TWI815798B - 透明皮膚試料之製造方法及透明皮膚試料 - Google Patents
透明皮膚試料之製造方法及透明皮膚試料 Download PDFInfo
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- TWI815798B TWI815798B TW106127127A TW106127127A TWI815798B TW I815798 B TWI815798 B TW I815798B TW 106127127 A TW106127127 A TW 106127127A TW 106127127 A TW106127127 A TW 106127127A TW I815798 B TWI815798 B TW I815798B
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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Abstract
本發明之課題在於提供一種透明皮膚試料。藉由酵素處理去除表皮。
Description
本發明係關於一種能夠利用光片顯微鏡(light-sheet microscopy)進行表皮正下方組織之觀察之透明皮膚試料、其製造方法、及皮膚切片之處理方法。
作為對組織進行三維分析之技術,自先前以來採用有使用螢光顯微鏡之技術,但於利用螢光顯微鏡觀察組織之情形時,必須將組織包埋後利用切片機進行切削而製作薄切片。此種方法適於對細胞結構等微細結構進行三維分析,但用於觀察更大之組織結構時,於試料製備及退色等方面產生問題。為了解決此種問題,開發有自試料側面照射片狀之激發光以獲得光學剖面之光片顯微鏡,從而能夠對活體試料或活體組織等更大之試料進行三維分析。斑馬魚或稻田魚之類的透明度較高之試料適宜使用光片顯微鏡,另一方面,哺乳動物之活體組織或組織切片之類的不透明之試料則須進行透明化處理。
近年來,開發有眾多組織試料之透明化技術(專利文獻1)。作為透明化技術,主要大致分為使用有機溶劑透明化劑之技術(非專利文獻1)及使用水溶性透明化劑之技術(非專利文獻2),但分別於透明化之困難性、透明化試料之折射率、抗原性之維持之方面存在問題。又,已知於使人類組織透明化之情形時,人類組織中基質豐富,光散射性較高,難以進行透明化。
[專利文獻1]日本專利特開2014-5231號公報
[非專利文獻1]Cell (2014) vol. 159, Issue 4, pp. 896-910
[非專利文獻2]Nature Neuro Science (2015), vol. 18, NO.10, pp1518-1529
[非專利文獻3]Cell (2014) vol. 157, Issue 3, pp. 726-39
[非專利文獻4]Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition Workshops: 29-37
本發明者等人嘗試將人類皮膚切片透明化,結果除了因皮膚中富存基質導致之透明化之困難性以外,還發現無法對經透明化之皮膚試料之表皮正下方進行觀察之問題。
因此,為了解決該課題,本發明者等人經過努力研究,結果發現表皮部分之透明化不充分(圖1)。對自皮膚切片僅除掉表皮部分之方法進行研究,結果發現藉由利用分散酶(Dispase)溶液之處理去除表皮部分,藉此,能夠進行表皮正下方之微血管結構之觀察,從而完成本發明。
具體而言,本發明係關於一種透明皮膚試料,其係能夠利用光片顯微鏡進行表皮正下方組織之觀察者,且不帶有表皮,抗原性經維持。
於另一態樣中,本發明亦關於一種由所獲取之皮膚切片製造透明皮
膚試料之方法、及藉由該製造方法製造之透明皮膚試料。
於又一態樣中,本發明亦關於一種皮膚切片處理方法、及利用光片顯微鏡觀察經該處理方法處理之皮膚試料之方法。
藉由使用本發明之透明皮膚試料,能夠進行表皮正下方之結構之觀察。
圖1係針對於未經表皮去除處理之情況下進行透明化處理而獲取之人類皮膚試料,使用抗CD31抗體作為一次抗體,使用AlexaFluoro594標記抗羊IgG抗體作為二次抗體,利用光片顯微鏡獲取之三維圖像。可知表皮正下方區域之結構無法可視化。
