Effect Of 280nm LED Ultraviolet Radiation On Proliferation Of SH-SY5Y Cell Line Under Hypoxia And Its Mechanism

ObjectiveThe aim of this experiment is to observe the effects of 280 nm Light Emitting Diode-Ultraviolet（LED-UV）on the morphological changes and proliferation,apoptosis and Bcl-2 gene expression of human neuroblastoma cell line（SH-SY5Y）under hypoxic condition,and to further explore its mechanism,so as to provide theoretical basis for the purification of NB in vitro.Methods1.In this study,human neuroblastoma cell line（SH-SY5Y）in logarithmic phase was used as the research object.A chemical hypoxic cell culture system was established by using 280nm LED-UV as light source and cobalt chloride（C_OCl₂）with final concentration of 150μmol/L.2.We randomly divided SH-SY5Y cells into control group and experimental group（control group A and experimental groups B,C,D）.Cells in group A which was controled group were untreat and experimental group B was treated with hypoxia（which was treated with C_OCl₂ with final concentration of 150 micromol/L）,experimental group C was ultraviolet group（which was irradiated with 280 nm LED-UV with dose of 30J/m²）and experimental group D was hypoxic combining with ultraviolet group（which was treated with C_OCl₂ and then irradiated with 280 nm LED-UV with dose of 30J/m²）.3.All cells were cultured inculture in DMEM/F-12 containing 10%fetal bovine serum and cultured in a constant temperature incubator for 48 hours at 37℃,5%CO₂and saturated humidity condition.4.Then we observe the morphology and growth of SH-SY5Y cells by invert microscope and test the cell inhibition rate of hyperplasia by CCK-8,detect apoptosis by flow cytometry and detect the expression of Bcl-2 gene by Real-time PCR.Results1.Morphological and density changes of SH-SY5Y cells:After 48 hours of culture,we observed cell density increased,about（21.10±1.41）*10⁴/ml in group A which was controled grup.Cells grew adherently and arranged in an orderly manner.The cells had complete morphology,obvious protrusion,abundant cytoplasm and large nucleus.The cell densities of experimental groups B-D were lower than that in group A（all P<0.01）,which were about（17.43±1.17）*10⁴/ml,（6.83±0.76）*10⁴/ml,（2.33±0.85）*10⁴/ml respectively.Some cells floated and some processes retracted or broke.The cell body was solid and the network disappeared.There were significant differences among the groups（F=199.53,P<0.01）.The density of cells decreased,thenumber of adherent cells decreased and the number of floating cells increased in experimental groups C and D.The change was most evident in group D.There were significant differences among the experimental groups（all P<0.05）.2.The inhibition rate of SH-SY5Y cell proliferation:The inhibitories rate of cells in experimental groups B-D were higher than controled group A（P<0.01）,which were（16.93±1.19）%,（67.93±7.35）%,（89.69±2.32）%respectively.There were significant differences among the groups（F=206.35,P<0.01）.The inhibition rates of proliferation of cells of experimental groups C and D were significantly higher than that of cells in experimental group B,especially in experimental group D.There were significant differences among experimental groups（all P<0.05）.3.Apoptosis rate of SH-SY5Y cell:The apoptotic rate of cells in control group A was（4.41±0.31）%.The apoptotic rates of cells in experimental groups B-D were significantly higher than controled group A（all P<0.01）,which were（4.41±0.31）%,（9.10±0.32）%,（22.93±0.33）%,（25.33±0.55）%.There were significant differences among the groups（F=2071.78,all P<0.05）.The apoptotic rate of cells of experimental groups C and D were significantly higher than that of cells in experimental group B,especially in experimental group D.There were significant differences among experimental groups（all P<0.05）.4.The expression of Bcl-2 gene in SH-SY5Y cells:The expression of Bcl-2 of cells in experimental groups B-D were significantly lower than that of cells in controled group A（all P<0.01）,which were（0.7073±0.0176）,（0.3987±0.0070）,（0.2137±0.0037）respectively.There were significant differences among the groups（F=1504.84,all P<0.01）.The expressions of Bcl-2 gene of cells of experimental groups C and D were significantly higher than that of cells in experimental group B,especially in experimental group D.There were significant differences among experimental groups（all P<0.05）.Conclusions1.Hypoxia and 280 nm LED-UV can inhibit the proliferation and promote apoptosis of SH-SY5Y cells,and 280 nm LED-UV can inhibit the proliferation and promote apoptosis of SH-SY5Y cells with lower oxygen intensity.The combination of them has the most significant inhibitory effect on the proliferation and apoptosis of SH-SY5Y cells.It is speculated that hypoxia and ultraviolet radiation inhibit cell proliferation by promoting SH-SY5Y cell apoptosis.280nm LED-UV and hypoxia could induce apoptosis.The effect that 280nm LED-UV induced apoptosis was stronger than hypoxia.The combined effect of SH-SY5Y cells is stronger than that of single factor.2.Hypoxia and 280 nm LED-UV could inhibit Bcl-2 gene expression in SH-SY5Y cells.The inhibition effect of 280 nm LED-UV on Bcl-2 gene expression was stronger than that of hypoxia.The most significant inhibition effect of 280 nm LED-UV on Bcl-2gene expression was under hypoxia.It is suggested that the decrease of Bcl-2 gene expression is one of the main mechanisms of SH-SY5Y cell apoptosis induced by 280nm LED-UV irradiation under hypoxia.