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系統識別號 U0026-2107201019024000
論文名稱(中文) 內生性類固醇在人體皮膚角質層之分布
論文名稱(英文) Distribution of Endogenous Steroids in Human Stratum Corneum
校院名稱 成功大學
系所名稱(中) 臨床藥學研究所
系所名稱(英) Institute of Clinical Pharmacy
學年度 98
學期 2
出版年 99
研究生(中文) 曾淑萍
研究生(英文) Shu-Ping Tseng
學號 s6696108
學位類別 碩士
語文別 中文
論文頁數 85頁
口試委員 指導教授-蔡瑞真
口試委員-廖寶琦
口試委員-許漢銘
口試委員-劉主成
中文關鍵字 角質層  內生性類固醇  液相層析串聯質譜儀 
英文關鍵字 SC  endogenous steroids  LC/MS/MS 
學科別分類
中文摘要 皮膚之生長與功能受到荷爾蒙影響,本身亦能合成多種荷爾蒙(CRH, ACTH, thyroid hormone, sex hormone, corticosteroid, etc.)及神經傳遞物質(neurotransmitters)。皮膚可依局部之需求進而代謝或形成性荷爾蒙(autonomous control)。在停經後婦女(post-menopausal women)性荷爾蒙之來源主要來自於皮膚。皮膚可視為一固醇類生成之組織(steroidogenic tissue),由於固醇類生成酵素(steroidogenic enzyme)及輔酶(cofactor)存在於表皮及皮脂細胞。荷爾蒙對皮膚生理活性影響甚遠,對於皮膚之修護功能亦相關。
至目前為止未有文獻報告角質層上內生性類固醇之存在,因此本研究之目的在探討角質層內生性類固醇之存在與分布。首先建立高靈敏度之液相層析串聯質譜儀(LC/MS/MS)分析方法,並加以確效,接著藉由非侵入性角質層取樣技術,比較不同性別、年齡、部位及角質層深度內生性類固醇含量之差異性。利用皮膚層撕法取得角質層,以reportor lysis buffer破壞角質細胞,並經二氯甲烷萃取後吹乾,回溶後注入液相層析串聯質譜儀,搭配MRM偵測模式進行分析。液相層析儀使用C18管柱,移動相為水及乙腈(95:5, v/v)、乙腈及水(95:5, v/v),利用梯度沖提方式於15分鐘內完成分析。
研究結果顯示,三種類固醇成分之檢量線於0.3-50 ng/mL濃度具有良好之線性相關係數(0.9999),添加於膠帶之回收率介於20-40% 之間,最低檢測限量及最低定量值分別介於0.1-0.8 ng/mL 及 0.3-1.0 ng/mL之間。
依此方法分析32位受試者,結果顯示僅有一位偵測到Testosterone成分。在Group 1中,Hydrocortisone於手臂部位檢出個數較多,Cortisone於背部部位檢出個數較多;Group 2中Cortisone於背部部位並未檢出。在前額、手臂及背部部位皮膚角質層之Hydrocortisone含量分別介於0.13-0.37 ng/cm2,0.02-0.96 ng/cm2及0.06-0.49 ng/cm2之間;在前額、手臂及背部部位皮膚角質層之Cortisone含量分別介於0.02-0.20 ng/cm2,0.03-0.12 ng/cm2及0.02-0.06 ng/cm2之間。Hydrocortisone及Cortisone含量在不同性別、年齡及部位均無統計上之顯著差異性;而Group 2中在角質層表層有較多之Cortisone存在(P < 0.05)。至於角質層上所偵測出之內生性類固醇是否與受試者內生性荷爾蒙之血中濃度具有相關性,仍待進一步研究釐清。
英文摘要 Hormones influence the development and function of human skin which also produces hormones and neurotransmitters. The local formation of sex steroids provides autonomous control to human skin according to local needs. In addition, in post-menopausal women, all sex steroids made in the skin are from adrenal steroid precursors. Human sebocytes and keratinocytes express steroidogenic enzymes and cofactors. Hormones exhibit a wide range of biological activities on the skin and regulate cutaneous wound healing.
Stratum corneum (SC) is the outmost and the terminal differention layer of the skin. The endogenous steroids content of the human SC has never been reported on the literature. The aim of the present study was to investigate the distribution of endogenous steroids in human SC with respect to gender, age, anatomical site, and depth into SC, using a non-invasive sampling technique and a sensitive analytic method for quantitation of endogenous steroids.