圖2(A)係於未經表皮去除處理之情況下進行透明化處理而獲取之透明之人類皮膚試料切片之照片。於表皮側殘留不透明之層。圖2(B)係進行表皮去除處理繼而進行透明化處理而獲取之透明之人類皮膚試料切片之照片。
圖3係針對進行表皮去除處理繼而進行透明化處理而獲取之人類皮膚試料,使用抗CD31抗體作為一次抗體,使用AlexaFluoro594標記抗羊IgG抗體作為二次抗體,利用光片顯微鏡獲取之三維圖像。表皮正下方之微血管之結構能夠可視化。圖3(A)係自人類背部獲取之皮膚切片之相關圖像,圖3(B)係自人類臉部獲取之皮膚切片之相關圖像。於如下方面存在差異:於背部,微血管形成環狀結構,另一方面,於臉部,微血管形成無規結構。
圖4係針對進行表皮去除處理繼而進行透明化處理而獲取之人類皮膚
試料,使用抗CD31抗體及Cy3標記抗αSMA(Smooth Muscle Actin,平滑肌肌動蛋白)抗體作為一次抗體,使用AlexaFluoro488標記抗羊IgG抗體作為二次抗體,利用光片顯微鏡獲取之三維圖像。圖4(A)係自人類背部獲取之皮膚切片之相關圖像,圖4(B)係自人類臉部獲取之皮膚切片之相關圖像。α-SMA係表現於平滑肌之蛋白質,CD31係表現於血管內皮細胞之蛋白質。圖4A及B中示出包圍血管內皮細胞之平滑肌,於背部及臉部之皮膚,微血管之結構不同。背部皮膚於俯視觀察微血管時,多數情況下看起來像點,另一方面,臉部皮膚之微血管於俯視觀察時,看起來形成網狀結構。即,於如下方面存在差異:於背部皮膚,環狀之微血管朝上延伸,另一方面,臉部皮膚之微血管橫向擴展。
圖5係表示進行表皮去除處理並使用各種透明化處理方法進行透明化之人類皮膚試料的透明度之圖。
圖6A係進行表皮去除處理並使用各種透明化處理方法進行透明化之人類皮膚試料之照片。圖6B係表示於各種透明化處理方法下透明化之皮膚試料之霧度(Haze率)之圖表。
圖7係表示外眼角之皮膚、皮下脂肪組織及環形肌成為一體之皮膚樣品的CD31(Cluster Of Differentiation 31,分化簇31)與LYVE1(Lymphatic Vessel Endothelial Hyaluronan Receptor 1,淋巴管內皮玻尿酸受體-1)共標記圖像、CD31與脂滴包被蛋白(Perilipin)共標記圖像、CD31與肌縮蛋白(dystrophin)共標記圖像。
圖8係針對年輕之受驗者群與年老之受驗者群之來自臉頰、外眼角及背部之經去除表皮之皮膚試料,利用CD31抗體使血管可視化所得之圖像。
圖9A~C係對可視化之血管之體積、直徑及分支數進行測定,將年輕之受驗者群與年老之受驗者群加以比較之圖表。
本發明係關於一種透明皮膚試料,其係能夠利用光片顯微鏡進行表皮正下方組織之觀察者,且不帶有表皮,抗原性經維持。
於本發明中,皮膚試料可為自任意之動物種類獲取之皮膚試料,亦可為藉由三維培養技術培養之培養皮膚組織。作為動物種類,可列舉任意之哺乳動物,例如人類、豬、馬、牛、小鼠、大鼠、兔、倉鼠、猴、黑猩猩等,但並不限定於該等。就有助於美容之觀點而言,皮膚試料較佳為取自人類。皮膚試料之獲取部位可為任意之部位,例如可列舉臉面、腕部、腹部、臀部等。又,就對存在暗沉、粗糙、黃褐斑、皺紋、皮膚炎等皮膚問題之皮膚區域之結構進行分析之觀點而言,可自存在皮膚問題之皮膚區域獲取皮膚試料。
所謂「透明」,只要為能夠利用光片顯微鏡進行觀察之透明度,則可為任意之透明度。就能夠利用光片顯微鏡進行觀察之觀點而言,所謂透明係指波長380nm~780nm之光線能夠透過,較佳為波長450nm~750nm、更佳為波長490nm~650nm之光線能夠透過。「能夠利用光片顯微鏡進行表皮正下方組織之觀察之透明」皮膚試料並非僅可供進行光片顯微鏡觀察之試料,只要為具有該透明性之皮膚試料,則當然亦能夠利用通常之螢光顯微鏡、共聚聚焦顯微鏡等進行觀察。透明性可由任意之指標確定,例如可使用下述式:[數1]Tp=(Tt-(1-s1)×α)/s1-Td/s1
{式中,Td係擴散透過率,Tt係全光線透過率,s1係皮膚組織之面積率,α係覆蓋玻璃之全光線透過率(0.77),Tp係皮膚組織之平行光線透過率}
