TWI444197B - 用於改善皮膚老化之組合物 - Google Patents
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- TWI444197B TWI444197B TW101114923A TW101114923A TWI444197B TW I444197 B TWI444197 B TW I444197B TW 101114923 A TW101114923 A TW 101114923A TW 101114923 A TW101114923 A TW 101114923A TW I444197 B TWI444197 B TW I444197B
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Description
本發明係關於一種用於改善皮膚外觀之組合物，尤其關於一改善皮膚老化之組合物，其係包含由顆粒白血球刺激因子(Granulocyte Colony Stimulating Factor，GCSF)所誘導生成之周邊單核球細胞。
皮膚老化的途徑可區分為自然性老化與光老化兩種途徑，其中，皮膚的自然性老化主要是由染色體控制，但目前的醫學科技尚無法從改變染色體來阻止其進行，只能由避免已知的惡化因素著手，例如避免使皮膚暴露於有害物質等。兩種老化途徑可由穿衣覆蓋部位與陽光經常暴曬部位區別，表現的皮膚老化程度也有所不同。自然性老化可觀察到角質幹細胞(keratinocyte stem cells)減少的趨勢，而且在顯微結構的老化表現為例如表皮中天然保濕因子流失、角質層增厚堆積、基底層細胞紊亂、黑色素增加而呈現老人斑、色素斑及暗沈、真皮中乳頭層平坦、玻尿酸減少、彈力纖維變性、彈力蛋白質排列紊亂、以及膠原蛋白降低等，造成皮膚日漸不平坦與張力及彈性喪失，於是皺紋及鬆弛接踵而至，導致皮下脂肪變薄、皮脂腺分泌下降、皮脂膜功能趨低、皮膚保護功能以及保水的能力變差等。
近年來各先進國家推陳出新各種醫學美容方式來改善皮膚老化現象，包括藥物治療、化妝品、外科治療、剝脫性治療、注射性治療、非剝脫性嫩膚治療等。但藥物及部份保養品所含膠原蛋白、彈力蛋白、水解蛋白質、黏多醣、玻尿酸、維他命E等因為分子結構太大，無法經皮吸收進入真皮，只能停留在表皮上扮演保濕的角色；外科治療、剝脫性治療及注射性治療(例如果酸換膚、微晶磨皮、雷射除斑、雷射磨皮、及除皺)可以更新老化膚質，達到再生新皮的目的，但會產生創傷，皮膚恢復緩慢且術後併發症多，而膠原蛋白注射只能暫時性填充真皮組織消除皺紋，維持時間不超過九個月；非剝脫性嫩膚治療的激光及射頻容易造成炎症後色素沉著及瘢痕等併發症。
細胞治療是使用幹細胞改善受損的器官，於醫學領域為最新的治療方式。造血幹細胞(hematopoiesis stem cell，HSC)是存在於骨髓內的多能性幹細胞，可分化成單核球、顆粒球、淋巴球、紅血球及血小板。在造血幹細胞來源中，周邊血液幹細胞(peripheral blood stem cells，PBSC)相對容易取得，對捐贈者之物理性傷害較小。迄今，周邊血液幹細胞尚未被應用於改善皮膚老化。
本案發明人發現來自周邊血液幹細胞之周邊單核球細胞具有改善皮膚老化之功效，其可用於淡化線紋(如抬頭紋、法令紋)、減少或預防皺紋、減少皮膚變色作用、改善皮膚色調、減少掉髮、促進毛囊細胞活化、或促進毛髮生長等。
本發明之一目的在於提供一種用於改善皮膚老化之組合物，其係包含由顆粒白血球刺激因子(Granulocyte Colony Stimulating Factor，GCSF)所誘導生成之周邊單核球細胞。
本發明之另一目的在於提供一種使用由顆粒白血球刺激因子所誘導生成之周邊單核球細胞於改善皮膚老化之用途。
