Os efeitos biológicos da laserterapia de baixa intensidade em células-tronco isoladas da papila apical humana

Abstract

The aim of this study was to analyze the effects of low-intensity laser therapy (LILT) in stem cells from apical papilla (SCAPs) proliferation. Stem cells isolated from apical papilla cells were donated by the cell bank from Pharmaceutical Nanotechnology Laboratory and Drug Delivery Systems, Federal University of Goiás (UFG). Cells were cultured in 75cm^2 bottles and kept in a humidified stove at 37 °C in a saturated atmosphere at 5% CO2. Standard culture medium used was composed of Dulbecco's modified Eagle's medium nutrient mixture F-12 (DMEM / F12), 10%, fetal bovine serum ,100 ug / ml streptomycin and 100 U / ml penicillin. For irradiation, the semiconductor laser diode(InGaAlP) was used at a wavelength of 660 nm, 100mW power, 6J energy, 60 seconds for irradiation time and 200 J / cm². Dose. Initially, the intensity of mitochondrial metabolism was evaluated by immunofluorescence. For this, we used Mitotraker Red in a concentration of 100mM. Intensities of mitochondrial activity was compared in non-irradiated cells, and after two irradiation times: immediately to the application of LILT and 24h after application. In order to assess the immunoreactivity of proteins involved in cell proliferation, it performed immunocytochemistry using Ki-67, p53, Bcl-2, Cyclin B1, TGF-β2 and TGF-β3 .The incubation time for the primary antibody exposure was standardized at 2hours. For each antibody, two different groups were analyzed: non-irradiated (CNT) and irradiated (G6J). The progression of the cell cycle analysis was performed, by flow cytometry. The results obtained in the groups irradiated and non-irradiated were compared. The normality of the data distribution was tested using the Kolmogov- Sminorv test. To check the diferences in the immunoreactivity between the groups, were used the Mann-Whitney test and to check whether there was a statistical difference between the groups regarding the progression of the cell cycle, we used the nonparametric T test. In both cases, it was adopted significance of 5%. Increased mitochondrial activity was evident immediately from the time of irradiation, and this metabolic activity increase even more after 24 hours of exposure to LILT The irradiated group showed greater median percentage of immunopositive Ki-67 and Cyclin B1. However, in both cases, there was no statistical significance when compared to control groups. There was no difference in TGF-β expression. Likewise, it was also no difference obtained as the p53 and Bcl-2, which indicates that the LILT does not deregulating the maintenance of cell cycle progression and control of apoptosis. There was no disagreement on the chronology of cell cycle progression between the groups irradiated and non-irradiated. Thus, it is possible to conclude that the LILT increases mitochondrial metabolism of isolated stem cells of the apical papila and it can influence the imunoexpression of proliferation marker. Furthermore, the irradiation of SCAPs does not result in any damage to regulation of cell proliferation and apoptosis functions.