Mechanism Study Of The Biological Effects Of Laser Irradiation In Living Cells

Low-power laser irradiation (LPLI) can modulate various biological processes. For instance, LPLI can induce cell proliferation, differentiation and apoptosis. Yet, the mechanism of LPLI biological effects remains unclear. In this dissertation, the effect of different fluence LPLI on human lung adenocarcinoma cells (ASTC-a-1) growth was analyzed using Cell Counting Kit-8; the mechanism of the effect was investigated using specific fluorescent probes staining and fluorescence resonance energy transfer (FRET) techniques. Our studies help to clarify the mechanism of LPLI biological effects and guide the clinic therapy.First in prolegomenon, the paper introduced the research status quo of the biological effects and the mechanisms of LPLI. At the same time, the paper generalized the study work in this dissertation and explained the aim and meaning of the study. And then, this paper recapitulative introduced the principle and application of laser confocal scanning microscope (LCSM) and FRET.In the first experimental study, the effect of different fluence LPLI on ASTC-a-1 cells growth was analyzed using Cell Counting Kit-8. The results show that low fluence LPLI (0.48J/cm~2-0.8J/cm~2) induces ASTC-a-1 cells proliferation, while high fluence LPLI (60J/cm~2-120J/cm~2) induces apoptosis.In the second experimental study, the spatio-temporal dynamics of Ras/Raf activity during 0.8J/cm~2 low fluence LPLI-induced ASTC-a-1 cells proliferation was monitored in single living cell using FRET imaging based on RaichuRas genetic reporter. The results show that low fluence LPLI induces activation of Ras/Raf. These results suggest activation of Ras/Raf plays an important role during low fluence LPLI-induced proliferation.In the third experimental study, the mechanism of high fluence LPLI-induced ASTC-a-1 cells apoptosis was investigated using specific fluorescent probes stainingand FRET techniques. We have measured and analyzed the dynamics of generation of reactive oxygen species (ROS), alteration of mitochondrial membrane potential (△Ψm) using H2DCFDA and Rhodaminel23 fluorescence probes respectively, the dynamics of activity of caspase-3 and Ras/Raf using FRET imaging based on SCAT-3 and RaichuRas genetic reporters, and the activity of caspase-8 during 120J/cm2 high fluence LPLI-induced apoptosis, respectively. The spatio-temporal dynamics of cells events can be monitored in real-time in single living cell by the use of LCSM imaging. The results show a temporal sequence of events during apoptosis induced by high fluence LPLI: intracellular ROS generation initiates during laser irradiation, △Ψm begins to decrease about 10min after laser treatment, and caspase-3 activation initiates about 30min after laser treatment. Our results also show high fluence LPLI deregulates the Ras/Raf activity and has no effect on caspase-8 activity. These results suggest high fluence LPLI-induced cells apoptosis is initiated directly from intracellular ROS generation and △Ψm decrease, and is independent of caspase-8 activation.Last, the paper prospected the foreground of the application of LPLI treating illness in the fields of biology and physics.