本發明之詳細技術及較佳實施態樣，將描述於以下內容中，以供本發明所屬領域具通常知識者據以明瞭本發明之特徵。
於本文中(尤其後附申請專利範圍中)，除非另外說明，所使用之「一」、「該」及類似用語應理解為包含單數及複數形式。
本案發明人發現周邊單核球細胞具有改善皮膚老化之功效，因此，本發明提供一種用於改善皮膚老化之組合物，其係包含由顆粒白血球刺激因子(Granulocyte Colony Stimulating Factor，GCSF)所誘導生成之周邊單核球細胞。特定言之，可藉由將GCSF注射至生物體內，以誘導單核球細胞之生成，再自生物體內之周邊血液中收集周邊單核球細胞，而取得本發明組合物中之周邊單核球細胞。
於本發明中，該周邊單核球細胞除可由GCSF誘導生成外，亦可由GCSF與其相關介白素(interleukin)之組合而誘導生成。其中，GCSF之相關介白素的例子包括介白素-3(IL-3)及介白素-6(IL-6)等。
於本發明之一實施態樣中，周邊單核球係於生物體內由GCSF或其與相關介白素之組合所誘導生成，並自血液中分離而得，故本發明組合物所含之周邊單核球可呈一活細胞萃取物之形式，該活細胞萃取物可包含如血液、體液、及其他細胞等成分。於此，以組合物之總重計，該活細胞萃取物之含量可為約0.001重量%至約20重量%；較佳地，為約0.05重量%至約10重量%；更佳地，為約0.5重量%至約5重量%。
較佳地，本發明組合物中的周邊單核球細胞為具表面抗原CD34之幹細胞。CD34抗原分子是高度糖基化的跨膜醣蛋白，選擇性地表現於人類或其他哺乳動物造血幹細胞(如周邊單核球細胞等)表面，並隨細胞的成熟而逐漸減少至消失。本發明組合物中具表面抗原CD34之幹細胞可來自骨髓、周邊血液、或其組合。
當本發明組合物中之周邊單核球細胞為具表面抗原CD34之幹細胞時，其於組合物中之含量可為約102
至約109
個，以每微升組合物計；較佳地，可為約105
至約109
個，以每微升組合物計。
本發明組合物可呈任何合宜的形式，並無特殊的限制。舉例言之，可呈供直接外用之乳液、乳霜或凝膠形式，例如保養品或化妝品等，經由塗敷於皮膚表面而施用本發明組合物。或者，可將本發明組合物製備成可供注射於皮下或靜脈內之輸注液形式，例如懸液劑、靜脈輸注液、乾粉注射劑、懸液注射劑及乾粉懸液注射劑等，經由皮下或靜脈內注射而施用本發明組合物。
本發明組合物可用以對抗皮膚老化現象，尤其可促進皮膚中玻尿酸生成、促進皮膚中彈性纖維生成、及/或降低皮膚中膠原蛋白耗損。此外，本發明組合物亦可改善皮膚堅實度或豐滿度、促進皮膚新陳代謝、及改善皮膚結構(包括使皮膚表皮層變薄及增加真皮層彈性)。透過上述機制，本發明組合物可達成以下功效：淡化線紋(如抬頭紋、法令紋)、減少或預防皺紋、減少皮膚變色作用、及改善皮膚色調。具體而言，咸信本發明組合物可用以減少毛孔大小、改善皮膚光彩、透明度或緊繃度、促進抗氧化劑活性、改良皮膚柔靭度或柔軟度、改善前膠原蛋白或膠原蛋白生成量、促進皮膚再組構作用、改善皮膚障蔽修護及機能、改善皮膚輪廓外觀、補充因老化或更年期而衰減之皮膚基本營養素或成分、改善皮膚細胞間之傳訊作用、增加細胞增殖或複製作用、增加因老化或更年期而衰減之皮膚代謝作用、改善皮膚濕度、促進或加速細胞更新、降低皮膚敏感度、及/或增加皮膚彈性及恢復力等。
此外，由於周邊單核球可聚集在毛囊處，並維持及促進毛囊活性及毛髮生長，因此，本發明組合物亦具有減少掉髮、促進毛囊細胞活化、促進毛髮生長之效果。
本發明組合物之用量，可依所施用對象之年紀及施用目的(例如減少皮膚皺紋或減少皮膚變色作用)而加以調整，亦可視需要而調整使用頻率。至於本發明組合物中之其它成分及其含量，端視組合物之最終形式而定。例如，當製備皮膚用之保養品時，可添加任何合宜且適量的乳化劑、香料及其它可改善膚質之活性成分等。原則上，只要所添加的其它成分之種類及含量，不會對組合物中之周邊單核球細胞有不利的影響即可。
