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研究生:翁仕明
研究生(外文):Shih-ming Weng
論文名稱:人類羊膜組織中的幹原/前驅細胞之分離與鑑定
論文名稱(外文):Isolation and Characterization of Stem/ Progenitor Cells from Human Amnion
指導教授:施子弼
指導教授(外文):Daniel T-B. Shih
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:細胞及分子生物研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:60
中文關鍵詞:羊膜人類間葉幹細胞(前驅細胞)神經分化
外文關鍵詞:amnionhuman mesenchymal stem/ progenitor cellmesenchymal & neural differentiation
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幹細胞擁有自我更新的特質及分化的能力。傳統上,幹細胞可以簡單區分成三類,即胚胎幹細胞、胎兒幹細胞以及體幹細胞。體幹細胞近年為一熱門研究題材,其可自不同人類成體組織分離而來。根據目前文獻報告,人類體幹細胞可從骨髓、腦組織、視網膜、頭皮、脂肪組織等分離而來。羊膜,乃是形成羊膜腔最主要的結構,將羊水及胎兒保護在內,提供新生命良好的成長環境。羊膜組織在懷孕前期由外胚層發育而來,在近來許多研究指出,羊膜層不但具有物理性的保護作用,羊膜組織內的細胞更能分泌許多生長因子及化學物質,對於胎兒的成長發育,應該亦有一定的影響力。同時,其他與胎兒發育相關的組織,如臍帶、羊水、胎盤等,經過許多科學家的努力,目前均能成功地分離出體幹細胞。因此,我們合理的假設,在羊膜層中亦有體幹細胞或前驅細胞的存在,由於羊膜組織容易在產後取得,於是,在本篇論文中,我們使用人類羊膜組織做為分離體幹細胞或前驅細胞的來源。羊膜組織於顯微鏡下,可以明顯的區分為表皮層、基底膜層以及基質層。羊膜上皮細胞可由表皮層分離而來,而羊膜間葉細胞則主要存在於基質層中。首先,在本實驗中,我們藉由調整不同酵素的作用時間,將兩種不同的細胞分離並於體外培養、增生。接下來,使用serial dilution的技術,建立由單一細胞增生而成的細胞株。我們在實驗的過程中,成功地建立了兩株細胞株,分別為8F3以及2B8。在完成細胞株的建立之後,我們進一步的分析其分化能力,以8F3細胞株為例,我們在分化實驗中發現其有分化成軟骨、脂肪以及類似神經細胞等組織之潛力。同時,分析細胞表面抗原所得之結果,發現有CD29、CDw90、CD105、SH2、SH4等表面抗原陽性反應,與其他人類成體組織所分離出來的間葉幹細胞表面抗原,相當一致。接著,由反轉錄聚合酶連鎖反應之分析,發現其所表現的基因,不但與來自其他人類成體組織之間葉幹細胞近似,同時可以發現其亦具有早期細胞的特性(由Oct-4及Rex-1的表現可得知)。由以上的結果可知,我們已成功地從人類羊膜組織中分離出具有間葉幹細胞特性的細胞株。另外,我們亦發現在HLA家族抗原分析中,此細胞株呈現HLA-DR陰性反應的結果。由於HLA家族抗原與組織移植後的排斥反應息息相關,也許在不久的將來,我們可以運用羊膜這種較易取得的組織中所分離的體幹細胞或前驅細胞,於再生醫療的領域中扮演舉足輕重的角色。
Stem cells exhibit the characteristic of self-renew and differentiation capacities. They are simply divided into three groups, embryonic, fetal and somatic stem cells. Somatic stem cells derived from various human adult tissues are deeply investigated in recent years. Many human somatic stem cells have been isolated from tissues such as bone marrow, brain, retina, scalp, fat tissue, etc. In this study, we described the isolation and characterization of stem/ progenitor cells from human amnion, which protects the fetus from mechanical force and also secrete some important factors to maintain the environment for fetal development. Due to the existence of two types of cells in the amnion tissue, we firstly modified the cell isolation methods to increase the purity of each type cell. Subsequently, we performed serial dilution for isolation of single cell clone. Then we examined the differentiation potentials after this clone was isolated. By functional assay, we concluded the isolated human amniotic mesenchymal stem/ progenitor cells can be differentiated into adipocyte, cartilage and neural cell lineages. With RT-PCR and cell surface marker analyses, we found these cells were similar to other MSCs. So, we have successfully isolated mesenchymal stem cells from human amnion. We found these cells were HLA-DR negative that they may have potential for clinical application. Since human amnion can be obtained during delivery, these cells may be useful for future therapies.
目次.........................第i頁
表目錄....................... 第iii頁
圖目錄....................... 第iv頁
Abbreviation List................... 第v頁
中文摘要.......................第1頁
Abstract....................... 第3頁

1. Introduction....................第4頁

2. Material and Methods
A. Cell culture...................第8頁
B. Flow cytometry analysis..............第9頁
C. Induction of osteogenic, adipogenic, and chondrogenic differentiation.................... 第10頁
D. Neural differentiation.............. 第11頁
E. RT-PCR analysis................第12頁
F. Histochemical staining..............第18頁

3. Results
A. Isolation condition optimization..........第19頁
B. Morphology of Amnion Cells...........第20頁
C. Single cell clone derived from HAM........ 第21頁
D. HAM derived single cell clone characterization (8F3)........................第22頁
E. Adipogenic, osteogenic, and chondrogenic differentiation potential of HAM derived single cell clone (8F3)...... 第24頁
F. Neural differentiation potential of 8F3........第25頁
4. Discussion.....................第27頁
5. Conclusion.................... 第34頁
Reference......................第50頁
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