近年研究指出在癌症細胞中具有一群稀有細胞會啟動並促使腫瘤 (tumor) 生長，並誘導腫瘤遠端轉移，且這些幹細胞具有抵抗化療與放射治療的特性，這一小群細胞稱之為「癌症幹細胞（亦稱腫瘤起始細胞）」 (Cancer Stem Cells) 。有些研究指出上皮-間質轉化 (epithelial-mesenchymal transition, EMT) 過程在誘導細胞侵入 (cell invasion) 及細胞轉移 (cell metastasis) 上扮演極關鍵的角色，更可進一步誘導癌細胞獲得幹細胞之特性。細胞內微環境又稱為利基 (niche) ，具有調控癌細胞存活、自我更新 (self-renewal) 及細胞分化 (differentiation) 的作用，且被證實對癌症的進展有重大的影響。近期研究更指出，在腫瘤形成時，骨髓間質幹細胞 (bone marrow-derived mesenchymal stem cells，BM-MSCs) 會被徵募 (recruit) 到產生癌症腫瘤的組織周圍，因此被認為是重要的微環境因素之一，且會透過多條路徑 (pathways) 來調控癌症的轉移，其中包括 IL6/STAT3 (signal transducer and activator of transcription 3, STAT3) 這條訊息傳遞路徑。然而由骨髓間質幹細胞 (Mesenchymal Stem Cells, MSCs) 所製造出的利基，如何誘導肺腺癌上皮細胞 (LM cells) 轉型為癌症幹細胞之過程，所扮演的角色仍然不清楚。
在我們的研究中，骨髓 (human bone marrows , hBMs) 來源為進行關節置換患者所提供，並使用Ficoll (d=1.077) 密度梯度離心法分離出骨髓間質幹細胞。隨後利用流式細胞儀 (flow cytometry) 檢測我們所分離出的骨髓間質幹細胞。接著，收集經由 BM-MSCs所培養的條件培養液 (MSC-conditional medium, MSC-CM) ，去探討是否 MSC-CM 會幫助建構出適合 CSC 生長的利基，並調控 CSC。我們的研究利用 RT-PCR或是 Western blotting 證明 BM-MSCs 所分泌的旁泌因子 (paracrine factors) 不管在中皆會促使低移動性肺腺癌細胞 (LM cell) 的間質標誌 (mesenchymal markers：vimentin及 Twist1) 表現量顯著上升。在抗藥性相關基因，ABC 運輸蛋白家族 (ATP-binding cassette transporters , ABC transporter family) 其表現量也顯著的上升 (包括：ABCA1、ABCB1、及 ABCG1)。而在functional assays中證實，肺癌上皮細胞在加入 MSC-CM 後，其爬行能力及腫瘤球體形成能力明顯的提升。綜合以上結果，經過 MSC-CM 培養後，會促進肺癌上皮細胞形成 CSC。此外，根據 RNA微陣列 (microarray)，我們發現到 JAK/STAT3 的標的基因再加入 MSC-CM 都有顯著的上升，藉此我們推論由間質幹細胞所分泌的旁泌因子，會透過 STAT3 這條訊息傳遞路徑建構 CSC 的利基並促進腫瘤發展。在我們研究中，確實發現， MSC-CM 會促使 STAT3 的 Tyr-705 位點磷酸化而活化 STAT3 訊息傳遞路徑。有趣的是，當我們使細胞表現點突變的 STAT3 (mutant STAT3, Y705F) ，可以觀察到由 MSC-CM所誘導的 EMT 現象及獲得 CSC 之性狀即會消失。綜合以上結論，本篇論文證實：骨髓間質幹細胞會透過活化 STAT3 signaling pathway，誘導 LM cells獲得癌症幹細胞性狀。根據此發現，在未來渴望可以找到直接標靶 CSC 的新治療方法及藥物，例如：抗體及蛋白酶抑制劑，藉此突破過去進行治療後仍會復發的現象，渴望成為新一代新穎的治療方式。

