The Preliminary Study Of NAD Against X-ray Induced Damage In L02 Cell

BackgroundRadiation therapy plays an important role in the treatment of malignant neoplasm. Radiation can kill tumor cells while normal tissue damage is inevitable, So cancer radiotherapy dose intensity is limited. The result is tumor cells can not be completely kill and Patient quality of life is reduced. At the same time, with the development of health cares and nuclear industy, Iatrogenic radiation and Non-iatrogenic radiation damage to normal tissues and cells is increasingly concerned. Therefore, It is one of emphasis and focus of molecular biology of radiation and Radiation Epidemiology that the mechanism of radiation damage and radiation damage protection.Domestic and foreign scholars suggest that the mechanism of cell damage induced by radiation focused on the following aspects:1) change the cell signaling pathway:Cells undergo radiation, a variety of intracellular or extracellular signaling molecules by its induction of changes in the signal pathway, ultimately leading to apoptosis.. p53 has a central role in the status.2) DNA damage:DNA damage caused by radiation, including single-strand breaks (SSB), double-strand break (DSB), base damage and protein cross-linking and other forms.3) cell cycle regulation:Primarily through the regulation of cell cycle G0/G1, S, G2/M control point, regulation of cell radiation sensitivity and radiation resistance. Cells after DNA damage, wild-type p53 gene in human cells into the G1 phase until DNA damage repair, If the damage is not repaired, p53 gene on the activation of gene transcription induced apoptosis, the cell apoptosis.4) micro-environmental changes:Cell survival is inseparable from their microenvironment, including cell oxygen supply, pH, nutrients, metabolites, ion balance, cytokines, etc. The micro-environment can cause changes in gene expression and cellular response to radiation.Molecular mechanism of cellular radiation damage was in-depth study, and research on radiation damage protection also made great progress. In recent years, research on radiation damage protectants have more reports, mainly related to the following categories:1) anti-oxidants, such as Amifostine, NAC, VitC, etc; 2) cytokines such as IL-2, IL-3, IL-6, TGF, etc; 3) trace elements such as selenium, zinc, etc; 4) herbs such as ginseng Gan, rosemary, etc. However, Antioxidant Amifostine and NAC drug side effects, Role of cytokines network characteristics and herbal ingredients are difficult to purify, Not an ideal radioprotective agents used in clinical. Find a new effective drug side effects of drugs on radiation protection radiation protection remain important.NAD+, chemical called nicotinamide adenine dinucleotide. It is an important cellular energy metabolism of coenzyme involved in cellular redox reactions and respiratory electron transport chain. NAD+accept electronic transfer reduced to NADH in the mitochondria. It is through the electron transfer can inhibit free radical generation and generate ATP needed for cell metabolism, So it may be to protect cells from radiation damage.In this study, Application of cell biology and molecular biology discussed the role of NAD+and its protective mechanism of radiation damage. It has theoretical and practical significance for further study of normal tissue radiation injury mechanism and explore a new and effective, and toxic side effects of radiation protection drugs.PurposeThis study observed the effects of oxidized coenzyme I (NAD+) to radiation damage of normal human liver cell line L02 cells, preliminary study of oxidized coenzyme I (NAD+) anti-radiation damage and its mechanism. Further study of iatrogenic and non-iatrogenic radiation injury protection, the new radiation protection agent to provide a new way of thinking and means.Methods1 cell culture and group:Normal human liver cell line L02 cells were cultured with 10% fetal bovine serum PRMI 1640 medium, placed in 37℃,5%CO2 incubator. The L02 cells were divided into 3 groups:treatment group and the irradiated cells were added with and without NAD (1000μg/ml) in RPMI-1640 medium after irradiation; control cells without exposure, only adding RPMI-1640 medium.2 X-ray irradiation Using Varian 6-MV X ray linear accelerator, the dose of 5 Gy, dose rate 500cGy/min, radiation source from the target center distance 100cm.3 MTT assay with different concentrations of NAD on cell proliferation Cells per well 3 x 105～5×105/ml were seeded in 96 tissue culture plate,100 ul/hole, cells adherent to the supernatant, adding 0.01mol/L PBS (pH 7.4), in accordance with the conditions X rays. Supernatant was tossed away after irradiation, by adding RPMI 1640 (containing 10% fetal bovine serum) dilution of different concentrations of NAD, respectively 0,200,400,600,800,1000,1200,1400 ug/ml,100 ul/hole at 37℃,5%CO2 incubator for 24h. MTT colorimetric assay with various concentrations of NAD on the L02 cell proliferation.4 Detection of apoptosis rate by flow cytometry L02 cells were prepared into single cell suspension after digested by trypsin. To each well 3×105～5×105/ml was inoculated in 6 well tissue culture plate, 1ml/hole until the cells adherent to the supernatant, adding O.Olmol/L PBS (pH 7.4), X-ray according to the above conditions irradiation. Supernatant was tossed away after irradiation. Treatment and control groups were added with and without NAD (1000μg/ml) in RPMI 1640 medium, at 37℃,5%CO2 incubator 24h. Collecting cells, regulation of cell concentration of 1 x 106～6 x 