所表示之平行光線透過率。於計算該平行光線透過率之情形時,為10%~100%。擴散透過率、全光線透過率及平行光線透過率可藉由使用霧度計(Haze meter)進行計算。更具體而言,可使用村上色彩技術研究所製造之HR-100製品而確定。就利用光片顯微鏡進行表皮正下方組織之觀察之觀點而言,平行光線透過率之下限較佳為20%以上,更佳為25%以上。就採用Focus法之觀點而言,透明度較佳為30%以上。就採用iDISCO法之觀點而言,較佳為40%以上之透明度,進而較佳為45%以上。上限無特別規定,但作為能夠達成之數值範圍,例如可列舉90%以下、更佳為80%以下、進而較佳為60%以下。
皮膚試料之透明化可藉由進行既知之透明化處理、例如非專利文獻1~2所記載之透明化處理而達成。透明化處理係藉由使皮膚試料與透明化劑接觸而進行。作為透明化劑,可列舉有機溶劑透明化劑及水溶性透明化劑,作為有機溶劑透明化劑之例,可列舉iDISCO、BABB等,作為水溶性透明化劑,可列舉CLARITY、CUBIC、Scale/S、FocusClear等。該等透明化劑可依照慣例而使用(非專利文獻1~2)。通常對經過固定處理及抗體標記處理之皮膚試料進行透明化處理,但亦可於透明化處理之過程中進行標記處理。
於透明化處理採用iDISCO法之情形時,本發明之透明皮膚試料之製造方法包括以下步驟:使皮膚切片與表皮去除用酵素溶液接觸之步驟;使該皮膚切片與固定溶液接觸而將其固定之步驟;與包含迭氮化鈉之溶液接觸之步驟;與甲醇溶液接觸之步驟;與二氯甲烷溶液接觸之步驟;及與苄醚溶液接觸之步驟。
亦可進而包括與包含抗體之溶液接觸之標記步驟。標記步驟可於與包含迭氮化鈉之溶液之接觸步驟之後進行。
於透明化處理採用BABB法之情形時,本發明之透明皮膚試料之製造方法包括以下步驟:使皮膚切片與表皮去除用酵素溶液接觸之步驟;使該皮膚切片與甲醇溶液接觸而將其固定之步驟;及與BABB溶液(苄醇與苯甲酸苄酯之混合溶液)接觸之步驟。
亦可進而包括與包含抗體之溶液接觸之標記步驟。標記步驟可於與甲醇溶液之接觸步驟之後進行。
甲醇溶液可自稀甲醇溶液(例如33%)至100%甲醇依序置換。
於透明化處理採用CUBIC法之情形時,本發明之透明皮膚試料之製造方法包括以下步驟:使皮膚切片與表皮去除用酵素溶液接觸之步驟;使該皮膚切片與固定溶液接觸而將其固定之步驟;與包含胺基醇、界面活性劑及尿素之CUBIC-1溶液接觸之步驟;及
與包含胺基醇、界面活性劑及糖類之CUBIC-2溶液接觸之步驟。
亦可進而包括與包含抗體之溶液接觸之標記步驟。標記步驟可於與CUBIC-1溶液之接觸步驟之後進行。作為CUBIC-1中使用之胺基醇,可列舉N,N,N',N'-四(2-羥丙基)乙二胺,作為界面活性劑,可列舉TritonX-100。作為CUBIC-2溶液中使用之胺基醇,可列舉2,2',2"-次氮基三乙醇,作為界面活性劑,可列舉TritonX-100,作為糖類,可列舉蔗糖。
於透明化處理採用Scale/S法或FocusClear法之情形時,本發明之透明皮膚試料之製造方法包括以下步驟:使皮膚切片與表皮去除用酵素溶液接觸之步驟;使該皮膚切片與固定溶液接觸而將其固定之步驟;及與Scale/S溶液或FocusClear溶液接觸之步驟。
亦可進而包括與包含抗體之溶液接觸之標記步驟。標記步驟可於固定步驟後進行。
所謂不帶有表皮之皮膚試料係指表皮經物理、化學或酵素處理去除之試料。表皮存在於皮膚之最外層,自最外層開始由角質層、顆粒層、有棘層及基底層構成,藉由表皮基底膜而與真皮隔開。並非意欲限定於理論,但表皮由於包含阻礙透明化之黑色素細胞或折射率較高之角質層,故難以藉由透明化處理實現透明化,因此,可知包含表皮之透明皮膚試料無法觀察表皮正下方之結構(圖1)。所謂不帶有表皮之皮膚試料,只要實質上不帶有表皮即可。所謂實質上不帶有表皮,只要能夠使本發明之目的即利用光片顯微鏡進行表皮正下方組織之觀察成為可能,則亦可一定程度上包含表皮細胞,只要為可使光片顯微鏡之雷射透過之表皮,則亦可包含於皮膚試料中。又,皮膚試料除包含皮膚以外,亦可一併包含存在於較真皮
層更深部之皮下脂肪組織或肌肉組織。