舉例言之，本發明組合物可視需要進一步包含選自以下群組之至少一者：膠原蛋白、玻尿酸、生長因子、幹細胞趨化因子、及一美容上可接受載劑。該生長因子與幹細胞趨化因子可維持或促進組合物中周邊單核球細胞之生長與活性，其可為例如血小板因子(platelet factor，PF)、GCSF、與GCSF之相關介白素(如介白素-3及介白素-6)、血管內皮生長因子(vascular endothelial growth factor，VEGF)、纖維母細胞生長因子(fibroblast growth factors，FGF)、及表皮生長因子(epidermal growth factor，EGF)等。
本發明亦提供一種使用由顆粒白血球刺激因子所誘導生成之周邊單核球細胞於改善皮膚老化之用途。其中，該周邊單核球細胞及其用量與使用態樣，係如上文中所描述者。
茲以下列具體實施態樣以進一步例示說明本發明。其中該些實施態樣僅提供作為說明，而非用以限制本發明之範疇。
[實驗設計]
以拉丁方格設計(Latin square design)實驗法，規劃實驗群組(此可參見Roger E. Kirk,1995，Latin Square Design,Corsini Encyclopedia of Psychology，該文獻全文併於此處以供參考)。以對抗平衡(counterbalancing)原理來安排實驗順序。
[實施例1] 豬種圈養
自臺東種畜繁殖場購得一隻蘭嶼公豬(五個月齡)及三隻蘭嶼母豬(年齡分別為六年、七年、九年)。每隻豬皆附有血統登錄證明書與生產履歷，圈養的豬皆為個別飼養並提供豬足夠的活動空間，且區分飼料、休息、糞便三區。豬舍水泥地面上有高架式條狀地板，牆邊富有自動給水裝置。圈養欄內沒有尖銳的突出物，屬於非密閉式飼養空間，讓豬可看、聞、聽和接觸外界。全採用最少病原等級之試驗動物管理方式飼養，每日提供兩次限量飼料，每月一至二次輪替藥品給予清潔消毒。該等豬隻係用於以下試驗。
[實施例2] 周邊血液幹細胞收集
周邊血液幹細胞是經由血液分離機取得。在分離前五天起，於公豬耳靜脈注射一劑顆粒白血球刺激因子(300微克/0.7毫升，Filgrastim M300)作為生長激素，以誘導生成周邊單核球細胞(即，具表面抗原CD34之幹細胞)，直至收集日的前一天為止，將骨髓中的造血幹細胞快速釋放於周邊血液中以便收集。採用頸靜脈抽血，取一管血液(BDCPTTM Tube)，緩慢搖晃與抗凝劑混合均勻，以1800 g離心15分鐘後，以700 g離心5分鐘，以100 g離心10分鐘三次，分離出富含白血球的白血球層細胞(buffy coat)，其含有具表面抗原CD34之幹細胞(即，周邊單核球)。
[實施例3] 流式細胞儀分析
將實施例2中取得之白血球層細胞以4℃ 1X PBS稀釋成1×106
個細胞後，取100微升細胞液，添加2微升抗CD34抗體，放置於4℃冰箱中30分鐘。接著，以4℃ 1X PBS清洗細胞液三次，每次以400 g離心5分鐘，添加1微升螢光標記的二級抗體(Alexa546 nm donkey anti-rabbit IgG，invitrogen)，並溶於200微升3% BSA/PBS，再存放於4℃冰箱30分鐘。然後，以4℃ 1X PBS清洗三次，每次以400 g離心5分鐘，取出離心後的細胞存放於4℃冰箱且避光。最後，使用流式細胞儀(BD LSRTM II flow cytometer)分析血液內CD 34表現量。試驗結果係顯示於表1及第1A至2D圖。
如表1及第1D圖所示，公豬施打GCSF後血液內之白血球含量升高，且中間型細胞(包含周邊單核球)較施打前增加6.9倍。第2C圖顯示具CD34表面抗原之幹細胞(即，周邊單核球)佔所有中間型細胞的20%；第2D圖顯示具CD34表面抗原之幹細胞佔所有中間型細胞的37%，因此，公豬施打GCSF後能增加具表面抗原CD34之幹細胞的量達17%。此試驗結果說明藉由施打GCSF可誘導具表面抗原CD34之幹細胞(即，周邊單核球)之生成。此經誘導生成之周邊單核球係用於以下實施例之試驗中。
