The Effect Of Reactive Oxygen Species Stimulated With 633nm Low-energy Red Light On Proliferation Of Human Keratinocytes

Objective: To investigate the effects of intracellular reactive oxygen species(ROS) levels stimulated with red light on cell proliferation activity in human keratinocyte line Ha Ca T.Methods: Human keratinocytes model in vitro was constructed with the following methods. Human keratinocytes cell line(Ha Ca T) was irradiated with 633 nm wavelength red light, and the energy densities were 0.082 J / cm2(10S), 0.164 J / cm2(20S), 0.245 J / cm2(30S), 0.491 J / cm2(60S), 1.472 J / cm2(180S), 2.453 J / cm2(300S), 4.91 J / cm2(600S) and 9.81 J / cm2(1200S), respectively. Control group was notirradiated. The cells were incubated with DCFH-DA for 1 hour, and fluorescence value was detected at 0h, 0.5h, 1h and 2h from incubation, respectively, so as to demonstrate the intracellular concentration of ROS; The human keratinocytes were exposed to the same conditions every 8 hours. Forty-eight hours later, incubated the cells with CCK-8 reagent 4 hours, and then fluorescence values were measured to verify the cells proliferation activity. Based on the above results, the cells exposed to different energy densities, including 0.082 J / cm2(10S)group, 0.491 J / cm2(60S), group 2.453 J / cm2(300S) group and 9.81 J / cm2(1200S)group, were stained with red mitochondria Mito-Tracker, and the empty cells and positive probes were regarded as the control, at the same time,cells were incubated with DCFH-DA probe. Confocal microscopy was used to observe the relationship between the origin of reactive oxygen species and mitochondrial.Results: Fluorescence values were enhanced in the experimental group at post-inc ubation 0h, 0.5h,1h and 2h except in 9.81 J/cm2 group. The value was higher in ex perimental group of 2.453J/ cm2 at post-incubation 1h than the control(t<0.05).Altho ugh a decreased trend of fluorescence value was shown in the 4.91 J / cm2 and mor e energy density of the experimental group, there was no statistically significance. A fter incubated for 4 hours with CCK-8, fluorescence values increased obviously in the 4.91 J / cm2 or less of the experimental group, there has a statistical difference co mpared with the control group, wherein 2.453 J / cm2 energy density of the group w as most obvious. While the fluorescence values of the energy density of 4.91 J / c m2 or more in experimental groups demonstrated a decline compared with the contr ol group, there was no statistical significance. The levels of ROS examined by confo cal microscopy were consistent with fluorescent display, and coincide with the mito chondria in each group.Conclusions: Promoting proliferation was observed by 633 nm low-energy red light in human keratinocyte, that had closely related which increased the levels of mitochondria intracellular ROS.