作為用以去除表皮之物理處理,可列舉熱處理、使用手術刀或鑷子等之剝離處理,於目視下或顯微鏡下去除表皮區域。作為酵素處理,可列舉利用蛋白質分解酵素之處理。作為蛋白質分解酵素,可使用非特異性酵素、特異性酵素之任一者,為了將表皮細胞分離,可使用分散酶、胰蛋白酶等。表皮與真皮交界處存在稱為乳頭層之複合結構,因此,就高效地去除表皮之觀點而言,較佳為酵素處理。若藉由酵素處理去除表皮,則能夠獲取維持乳頭結構且去除表皮之皮膚試料。又,熱處理等物理處理會使真皮之蛋白質失去抗原性,因此,就維持抗原性之觀點而言亦宜為酵素處理。
所謂抗原性經維持之皮膚試料係指於皮膚試料中所包含之各種蛋白質之表位部分能夠特異性地結合對應抗體之皮膚試料。抗原性經維持之皮膚試料並非一定指維持全部抗原性。原本於免疫染色領域,通常會於固定處理等處理期間失去一部分抗原性,即便為對純化蛋白質或表位具有特異結合性之抗體,亦未必於固定試料產生特異性結合。因此,於本發明中,所謂「抗原性經維持」係指至少1種或1種以上之抗體具有特異性而具有與表位之結合性。於透明化處理在抗體處理後進行之情形時,透明皮膚試料包含靶抗原上結合之標記抗體。並不意欲限定於以下者,但作為一例,較佳為維持選自由CD31、LYVE1、脂滴包被蛋白及肌縮蛋白所組成之群中之至少1種蛋白質之抗原性。就使皮膚正下方之脈管可視化之觀點而言,較佳為維持對於CD31、LYVE1而言之抗原性。
將透明皮膚試料供於光片顯微鏡,利用抗體識別皮膚試料中之特定蛋白質,藉此能夠使其可視化。又,藉由使用針對於皮膚所包含之任意結
構特異性地表現之蛋白質之抗體,而能夠對皮膚所包含之任意結構進行觀察。作為皮膚之結構,可列舉細胞外基質、淋巴管、靜脈、動脈、微血管、神經、汗腺、皮脂腺、細胞成分(例如肥胖細胞、漿細胞、成纖維細胞、蘭格漢氏細胞(Langerhans cell)、梅克爾細胞(Merkel cell)、血管內皮細胞、淋巴管內皮細胞、神經細胞、汗腺細胞)等,但並不限定於該等。例如,於使血管可視化之情形時,使用針對於血管之細胞特異性地表現之蛋白質、CD31、vWF(Von Willebrand factor,血管性假血友病因子)、CD34等之抗體。於使細胞外基質可視化之情形時,使用針對膠原蛋白、彈力蛋白、αSMA、纖維結合蛋白等之抗體。於使神經可視化之情形時,可使用針對PGP9.5等之抗體。於使淋巴管可視化之情形時,使用針對LYVE-1、腎小球足突細胞膜黏蛋白(Podoplanin)等抗體。該等抗體可單獨使用,亦可組合使用。
用於對透明皮膚試料進行觀察之抗體可為自任意之動物種類獲取之抗體,又,亦可為採用噬菌體呈現(phage display)法等基因工程製成之抗體。作為動物種類,可列舉小鼠、人類、大鼠、兔、山羊、駱駝、驢等,可藉由對該等動物導入抗原而獲取抗體。抗體可為單株抗體,亦可為多株抗體。亦可為該等抗體之組合即嵌合抗體。亦可代替抗體而使用具有結合性之抗體片段。作為抗體片段之例,可列舉Fab片段(fragment of antigen binding,抗原結合片段)、Fv片段(variable fragment,可變區片段)、F(ab')2片段、Fab'片段或scFv(single-chain variable fragment,單鏈可變區片段)。
可對與抗原直接結合之抗體本身標附能夠利用光片顯微鏡進行觀察之標記,亦可對與抗原直接結合之抗體之二次以後之抗體標附標記。所標
附之標記較佳為用於螢光顯微鏡之任意之螢光標記,例如可使用若丹明、螢光素、Cy色素、Alexa等任意之螢光標記。亦可使用分別與複數種抗原結合之抗體,並使用標附有不同標記之抗體作為區分各抗體而與之結合而之二次抗體,藉此使複數種抗原同時或連續地可視化。
除利用抗體進行標記以外,亦可代替該利用抗體之標記,而對透明皮膚試料中之細胞進行核染色或使細胞表現螢光蛋白質。作為核染色試劑,可使用公知之螢光試劑,例如可使用DAPI(4',6-diamidino-2-phenylindole,4',6-二脒基-2-苯基吲哚)、碘化丙錠(PI)、Hoechst 33342等。核染色或抗體之螢光標記較佳為以具有能夠各自區分識別到之螢光波長之方式進行選擇。螢光蛋白質、例如GFP(Green Fluorescent Protein,綠色螢光蛋白)等可藉由製作於所需啟動子之下游導入有GFP、YFP(Yellow Fluorescent Protein,黃色螢光蛋白)之基因的基因轉殖動物或藉由使載體於動物體內局部地表現而利用。
