使用寬範圍近紅外光對細胞色素c氧化酶活性的光生物調節

Abstract

光生物調節（Photobiomodulation）是一種新穎且具有潛力的治療方式，一方面光治療本身是無創的，且光治療使用的波長較長，對於人體的傷害較低；另一方面是LED的取得相當容易，不論是臨床治療或是居家應用，都非常容易實行。光生物調節的應用相當廣泛，根據治療的部位不同會達到不同的治療效果，例如：促進傷口復原、舒緩肌肉痠痛以及提升大腦的認知能力等，不只是使用在特定病患上，而是幾乎適用於每一個人。Cytochrome c Oxidase，電子傳遞鏈裡其中一個蛋白質複合體，是促進氧氣還原成水的關鍵酵素。在近期的研究中被認為是參與光生物調節中，吸收紅外光和近紅外光的重要的發色團（Chromophore）。 在本研究中，我們使用電極作為偵測氧氣消耗的工具，測量人類心肌細胞AC16在照射750 nm、810 nm、940 nm及1050 nm（約2 J/cm2，持續照射五分鐘）後得到的不同耗氧結果；從結果中得到750 nm及940 nm會造成粒線體耗氧率下降，810 nm及1050 nm則會造成粒線體耗氧率上升。同時本篇研究除了AC16之外，還對其他細胞株Panc-1、HEK-293、HepG2和IMR-32照射1050 nm（約2 J/cm2，持續照射五分鐘）光處理，並測量各株細胞對其控制組氧氣消耗的結果，從實驗結果發現，AC16及HepG2在照射1050 nm對上其對照組的氧氣消耗有顯著提升。本篇研究還使用了Cytochrome c Oxidase Assay做為粒線體活性測量的工具，對細胞照射810 nm及1050 nm（1.07～3.06 J/cm2，持續照射五分鐘），發現照射810 nm後，細胞活性有顯著提升。最後對粒線體染TMRM，使用Confocal拍攝螢光訊號來確認膜電位的高低，我們對細胞AC16、Panc-1、HEK-293、HepG2和IMR-32細胞照射1050 nm（6.37 mW/cm2），發現照射後能夠減緩AC16膜電位下降。儘管對於光生物調節的機制尚未完全了解，但從多篇研究及本篇實驗結果可以確定，使用近紅外光是一種很有潛力的治療手段。

Photobiomodulation has been applied as noninvasive intervention to regulate cellular functions in animal models, and clinical applications. Cytochrome c oxidase (CCO) is a protein complex of the electron transport chain that has potential chromophores for infrared light absorption, which can modulate mitochondrial respiration and energy production. The photobiomodulation mechanisms of CCO may come from direct changes in enzymatic activity or indirect responses involving the activation of transcription factors or secondary signaling pathways. In this thesis, we focused on acute infrared treatment effects on mitochondria respiration in human cell lines under different wavelengths and intensities. The oxygen consumption rate of suspended cultured cells was measured using a Clarke-type electrode under different light wavelengths and intensities that ranged from near-infrared to short-wavelength infrared. In the wavelength ranges, 810 nm and 1050 nm showed upregulation of mitochondrial respiration, while 750 nm and 940 nm showed downregulation. We observed the same trend in a parallel CCO activity colorimetric assay measuring the absorption of cytochrome c at 550 nm. Among five tested cell lines with different human organ origins ( AC16, PANC-1, HEK293, HepG2, and IMR32 cell lines), the HepG2 cell line showed the greatest activity increase under low energy light treatment of 1050 nm (1.07～3.06 J/cm2). The AC16 cells maintained the TMRM fluorescent intensity best among these five cell lines. Although the exact mechanisms of how infrared light modulates mitochondria respiration are not fully understood, short-wavelength infrared (> 1000 nm) is an attractive candidate for therapeutic intervention because it provides noninvasive drug-free photomodulation of mitochondrial energetics while indirectly controlling ΔΨm and ROS production with the benefit of better human tissue penetration than near-infrared light (< 1000 nm).