臺灣博碩士論文加值系統

幹細胞是應用於再生醫學、組織工程、細胞療法與基因療法的主要素材，最近的研究發現，人類間葉幹細胞已由不同的成體及胎兒組織中分離與鑑定，因此我們認為胎盤中可能存在較原始的間葉幹細胞。我們分離由胎盤絨毛組織取得的細胞，並鑑定其是否具有間葉幹細胞與胚胎幹細胞的特性。
由胎盤所取得的細胞呈現類似纖維母細胞之型態與可增生形成群落，表現ABCG2、CD9、CD29、CD44、CD73、CD90、 CD105、HLA-ABC，不表現HLA-DR、CD34、CD45細胞標誌，且經由適當的誘導分化為脂肪、成骨、軟骨細胞，顯示此細胞仍保有分化之潛能。此間葉幹細胞會表現胚胎幹細胞的細胞標誌Oct-4、Sox-2、SSEA-3、TRA-1-60、TRA-1-81但不表現SSEA-4，以RT-PC及real-time PCR分析也會表現HOX-b4、Bmi-1、Oct-4、Rex-1幹細胞相關的基因。
在分離、培養台盤取得的間葉幹細胞之後，進行較原始的胎盤間葉幹細胞之鑑定，利用流式細胞儀由胎盤取得之細胞中分選出CD90+/CD44+細胞，約2~3%的細胞有CD90和CD44表現，且此群細胞也會表現間葉幹細胞的標誌，而表現Oct-4、Sox-2、TRA-1-60、TRA-1-81但不表現SSEA-3、SSEA-4。CD90+/CD44+細胞分選後不能培養，而能增生形成群落的細胞都能同時表現CD90和CD44，所以將能增生形成群落的細胞當作CD90+/CD44+細胞來進行更進一步的分析。單一群落細胞具有間葉幹細胞的特性，並且保有能分化脂肪細胞、成骨細胞、軟骨細胞之潛能。所以我們認為CD90+/CD44+細胞可能為較原始的胎盤間葉幹細胞。

Stem cells could be useful for regeneration medicine, tissue engineering, gene therapy and cell therapy. Human mesenchymal stem cells (MSCs) can be isolated and identified from various adult and fetal tissues. We suggest that primitive mesenchymal stem cells may exist in placental villa. In this study, we isolate and characterize cells from placental villa as regards their MSC properties and embryonic stem cell (ESC）properties.
The placenta-derived cells display fibroblast-like morphology and grow into colonies. They express ABCG2, CD9, CD29, CD44, CD73, CD90, CD105 and HLA-ABC but not, CD34, CD45 and HLA-DR. Under appropriate culture conditions, these cells are able to differentiate into cells of the adipogenic, osteogenic and chondrogenic lineages. The MSCs express ESC markers, such as Oct-4, Sox-2, SSEA-3, TRA-1-60 and TRA-1-81 but not SSEA-4. The PDMSCs also express stem cell-related gene such as HOX-b4, Bmi-1, Oct-4, and Rex-1 by RT-PCR and real-time PCR.
After isolation and cultivation of MSCs from placenta we tried to identify the primitive MSCs. The CD90+/CD44+ cells were isolated from fresh placental cells by fluorescence activated cell sorting. There was only about 2~3% population of cells were found to be positive for CD90 and CD44, and expressed MSC markers. The CD90+/CD44+ cells expressed Oct-4, Sox-2, TRA-1-60 and TRA-1-81, but did not express SSEA-3 and SSEA-4. The CD90+/CD44+ cells could not be expended after sorting . Colony-forming are found to be positive for CD90 and CD44. So we considered colony-forming cells as criteria of the CD90+/CD44+ cells. Colony-forming cells have MSC proprieties and multilineage potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages. The subpopulation of CD90+/CD44+ cells could be regarded as primitive MSCs from fresh placental cells.

中文摘要 I
Abstract III
目錄 V
圖表目錄 VIII
第一章、文獻回顧 1
1.1 幹細胞（Stem cells） 1
1.2 幹細胞的應用 4
1.3 體幹細胞的分化 4
1.4 幹細胞發展之現況 6
1.5 間葉幹細胞（Mesenchymal stem cells, MSCs） 7
1.6 間葉幹細胞鑑定分析 9
1.7 胎盤間葉幹細胞之細胞標誌 11
1.7.1 CD90 11
1.7.2 CD44 12
1.7.3 ABCG2 12
1.7.4 CD9 12
1.7.5 CD29 13
1.7.6 CD34 13
1.7.7 CD73 13
1.7.8 CD105 13
1.8 胚胎幹細胞之細胞標誌 14
1.8.1 Stage-specific embryonic antigens 14
1.8.2 Tumor rejection antigens 14
1.8.3 SOX2 14
1.9 幹細胞相關基因 14
1.9.1 Oct4 14
1.9.2 Rex1 15
1.9.3 Bmi-1 15
1.9.4 Hoxb4 15
1.10 胎盤（placenta） 17
1.11 研究動機 19
第二章、實驗材料 21
2.1 實驗儀器 21
2.2 實驗藥品 22
第三章、實驗方法 25
3.1 胎盤絨毛組織細胞分離 25
3.2 初代細胞培養及繼代 27
3.3 細胞繼代 27
3.4 流式細胞儀分析 28
3.5 Cell array分析 28
3.6 細胞免疫染色 30
3.7 細胞生長曲線分析 31
3.8 細胞分化 32
3.9 RNA分離純化 34
3.10 反轉錄聚合酶連鎖反應（RT-PCR） 34
3.11 及時定量聚合酶連鎖反應（real-time PCR） 36
第四章、實驗結果 38
4.1 細胞分離、培養及生長情形 38
4.2 間葉幹細胞之細胞標誌表達分析 39
4.3 胎盤幹細胞之細胞標誌表達分析 39
4.4 PDMSC之分化能力分析 40
4.5 PDMSC之增生能力分析 41
4.6 Colony-forming cells之繼代培養 41
4.7 Colony-forming cells之MSC細胞標誌鑑定分析 41
4.8 Colony-forming cells之增生能力分析 42
4.9 Colony-forming cells之分化能力分析 42
4.10 CD90+/CD44+細胞分選 43
4.11 CD90+/CD44+細胞之鑑定分析 44
4.12 幹細胞相關基因分析 44
第五章、討論 46
5.1 初始分離之胎盤細胞 46
5.2 細胞標誌之表達 47
5.3 PDMSC之分化潛能分析 47
5.4 幹細胞相關基因分析及ES細胞標誌分析 49
5.5 CD90+/CD44+細胞鑑定分析 50
第六章、圖 53
第七章、參考文獻 89

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