於本發明之另一態樣中,本發明亦關於一種由皮膚切片製造透明皮膚試料之方法或皮膚切片之處理方法。皮膚切片可使用預先獲取之皮膚切片。該等方法可按照任意順序包括以下步驟:使皮膚切片與表皮去除用酵素溶液接觸而去除表皮之步驟;使皮膚切片與固定溶液接觸而將其固定之步驟;使皮膚切片與透明化試劑接觸而使其透明化之步驟。較佳為依序進行表皮去除步驟、固定步驟及透明化步驟。亦可進而包括於固定步驟之後使皮膚切片與標記抗體溶液接觸之標記化步驟。亦可包括於各步驟之前後進行洗淨之洗淨步驟。於本發明之又一態樣中,亦關於一種利用由皮膚切片製造透明皮膚試料之方法製造之透明皮膚試料。
表皮去除步驟可藉由例如於表皮去除用酵素溶液中,於室溫、加溫或冷卻下培養數小時~數天而進行。就促進酵素反應之觀點而言,較佳為37℃左右,例如33℃~40℃。另一方面,就防止蛋白質改性之觀點而言,較佳為於冷卻下培養,例如於0℃~5℃下、較佳為4℃~5℃下培養。就適當去除表皮之觀點而言,較佳為例如於冷卻下培養1小時~2天,更佳為培養3小時至12小時。為了僅去除表皮,較佳為使皮膚切片之表皮側與浸有酵素溶液之紗布等支持體接觸並培養。最佳為藉由以皮膚切片之表皮為下側將其載置於浸有酵素溶液之紗布等吸水性之支持體上而進行培養。培養條件可根據試料之獲取部位而不同,亦可根據試料獲取部位之狀態而改變。例如對於粗糙之皮膚,由於皮膚屏障功能下降,故而可選擇酵素溶液之滲透較快、較弱之培養條件,例如短時間及低溫度之條件。於與酵素溶液接觸後,使用鑷子等自真皮剝離表皮,藉此將表皮去除。
固定步驟可按照免疫染色領域中通常所用之方法進行。作為固定溶液,可使用多聚甲醛、甲醇等。作為一例,可藉由將皮膚切片浸於4%多聚甲醛溶液中,於室溫或冷卻下培養數分鐘~數天而進行。酵素處理不易對固定後之試料起作用,因此,較佳為於表皮去除步驟之後進行固定步驟。
標記步驟可按照免疫染色領域中通常所用之方法進行。例如,亦可對於經固定之試料,將針對靶抗原之抗體於一次抗體溶液中進行培養,洗淨後,於針對一次抗體之經標記之二次抗體之溶液中進行培養。抗體之稀釋率、培養時間、溫度可根據所使用之抗體而適當選擇。就避免退色之觀點而言,與標記二次抗體溶液之培養較理想為於暗處進行。較理想為標記步驟後之處理及保管全部於暗處進行。
透明化步驟係藉由將試料於既知之透明化試劑之溶液中加以培養而進行。作為透明化步驟之一例,可列舉利用iDISCO法(iDISCO:A Simple,Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging.Cell 159,896910,November 6,2014)、CUBIC法、Scale法或FocusClear法進行之處理。可適當選擇透明化試劑及培養時間等以獲取充分透明之試料。
經過標記步驟而製成之透明皮膚試料可供進行光片顯微鏡或螢光顯微鏡觀察。利用光片顯微鏡或螢光顯微鏡之觀察可藉由該等顯微鏡通常所用之方法進行。例如根據所標附之標記選擇入射光,選擇與激發光對應之濾光片,藉此能夠觀察來自標記之激發光。
藉由使用本發明之透明皮膚試料,能夠對皮膚內部之微細結構進行觀察,藉此達到皮膚內部結構相關知識積累之目的。藉由精細地觀察皮膚之問題、例如粗糙、黃褐斑、皺紋、黑斑(chloasma)、痤瘡等皮膚區域之皮膚內部結構,能夠有助於對其原因之瞭解或相關改善、治療方法之開發。雖然本發明必須使用侵入性獲取之皮膚切片,但該光片顯微鏡或螢光顯微鏡之觀察方法具有目前正在開發之非侵入性皮膚內部結構之觀察方法所無法比擬之優勢,又,其可視化對象之結構亦明確。因此,領先於將來可期待採用之非侵入式之皮膚內部結構之觀察,能夠積累有關皮膚內部結構之資料及知識。
本說明書中所言及之全部文獻係以引用之方式將其整體併入至本說明書中。
以下所說明之本發明之實施例僅以例示為目的,並不限定本發明之技術範圍。本發明之技術範圍僅由申請專利範圍之記載進行限定。可於不
脫離本發明之主旨之條件下對本發明進行變更,例如,本發明之構成要件之追加、刪除及置換。
酵素處理步驟
