臺灣博碩士論文加值系統

有絲分裂的過程中，以微管（microtubule）為基礎的大分子紡錘體扮演首當其衝要感謝的辨識黃筱鈞老師將同源染色體精準地分離至兩個子細胞中的重要角色。有絲分裂紡錘體的結構型態及方向調控深受位於細胞皮層及紡錘體赤道板的分子影響。我們的研究將探討有絲分裂紡錘體於對稱與不對稱細胞分裂中的調控。我們選擇數株分離自人類肺癌、乳癌及大腸癌細胞株作為對稱細胞分裂之研究模型。透過免疫螢光染色法（immunofluorescence），我們發現人工篩選出高侵略能力的肺腺癌子細胞株CL1-5具有較長的穩定細胞分裂中期紡錘體 （steady-state metaphase spindle），其型態與其原位癌細胞株CL1-0有顯著差異。此外，我們近期的研究發現，一參與細胞分裂時紡錘極體（spindle pole）及染色體分離的細胞骨架調控子驅動蛋白5 （kinesin-5）在CL1-5細胞中的表現量顯著上升。我們透過對CL1-5細胞加入低濃度驅動蛋白5抑制劑，進一步觀察驅動蛋白5與紡錘體長度縮放之間的關係。實驗結果顯示CL1-5細胞的紡錘體長度隨著加入驅動蛋白5抑制劑的濃度增加而縮短，並解具有劑量依存性。同時，我們也發現在體內轉移的大腸癌細胞株SW620與其原位癌細胞株SW480相較之下，具有較長的穩定細胞分裂中期紡錘體。這些研究結果顯示紡錘體長度縮放具有成為高轉移性癌症細胞標記的潛力，同時突顯了驅動蛋白5抑制劑在癌症療法中的附加優勢。另一方面，我們利用顯微縮時影像分析小鼠胚胎幹細胞不對稱分裂中紡錘體的方向調控。我們使用磁珠提供局部性Wnt3a訊號，建立促使細胞不對稱分裂的微環境，目前已成功建立活細胞影像平台並進一步驗證局部性的Wnt3a訊號調控細胞分裂方向。在哺乳動物不對稱細胞分裂中，位於細胞皮層調控紡錘體方向的分子目前仍在研究中。

During cell division, a microtubule-based macromolecular machine known as mitotic spindle plays an important role to segregate chromosomes to two daughter cells. Molecules locating at cell cortex and spindle equator regulate spindle architecture and orientation. In our study, mitotic spindles in symmetric and asymmetric cell division were investigated. Several human cancer cell lines from lung, breast and colon were chosen as models of symmetric division. By immunofluorescence, we found that the functionally selected highly invasive lung adenocarcinoma cell line, CL1-5, has lengthened steady-state metaphase spindle morphologically distinct from the original line, CL1-0. In addition, our recent study showed that in CL1-5, there was an up-regulation of kinesin-5, a motor protein involved in pole separation and chromosome segregation during mitosis. Therefore, we applied low dose kinesin-5 inhibitor to CL1-5 to reveal the relationship between kinesin-5 and spindle length scaling. As a result, dose-dependent change of spindle length was found. Similarly, an in vivo selected metastatic colon cancer cell line SW620 presented lengthened steady-state metaphase spindle compared to its in situ cell line SW480. These findings entitle spindle length scaling a candidate of cellular marker for metastatic cancer clone, and emphasize alternative benefits of kinesin-5 inhibitor in anti-cancer therapies. On the other hand, we analyzed spindle orientation in asymmetric cell division (ACD) by time-lapse imaging with mouse embryonic stem cells as our model. Microenvironment that promoted asymmetric cell division was built by localized Wnt3a beads. We have established live imaging platform and validated localized Wnt3a signals did orient cell division. We are currently investigating molecules locating at the cell cortex that mediate spindle orientation in asymmetric cell division.

誌謝 i
中文摘要 iii
Abstract v
List of figures ix
Chapter 1. Introduction 1
1.1 Mitosis and mitotic spindle 1
1.2 Cancer evolution 2
1.3 Spindle length scaling 3
1.4 The role of Kinesin-5 in mitosis 4
1.5 Symmetric cell division and Asymmetric cell division 5
1.6 Spindle orientation 6
1.7 The canonical Wnt signaling pathway 7
1.8 The thesis organization 8
Chapter 2. Materials and methods 10
2.1 Cell Lines, Drugs, and Synchronization 10
2.2 Immunofluorescence 11
2.3 Immobilized Wnt3a onto beads 11
2.4 Time-lapse Imaging 12
2.5 Plasmids and transfection 12
2.6 Mouse intestinal stem cells isolation, sorting and culture 13
Chapter. 3 Results and discussion 15
3.1 CL series cells presented a miniature of cancer evolution 15
3.2 CL1-5 evolved lengthened spindle in relatively small cell size. 15
3.3 The spindle of CL1-5 morphologically distinct from CL1-0. 16
3.4 Kinesin-5 inhibition decreased spindle aspect ratio in a dose-dependent manner in CL1-5. 17
3.5 The aspect ratio of in vivo metastatic cancer cell line was significantly higher than that of cell line from tumor in situ. 18
3.6 Wnt3a beads regulate cell division direction in mouse embryonic stem cells. 18
Chapter 4. Conclusion and future works 20
4.1 Spindle scaling in symmetric cell division during cancer evolution 20
4.2 Spindle orientation in asymmetric cell division 21
4.2.1 A live imaging platform was established for investigating asymmetric cell division 21
4.2.2 Ongoing work for investigating cell cortex-located molecules 22
4.2.3 Ongoing work for investigating asymmetric cell division in mouse intestinal stem cells. 22
Chapter 5. References 24

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