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研究生:鐘毓棠
研究生(外文):Jong, Yu-Tang
論文名稱:脂肪幹細胞與富含血小板纖維蛋白萃取液對兔子眼角膜損傷治療之影響
論文名稱(外文):The effect of Platelet-rich fibrin (PRF) extract and Adipose-derived stem cells (ADSCs) reconstruction on rabbit corneal damage reconstruction
指導教授:許岩得
指導教授(外文):Hsuuw, Yan-Der
口試委員:林昇全莊國賓
口試委員(外文):Lin, Sheng-Chuan Chuang, Kuo-Pin
口試日期:2016-07-14
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技系所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:59
中文關鍵詞:富血小板纖維蛋白脂肪幹細胞眼角膜
外文關鍵詞:Platelet-rich fibrinAdipose-derived stem cellsRabbitCornea
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富血小板纖維蛋白(Platelet-rich fibrin ,PRF)是將血漿離心後取得,富含多種傷口癒合相關的蛋白與生長因子的生物材料。幹細胞(stem cells)是生物體內尚未分化的細胞,具有潛力可分化、增生成各種類型細胞。自1986年在眼角膜與鞏膜交界處(limbus)發現眼角膜幹細胞(corneal stem cells)的存在後,角膜的幹細胞再生治療相關研究便開始大幅增加。在本實驗室的前期研究中,發現PRF有促進幹細胞移動、增生、分化、血管新生、膠原蛋白合成與再生新組織的效果。
在本試驗中,以NaOH破壞兔子的眼角膜製造模擬損傷。被強鹼破壞的角膜,外觀上變的不透明;從切片可看到角膜的Epithelium與Stroma受到破壞。接著,從兔子心臟採血、離心取得PRF萃取液。接著,從兔子的腹部手術切開取得脂肪,純化脂肪幹細胞(adipose-derived stem cells, ADSCs)。以ADSCs搭配PRF萃取液,治療損傷24小時後之眼角膜,連續14天。

Platelet-rich fibrin (PRF), a biologic material isolated from centrifugal blood contains many cytokines and growth factor. Stem cells is undifferentiated cell which has potential to differentiate into different cell types. In 1986, Schermer found corneal stem cells in the limbus and the reports of stem cells reconstruct on cornea was increased. In our studies, we found PRF that can help stem cells express migration, proliferation, differentiation, angiogenesis, collagen synthesis and even regeneration of new tissue.
In this study, cornea damage model were induced by immersion in NaOH. Cornea becomes nontransparent. The cornea’s epithelium and stroma were measured with microscope. Then, PRF was isolated from blood by cardiac puncture. The adipose-derived stem cells (ADSCs) was obtained from rabbit’s visceral fat. The damaged corneas were reconstructed with ADMSCs and PRF extract during 14 days treatment.

目錄
中文摘要……………………………………………………………………I
英文摘要……………………………………………………………………II
謝誌…………………………………………………………………………III
圖目錄………………………………………………………………………VI
表目錄……………………………………………………………………VII
第一章 前言…………………………………………………………………1
第二章 文獻回顧
(一) 幹細胞
1 分類………………………………………………………2
2 間質幹細胞………………………………………………3
3 脂肪幹細胞………………………………………………5
3.1 脂肪………………………………………………5
3.2 脂肪幹細胞取得…………………………………5
3.3 分化與治療………………………………………7
4 幹細胞治療………………………………………………9
4.1 脂肪幹細胞的治療………………………………9
4.2 異體幹細胞移植…………………………………9
4.3 外國狀況…………………………………………10
4.4 國內狀況…………………………………………11
(二) 角膜
1 結構………………………………………………………11
2 疾病………………………………………………………13
3 角膜移植…………………………………………………13
4 角膜幹細胞………………………………………………15
4.1 角膜幹細胞損傷…………………………………17
4.2 幹細胞治療………………………………………17
5 兔子與角膜研究…………………………………………18
(三) 富血小板纖維蛋白
1 成分與功能………………………………………………19
2 富血小板血漿、富血小板生長因子……………………19
3 優缺點……………………………………………………20
第三章 研究目的…………………………………………………………21
第四章 材料與方法
1 富血小板纖維蛋白萃取液製作………………………………24
2 脂肪幹細胞取得………………………………………………26
3 角膜損傷模型…………………………………………………28
4 治療……………………………………………………………29
5 標本切片觀察…………………………………………………29
6 實驗耗材、實驗器材、藥品來源……………………………31
第五章 結果
1 正常與受傷角膜………………………………………………32
2 PRF治療………………………………………………………34
3 ADSC注射治療………………………………………………37
4 PRF與ADSC注射混合治療…………………………………39
5 統計……………………………………………………………41
第六章 討論
1 PRF……………………………………………………………44
2 幹細胞注射……………………………………………………44
3 幹細胞與PRF …………………………………………………45
第七章 結論………………………………………………………………46
第八章 參考文獻…………………………………………………………47
附圖…………………………………………………………………………54
作者簡介……………………………………………………………………59
圖目錄
圖1. 內皮幹細胞的生成假說……………………………………………6
圖2. 脂肪幹細胞的分化能力與分化相關因子示意圖…………………7
圖3. 人類角膜結構……………………………………………………12
圖4. 角膜疾病…………………………………………………………14
圖5. 角膜上皮層生長模型……………………………………………16
圖6. 血離心後分層示意圖……………………………………………25
圖7. 細胞外型…………………………………………………………27
圖8. 脂肪幹細胞取得與………………………………………………27
圖9. 兔子麻醉…………………………………………………………28
圖10. 角膜………………………………………………………………30
圖11. 正常角膜與切片…………………………………………………32
圖12. 強鹼破壞後角膜與切片…………………………………………33
圖13. PRF治療外觀照…………………………………………………34
圖14. PRF治療後角膜切片………………………………………………36
圖15. ADSC注射外觀照…………………………………………………37
圖16. ADSC治療的角膜切片……………………………………………38
圖17. 混合治療:ADSC注射加上PRF滴眼外觀照……………………39
圖18. 混合治療:ADSC注射加上PRF滴眼角膜切片…………………40
附圖1. 連續14天的角膜外觀:PRF治療組3組…………………………54
附圖2. 連續14天的角膜外觀:ADSC治療組3組………………………55
附圖3. 連續14天的角膜外觀:ADSC + PRF治療組3組………………56
附圖4. 角膜厚度統計: 正常………………………………………………57
附圖5. 角膜厚度統計: 破壞………………………………………………57
附圖6. 角膜厚度統計: PRF………………………………………………57
附圖7. 角膜厚度統計: ADSC……………………………………………58
附圖8. 角膜厚度統計: ADSC + PRF……………………………………58
表目錄
表1. 間質幹細胞來源與分化可能組織…………………………………4
表2. 脂肪幹細胞的表現與不表現特定抗原……………………………8
表3. 富血小板纖維蛋白、富血小板血漿與富血小板生長因子比較表20
表4. 正常與破壞24hr後的切片厚度………………………………….41
表5. 破壞24hr後角膜總厚度變化……………………………………41
表6. 14天療程的切片厚度統計………………………………………42
表7. 14天治療後角膜總厚度變化……………………………………43
表8. 角膜表面厚度變化………………………………………………43


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