使分散酶(Roche)(38U/vial)溶解於milliQ水38ml中。於培養皿中鋪覆Kimwipe,並使之含浸分散酶。將0.5mm見方之人類皮膚切片之表皮側朝下,浸於分散酶中,於4℃下培養一晚。次日使用鑷子將角質層剝離。
使用CUBIC之透明化處理
將5mm見方之人類皮膚切片浸漬於4%多聚甲醛(PFA)中,利用旋轉器(Rotator)RT-50(TAITEC)進行旋轉,於4℃下培養一晚而將其固定。將經固定之皮膚切片試料於CUBIC-1溶液中浸透1週。於CUBIC-1處理後,利用PBS(Phosphate-buffered saline,磷酸鹽緩衝液)清洗3次,於20%蔗糖溶液中於4℃下旋轉一晚。其後,將其包埋於O.C.T冷凍切片包埋劑(O.C.T compound)(Sakura finetek)中進行冷凍。解凍後,利用PBS清洗3次後,於一次抗體溶液中於37℃下浸透3天。利用PBST(PBS+0.1%triton)清洗3次後,於二次抗體溶液中於37℃下浸透一晚。利用PBST(PBS+0.1%triton)清洗3次後,於20%蔗糖中浸泡一晚,放入至CUBIC-2溶液中,於4℃下培養1週。CUBIC-1及CUBIC-2之組成如Susaki et al.,Cell.2014 Apr 24;157(3):726-39之記載。具體而言,CUBIC-1溶液係包含N,N,N',N'-四(2-羥丙基)乙二胺、TritonX-100及尿素之溶液,CUBIC-2溶液係包含2,2',2"-次氮基三乙醇、TritonX-100及蔗糖之溶液。
使用Scale或FocusClear之透明化處理
將5mm見方之人類皮膚切片於4%多聚甲醛(PFA)中於4℃下進行固定。固定後,利用PBS清洗3次後,於一次抗體中於4℃下浸透7天,利用PBST清洗3天,於二次抗體中於4℃下浸透7天。利用PBST清洗後,於FocusClear(銷售商:CEDARLANE)或Scale(銷售商:Olympus公司)中於4℃下浸透7~14天。
使用iDISCO之透明化處理
將5mm見方之皮膚樣品於4%PFA中進行固定。利用PBS清洗3次。於5%Triton、2.5%Tween20之PBS溶液中振盪後,利用PBS清洗3次。其後,於Perm Block溶液(放入有0.5g之BSA(Bovine Serum Albumin,牛血清白蛋白)、50μl之Tween20、300μl之5%迭氮化鈉之PBS溶液50ml)中振盪。繼而,於包含一次抗體之溶液中於37℃下振盪3天,利用PBST(0.1%Tween20)清洗3天。於包含二次抗體之溶液中於37℃下振盪3天,其後,利用PBST(0.1%Tween20)清洗3天。其後,於50%MeOH中浸泡3小時、於70%MeOH中浸泡3小時、於100%MeOH中浸泡一晚並振盪。最後,於二氯甲烷(Sigma)中振盪15分鐘×2次,於二苄醚(Sigma)中靜置一晚。
作為皮膚樣品,使用自臉部獲取之皮膚切片及自背部獲取之皮膚切片。製備經酵素處理而去除了表皮之皮膚樣品及未經酵素處理而殘留有表皮之皮膚樣品。利用iDISCO法對皮膚樣品進行透明化處理。將進行透明化處理後之試料示於圖2。使用經PBS稀釋100倍之抗CD31綿羊抗體(銷售商:R&D systems)作為一次抗體,使用經PBS稀釋200倍之AlexaFluoro594標記抗綿羊IgG抗體(銷售商:Invitrogen)作為二次抗體。又,作為共染色,使用經PBS稀釋之Cy3標記抗αSMA抗體(銷售商:
Sigma)進行染色。於光片顯微鏡(製造商:Carl Zeiss公司)下觀察經染色之皮膚試料。將殘留有表皮之皮膚樣品之CD31可視化照片示於圖1。將已去除表皮之皮膚樣品(自背部獲取之皮膚切片及自臉部獲取之皮膚切片)之CD31可視化照片示於圖3(A)及圖3(B)。又,將已去除表皮之皮膚樣品(自背部獲取之皮膚切片及自臉部獲取之皮膚切片)之CD31及αSMA可視化之構建三維結構照片示於圖4(A)及(B)。
透明度測定法