[實施例4]
周邊單核球之施用
依第3圖所示方式將實施例2中製得之含有具表面抗原CD34之幹細胞的白血球層細胞施打至豬隻真皮層中，其中，進針角度與表皮呈15度，且進針深度為0.3公分。於施用後一週及二週，以下述生化分析方法觀察豬隻之皮膚變化。
[實施例5]
皮膚組織處理及切片
取樣部位先用電動剃毛刀將豬皮膚毛髮剃除，再以75%酒精消毒手術部位，輕輕用解剖刀割取約1公分平方大小的皮膚組織，深度約8毫米，以達可見脂肪層為主。將取樣後之樣品以10%福馬林(neutral buffered formalin，Surgipath)作為固定液浸泡組織，使組織的蛋白質凝固保持原始狀態，移至組織脫水機(Leica TP1020)進行脫水。首先將含有皮膚組織的包埋盒(casette)，放入組織脫水機中依序將組織作固定、脫水、清洗及浸潤(灌蠟)處理，處理後組織中的水份將會被石蠟(paraffin)所取代。處理過程為：10%福馬林1小時(固定)，二次水30分鐘(浸洗)，80%酒精1小時(脫水)，95%酒精1小時(脫水)，95%酒精1小時(脫水)，100%酒精1小時(脫水)，100%酒精1小時(脫水)，100%酒精2小時(脫水)，二甲苯(xylene)1小時(清洗)，二甲苯2小時(清洗)，石蠟1小時(浸潤)，石蠟2小時(浸潤)。將脫水完成的組織進行石蠟包埋(tissue block system TBS 88，Medite)成石蠟塊(paraffin block)後，以手搖式石蠟切片機(Shandon AS325，Leica，德國)進行組織切片，每片厚度為3至5微米。
[實施例6] 皮膚組織染色
核酸帶負電，與帶正電鹼性染料(蘇木紫染液)的親和力較強，而細胞漿則相反，其含有鹼性物質因而與帶正電酸性染料(伊紅染液)的親和力較大。因此，經由蘇木紫及伊紅染色，細胞核被染成紫藍色，細胞漿、肌纖維、膠原纖維等染成不同程度的紅色，紅細胞則呈橙紅色。染色步驟如下：將皮膚組織切片(5微米/片(section))，放置55℃烤30分鐘，以二甲苯脫蠟後使用酒精使組織回水(rehydration)，再以蘇木紫溶液(Sigma，MS-16)染色2分鐘，再以水流沖洗15分鐘。接著以伊紅Y(Sigma，E4382)染色1分鐘，再以水流沖洗15分鐘。最後以酒精脫水後以封片膠封片。於抽風櫃乾燥一天後在顯微鏡下觀察結果並以CCD數位照相系統(OLYMPUS,DP70)照相存檔。
梅生三色染色/膠質纖維染色為組織化學染色的一種，主要用以區別膠原纖維。在酸性環境下，對膠原纖維與肌肉纖維有選擇性染色。膠原纖維經酸處理後使比布裡希猩紅(Biobrich Scarlet)由膠原滲出而被苯胺藍(aniline blue)染色，呈現藍色。染色步驟如下：將皮膚組織切片(5微米/片(section))，放置55℃烤30分鐘，以二甲苯脫蠟後使用酒精使組織回水(rehydration)，以媒染劑(Mordant)浸泡30分鐘，再以Carrazi’s蘇木紫溶液染色40分鐘。完成後以水流沖片沖洗10分鐘，再以0.75% Orange G溶液染色1分鐘後，以1%冰醋酸清洗兩次，Masson B stain溶液染色10分鐘，以1%冰醋酸洗兩次，2.5%磷鉬酸-磷鎢酸(Phosphomolybdic-Phosphotungstic acid)染色10分鐘，以1%冰醋酸洗兩次，以苯胺藍(anilin blue)染色15分鐘，浸泡1%冰醋酸兩次後以酒精脫水後以封片膠封片。於抽風櫃乾燥一天後以顯微鏡觀察並以CCD數位照相系統(OLYMPUS，DP70)照相存檔。
