/ml脂聯素,1 ng/ml脂聯素與1 mM MNX,以及1 ng/ml脂聯素、1 mM MNX、10 nM維生素D3;前述四組中,各組再分為施予及未施予10%多胜肽組成物,該10%多胜肽組成物係由製備例6所製得之多胜肽組成物添加至各組中達10%用量。經過72小時後,細胞生長的情形以細胞活性分析(MTS Assay,同上)進行量化分析,以檢測存活細胞的增生情形。 表4、各組施予與未施予10%多胜肽組成物於第3天真皮乳頭細胞存活狀態。 The dermal papilla cells of Preparation Example 8 were also planted in a 96-well plate at a rate of 3 x 10 3 per well, and 0.1 ml of hair follicle dermal papilla cell culture medium was added to each well. After the medium was removed every other day, a new hair follicle dermal papilla cell culture medium was added. The experiment was divided into control group (only hair follicle dermal papilla cell culture medium), 1 ng/ml adiponectin, 1 ng/ml adiponectin and 1 mM MNX. And 1 ng/ml adiponectin, 1 mM MNX, 10 nM vitamin D3; in the above four groups, each group is subdivided into administered and not administered 10% multi-peptide composition, the 10% multi-peptide composition The multi-peptide composition prepared in Preparation Example 6 was added to each group in an amount of 10%. After 72 hours, the cell growth was quantified by cell activity assay (MTS Assay, supra) to detect the proliferation of viable cells. Table 4. Survival status of dermal papilla cells on day 3 of each group administered with and without administration of 10% polypeptide composition.

請參閱圖4及表4所示,就1 ng/ml脂聯素組而言,合併添加10%多胜肽組成物後,真皮乳頭細胞存活百分比差為(105.04%),顯著大於單獨施予1 ng/ml脂聯素(5.42%)以及10%多胜肽組成物(78.94%)的總和,因此脂聯素與多胜肽組成物的組合具有促進真皮乳頭細胞生長之加乘效果,得到了無法預期的功效。Referring to Figure 4 and Table 4, in the 1 ng/ml adiponectin group, the percentage difference in survival of dermal papilla cells was (105.04%) after the addition of 10% polypeptide composition, which was significantly greater than that of the single administration. The sum of 1 ng/ml adiponectin (5.42%) and 10% polypeptide composition (78.94%), so the combination of adiponectin and polypeptide composition has the effect of promoting the growth of dermal papilla cells. Unexpected effects.

此外,就1 ng/ml脂聯素與1 mM MNX組而言,合併添加10%多胜肽組成物後,真皮乳頭細胞存活百分比差為(120.30%),亦顯著大於單獨施予1 ng/ml脂聯素與1 mM MNX (15.51%)以及10%多胜肽組成物(78.94%)的總和;就1 ng/ml脂聯素與1 mM MNX與10 nM維生素D3組而言,合併添加10%多胜肽組成物後,真皮乳頭細胞存活百分比差為(107.94%),亦顯著大於單獨施予1 ng/ml脂聯素與1 mM MNX與10 nM維生素D3 (0.76%)以及10%多胜肽組成物(78.94%)的總和。因此除了脂聯素與多胜肽組成物的組合具有加乘效果外,再分別添加MNX、或同時添加MNX與維生素D3皆有促進細胞生長的加乘效果。In addition, in the case of 1 ng/ml adiponectin and 1 mM MNX, the percentage difference in survival of dermal papilla cells was (120.30%) after the addition of 10% polypeptide composition, which was also significantly greater than 1 ng administered alone. The sum of ml adiponectin with 1 mM MNX (15.51%) and 10% polypeptide composition (78.94%); for 1 ng/ml adiponectin and 1 mM MNX and 10 nM vitamin D3, combined addition After 10% polypeptide composition, the percentage difference in survival of dermal papilla cells was (107.94%), which was also significantly greater than 1 ng/ml adiponectin alone and 1 mM MNX and 10 nM vitamin D3 (0.76%) and 10%. The sum of the multi-peptide compositions (78.94%). Therefore, in addition to the combination of adiponectin and multi-peptide composition, the addition of MNX, or the simultaneous addition of MNX and vitamin D3 have the effect of promoting cell growth.