對已去除角質層之皮膚組織,分別進行CUBIC處理、FocusClear處理、Scale處理及iDisco處理。針對經過透明處理之皮膚組織,參照Tainaka et al.,Cell.2014 Nov 6;159(4):911-24,加以改編而進行透明度測定。利用霧度計(公司名:製品編號:),測定人類皮膚組織之Td:擴散透過率、Tt:全光線透過率。由於皮膚組織並未將試片全部覆蓋,故利用面積率(s1)進行修正,算出皮膚組織之平行光線透過率(Tp)。具體而言,藉由下述式:Tp=(Tt-(1-s1)×α)/s1-Td/s1
{式中,Td係擴散透過率,Tt係全光線透過率,s1係皮膚組織之面積率,α係覆蓋玻璃之全光線透過率(0.77),Tp係皮膚組織之平行光線透過率}
算出。將結果示於圖5。
又,藉由將擴散透過率除以全光線透過率而算出霧度(%)。
人類皮膚樣品
自ILSBIO LLC(Chestertown,MD)或(Gakugeidai-Nishiguchi Clinic)獲取人類皮膚樣品。自ILSBIO獲取之亞洲人之臉面及背部之皮膚試料全部基於美國及國際倫理指南而獲取。ILSBIO協定已得到健康和人類服務認證臨床試驗審查委員會(Health and Human Services registered Institutional Review Board(IRB))之承認。於收集之前已獲得知情同意。
又,從學藝大西口診所獲取來自日本人男性及女性之外眼角之皮膚、環形肌及皮下脂肪組織成為一體之皮膚樣品。確認該等對象未患有異位性皮膚炎或酒渣鼻。將全部樣品急速冷凍,供其後進行組織學分析。包含人類對象之全部方法已得到資生堂全球創新中心(Shiseido Global Innovation Center)之臨床試驗審查委員會之承認,並且已自全部對象獲得書面之知情同意。
將所獲得之皮膚樣品供於上述酵素處理步驟,剝離角質層。將角質層被剝離之皮膚樣品浸漬於4%多聚甲醛(PFA)中,藉此將其固定。
透明化
將經固定之來自臉面之皮膚樣品供於Cubic、Focus Clear、BAAB及iDISCO法,進行透明化。拍攝經透明化之皮膚樣品之照片(圖6A)。如上所述,對經透明化之皮膚樣品測定光透過率,並測定霧度(%)。將結果示於圖6B。
免疫標記
皮膚、環形肌及皮下脂肪組織之多重標記
將經PFA溶液固定之皮膚、環形肌及皮下脂肪組織成為一體之皮膚樣品利用PBS洗淨,利用PBS中之0.5%TritonX-100進行浸透處理,其次與
PBS中之1%TritonX-100/0.5%Tween-20進行培養。於與封閉溶液培養3天後,將試料與封閉溶液中之一次抗體於37℃下培養3天。此處,使用針對CD31之抗多株綿羊抗體(R&D systems,Minneapolis,MN)與針對LYVE-1之多株兔抗體(Angiobio,San Diego,CA)、或與針對脂滴包被蛋白之多株豚鼠抗體(Progen,Heidelberg,Germany)、或與針對肌縮蛋白之多株兔抗體(Santa Cruz biotechnology,Dallas,Texas)作為一次抗體。此處,獲得利用抗CD31抗體與抗LYVE1抗體進行共標記之皮膚樣品、利用抗CD31抗體與抗脂滴包被蛋白抗體進行共標記之皮膚樣品、及利用抗CD31抗體與抗肌縮蛋白抗體進行共標記之皮膚樣品。將該等進行共標記之皮膚樣品利用PBS-T清洗2天,其後使用經稀釋之AlexaFluoro594標記抗綿羊IgG抗體(銷售商:Invitrogen)、及AlexaFluoro488標記抗兔IgG抗體或AlexaFluoro488標記抗豚鼠IgG抗體作為二次抗體,於封閉溶液中於37℃下培養3天,藉此進行多重標記。
針對來自臉頰、外眼角及背部之皮膚樣品之CD31之單獨標記
將經PFA溶液固定之來自臉頰、外眼角及背部之皮膚樣品利用PBS洗淨,利用PBS中之0.5%TritonX-100進行浸透處理,其次與PBS中之1%TritonX-100/0.5%Tween-20進行培養。於與封閉溶液培養3天後,將試料與封閉溶液中之一次抗體於37℃下培養3天。此處,使用針對CD31之多株綿羊抗體(R&D systems,Minneapolis,MN)作為一次抗體。將試料利用PBS-T清洗2天,其後使用經稀釋之AlexaFluoro594標記抗綿羊IgG抗體(銷售商:Invitrogen)作為二次抗體,於封閉溶液中於37℃下培養3天,藉此進行單獨標記。