彈性蛋白染色為用於區別彈性蛋白之染色法，彈性蛋白經染色後呈現藍黑色。染色步驟如下：將皮膚組織切片(3微米/片(section))，放置55℃烤30分鐘，以二甲苯脫蠟後使用酒精使組織回水，以活化的彈性蛋白染色溶液(working elastic stain solution)染色15分鐘，以流水沖洗後，以2%氯化鐵區分溶液(Ferric Chloride Differentiating Solution)染色20秒，流水沖洗後，以5%硫代硫酸納溶液染色1分鐘，流水沖洗後，以Van Gieson’s溶液染色3分鐘，以酒精脫水後以封片膠封片。於抽風櫃乾燥一天後以顯微鏡觀察並以CCD數位照相系統(OLYMPUS，DP70)照相存檔。
黏液素胭脂紅染色係對黏液素(mucine)具專一性之染色法，黏液素由上皮細胞與結締組織細胞分泌。胭脂紅(carmine)與鋁染媒(氯化鋁)劑形成胭脂紅-鋁，其與帶負電荷的硫酸基及羧基結合而呈紅色。染色步驟如下：將皮膚組織切片(5微米/片(section))，放置55℃烤片30分鐘，以二甲苯脫蠟後使用酒精使組織回水，以二次水沖洗，以蘇木紫染色3分鐘，流水沖洗2分鐘，以Bluing試劑染色30秒，浸泡二次水，以活化的Mucicarmine溶液(33% Mucicarmine)染色1小時，快速以流水沖洗後浸泡於二次水，以酒石黃溶液染色1分鐘，快速以流水沖洗後浸泡於二次水，以酒精脫水後以封片膠封片。於抽風櫃乾燥一天後在顯微鏡下觀察結果並以CCD數位照相系統(OLYMPUS，DP70)照相存檔。
利用免疫組織螢光染色(Immunofluorescence，IF)偵測組織中特殊抗原表現。染色步驟如下：將皮膚組織切片(3微米/片(section))，放置55℃烤片30分鐘，以二甲苯脫蠟後以酒精使組織回水，以抗原恢復緩衝液於121℃下加熱10分鐘，以厭水筆(DAKO pen)將組織區域圈住，取3%雙氧水於1X PBS溶液中室溫下浸泡組織10分鐘，除去內生性還原酶之活性，以清洗液(rinse buffer，1X PBS+0.3% TritonX-100)清洗一次，再以5% FBS於1X PBS中在室溫下處理組織30分鐘，阻斷抗體非專一性之鍵結，以清洗液(1X PBS+0.05% TritonX-100)清洗一次，加入適當稀釋(1：100至1：200)一級抗體(玻尿酸及CD44，Abcam)，於室溫下作用2小時或於4℃下作用至隔夜，以清洗液(1 X PBS+0.05% TritonX-100)清洗三次；加入適當稀釋之二級抗體(1：5000)，於室溫下作用1小時，以清洗液(1X PBS+0.05% TritonX-100)清洗三次；加入DAB呈色劑(3,3’-Diaminobenzidine)(LSAB2 Kit，DAKO，加州，美國)呈色10分鐘。陽性反應呈紅棕色，若未有顏色出現即為陰性(negative)結果，以蘇木紫染1分鐘做對比染色(counter stain)，洗去過多之蘇木紫，再以自來水浸泡10分鐘，以酒精脫水後以封片膠封片。於抽風櫃乾燥後在顯微鏡下觀察結果並以CCD數位照相系統(OLYMPUS，DP70)照相存檔。
[統計分析]
於三隻蘭嶼母豬中，每群組的實驗分析結果以平均值±標準差(mean±SD)之形式表示。常態分布之資料採用Student’s t-test分布實驗資料相異性之意義；非常態分布之資料採用Mann-Whitney U test分析實驗資料之相關性。統計結果之p值<0.05視為有統計上之意義。
[試驗結果]
實施例5及6之分析結果顯示於第4A至7B圖中。第4A圖顯示蘭嶼公豬施打GCSF後，以螢光染劑標定中間型細胞(包含周邊單核球)之結果。第4A圖左圖為可見光視野下的細胞型態，右圖則為螢光顯微鏡下的細胞型態。