實施例3 角質細胞增生實驗Example 3 Keratinocyte proliferation experiment

先於96孔盤以塗佈物質(coating matrix)塗佈大於30分鐘後,將製備例9的角質細胞以每孔3x103 的數量種植於96孔盤,每孔添加0.1 ml的角質細胞培養基以及人類角質細胞生長補充劑(human keratinocyte growth supplement,HKGS,購買自Thermo Fisher公司)。隔天移除培養基後添加新的角質細胞培養基,實驗分為控制組(僅角質細胞培養基)、0.001%咖啡因、以及1 ng/ml脂聯素;前述三組中,各組再分為施予及未施予10%多胜肽組成物,該10%多胜肽組成物係由製備例6所製得之多胜肽組成物添加至各組中達10%用量。經過72小時後,細胞生長的情形以細胞活性分析(MTS Assay,同上實施例2)進行量化分析,以檢測存活細胞的增生情形。 表5、各組施予與未施予10%多胜肽組成物於第3天角質細胞存活狀態。 After coating with a coating matrix for more than 30 minutes before the 96-well plate, the keratinocytes of Preparation Example 9 were planted in a 96-well plate at a rate of 3×10 3 per well, and 0.1 ml of keratinocyte medium was added per well and Human keratinocyte growth supplement (HKGS, purchased from Thermo Fisher). New keratinocyte culture medium was added after removing the medium every other day. The experiment was divided into control group (keratinocyte culture medium only), 0.001% caffeine, and 1 ng/ml adiponectin. In the above three groups, each group was subdivided into The 10% multipeptide composition was administered without addition, and the multi-peptide composition prepared in Preparation Example 6 was added to each group to an amount of 10%. After 72 hours, the cell growth was quantified by cell activity assay (MTS Assay, same as in Example 2 above) to detect the proliferation of viable cells. Table 5, keratinocyte survival status on day 3 of each group administered and not administered 10% multi-peptide composition.

請參閱圖5及表5所示,就1 ng/ml脂聯素組而言,合併添加10%多胜肽組成物後,角質細胞存活百分比差為(9.89%),顯著大於單獨施予1 ng/ml脂聯素(5.10%)以及10%多胜肽組成物(1.01%)的總和,因此脂聯素與多胜肽組成物的組合具有促進角質細胞生長之加乘效果,得到了無法預期的功效。值得留意的是,觀察0.001%咖啡因組中,添加10%多胜肽組成物相較於單獨添加0.001%咖啡因而言,反而產生抑制角質細胞生長效果的反向教示。Referring to Figure 5 and Table 5, for the 1 ng/ml adiponectin group, the percentage difference in keratinocyte survival was (9.89%) after the addition of 10% multi-peptide composition, which was significantly greater than that of 1 alone. The sum of ng/ml adiponectin (5.10%) and 10% multi-peptide composition (1.01%), so the combination of adiponectin and multi-peptide composition has the effect of promoting the growth of keratinocytes, and it is impossible to Expected efficacy. It is worth noting that in the 0.001% caffeine group, the addition of a 10% polypeptide composition resulted in a reverse teaching of inhibiting keratinocyte growth compared to the addition of 0.001% caffeine alone.

根據本發明可作之不同修正及變化對於熟悉該項技術者而言均顯然不會偏離本發明的範圍與精神。雖然本發明已敘述特定的較佳具體事實,必須瞭解的是本發明不應被不當地限制於該等特定具體事實上。事實上,在實施本發明之已述模式方面,對於熟習該項技術者而言顯而易知之不同修正亦被涵蓋於下列申請專利範圍之內。It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention should not be In fact, the various modifications that are apparent to those skilled in the art are also contemplated by the scope of the invention.

圖1為本發明之組合物用於毛髮增長實驗第5天至第25天之柱狀圖;「-」表示未施予10%多胜肽組成物;「+」表示施予10%多胜肽組成物;MNX為米諾地爾(minoxidil的縮寫)。 圖2為本發明之組合物用於毛髮增長實驗第5天與第10天之柱狀圖;「-」表示未施予10%多胜肽組成物;「+」表示施予10%多胜肽組成物;MNX為米諾地爾。 圖3為不同濃度之多胜肽組成物、米諾地爾、維生素D3、咖啡因與脂聯素於真皮乳頭細胞存活試驗之柱狀圖。 圖4為本發明之組合物用於真皮乳頭細胞存活試驗之柱狀圖;「-」表示未施予10%多胜肽組成物;「+」表示施予10%多胜肽組成物;MNX為米諾地爾。 圖5為本發明之組合物用於角質細胞存活試驗之柱狀圖;「-」表示未施予10%多胜肽組成物;「+」表示施予10%多胜肽組成物;MNX為米諾地爾。Figure 1 is a bar graph of the composition of the present invention for the 5th to 25th day of the hair growth experiment; "-" means that 10% of the peptide composition is not administered; "+" means that 10% is awarded. Peptide composition; MNX is minoxidil (abbreviation of minoxidil). Figure 2 is a bar graph of the composition of the present invention for the 5th and 10th day of the hair growth experiment; "-" means that 10% of the peptide composition was not administered; "+" means that 10% of the victory was administered. Peptide composition; MNX is minoxidil. Figure 3 is a bar graph of the multi-peptide composition, minoxidil, vitamin D3, caffeine and adiponectin in dermal papilla cell survival assays at various concentrations. Figure 4 is a bar graph of the composition of the present invention for dermal papilla cell survival test; "-" means that 10% of the peptide composition was not administered; "+" means that 10% of the peptide composition was administered; MNX For Minoxidil. Figure 5 is a bar graph of the composition of the present invention for keratinocyte survival test; "-" means that 10% of the peptide composition was not administered; "+" means that 10% of the peptide composition was administered; Minoxidil.

