Effect Of 280nm-LED Ultraviolet On The Proliferation Of HL-60 Cells Under Hypoxia And Its Mechanism

ObjectiveThe aim of this experiment is to observe the effect of 280nm Light Emitting Diode-Ultraviolet（LED UV）on human acute early myocyte leukemia（HL-60）cells proliferation under hypoxic conditions and explore its mechanism.Methods1.In this study,HL-60 cell lines in logarithmic growth phase were selected as the research object,280 nm LED UV was used as light source,and cobalt chloride（CoCl₂）was used to simulate hypoxia2.We randomly divided HL-60 cells into control group and experimental group（control group A and experimental groups B,C,D,E,F）.The HL-60 cells were routinely cultured in group A,and cells in group B were treated with CoCl₂（final concentration150μmol/L）as a hypoxic group,those in the ultraviolet group were irradiated by 280nm-LED UV with energy intensity of 30J/m²,and cells in hypoxia combining ultraviolet group were treated with cobalt chloride and then irradiated by 280nm-LED ultraviolet.The cells in twice ultraviolet group were irradiated again after 24h incubation on the basis of irradiating by 280nm-LED UV with energy intensity of 30J/m²,and cells were irradiated again after 24h incubation on the basis of the hypoxia combining ultraviolet group as hypoxia combining twice ultraviolet group.3.Cells in each group were cultured in IMDM medium containing 10%fetal bovine serum for 48 hours at 37 C,5%CO₂ and saturated humidity4.Detection of the following indicators:cell number and morphological changes under inverted microscope;CCK-8 detection of cell proliferation inhibition rate;AnnexinV-FITC/PI double staining flow cytometry detection of apoptosis;real-time fluorescence quantitative PCR detection of Bcl-2 gene expression;Western Blot detection of Bcl-2protein expression.Results1.Changes of growth,morphology and density of HL-60 cell line:After 48 hours of culture,HL-60 cells were observed under inverted microscope.The cells in control group were arranged neatly,round,transparent,and intact.The number of cells increased,and the number of cells was about（201.67±6.51）*10⁴/ml.In experimental group,the number of cells was disordered,shrinkage,transparency decreased,and the number of cells decreased,and the number of cells from high to low was about hypoxic group（161.67±3.51）*10⁴/ml,ultraviolet group（112.00±2.65）*10⁴/ml,hypoxia combining ultraviolet group（71.33±2.52）*10⁴/ml,twice ultraviolet group（52.67±1.53）*10⁴/ml,hypoxia combining twice ultraviolet group（24.67±1.53）*10⁴/ml.There was a statistically significant difference in cell density between the groups（F=1135.695,P<0.01）.The differences between the groups were statistically significant（P<0.05）.The growth status and cell morphology of B-F cells in the experimental group gradually deteriorated:the growth status of B-F cells in the ultraviolet group was worse than that in the low oxygen group;the growth status of cells in the hypoxia combining ultraviolet group was worse than that in the ultraviolet group;a certain proportion of dead cells and cytoplasmic bodies were observed in the twice ultraviolet group,and the growth status of cells in the low oxygen combining ultraviolet group was worse;cells growth in the hypoxia combining twice ultraviolet group was the worst,with a large number of lysed cells.2.Inhibitory rate of HL-60 cell proliferation:Compared with the control group,the inhibition rate of B-F group in the experimental group increased significantly（all P<0.01）.The order from low to high was hypoxia group（20.51±1.01）%,ultraviolet group（44.32±2.07）%,hypoxia combining ultraviolet group（64.67±3.34）%,twice ultraviolet group（74.53±1.01）%,hypoxia combining twice ultraviolet group（87.47±0.68）%.The cells in hypoxia combining twice ultraviolet group had the highest inhibition rate.There were significant differences in the inhibition rate of cell proliferation among groups.（F=581.561,P<0.01）,There were significant differences among the groups（all P<0.05）.3.Apoptotic rate of HL-60 cells:The apoptotic rate of the control group was（2.64±1.16）%,and the apoptotic rate of each experimental group was significantly higher than that of the control group（all P<0.01）.The apoptotic rates in the experimental group was increased from low to high in order of hypoxia group（10.65±0.73）%,ultraviolet group（14.47±1.46）%,hypoxia combining ultraviolet group（31.71±1.27）%,twice ultraviolet group（39.30±1.39）%,hypoxia combining twice ultraviolet group（72.10±1.44）%,In addition,cells in hypoxia combining twice ultraviolet group had the highest apoptosis rate.There was significant difference in apoptotic rate among groups.（F=1195.466,P<0.01）,There were significant differences among the experimental groups（all P<0.05）.4.Bcl-2 gene expression in HL-60 cells:Compared with the control group,the Bcl-2 gene expression in the experimental group was down-regulated（all P<0.01）.From high to low,the Bcl-2 gene expression in the experimental group was decreased in the following order:hypoxia group（0.7374±0.0184）,ultraviolet group（0.6274±0.01894）,hypoxia combining ultraviolet group（0.4616±0.0230）,twice ultraviolet group（0.3810±0.0262）,hypoxia combining twice ultraviolet group（0.2575±0.0268）.Bcl-2 gene expression was down-regulated most significantly in hypoxia combining twice ultraviolet group.There were significant differences in Bcl-2 gene expression among different groups.（F=652.230,P<0.01）.There were significant differences among the experimental groups（all P<0.05）.5.The expression of Bcl-2 protein in HL-60 cells:Compared with the control group,the Bcl-2 protein expression in the experimental group was down-regulated（all P<0.01）.From high to low,the Bcl-2 protein expression in the experimental group was decreased in the following order:hypoxia group（0.7418±0.0186）,ultraviolet group（0.6523±0.0145）hypoxia combining ultraviolet group（0.4948±0.0156）,twice ultraviolet group（0.4061±0.0156）,hypoxia combining twice ultraviolet group（0.3236±0.0109）.Bcl-2protein expression was down-regulated most significantly in hypoxia combining twice ultraviolet group.The expression of Bcl-2 protein in each group was significantly different.（F=925.827,P<0.01）.There were significant differences among the experimental groups（all P<0.05）.Conclusions1.When HL-60 cells were simulated by hypoxia and 280 nm LED UV irradiation,the growth of HL-60 cells was significantly inhibited.The growth of HL-60 cells was inhibited by increasing the dose of 280 nm LED UV irradiation under hypoxia,which indicated that hypoxia and hypoxia combining LED UV irradiation could effectively inhibit the proliferation of HL-60 cells.2.The apoptosis rate of HL-60 cells induced by 280 nm LED UV irradiation under hypoxic condition was higher than that under normal oxygen condition;the apoptosis rate of HL-60 cells was significantly higher after twice 280 nm LED UV irradiation than that of single irradiation;Increasing the dose of 280 nm LED UV irradiation while under hypoxic condition,the cells were mainly necrotic,suggesting that inhibiting the proliferation of HL-60 cells by hypoxia,hypoxia combining LED UV irradiation was achieved by promoting apoptosis or necrosis of HL-60 cells.3.The expression of Bcl-2 gene and protein in HL-60 cells was inhibited by 280 nm LED UV irradiation under hypoxic conditions.With the increase of 280 nm LED UV irradiation dose,the expression of Bcl-2 gene and protein was down-regulated more obviously,suggesting that the expression of Bcl-2 gene and protein is one of the main mechanisms of apoptosis induced by 280 nm LED UV irradiation under hypoxic conditions.