A liquid chromatograph-tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous identification and quantitation of three endogenous steroids (hydrocortisone, cortisone, and testosterone) in human SC. SC was removed by sequential tape stripping and the samples were incubated with reporter lysis buffer followed by extraction with dichloromethane. The extractants were evaporated to dryness, resuspended in 0.1 mL methanol, and then detected using LC/MS/MS in the multiple reaction monitoring mode (MRM). The chromatography was operated on a reversed phase C18 column using a linear gradient of water/acetonitrile (95/5, v/v) and acetonitrile/water (95/5, v/v) in 15min. The detector’s response was linear for hydrocortisone, cortisone, and testosterone concentrations ranging from 0.3 to 50 ng/mL. Extraction recovery ranged from 20% to 40%. Limits of detection and limits of quantification for three steroids were in the ranges of 0.1-0.8 ng/mL and 0.3-1.0 ng/mL, respectively.
32 healthy volunteers participated in the study. The result showed that testosterone was detected in only one volunteer. The frequency of hydrocortisone detection was higher in the SC of the forearm, while cortisone was detected more frequently in the SC of the back from Group 1 subjects (years 20-35). However, cortisone was not observed in the back from Group 2 subjects (years 50-65). Amounts of hydrocortisone and cortisone in SC of forehead ranged 0.13-0.37 ng/cm2 and 0.02-0.20 ng/cm2, respectively. Amounts of hydrocortisone and cortisone in SC of forearm ranged 0.02-0.96 ng/cm2 and 0.03-0.12 ng/cm2, respectively. Amounts of hydrocortisone and cortisone in SC of back ranged 0.06-0.49 ng/cm2 and 0.02-0.06 ng/cm2, respectively. The amounts of hydrocortisone and cortisone were not statistically different between genders, age groups and anatomical sites. Statistically higher amounts of cortisone were found in the surface layer of SC than deeper layers in Group 2 subjects. Whether the amounts of hydrocortisone and cortisone found in SC are related to their plasma levels remains to be clarified in future studies.
論文目次 中文摘要.................................................i
英文摘要.................................................iii
誌謝.....................................................v
目錄.................................................... vi
表目錄.................................................. x
圖目錄.................................................. xii
縮寫表................................................xiv
第一章 文獻回顧......................................... 1
第一節 人體內類固醇荷爾蒙之來源與功能................1
第二節 人體皮膚為一固醇類生成組織...................2
第三節 影響人體內類固醇分泌之因素...................8
第四節 非侵入性方法在偵測皮膚上成分之應用............9
第五節 體內類固醇荷爾蒙樣本來源與分析方法............11
第二章 研究目的..........................................16
第三章 實驗及儀器........................................17
第一節 材料......................................17
第二節 儀器......................................18
第三節 分析軟體...................................19
第四章 研究設計..........................................20
第一節 內生性類固醇之分析方法.......................20
1.溶液之配製................................20
2.檢量線之製作..............................21
3.建立質譜儀及液相層析儀之最適分析條件.........21
4.膠帶之選擇................................22
5.液相層析串聯質譜儀方法之確效................23
第二節 預試驗.....................................24
1.角質層內生性類固醇取樣方法與樣品前處理.......24
2.角質層上內生性類固醇之初步試驗..............24
3.角質層層撕法之確效.........................25
第三節 正式人體試驗................................26
第四節 角質層內生性類固醇含量於不同時間與位置之分析....26
第五節 數據處理...................................27
第六節 統計分析...................................27
第五章 研究結果..........................................29
第一節 質譜儀及液相層析儀之最適分析條件..............29
1.質譜儀之最適分析條件......................29
2.液相層析儀之最適分析條件...................30
第二節 液相層析串聯質譜儀方法之確效.................31
1.檢量線...................................31
2.回收試驗.................................31
3.最低檢測限量及最低定量值...................31
4.同日(Inter-day)與異日(Intra-day)試驗......32
第三節 角質層層撕法之確效..........................32
1.檢量線...................................32
2.角質層蛋白質含量分析.......................32
第四節 角質層內生性類固醇含量分析...................33
第五節 角質層內生性類固醇含量於不同時間與位置之分析....34
第六章 討論..............................................71
第一節 分析方法....................................71
第二節 角質層層撕法之確效...........................72
第三節 正式人體試驗................................72
第四節 角質層內生性類固醇含量於不同時間與位置之分析....74
第七章 結論..............................................76
參考文獻..................................................77
附錄 (一) 人體試驗委員會同意臨床試驗證明書.............83
附錄 (二) 人體試驗同意書....................................84
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