Claims (9)

一種促進毛髮增長的組合物,其包含一多胜肽組成物以及脂溶性化合物,其中該脂溶性化合物為脂聯素(adiponectin)或維生素D3;該組合物包含體積百分比為1%至20%的多胜肽組成物,該脂聯素為每毫升0.1奈克(ng/ml)至100ng/ml,該維生素D3為1奈莫耳濃度(nM)至1000nM,且該多胜肽組成物,以該多胜肽組成物的體積為基準,包含20ng/ml至200ng/ml之血管生成素(angiogenin)、2ng/ml至20ng/ml之血小板衍生生長因子(platelet-derived growth factor,PDGF)和2ng/ml至20ng/ml之纖維母細胞生長因子7(fibroblast growth factor7,FGF7)。 A composition for promoting hair growth comprising a multi-peptide composition and a fat-soluble compound, wherein the fat-soluble compound is adiponectin or vitamin D3; the composition comprises 1% to 20% by volume a multi-peptide composition, the adiponectin is 0.1 ng/ml to 100 ng/ml per ml, the vitamin D3 is 1 nanomolar (nM) to 1000 nM, and the multi-peptide composition is Based on the volume of the multipeptide composition, comprising 20 ng/ml to 200 ng/ml of angiogenin, 2 ng/ml to 20 ng/ml of platelet-derived growth factor (PDGF) and 2 ng /ml to 20 ng/ml of fibroblast growth factor 7 (FGF7). 如請求項1所述之促進毛髮增長的組合物,其中該組合物包含體積百分比為10%的多胜肽組成物以及1ng/ml的脂聯素。 The hair growth promoting composition according to claim 1, wherein the composition comprises a 10% by volume multi-peptide composition and 1 ng/ml of adiponectin. 如請求項1所述之促進毛髮增長的組合物,其中該組合物包含體積百分比為10%的多胜肽組成物以及10nM的維生素D3。 The hair growth promoting composition according to claim 1, wherein the composition comprises 10% by volume of a multi-peptide composition and 10 nM of vitamin D3. 一種如請求項1至3中任一項所述之組合物用於製備促進毛髮增長的醫藥品的用途,其中將有效劑量之該醫藥品施予受體以促進受體之毛髮增長。 A use of a composition according to any one of claims 1 to 3 for the manufacture of a medicament for promoting hair growth, wherein an effective amount of the medicament is administered to a recipient to promote hair growth of the recipient. 一種包含多胜肽組成物以及脂聯素之組合物用於製備促進毛髮細胞生長的醫藥品的用途,其中將有效劑量之該醫藥品施予受體以促進受體之毛髮細胞生長;其中該組合物包含體積百分比為1%至20%的多胜肽組成物,該脂聯素為0.1ng/ml至100ng/ml,且該多胜肽組成物,以該多胜肽組成物的體積為基準,包含20ng/ml至200ng/ml之血管生成素、2ng/ml至20ng/ml之血小板衍生生長因子和2ng/ml至20ng/ml之纖維母細胞生長因子7。 A use of a composition comprising a multi-peptide composition and adiponectin for the preparation of a medicament for promoting growth of hair cells, wherein an effective amount of the medicament is administered to a recipient to promote hair cell growth of the recipient; The composition comprises a multi-peptide composition having a volume percentage of from 1% to 20%, the adiponectin is from 0.1 ng/ml to 100 ng/ml, and the multi-peptide composition has a volume of the multi-peptide composition The reference comprises 20 ng/ml to 200 ng/ml of angiogenin, 2 ng/ml to 20 ng/ml of platelet-derived growth factor, and 2 ng/ml to 20 ng/ml of fibroblast growth factor 7. 如請求項5所述之用途,其中該多胜肽組成物之體積百分比為10%,該脂聯素為1ng/ml。 The use according to claim 5, wherein the multi-peptide composition has a volume percentage of 10% and the adiponectin is 1 ng/ml. 如請求項5所述之用途,其中該組合物更包含0.1微莫耳濃度(μM)至100μM的米諾地爾(minoxidil,MNX)。 The use of claim 5, wherein the composition further comprises 0.1 micromolar (μM) to 100 μM minoxidil (MNX). 如請求項7所述之用途,其中該米諾地爾為1μM。 The use of claim 7, wherein the minoxidil is 1 μM. 如請求項5至8中任一項所述之用途,其中該組合物更包含1nM至1000nM的維生素D3。The use of any one of claims 5 to 8, wherein the composition further comprises from 1 nM to 1000 nM of vitamin D3.
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