| Summary: | 碩士 === 國立臺灣大學 === 生醫電子與資訊學研究所 === 107 === 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.
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