- 學位論文
探討在癌細胞中演化幹性與不對稱細胞分裂
Evolution of Stemness and Asymmetric Cell Division in Cancer
摘要
生命演化的歷史中，幹性（stemness）的出現是最顯著最突破的轉淚點。而演化的探討，除了利用演化譜系進行基因體學或基因篩選的方法外，許多科學家也嘗試利用實驗演化學（experimental evolution）進行研究。此外，實驗演化學上，科學家也成功地將單細胞酵母菌演化出多細胞狀態與簡單的細胞分工。根據這兩個理由，加上癌細胞基因體不穩定的特性，我們想見也能在一定可行時間內，在實驗室中利用癌細胞，將其演化出具有幹性的狀態。我們選擇人類大腸癌細胞株HCT116作為演化之研究模型，並透過球形成試驗（sphere-forming assay）作為篩選壓力。由球形成試驗中，得到具有較高幹性的細胞，我們稱之為來自球體的癌幹細胞（SDCSCs），並將其養回一般培養環境中，而這樣的細胞稱為來自球體的貼盤癌細胞（SDACs）。首先，我們發現幹細胞標記Nanog, Oct4 和Lgr5基因，在第一代來自球體的癌幹細胞中，表現量有顯著上升的趨勢。相反的，在來自球體的貼盤癌細胞中，三個幹細胞基因表現量會隨著培養時間而逐漸下降。此外，每代篩選中，幹細胞標記Nanog、Oct4、Lgr5基因表現量在來自球體的癌幹細胞，均有顯著上升。由此可證明，我們系統的可行性。經過八次篩選後，我們發現相較於前期的癌細胞株，具有較高形成球態的能力。我們目前正在利用轉錄體定序（RNA-Seq）的方法，全面性探測我們演化出的細胞株中，幹性基因表達的差異。另一方面，我們利用顯微縮時影像分析，建立小鼠胚胎幹細胞不對稱分裂（asymmetric cell division）中紡錘體方向調控（spindle orientation）的平台。在細胞外，使用磁珠給予細胞局部性Wnt3a訊號，建立促使細胞進行不對稱分裂的微環境。我們證實局部性的Wnt3a訊號可調控細胞分裂方向。此外，在我們平台中，動力蛋白（dynein）抑制劑（Ciliobrevin D）會降低Wnt3a訊號調控細胞分裂的能力。由此可知，Wnt3a訊號可能透過動力蛋白來調控紡錘體方向，進而促使細胞進行不對稱分裂。
並列摘要
In the history of life, the appearance of stemness is one of the most remarkable and transformative advances. Experimental evolution approaches have been used to successfully evolve multicellularity in the unicellular budding yeast, Saccharomyces cerevisiae. We reason, given the genome instability of cancer, stemness can also arise from in vitro cancer evolution in the laboratory within a reasonable timescale. We approached the question with a colorectal cancer cell line, HCT116, and used sphere forming as the selective pressure. Sphere-derived cancer stem cells (SDCSCs) were repeatedly isolated and then re-plated onto a regular dish as adherent cells (sphere-derived adherent cells, or SDACs). We first confirmed that the level of canonical stemness markers, Nanog, Oct4, and Lgr5, were elevated in first-generation SDCSCs and were gradually down to the original level in SDACs from day 0 to day 14. Nanog, Oct4, and Lgr5 were up-regulated in SDCSCs in every generation, further confirming the reliability of our assay. We found that sphere-forming capability is higher after 8 generations of selection. We are currently using RNA-seq to probe potential global difference in the expression of stemness genes in our evolved cancer clone. In addition, we established a platform to analyze spindle orientation in cell division with mouse embryonic stem cells. We built a microenvironment that promoted asymmetric cell division with localized Wnt3a beads and validated that localized Wnt3a signals can indeed orient cell division. Furthermore, dynein inhibitor, Ciliobrevin D, decreased Wnt3a’s ability to orient spindles on our platform, suggesting that dynein may participate in the Wnt3a-mediated pathway in asymmetric cell division.