臺灣博碩士論文加值系統

幹細胞具有自我更新以及分化成各種不同細胞型態的能力。因為脂肪組織在人體中具有相當多的含量，且能以較低風險的方式取得，近來也成為引起興趣的成體幹細胞來源。取得組織後，藉由酵素作用與離心的方式分離出脂肪幹細胞。離心後的沉積團塊被認為是含有幹細胞的存在。能被直接使用成為幹細胞的來源也能被培養數個世代。實驗室著眼於眼眶周圍的脂肪組織作為成體幹細胞的來源。通常身體的脂肪組織均發育自中胚層，但以脊椎動物的顱顏部發育過程來看，顱部的神經嵴細胞依其獨特能力能分化成為顱部的結締組織。因此我們預測由神經嵴細胞產生的幹細胞比一般的成體幹細胞更適合用在神經修復的移植實驗。我們將分離自眼瞼脂肪組織的脂肪幹細胞作特性確認，進行免疫表現型、增殖能力、分化能力等分析。結果發現脂肪幹細胞不僅表現胚胎幹細胞標定物Oct-4，也會表現各胚層的標定物BMP-4和nestin，而根據成長曲線圖以及端粒長度分析的結果也能證實其自我更新的能力。在分化能力上也證實了其往脂肪、軟骨、神經、角質細胞各種細胞型態的發展。證實從人類眼瞼脂肪組織所取得的脂肪幹細胞仍維持相當早期的幹細胞特性，穩定的成長速率以及多重的分化潛能。實驗結果除了建議以眼瞼脂肪組織來當作脂肪幹細胞的較佳來源，除了在自體移植的應用外，也建議能使用在彈性軟骨的替代。

Stem cells are characterized for the ability to undergo self-renewal and differentiation into multilineage cell type. Owing to easy to harvest with low risk and highly abundant in the human body, adipose-derived stem cells (ASCs) are an attractive source of the adult stem cells. ASCs are then isolated using enzymatic digestion and centrifugation. The pellet is considered containing the stem cells. It can be used directly as a source of stem cells and expanded in culture through many passages. We directed our attention to periocular adipose tissue as a potential source of adult stem cells. Adipose tissue throughout the body is generally derived from mesodermal tissues. But based on the vertebrate craniofacial evolution, cranial neural crest cells exhibit a unique ability to differentiate into cranial connective tissue. Thus, we speculate stem cells derived from neural crest cell probably be a better source to the neural repair transplantation. In this study, we characterized ASCs derived from human eyelid adipose tissue in terms of immunophenotypic characterization, proliferation capacity, differentiation ability. ASCs from the human eyelid adipose tissue expressed stem cell markers (Oct-4) and germ layer markers (BMP-4 and nestin). On the basis of growth curve and telomere length assay, ASCs possess the high proliferation potential and self-renewal capacity. ASCs also differentiate toward the adipogenic, chondrogenic, neuogenic and keratinogenic lineages. The results demonstrated that adult human eyelid ASCs were stayed in early stage, with continually stable growth rate, and revealed multi-lineage potential. According to this study, the human eyelid adipose tissue is beneficial source to isolate ASCs for clinical autologous applications, such as elastic cartilage replacement.

目 錄 i
圖目錄 iv
中文摘要 vii
英文摘要 viii
第一章、緒論 1
第二章、材料與方法 6
第一節、實驗架構 6
第二節、初級培養 7
第三節、生長速率 7
第四節、端粒長度分析 8
第五節、免疫細胞化學染色 8
第六節、誘導分化 10
壹、脂肪細胞誘導分化 10
貳、硬骨細胞誘導分化 10
参、軟骨誘導分化 11
肆、神經性誘導分化 11
伍、角質細胞誘導分化 11
第七節、化學染色 12
壹、油紅染色 12
貳、茜素紅染色 12
参、亞爾襄藍染色 12
第八節、流式細胞儀分析 13
第三章、實驗結果 14
第一節、幹細胞培養 14
第二節、細胞定性：生長速率及自我更新能力 14
壹、生長速率 14
貳、端粒長度 15
第三節、細胞定性：免疫細胞化學染色及流式細胞儀分析 15
壹、巢蛋白(nestin) 15
貳、骨型態生成蛋白-4 (BMP-4) 16
参、Octamer-4 17
第四節、分化能力：免疫細胞化學染色及化學染色 18
壹、脂肪細胞誘導分化 18
貳、硬骨細胞誘導分化 19
参、軟骨誘導分化 19
肆、神經性誘導分化 19
伍、角質細胞誘導分化 20
第四章、結論 22
第五章、討論 23
第六章、圖 26
第七章、參考文獻 48

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