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研究生:高安佩
研究生(外文):An-Pei Kao
論文名稱:人類子宮內膜與異位內膜間葉幹細胞的分離與鑑定並探討幹細胞於內膜異位症之角色
論文名稱(外文):Isolation and characterization of human mesenchymal stem cells derived from eutopic and ectopic endometrium to elucidate the role of endometrial stem cells in endometriosis
指導教授:蔡英美蔡英美引用關係
指導教授(外文):Eing-Mei Tsai
學位類別:博士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
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:2011
畢業學年度:100
語文別:中文
論文頁數:153
中文關鍵詞:子宮內膜異位症間葉幹細胞白細胞介素-1β細胞侵犯作用動物模式
外文關鍵詞:endometriosismesenchymal stem cellsinterleukin-1βcell invasionanimal model
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「內膜異位症」對育齡婦女造成日常生活的困擾,主要症狀包括慢性骨盆腔疼痛、生理痛,性交疼痛甚致不孕。雖然已經陸續有子宮幹細胞的理論被提出,但幹細胞在子宮內膜異位症的致病機轉仍不甚清楚。為了探討幹細胞與子宮內膜異位症的關係,本研究由相同的內膜異位症患者分離、鑑定與比較子宮正位和異位內膜間葉幹細胞(endometrial mesenchymal stem cells, EN-MSCs);此研究平台可屏除個體差異造成的誤差,進而釐清子宮異位內膜間葉幹細胞在內膜異位症的致病機轉。研究結果顯示,雖然子宮正位和異位內膜間葉幹細胞均表現出相似的幹細胞特性,包括:表現細胞自我更新能力、多潛能幹細胞Oct-4和間葉幹細胞生物標誌及細胞分化能力,但子宮異位內膜間葉幹細胞卻展現高度的細胞遷移和侵襲作用;進一步,於裸鼠體內(in vivo)建立細胞侵襲的動物模式,結果除了證實子宮異位內膜間葉幹細胞具有侵襲周遭組織的能力,還可促進血管新生。比較兩者基因表現,可知子宮異位內膜間葉幹細胞的白細胞介素-1β(IL-1β)顯著高於子宮正位內膜間葉幹細胞的基因表現,且IL-1β可促進環氧酵素-2(COX-2)表現進而增強子宮異位內膜間葉幹細胞的細胞遷移和侵襲作用;為了直接觀察IL-1β刺激子宮異位內膜間葉幹細胞的作用,在體外建立一擬體內(ex vivo)的立體培養模式,由此實驗可證實子宮異位內膜間葉幹細胞在IL-1β刺激下,細胞會形成觸手狀結構延展,展現侵襲周遭環境的能力。本研究結果證實子宮內膜異位症屬於一種幹細胞相關疾病,也提供了一新的內膜異位症治療方向。

Endometriosis affects women during the reproductive years and may cause chronic pelvic pain, dysmenorrhea, dyspareunia and infertility. The role of endometrial stem/progenitor cells in endometriosis has been proposed, although the nature of these stem cells has not been well characterized. To obtain relevant information to this issue, this study attempted to isolate and characterize human eutopic and ectopic endometrial mesenchymal stem cells (EN-MSCs) from the same patient and explore by cell function and the genome-wide mRNA expression analysis. This provides an opportunity for comparative study of these two types of EN-MSCs with homogeneous genetic background. The results showed that both eutopic and ectopic EN-MSCs which showed high proliferation ability expressed the pluripotent stem cell and the mesenchymal stem cell surface markers and are capable of differentiation and transdifferentiation. Although both showed many similar stem cell phenotypes, but ectopic EN-MSCs showed distinctly greater ability of cell migration and invasion ability in vitro. Furthermore, in an in vivo cell invasion model using cells grown in scaffolds and transplantation in immune-deficient mice, the ectopic EN-MSCs were found to form many new blood vessels, and to invade surrounding tissue. These ectopic EN-MSCs were significantly higher than eutopic EN-MSCs in interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) in gene expression and expression of COX-2 in eutopic and ectopic EN-MSCs is distinctly regulated by IL-1β; IL-1β has more effect on ectopic EN-MSCs. The abilities of cell migration and invasion were significantly enhanced by IL-1β in ectopic EN-MSCs. Using an ex vivo invasion model, the cell mass of IL-1β-treated ectopic EN-MSCs was found to form many tentacle-like structures that extended and invaded surrounding area. These results shed light on a pathogenesis mechanism of endometriosis as a stem cell disease and identified potential new therapeutic targets for this multifaceted disease.