第4B圖顯示經標定之中間型細胞(包含周邊單核球)依第3圖所示方式打入90月齡的蘭嶼母豬後，一週後取下皮膚組織以螢光顯微鏡觀察(右圖)並比較組織型態(左圖)。結果顯示施打後一週，中間型細胞聚集在毛囊處並散佈於真皮層中。
第4C圖顯示經標定之中間型細胞(包含周邊單核球)細胞依第3圖所示方式打入90月齡的蘭嶼母豬後，二週後取下皮膚組織以螢光顯微鏡觀察(右圖)並比較組織型態(左圖)。結果顯示施打後二週，中間型細胞聚集在毛囊處及表皮層，並散佈於真皮層中。
由於毛囊為毛囊幹細胞的存在地點(niche)，毛囊幹細胞與毛囊的活性及毛髮的生長有密切關係。第4B及4C圖顯示包含周邊單核球之中間型細胞聚集在毛囊處，此現象說明原本毛囊中因生長因子缺乏而釋放出歸化因子(homing factor)以吸引幹細胞進駐，進而維持毛囊活性及毛髮生長，而中間形細胞本身具備多種生長因子且能受到歸化因子驅使進駐毛囊。因此，本發明組合物有助於維持毛囊健康、減少掉髮、以及促生新髮。
第5A圖比較施打包含周邊單核球前後之90月齡蘭嶼母豬的皮膚結構變化，由第5A圖可看出施打前的皮膚(中間圖)，表皮層皺折明顯，且皮膚結構鬆散排列不均，但施打後(右圖)，表皮層細緻，皮膚堅實度與豐滿度獲得改善，5月齡的小豬皮膚(左圖)則作為年輕膚質之對照組。
第5B圖顯示母豬各皮層之厚度，可看出施打周邊單核球可大幅降低真皮層厚度，改善皮膚結構。
第6圖顯示利用免疫組織化學染色分析施打周邊單核球前後90月齡的蘭嶼母豬皮膚，並利用特定抗原分析膠原蛋白、彈性蛋白(皮膚彈性指標)、多醣(皮膚保水性指標)、及玻尿酸與CD44(消除疤痕能力指標)。表2為第6圖之量化結果。
表2及第6圖顯示施打周邊單核球後的90月齡蘭嶼母豬皮膚有以下現象：(1)降低膠原蛋白耗損；(2)改善皮膚堅實度與豐滿度；(3)改善皮膚保水性；以及(4)減少線紋及皺紋。
第7A及7B圖顯示利用免疫組織化學染色分析施打周邊單核球前後90月齡的蘭嶼母豬皮膚，並利用特定抗原分析內披蛋白(involucrin)(角質化程度指標)與角質細胞之β1整合蛋白(β1 integrin)。第7A及7B圖顯示蘭嶼母豬於施打周邊單核球前之皮膚的角質化程度與角質細胞比值較高，而施打後角質細胞增生程度遠高於角質化程度，使得皮膚代謝速率增加，因此，周邊單核球可促進皮膚新生及代謝。
以上實施例之結果顯示，就生化功能而言，本發明組合物可促進皮膚中玻尿酸生成、促進皮膚中彈性蛋白(纖維)生成、及降低皮膚中膠原蛋白耗損；就生理效果而言，則可改善皮膚堅實度或豐滿度、促進皮膚新陳代謝、及改善皮膚結構(包括使皮膚表皮層變薄及增加真皮層彈性)。因此，經由上述機制，本發明組合物可達成淡化線紋、減少或預防皺紋、減少皮膚變色作用、及/或改善皮膚色調之功效。
上述實施例僅係用以例示說明本發明之原理及功效，而非用於限制本發明。任何熟於此項技藝之人士均可在不違背本發明之技術原理及精神的情況下，對上述實施例進行修改及變化。因此，本發明之權利保護範圍應如後附申請專利範圍所界定者。
第1A圖所示為蘭嶼公豬之全血細胞抹片圖；
第1B圖所示為經施打GCSF之蘭嶼公豬之全血細胞抹片圖；
第1C圖所示為顯示蘭嶼公豬之全血液中白血球含量的分析圖；
第1D圖所示為顯示經施打GCSF之蘭嶼公豬之全血液中白血球含量的分析圖；
第2A圖所示為針對蘭嶼公豬之全血細胞進行細胞特性(CD34抗原分子)分析之流式細胞儀分析圖，其中，圈選區域(P1)為中間型細胞；
第2B圖所示為針對經施打GCSF之蘭嶼公豬之全血細胞進行細胞特性(CD34抗原分子)分析之流式細胞儀分析圖，其中，圈選區域(P1)為中間型細胞；
第2C圖所示為第2A圖之流式細胞儀分析之結果圖；
第2D圖所示為第2B圖之流式細胞儀分析結果圖；
第3圖所示為本發明周邊單核球之施打方式示意圖；
第4A圖所示為蘭嶼公豬施打GCSF後，以螢光染劑標定中間型細胞之螢光顯微鏡圖；
