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標題: | 整合微流道系統與超解析顯微鏡進行細胞主纖毛之力學刺激反應研究 A Microfluidic Device Integrated Superresolution Microscopy for In-situ Fixation and Mechanosensation of Primary Cilia |
作者: | Sheng-Han Chu 朱聖瀚 |
指導教授: | 黃念祖(Nien-Tsu Huang) |
關鍵字: | 微流道,主纖毛,超解析顯微鏡, Microfluidic,Primary Cilia,Superresolution Microscopy, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 在哺乳動物細胞中常見的主纖毛 (primary cilium) ,是一種負責偵測環境中各種變化的胞器,從研究顯示流體、化學分子、光等刺激都需要透過主纖毛來傳遞刺激訊號至細胞內。其中,物理刺激的偵測 (mechanosensation) 為主纖毛的主要功能,且與許多疾病相關聯。然而,由於主纖毛的尺寸在微米等級,一般研究方法無法給予精準的刺激,同時,由於繞射極限,直接觀察主纖毛在刺激下的變化更為困難,目前研究多利用主纖毛的長度、細胞內鈣離子濃度、基因表現來觀測主纖毛的功能。
微流道 (microfluidics) 是一種在微米等級下控制流體的技術,具有體積小、價格低廉、低樣本需求量等優點。其中精準的流體控制,為微流道特別適合運用在主纖毛研究上的原因,無論是給予流場刺激、液體的切換,都較傳統的研究方法更加方便。而在超解析顯微鏡 (superresolution microscopy) 的發展下,主纖毛內部的蛋白質變化得以被觀測到,不論是蛋白質的組成、移動或是數量,都能透過此技術來進行分析,進而得知主纖毛是如何調控及傳輸這些外部刺激到細胞裡。 為提供一個能給予適當刺激、在活細胞以及免疫染色下都方便觀察的實驗架構,本研究開發出結合多流速、可拆卸式等優點的微流道系統,透過給予精準的流場刺激,可用於觀察活細胞下的主纖毛的變化,同時也能在拆解之後針對想觀察的蛋白質進行免疫染色,最後透過螢光顯微鏡或是超解析顯微鏡來分析刺激之後的結果。在實驗中,我們最佳化細胞培養的參數來產生高密度的主纖毛,也觀測到在活細胞下主纖毛受到流場刺激的變化。透過免疫染色以及觀察大量的主纖毛,我們進一步發現主纖毛在統計上的變化。最後,在超解析顯微鏡的幫助下,我們發現主纖毛中的蛋白質分佈會受到流場的刺激而改變,我們相信此結果與主纖毛的訊息傳遞功能高度相關。未來期望此系統除了能完成更多有關上述的實驗之外,亦具有潛力與蛋白質譜 (protein mass spectrometry) 結合,進行高通量的刺激同時針對主纖毛特有蛋白進行更深入的研究。 Primary cilium is an organelle found in most mammalian cells. It is known that primary cilium can sense fluid flow, molecules and light in order to transfer these stimulations into cells. Mechanosensation which senses the mechanical stimulation and transfers to cells is the main function of primary cilia and associated with various syndrome. Due to the size of primary cilium is around few micrometers, conventional research methods cannot provide precise stimulation. At the same time, diffraction limit also makes direct observation of primary cilia more difficult. Presently, primary cilium length, intercellular calcium concentration and gene expression are commonly used to observe the function of primary cilium. Microfluidics is a method which control fluid under micrometer scale. It has the advantages of small volume, low cost and low sample demand compare to conventional methods. The ability of applying flow stimulation and switching fluid accurately makes microfluidic the most suitable tools to study primary cilia. Under the development of superresolution microscopy, protein composition, movement and numbers can be observed inside the primary cilium. This powerful tool help scientists study more detailed about mechanosensation of primary cilium. A device which can provide proper stimulation, real-time cilia bending and ability to do immunostaining is needed. We develop a multiple flow velocity and resealable microfluidic system. By applying precise flow velocity, it is capable to observe primary cilia change in live cell, do immunostaining of the desired protein after stimulation and analyze the result using epifluorescence or superresolution microscopy. In the experiments, we optimized the protocol to promote ciliogenesis, observed the bending in both live and immunostained images under the flow stimulation. Last but not least, by superresolution microscopy, we found the protein distribution inside primary cilia has huge differences before and after flow stimulation. We believe that the result is highly correlated to the signal transduction of primary cilia. In future work, we plan to do more relative experiments can be done by using our system. Also, the system has the potential to integrate with protein mass spectrometry to do high throughput protein analysis for primary cilia specific proteins. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71367 |
DOI: | 10.6342/NTU201900442 |
全文授權: | 有償授權 |
顯示於系所單位: | 生醫電子與資訊學研究所 |
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