光基因學是一個新興的領域，結合了光學與基因遺傳工程之應用，主要的概念是利用病毒將光基因蛋白轉染至哺乳類或其他動物；例如將ChR2與NpHR同時感染至特定的神經細胞中，即可藉由二種不同波長的色光加以控制神經細胞的活性。然而ChR2與NpHR這類的光基因蛋白需要適當的波長並且足夠功率的光源，根據此需求，我們發展了一以LED作為光刺激源，並與記錄電極結合之探針，經由簡單的硬體及軟體進行控制，可長期置入活體動物中。
本文中，我們完成了Lentivirus的製備，並將光基因蛋白表現在HEK 239T細胞中，記錄其受光反應而分別產生去極化及超極化的電生理特性；藉此確認我們所研製之LED探針，具備活化及抑制神經細胞之功能。我們預期此置入性LED探針比一般表面光照更為有效，可為將來神經肌肉疾病之修復與發展人工腦皮質……等臨床醫學預作準備。光基因學方法確實提供了一個嶄新的機會，可針對特定的神經細胞進行暫時性的刺激，這是一個對於神經修復及其研究相當有前瞻性的技術。

關鍵字：　光基因學、神經元活性、神經生理學、腦機介面

Optogenetics is an emerging field combining optical and genetic techniques. Utilizing lentivirus vectors to transfect light-sensitive ion channels and pumps (ChR2 and NpHR) into mammals and other animals, it is now possible to turn genetically specific neurons on and off with millisecond time precision. However, ChR2 and NpHR proteins need adequate light power at appropriate wavelengths to activate. It remains unknown how optogenetic stimulation compares with traditional electrical stimulation. Accordingly we have developed an LED stimulating-recording chronic implantable multielectrode array and associated control hardware and software.
In summary, we have achieved lentivirus production, infection in 293T cells and electrophysiological testing of ChR2 and NpHR in vitro. Also, we have confirmed that our device has the appropriate power and functionality to permit excitation or inhibition using two different light wavelengths of the LED array. We expect that the LED array will allow deeper stimulation compared to surface illumination, and its basic design could be applied towards future development in neuromuscular rehabilitation and cortical prosthetics. The optogenetic approach offers new opportunities for minimally-invasive temporal- and cell-specific neuronal stimulation and is a promising technology for neural rehabilitation and research.

Key Words:　Optogenetics, neuronal activity, neurophysiology, brain machine interface.

中文摘要………………………………………………………………………… I
Abstract ………………………………………………………………………… II
目錄……………………………………………………………………………… III
圖目錄…………………………………………………………………………… IV
第一章 導論 (Introduction)…………………………………………………… 01
第二章 研究方法 (Research Method)………………………………………… 05
第一節 ChR2與NpHR的基因轉染與病毒感染……………………… 05
第二節 病毒感染至活體動物內的程序………………………………… 06
第三節 電生理測量儀器………………………………………………… 06
第四節 微機電製程之探針……………………………………………… 07
第五節 繼電器系統……………………………………………………… 09
第六節 系統雜訊測試…………………………………………………… 11
第三章 研究結果 (Results) …………………………………………………… 12
第一節 生物螢光影像…………………………………………………… 12
第二節 電生理的結果…………………………………………………… 12
第三節 探針的功能測試………………………………………………… 13
第四節 繼電器分線器的功能…………………………………………… 13
第四章 結論與未來展望 (Conclusion and future perspectives) …………… 15
第五章 圖 (Figures) …………………………………………………………… 17
參考文獻 (References) ………………………………………………………… 41

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