<目錄>
摘要
中文摘要 ………………………………………………………… 1
英文摘要 ………………………………………………………… 2

縮寫表 ………………………………………………………… 3

前言 ………………………………………………………… 4

材料與方法
A. 細胞培養
1.人類子宮內膜細胞的分離與培養………………………… 20
2.人類子宮內膜間質幹細胞的分離、培養與純化…………… 21
3.細胞培養與繼代……………………………………………… 22
4.細胞冷凍保存………………………………………………… 22
5.冷凍細胞之活化……………………………………………… 23
6.細胞計數 …………………………………………………… 23
B. 幹細胞分化
1.累計細胞複製次數…………………………………………… 25
2.細胞群落試驗………………………………………………… 26
3.細胞間隙交流………………………………………………… 26
4.間質幹細胞之誘導分化 ……………………………………… 27
C. RNA備製、純化與分析
1.總量RNA抽取……………………………………………… 28
2.定量RNA…………………………………………………… 29
3.cDNA的合成………………………………………………… 29
4.聚合酵素連鎖反應…………………………………………… 30
5.DNA洋菜膠體電泳法………………………………………… 31
6.定量即時聚合酶鏈鎖反應…………………………………… 32
D. 蛋白質備製、純化與分析
1.細胞免疫螢光染色…………………………………………… 32
2.流式細胞儀測定……… ……………………………………… 33
3.細胞總蛋白萃取……………………………………………… 33
4.蛋白質標準檢量線…… ……………………………………… 34
5.蛋白質定量…………………………………………………… 34
6.西方墨點法…………………………………………………… 35
E. 細胞遷移與侵襲
1.細胞遷移實驗………………………………………………… 37
2.細胞侵襲實驗………………………………………………… 38
F. 體外類器官培養方式
體外細胞侵襲實驗…………………………………………… 39
G. 動物實驗
體內細胞侵襲實驗…………………………………………… 40
H. 統計分析 41

結果
1. 人類子宮內膜與異位內膜間葉幹細胞的分離與鑑定
1-1 人類子宮內膜細胞的分離 …………………………………… 42
1-2 人類子宮內膜細胞的鑑定 …………………………………… 42
1-3 人類子宮內膜間葉幹細胞的分離…………………………… 43
1-4 人類子宮內膜間葉幹細胞的鑑定…………………………… 45
2. 人類子宮內膜與異位內膜間葉幹細胞的比較
2-1 人類子宮內膜與異位內膜間葉幹細胞有相似的細胞群落形成能 49
2-2 人類子宮異位內膜間葉幹細胞表現較強的細胞遷移與侵襲作用 50
2-3 動物體內研究證實人類子宮異位內膜間葉幹細胞的具細胞侵犯作用與促進血管新生能力 …………………………………………………… 51
2-4 人類子宮內膜與異位內膜間葉幹細胞的基因特性分析人類子宮內膜與異位內膜間葉幹細胞的基因特性分析 ……………………………… 53
3. 白細胞介素-1β對人類子宮異位內膜間葉幹細胞的影響
3-1 人類子宮異位內膜間葉幹細胞高度表現IL-1β與COX-2基因… 54
3-2 COX-2促進人類子宮異位內膜間葉幹細胞的細胞遷移與侵襲能力 55
3-3 IL-1β調節人類子宮異位內膜間葉幹細胞的COX-2表現………… 56
3-4體外類器官培養方式(ex-vivo organoid culture)觀察子宮異位內膜間葉幹細胞的細胞侵犯作用………………………………… 57

討論與結論………………………………………………………… 59

參考文獻 ………………………………………………………… 64

表 ………………………………………………………… 90

圖 ………………………………………………………… 93

附錄
附錄 一、發表文章 ………………………………………………… 106
Kao AP, Wang KH, Chang CC, Lee JN, Long CY, Chen HS, Tsai CF, Hsieh TH, Tsai EM. Comparative study of human eutopic and ectopic endometrial mesenchymal stem cells and the development of an in vivo endometriotic invasion model. Fertil Steril. 2011 Mar 15;95(4):1308-15.e1. Epub 2010 Nov 3.

附錄 二、發表文章 ………………………………………………… 116
Kao AP, Wang KH, Long CY, Chai CY, Tsai CF, Hsieh TH, Hsu CY, Chang CC, Lee JN, Tsai EM. Interleukin-1β induces cyclooxygenase-2 expression and promotes the invasive ability of human mesenchymal stem cells derived from ovarian endometrioma. Fertil Steril. 2011 Sep;96(3):678-684.e1. Epub 2011 Jul 16.



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