第4B圖所示為將經螢光染劑標定之中間型細胞依第3圖方式打入90月齡的蘭嶼母豬達一週後之皮膚組織的螢光顯微鏡圖(右圖)及組織型態圖(左圖)；
第4C圖所示為經螢光染劑標定之中間型細胞依第3圖方式打入90月齡的蘭嶼母豬達二週後之皮膚組織的螢光顯微鏡圖(右圖)及組織型態圖(左圖)。
第5A圖所示為5月齡蘭嶼母豬之皮膚結構圖(左圖，對照組)及施打周邊單核球前後之90月齡的蘭嶼母豬皮膚結構變化圖(中間圖及右圖)；
第5B圖所示為第5A圖中各皮膚層之量化直條圖；
第6圖所示為利用免疫組織化學染色分析施打周邊單核球前後90月齡蘭嶼母豬皮膚之膠原蛋白、彈性蛋白、多醣、玻尿酸及CD44之染色圖；以及
第7A及7B圖所示為利用免疫組織化學染色分析施打周邊單核球前後90月齡蘭嶼母豬皮膚之內披蛋白與角質細胞之β1整合蛋白之染色圖。
Claims (14)
- 一種使用由顆粒白血球刺激因子(Granulocyte Colony Stimulating Factor，GCSF)所誘導生成之周邊單核球細胞於製造用於改善皮膚老化之保養品、化妝品或藥劑的用途。
- 如請求項1之用途，其中該周邊單核球細胞係由顆粒白血球刺激因子及其相關介白素(interleukin)所誘導生成。
- 如請求項2之用途，其中該介白素為介白素-3(IL-3)及介白素-6(IL-6)。
- 如請求項1之用途，其中該周邊單核球細胞係呈一活細胞萃取物之形式。
- 如請求項1之用途，其中該保養品、化妝品或藥劑更包含選自以下群組之至少一者：膠原蛋白、玻尿酸、生長因子、幹細胞趨化因子、及一美容上可接受載劑。
- 如請求項1至5中任一項之用途，其中該周邊單核球細胞為具表面抗原CD34之幹細胞。
- 如請求項6之用途，其中該具表面抗原CD34之幹細胞係來自骨髓、周邊血液、或其組合。
- 如請求項6之用途，其中該具表面抗原CD34之幹細胞的含量為約102 至約109 個，以每微升組合物計。
- 如請求項8之用途，其中該具表面抗原CD34之幹細胞的含量為約105 至約109 個，以每微升組合物計。
- 如請求項1至5中任一項之用途，其中該保養品、化妝 品或藥劑係施用於皮膚表面或經靜脈或皮下施用。
- 如請求項1至5中任一項之用途，其中該保養品、化妝品或藥劑係用於選自以下群組之至少一者：促進皮膚中玻尿酸生成、促進皮膚中彈性纖維生成、及降低皮膚中膠原蛋白耗損。
- 如請求項1至5中任一項之用途，其中該保養品、化妝品或藥劑係用於選自以下群組之至少一者：改善皮膚堅實度或豐滿度、促進皮膚新陳代謝、及改善皮膚結構。
- 如請求項1至5中任一項之用途，其中該保養品、化妝品或藥劑係用於選自以下群組之至少一者：淡化線紋、減少或預防皺紋、以及改善皮膚色調。
- 如請求項1至5中任一項之用途，其中該保養品、化妝品或藥劑係另用於選自以下群組之至少一者：減少掉髮、促進毛囊細胞活化及促進毛髮生長。
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| TW101114923A TWI444197B (zh) | 2012-04-26 | 2012-04-26 | 用於改善皮膚老化之組合物 |
| US13/527,984 US9044444B2 (en) | 2012-04-26 | 2012-06-20 | Method for improving skin appearance and reducing hair loss |
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| TW201343175A (zh) | 2013-11-01 |
| US20130287717A1 (en) | 2013-10-31 |
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