106/ml, using Annexin V/PI staining to detect cell apoptosis.5 Detection of cell cycle by flow cytometry Cells per well 3×105～5×105/ml was inoculated in 6 well tissue culture plate, lml/hole, Supernatant was tossed away after the cells attached to the wall, adding 1.01mol/L PBS (pH 7.4), X-ray irradiation in accordance with the above conditions. Supernatant was tossed away after irradiation. Treatment and control groups were added with and without NAD+ (1000μg/ml) in RPMI 1640 medium, at 37℃,5%CO2 incubator 24h. Collecting cells, regulation of cell concentration of 3×106～6×106/ml, After centrifugation, PBS wash, adding ice-cold 70%ethanol, detecting the percentage of cells in each cell cycle phase by flow cytometry.6 Detection of p53, bax, bcl-2 protein percentage by flow cytometry cells were collected at three groups After the above treatments. With 0.5%paraformaldehyde solution lml fixed 30min, breaking Remover cells in charge of cracking, washing, discard supernatant after centrifugation. The tubes were added mouse anti-human p53, bax, bcl-2 monoclonal antibody, and mix, Incubated 1h at 37℃, Then were added FITC labeled anti-mouse secondary antibodies,37℃incubated 1h, the protein was detected by flow cytometry percentage.7 Caspase-3, Caspase-8, Caspase-9 activity assay cells were collected at three groups After the above treatments. Press kit instructions for each step. Plus a separate cell while other operations are not the same hole as a blank control group, measured by microplate reader 405nm Department OD absorbance value.8 L02 cell morphology observed by transmission electron microscopy We manufacture agarose conical tube with a conical hole plasma. Join the collection and processing of cells, centrifugation,2.5%glutaraldehyde,1%osmium tetroxide fixed double fixed, gradient dehydrated alcohol, propylene oxide saturated, fat-embedded, thin sections, electron stain electron microscopy.9 Statistical analysis Processing experimental data using statistical software SPSS 13.0. Experimental data is expressed with x±s, Statistical analysis using ANOVA, P<0.05 as statistically significant difference. Results1 MTT assay with different concentrations of NAD+on cell proliferation L02 cells in the acceptance of 5.0Gy X ray irradiation, by adding RPMI 1640 (containing 10%fetal bovine serum) dilution of different concentrations of NAD+, respectively 0,200,400,600,800,1000,1200,1400 ug/ml, at 37℃,5%CO2 incubator for 24h. MTT cell proliferation test, with the increase in NAD concentration, cell proliferation activity increased, the concentration of the NAD+1000～1400 ug/ml, the irradiated cells increased activity reached a plateau. Therefore, NAD+concentration 1000 ug/ ml, as subsequent experimental conditions.2 the apoptosis rate in each group Cell culture 24h after irradiation, the apoptosis rate were detected by flow cytometry. The control group (1.50±0.67) and treatment group (12.85±1.59) were significantly lower than the radiation group (31.72±3.07) (P<0.05). Description NAD significantly decreased apoptosis rate of L02 after X-irradiation, with anti-X-induced apoptosis.3 NAD+regulate irradiated the cell cycle In L02 cells were X-irradiated 24h time, Irradiated group G1, S phase cells was significantly increased compared with control group, G2/M phase cells decreased. This showed G1 arrest. Treatment group compared with the irradiation, G1 phase cells decreased, S phase and G2/M phase cells increased, reflected in cell division and cell DNA synthesis on the increase in the number of cells.4 NAD+on the X-ray-induced apoptosis in the regulation of apoptosis-related protein L02 cells were cultured 24h after 5.0Gy X-ray irradiation, Detection of apoptosis-related protein three groups of cells. Treated cells p53, bax expression lower than the radiation group (P<0.05), while the irradiation control group (P<0.05). This indicates that the cell p53, bax protein increased after irradiation, NAD+can reduce the Irradiated cells p53, bax protein expression. Three groups of cells in the bcl-2 expression in the opposite. Treated cells was higher than the expression of bcl-2 irradiated group (P<0.05), while the irradiated group than the control group (P<0.05), Show that the cells after irradiation decreased expression of bcl-2, NAD+can Increase the Irradiated cells bcl-2 protein expression.5 Cell Caspase-3, Caspase-8, Caspase-9 activity L02 cells were cultured 24h after 5.0Gy X-ray irradiation. Treated cells Caspase-3, Caspase-8, Caspase-9 activity than the irradiated control group decreased significantly (P<0.05). NAD+can inhibit Caspase-3, Caspase-8, Caspase-9 activity, thus inhibiting L02 X-ray irradiation induced apoptosis.Conclusion1 X ray can induce L02 cell proliferation decreased, NAD+can inhibit X-ray induced L02 cells decreased proliferation.2 NAD+can reduce the X-ray induced L02 cells increased apoptosis rate.3 X-ray induced L02 cells arrest in G0/G1 phase. NAD+allows cells to enter S phase for DNA replication.4 NAD+can reduce the cell p53, bax Protein expression after irradiation, Increases the expression of bcl-2 protein.5 NAD+Can reduce the expression of Caspase-3,Caspase-8,Caspase-9 After irradiation in cells.The experiment by observing the oxidized coenzyme I (NAD+) to radiation damage human normal liver cell line L02 cells showed that:NAD+is able to resist X-ray induced L02 cells increased, Restoration of cell proliferation. The approach may be associated with reduced p53, bax protein, increased bcl-2 protein expression, reducing Caspase-3, Caspase-8, Caspase-9 activity.