光遺傳學與磁遺傳學在細胞電生理反應之探討

Abstract

近二十年來光遺傳學已逐漸廣為人們所知，其中它被認為可能會是新一代的神經控制的工具。其中，Channelrhodopsin 2 (ChR2)更已經被證實可以在藍光的刺激下產生類似電流訊號，藉此影響細胞的膜電位。而另一方面，由於一些在光遺傳學發展上目前仍無法解決的限制，近幾年來，磁遺傳學的相關研究也應運而生。磁遺傳學的主要概念是來自於候鳥遷徙中，候鳥能夠依靠自體對於地磁感應而判斷出正確方向，科學家們將此一對磁場敏感的蛋白稱之為磁性蛋白(MagR)。 本篇研究主要分為兩部分，光遺傳學與磁遺傳學。我們透過病毒與脂質體包覆等方式，將我們的目標蛋白(ChR2或MagR)轉染至新生大鼠的心肌細胞中，我們期待透過比較藍光刺激或磁刺激前後，細胞是否會有任何電生理方面的差異。 首先，我們透過螢光顯微鏡與流式細胞儀得出，透過使用2x107vg/ml的AAV感染四十八小時後可以得到最佳的轉染效率。除此之外，在電生理方面，我們也得出在藍光的刺激下，動作電位持續時間(APD)有至少18%的延長。然而，在磁遺傳學研究方面，我們並未發現有任何顯著的差異，這也間接證實，磁性蛋白並不能單獨在細胞中造成細胞電生理上的影響。

Optogenetics has been widely studied in recent twenty years, it is considered as a brand new way as neuron or nerve control. Channelrhodopsin 2 (ChR2) has been proved to generate photocurrent under blue light stimuli, which is able to influence the membrane potentials. On the other hand, because of some limitations against optogenetics developments, magnetogenetics was born in recent years. The concept of this article is from the biocompass of migratory birds, and scientists name the protein as magnetoreceptor (MagR), which is sensitive to magnetic stimuli. Our study was divided into two parts: optogenetic and magnetogenetic experiments. We transfected the target protein (ChR2 or MagR) into neonatal rat cardiomyocytes, we expected to see the changes of any electrophysiological properties before and after (during) the blue light stimuli or magnetic stimuli. We utilized fluoresce images and flow cytometry to find out that the AAV infection in 2x107vg/ml for 48 hours had the best transfection efficiency. Besides, we also found that at least 18% increase of APD under or during blue light stimuli; however, there was no significant changes of APD in magnetogenetic experiment, which indicated that MagR alone may be insufficient to cause cellular responses.