在球形圓洞中單顆人類間葉幹細胞的細胞週期、形態特徵與牽引力之量化研究

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姓名

吉潘尼(Giovanni Jariol Paylaga) 查詢紙本館藏

畢業系所

物理學系

論文名稱

在球形圓洞中單顆人類間葉幹細胞的細胞週期、形態特徵與牽引力之量化研究
(Quantitative investigations on cell-cycle, morphological features, and traction stresses of single human mesenchymal stem cells in spherical microwells)

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摘要(中)

我們對細胞生物學的理解大多來自於細胞培養在二維 (2D) 平面基質的研究，但實際上體內的微環境是三維 (3D) 的。已知的是，細胞會通過牽引力 (traction force) 來感知微環境的機械性質並且做出反應，這是機械生物學這一快速發展領域的研究重點。然而，細胞如何感知維度 (dimensionality) 仍然未知。前人的研究顯示，如細胞貼覆面積、硬度、拉伸壓力和細胞體積等許多參數與培養在2D基質上有密切的關聯，這些參數會通過影響轉錄啟動子yes-associated-protein (YAP) 的所在位置而調控細胞增殖或幹細胞分化。相反地，這個機制在3D環境下會改變，因此3D細胞培養成為瞭解機械生物學的理想測試平臺，並為3D細胞培養系統提供更深入的理解。雖然3D細胞培養是為再生醫學和藥物篩選更佳的體內模擬微環境，但其複雜性和多樣性也使得數據的系統化和量化相當困難。這使得簡化3D細胞培養系統但保持維度性質有其必要性。在本篇論文中，我證明球形圓洞是研究3D機械生物學的有力工具。我們基於球形圓洞開發了3D拉伸力顯微鏡技術，並發現人類間充質幹細胞 (hMSCs) 培養在直徑較小的圓洞中，會通過減少核內轉錄啟動子 YAP 而出現細胞週期停滯。我們進行量化以了解在2D調節YAP的機制是否在3D系統也會發生。我們的數據顯示，拉伸壓力不會通過細胞核周圍的肌動蛋白傳遞，且不會導致細胞核扁平化。此外，在直徑較大的圓洞中，細胞具有更大的細胞體積、更大的拉伸應力，並且細胞貼覆面積與其細胞核體積之間有正相關。細胞體積的量化趨勢與在2D環境的前人研究結果不相同 [1]。然而，細胞核扁平度與核內 YAP 遷移和拉伸壓力呈負相關，表示出類似的趨勢但不如 2D 的情況那麼強。細胞形狀指數(cell shape index, CSI)與核內YAP遷移呈反相關，並且在直徑較小的圓洞中，CSI與拉伸壓力呈正相關。此外，主成分分析 (principal component analysis, PCA) 顯示在每種圓洞大小都有兩種表型(拉伸型和貼覆型)，但會表現出不同的機械反應。我們進一步使用藥物blebbistatin 來降低拉伸壓力，並觀察到 hMSCs 展現出較小的突起並傾向於橫跨圓洞。Blebbistatin 主要影響在 100 微米圓洞內的 hMSCs，導致細胞體積、細胞核體積和 YAP 細胞核遷移降低，但在 60 微米圓洞內的 hMSCs 影響不大。綜上所述，我們的結果顯示出在機械生物領域中常研究特徵的新機制。球形圓洞可以控制基質曲率而影響細胞的生長和機械反應，並且可以輕鬆地與拉伸力顯微鏡配合使用，是今後3D機械生物學更多研究的良好模型系統。

摘要(英)

