上转换纳米颗粒结合近红外光进行心脏光遗传学研究的可行性探究

Effectiveness and feasibility of upconversion nanoparticles combined with near infrared laser in cardiac optogenetics

目的探讨上转换纳米颗粒(upconversion nanoparticles,UCNPs)结合980 nm近红外光(near infrared,NIR)能否通过激发可见光激活光敏蛋白(channelrhodopsin-2,ChR2)成功夺获心肌。方法15只SD幼鼠按随机数字表法随机分为光敏蛋白(ChR2)组(5例)、红色荧光蛋白(mCherry)组(5例)和对照组(5例),分别经颈静脉注入带有相应基因的腺相关病毒(adeno-associated virus,AAV)载体AAV9-CAG-hChR2(H134R)-mCherry、AAV9-CAG-mCherry和磷酸盐缓冲盐溶液(PBS),8周后开胸暴露心脏实施在体实验。自制包含UCNPs的聚二甲基硅氧烷(polydimethylsiloxane,PDMS)透光薄膜,将薄膜贴附于右心室游离壁,980 nm NIR经程序设置脉冲参数,由400μm芯径光纤输出,至于薄膜上方,同步记录NIR输出信号及体表心电图,观察NIR夺获右心室心肌的情况。结果ChR2组大鼠心肌可被NIR激发转换出的可见光(包含波长450 nm和475 nm的蓝光)夺获,并且随着NIR输出功率的增加1~6 W,心肌夺获率可增加至100%,随着NIR脉冲宽度的增加(5、10、20、50 ms),夺获心脏的NIR输出功率逐渐减小。而mCherry组和对照组大鼠心脏对980 nm NIR经UCNPs薄膜上转换的可见光无上述反应。结论UCNPs结合980 nm NIR用于光遗传学技术可成功夺获心肌,利用NIR的组织穿透性优势,有可能为心脏光遗传学研究提供新的实施策略。

Objective To investigate whether upconversion nanoparticles(UCNPs)combined with 980 nm near infrared(NIR)could stimulate visible light and activate the photosensitive protein channelrhodopsin-2(ChR2)for cardiac optogenetics study.Methods Sprague Dawley young rats were randomly divided into three groups using random number table:ChR2 group,mCherry group and control group(n=5 each).AAV9-CAG-hChR2(H134R)-mCherry,AAV9-CAG-mCherry or phosphate buffered saline(PBS)were injected via jugular vein respectively.The heart was exposed after 8 weeks.Polydimethylsiloxane(PDMS)film containing UCNPs was made and attached to the free wall of right ventricle in the open-chest rats.980 nm NIR through the optic fiber was focused above the UCNPs-PDMS film,and the NIR parameters were set by program.NIR output signals and electrocardiogram(ECG)were recorded synchronously to evaluated the cardiac capture by NIR.Results Under NIR illumination,UCNPs-PDMS film could upconvert visible light,including the wavelengths at 450 nm and 475 nm.As the NIR output power gradually increased from 1-6 W,the heart capture rate increased to 100%.With the increase of NIR pulse width(5,10,20,50 ms),the NIR output power of cardiac capture gradually decreased.Conclusion UCNPs can mediate NIR to capture the heart,which may provide a new strategy for tissue-penetrating cardiac optogenetics research.