Recent evidences indicate that a small subpopulation of cancer cells called cancer stem cells (CSCs) is possible to initiate tumor growth, drive tumor metastasis and lead to drug resistance. In addition, some reports suggest that EMT (epitheli-al-mesenchymal transition), playing an important role in the cancer invasion and me-tastasis, can induce cancer cell to acquire stem-like phenotype. An innovative concept is that the niche has a profound influence on the cancer progression. The niche is a specific microenvironment that regulates cancer cell survival, self-renewal, and dif-ferentiation. In particular, studies have shown that bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited into tumors and considered regulating cancer metastasis by several signal pathways, including IL6/STAT3 signal. However, the ex-act network of CSC niche created by BM-MSCs is poorly understood.
In this study, human bone marrows (hBMs) were obtained from volunteers, without the history of cancer, undergoing joint arthroplasty surgeries. After isolating hBM-MSCs , we identified the characteristics of isolated BM-MSCs by flow cytome-try. Then, we collected MSC-conditional medium (MSC-CM) to investigate if MSCs help the establishment of CSC niche and promote the CSC properties. The RT-PCR and Western blotting showed that paracrine factors in MSC-CM secreted by BM-MSCs would upregulate mesenchymal markers in epithelial-type cells (LM cells). We also found the ABC transporter genes, related to the ability of drug resistance, were distinctly upregulated (including ABCA1, ABCB1, and ABCG1) after MSC-CM treatment. In the functional assays, we suggested MSC-CM treatment ob-viously promoted migration abilities and sphere forming abilities in LM cells. Taken together, we demonstrate that MSC-CM treatment can elicit the CSC phenotype. Furthermore, according to the microarray data, the target genes of JAK/STAT3 signal pathway were upregulated in LM cells treated with MSC-CM. Therefore, we hypoth-esized paracrine factors secreted by MSCs might establish the CSC niche to promote cancer progression via STAT3 signal pathway. Here, we illustrated that MSC-CM treatment predominantly activated STAT3 transcriptional activity through enhancing phosphorylation of STAT3 (Tyr705). Surprisingly, the EMT and CSC properties in-duced by MSC-CM would be eliminated, after the transfection of mutant STAT3 (Y705F) in LM cells. In conclusion, BM-MSCs can trigger STAT3-elicited promotion of the CSC phenotype. Based on these findings, we look forward to developing new strategies and drugs, such as antibodies and pharmacologic inhibitors, directly target-ing CSCs to improve tumor therapies.

I. 緒論 8
一、 癌症幹細胞 (Cancer stem cells) 簡介 8
1. 前言 8
2. 癌症起源 8
3. 癌症幹細胞的特性 9
4. 癌症幹細胞的篩選 9
二、 上皮-間質轉化 (Epithelial-mesenchymal transition, EMT) 和癌症及癌症幹細胞的關係 10
三、 間質幹細胞 (Bone marrow-derived mesenchymal stem cells, BM-MSCs) 11
1. 前言 11
2. 間質幹細胞與癌症的關係 11
II. 研究動機 13
III. 材料與方法 14
一、 細胞培養 (cell culture) 14
1. 骨髓間質幹細胞萃取 (isolation) 14
2. 細胞繼代培養 14
3. 凍細胞 15
4. 細胞轉染 (cell transfection) 15
二、 流式細胞儀分析間質幹細胞表面抗原 (surface marker) 16
1. 間質幹細胞之抗體 16
2. 流式細胞儀前置步驟 17
三、 骨髓間幹細胞條件培養液 (MSC-conditional medium, MSC-CM) 收集 17
四、 西方墨點法 (Western blotting) 17
1. 萃取細胞總蛋白質 (total cell lysate) 17
2. 蛋白質濃度定量 18
3. 西方墨點法 (Western blotting) 18
反轉錄聚合酶連鎖反應 (RT-PCR) 19
五、 19
1. 總核糖核酸萃取 (total RNA extraction) 19
2. RNA定量分析 (quantification of total mRNA) 19
3. mRNA反轉錄 (reverse transcription) 及聚合酶鏈鎖反應 19
六、 螢火蟲冷光活性方法 (luciferase reporter assay) 20
七、 傷口癒合試驗 (wound healing assay) 20
腫瘤球體形成篩選 (sphere forming assays) 21
八、 21
IV. 實驗結果 22
一、 人體萃取之骨髓間質幹細胞的鑑定 22
二、 經骨髓間質幹細胞條件培養液 (MSC-conditional medium, MSC-CM) 培養之肺腺癌上皮細胞 (LM cells) 其RNA微陣列數據分析 23
三、 骨髓間質幹細胞於條件培養液中所分泌之旁泌因子 (paracrine factors) 促使低移動性肺癌上皮細胞 (LM cells) 進行上皮-間質轉化 (Epithelial-mesenchymal transition，EMT) 且增加其移動能力 (migration ability) 24
四、 骨髓間質幹細胞於條件培養液中所分泌之旁泌因子 (paracrine factors) 促使肺腺癌上皮細胞 (LM cells) 獲得癌症幹細胞之性狀 (cancer stem cells phenotype) 25
五、 透過基因調控網路之建構，鎖定JAK/STAT3訊息傳遞路徑 (signal transduction pathway)，可能於骨髓間質幹細胞所誘導之癌症幹細胞形成過程中扮演關鍵角色 26
六、 骨髓間質幹細胞透過活化 STAT3 此訊息傳遞路徑，使肺癌上皮細胞 (LM cells) 進行上皮-間質轉化 (Epithelial-mesenchymal transition, EMT) 且增加其移動能力 (migration ability) 27
七、 骨髓間質幹細胞透過活化 STAT3 訊息傳遞路徑，誘導肺癌上皮細胞 (LM cells) 獲得癌症幹細胞之性狀 (CSCs phenotype) 29
V. 實驗討論 31
VI. 文獻參考 36
VII. 圖表與圖表說明 40

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