基于单细胞荧光成像技术的昼夜节律监测分析

Application of single-cell bioluminescence imaging on the monitoring and analysis of circadian rhythm

昼夜节律作为机体的无形时钟精密调控多种生理功能,包括睡眠、觉醒、大脑认知、免疫力等。下丘脑视交叉神经上核(suprachiasmatic nucleus, SCN)作为昼夜节律系统的主起搏器,能够自主产生节律性输出信号,协调维持全身多个组织、器官的时钟变化。同时,SCN通过神经元间的耦合作用,实现不同神经细胞节律性变化的步调一致性,产生强劲的振荡性规律,以维持机体内部节律稳定、抵抗外界环境因素干扰。为直观的研究SCN区在机体昼夜节律中的功能,实现单细胞尺度观测SCN神经元节律性变化,本研究搭建了单细胞荧光成像系统,通过深度制冷CCD相机,利用荧光素酶报告基因系统,对体外分离培养的SCN脑片进行长周期实时成像,原位观察SCN区不同细胞节律基因的振荡性表达规律。利用Eviews、IgorPro等软件,对荧光强度在单细胞尺度及时间尺度上进行量化分析,最终实现了在单细胞水平监测观察节律基因的振荡性变化。利用河豚毒素(TTX)能够可逆性的破坏SCN细胞间的耦合作用,验证了该系统的可行性。综上,本研究成功搭建了基于荧光素酶报告基因系统的单细胞实时荧光成像平台及分析技术,实现了对SCN神经元基因表达节律性变化的实时监测。

As an invisible clock of the body, circadian rhythm accurately regulates many physiological functions, including sleep, wake, brain cognition, immunity and so on. As the main pacemaker of the circadian rhythm, the suprachiasmatic nucleus(SCN) can autonomously generate rhythmic output signals and coordinate the peripheral clocks in multiple tissues and organs. At the same time, intercellular coupling in the SCN achieves the consistency of the rhythms in different cells, and generates robust oscillation to maintain the stability of the internal rhythm and resist the interference of external cues. In order to visually study the function of SCN and observe the rhythm of SCN neurons at the single-cell level, we constructed a single-cell luminescence imaging system. By using a deep-cooled CCD camera and luciferase reporter gene system, long-term real-time imaging was performed on SCN slices. The oscillatory expression patterns of circadian gene in different cells were observed in situ. Eviews, IgorPro and other software were used to quantitatively analyze the luminescence intensity at the single-cell scale and the time scale. Tetrodotoxin(TTX) could reversibly destroy the coupling between SCN cells, which verified the feasibility of the system. In conclusion, this study successfully built a single-cell bioluminescence imaging technology, and realized the real-time monitoring of the circadian rhythm in SCN neurons.