Most of our understanding on cell biology is based on two-dimensional (2D) flat substrates while an in vivo microenvironment is intrinsically three-dimensional (3D). How cells sense and respond to the dimensionality is still largely under-explored. Cells sense the microenvironment through traction force and a lot of work has been carried out on 2D. It has been shown that cell-spread area, rigidity, traction stress, and cell volume are strongly coupled on 2D, and these factors affect the cell proliferation or stem cell differentiation through affecting the translocation of transcriptional activator YAP. In contrast, some of the coupled relationships change in 3D and thus make a 3D cell culture an ideal testbed for the current understanding on 2D mechanobiology and provides deeper understanding for 3D cell culture. Though 3D cell culture is a better in vivo mimicking microenvironment for the regenerative medicine and drug screening, its complexity and diversity also make systematic and quantitative studies difficult. It is crucial to simplify 3D cell culture system but keep the essential features of dimensionality. In this thesis, I demonstrate that spherical microwells is a novel tool to study 3D mechanobiology. We developed 3D traction force microscopy based on spherical microwells and identified that human mesenchymal stem cells (hMSCs) exhibit cell-cycle arrest in smaller microwells through reducing transcriptional activator YAP in nucleus. We performed quantitative measurements to test whether the proposed mechanism on 2D regulates YAP translocation in 3D. We examined several quantities in 3D and found some relationships are consistent with the relationships on 2D and some are not. For example, the traction stress is not directly transmitted through perinuclear actin to induce nuclear flattening. We also found that cells in larger microwells have larger cell volume, greater traction stress, and their cell adhesion area has positive correlation with both their cellular and nuclear volume. The relationships between cell volume with traction stress and adhesion area do not agree with previous work on 2D [1]. Nuclear flatness is inversely correlated with both the nuclear YAP translocation and traction stress, indicating a similar trend but not as strong as the 2D counterparts. Their cell shape index (CSI) has anti-correlation with nuclear YAP translocation, and in smaller microwells, CSI has correlation with traction stress. Additionally, principal component analysis shows that two phenotypes (stretchers and stickers) demonstrate distinct mechanical responses inside the microwells despite sharing comparable morphological traits in each microwell size. We further performed blebbistatin to reduce traction stress and we observed that hMSCs show smaller protrusions and they tend to straddle more across the microwell. Blebbistatin treatment affects mostly on hMSCs in 100-µm microwells resulting in reduced cell volume, nuclear volume, and YAP nuclear localization but not so much on hMSCs in 60-µm microwells. Taken together, our investigations show new relationships between quantities commonly investigated in mechanobiology. Spherical microwells provide control on substrate curvature, which affects how cells grow and mechanically respond, and can be implemented with traction stress microscopy easily. It serves as a good model system for more investigation in 3D mechanobiology.

關鍵字(中)

★ 三維細胞培養
★ 三維牽引力顯微鏡
★ 力生物學
★ 利基質曲率
★ 細胞遷移
★ 細胞增殖

關鍵字(英)

★ 3D cell culture
★ 3D TFM
★ mechanobiology
★ substrate curvature
★ cell migration
★ cell proliferation

論文目次

Chinese Abstract i
English Abstract iii
Acknowledgements v
Chapter I. Introduction 1
Chapter II. Results 6
2.1 The proliferation of hMSCs in microwells depends on the size of microwells. 6
2.2 Quantitative morphological features 8
2.3 Average traction stress, strain energy, and contractility is higher in larger microwells 16
2.4 Cell volume, nuclear volume and YAP N/C ratio of stickers and stretchers are similar, but their nuclear flatness, cell surface area, adhesion area, and their mechanical responses in the microwells are distinct. 24
2.5 Blebbistatin treatment alters morphology of hMSCs in 100-μm microwells. 30
Chapter III. Materials and Methods 32
3.1 Fabricating spherical microwells 32
3.2 Cell culture and cell seeding to spherical microwells 33
3.3 EdU proliferation assay 33
3.4 Immunofluorescence staining 33
3.5 Image processing, segmentation and quantification 34
3.7 Particle tracking and surface boundary conditions 39
3.8 Traction force computation and simulation 40
3.9 Traction force microscopy and validation 42
Chapter IV. Discussion 45
References 49
Appendix 51

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指導教授

林耿慧(Keng-hui Lin)

審核日期

2023-2-1

推文