將免疫標記後之樣品如上所述般供於iDISCO法,進行透明化。將經
透明化之皮膚樣品供於顯微鏡及成像分析。
顯微鏡及成像分析
針對經免疫標記之皮膚樣品,使用光片螢光顯微鏡(Lightsheet microscopy Z.1,Carl Zeiss,Germany)獲取圖像。使用軟體Zen(Carl Zeiss)進行最大投影。使用軟體Imaris(Bitplane,Concord,MA)進行3D處理。針對利用抗CD31抗體與抗LYVE1抗體進行共標記之皮膚樣品,使皮膚區域之CD31與LYVE1可視化,針對利用抗CD31抗體與抗脂滴包被蛋白抗體進行共標記之皮膚樣品,使皮下組織區域之CD31與脂滴包被蛋白可視化,針對利用抗CD31抗體與抗肌縮蛋白抗體進行共標記之皮膚樣品,使肌肉層區域之CD31與肌縮蛋白可視化,分別獲取三維圖像(圖7)。
將臉頰、外眼角及背部之皮膚分為2個年齡群,即「年輕群(關於臉頰之皮膚,平均年齡20.8±4.8歲,年齡範圍18~29歲,n=4;關於外眼角之皮膚,平均年齡20.5±5.4歲,年齡範圍12~27歲,n=4;關於背部之皮膚,平均年齡19.3±6.4歲,年齡範圍18~20歲,n=3)」以及「年老群(關於臉頰之皮膚,平均年齡46.3±2.2歲,年齡範圍45-50歲;關於外眼角之皮膚,平均年齡51.3±9.7歲,年齡範圍35-61歲,n=4;關於背部之皮膚,平均年齡44.3±4.1歲,年齡範圍39-49歲,n=3)。藉由上述使用抗CD31抗體之標記,使該等對象之臉頰、外眼角、背部之皮膚之血管可視化並拍攝照片(圖8)。如先前報告(非專利文獻4:Bise R,Sato I,Kajiya K,et al.(2016)3D Structure Modeling of Dense Capillaries by Multi-Objects Tracking.Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition Workshops:29-37)般進行形態三維分析,根據圖像,測定血管之體積、尺寸及微血管之分支數(圖9)。
統計分析
全部統計均以平均±SD表示。使用ANOVA,繼而使用Posthock檢定,進行統計分析。為了評價對照群與複數群之比較中之統計上有意義差,使用圖基(Tukey)檢定,其次使用巴特利特(Bartlett)檢定。將P<0.05作為有意義差。
Claims (6)
- 一種由所獲取之皮膚切片製造透明皮膚試料之方法,其包括:使該皮膚切片與表皮去除用分散酶溶液接觸,以去除表皮之步驟;使該皮膚切片與多聚甲醛溶液接觸而將其固定之步驟;及使該皮膚切片與選自由iDISCO、Scale、Focus Clear、及BAAB所組成群中之透明化處理法之透明化試劑接觸之步驟。
- 如請求項1之方法,其進而包括於上述固定步驟之後使上述皮膚切片與標記抗體溶液接觸之步驟。
- 如請求項1或2之方法,其中上述皮膚試料來自人類。
- 一種透明皮膚試料,其係藉由如請求項1至3中任一項之方法製造者,且不帶有光不透過性之表皮,抗原性經維持。
- 如請求項4之透明皮膚試料,其中於對下式:[數1]Tp=(Tt-(1-s1)×α)/s1-Td/s1{式中,Td係擴散透過率, Tt係全光線透過率,s1係皮膚組織之面積率,α係覆蓋玻璃之全光線透過率(0.77),Tp係皮膚組織之平行光線透過率}所表示之透明度進行測定之情形時,透明皮膚試料之平行光線透過率為10%~100%。
- 如請求項5之透明皮膚試料,其中上述透明皮膚試料能夠利用光片顯微鏡進行表皮正下方組織之觀察。
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期刊 Renier et al., "iDISCO: A Simple, RapidMethod to Immunolabel Large Tissue Samples for Volume Imaging" Cell, vol. 159, November 6, 2014 p.896-910 |
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