| Objective: Vitamin D3can regulate the calcium andphosphorus metabolism in the body，which is also involved in thedifferentiation of the immune system regulation and the cell cycleHowever，the role of vitamin D3in oxidative stress mechanism is stillunclear. Our research is to study the effect of vitamin D3on DNAdamage and proliferation induced by LPS in human B-lymphoblastoidcell (Hmy2cir). Methods: Grouping of human B-lymphoblastoid celllines, PBS control group, LPS stimulation group, in LPS+vitamin D3stimulation group established under the successful models of oxidativestress injury. Detection of ROS reactive oxygen species,LPS acting on thehuman B-lymphoblastoid cell (Hmy2cir) expression in reactive oxygenspecies ROS.2comet assay (COMET SOP) comet assay kit to measurethe human B-lymphoblastoid cell DNA damage and tailing expression ofDNA damage in LPS-stimulated group and joined the group of vitaminD3stimulation and tailing situation.3, apoptosis experiments. Annexinv-FITC kit to detect apoptotic cells. Using flow cytometry andconfocal immunofluorescence microscopy microscope. LPS1ug/ml andthe LPS10ug/ml apoptosis in human B-lymphoblastoid cell was observed.4,cell proliferation assay. Use Edu proliferation kit, and CCK8 proliferation kit to detect the PBS control group, LPS stimulation group,LPS+Vitamin D3stimulate expression of group. Edu cell theproliferation and cck8microplate proliferation of the OD values are thenobserved under the confocal immunofluorescence microscopy.5, theobservation of human B-lymphoblastoidcells of cell proliferation in thephosphorylation of NF-kB and NF-kB. Edu proliferation kit in confocalimmunofluorescence microscopy observation PBS control group, LPSstimulation group and he of LPS+Vitamin D3stimulate group of cellsproliferation. Western Blot method to detect NF-Kb phosphorylation ofNF-KB and TNF-α protein expression in human B-lymphoblastoid cells,causing oxidative stress, DNA damage, cell proliferation was observedafter PBS control group, stimulation with LPS and LPS+vitamin D3stimulation group, NF-Kb phosphorylation, NF-KB and TNF-α proteinprotein expression. Results:1.Microplate Spectrophotometer(microplatereader)was used to determine the oxidation damage on human Blymphoblasts (Hmy2-cir) which was caused by reactive oxygen species(ROS). The OD value of LPS stimulation group was significantly higherthan the control group of PBS and the vitamin D3group.The OD valueof LPS+vitaminD3stimulation group was significantly lower than thegroup of LPS, and the value of PBS control group was the lowest one.There was a statistically significant difference between the three sets ofpairwise comparisons in each group with the SPSS statistical software for analysis (P <0.05).Under Confocal immunofluorescence microscopy, thefluorescence intensity of LPS stimulation group was obviously higherthan the PBS control group and LPS+vitamin D3stimulation group. Thegroup of LPS-stimulated had the highest fluorescence intensity whichmeans the oxidative damage was the most serious. The fluorescenceintensity of LPS+vitamin D3stimulation group was weaker, and theoxidative damage was lighter.The PBS control group had the weakestfluorescence intensity, and also had no significant oxidative damage.2.Comet assay was found no marked smearing of human B lymphoblastcells in the PBS control group, which was signified no significant injuryon DNA. The group of LPS stimulated cells trailed the longest tail, higherdamage of DNA than the PBS control group and the LPS+vitamin D3stimulation group obviously. Tails of the LPS+vitamin D3stimulationgroup were inconspicuous, which was implied that the DNA damagelightly. There was a statistically significant difference between the threegroups of pairwise comparisons in each group with the SPSS statisticalsoftware for analysi（sP<0.01）.3. Apoptosis experiments: Flow cytometryand Confocal immunofluorescence microscopy was used for detection.The ratio of flow cytometry of the three groups (The PBS group, the1ug/ml LPS stimulation group and the10ug/ml LPS stimulation group)have had no marked difference. Apoptosis rate was analyzed with theSPSS statistical software, a pairwise comparison of each group was implied that there were no significant differences statistically (P>0.05).The apoptotic cells were observed with the Con focalimmunofluorescence microscopy and the SPSS statistical software wasused for analysis of the number of apoptotic cells. No statisticallysignificant differences were shown between the three sets (The PBSgroup, the1ug/ml LPS stimulation group and the10ug/ml LPSstimulation group) of pairwise comparisons in each group （P＞0.05）.4.Cell proliferation assay：Cells of all groups that stained by ShuiboEdu cell proliferation reagent kit were detected by confocalimmunofluorescence microscopy.Edu stained cells observed throughconfocal immunofluorescence microscopy were regarded as proliferatingcells.The amount of proliferatiing cells in the PBS control group was theleast.The amount of Proliferating cells in the LPS group was the most.Afew number of proliferating cells were detected in the LPS+Vitamin D3Group. The proliferating cells were analysed by statisticalmethod.Analysis was performed using software package SPSS. Statisticalsignificance was determined using the t-test and P values(P<0.05).CCK8enzyme standard instrument were used to detect cell OD value.The ODvalue of three group compared to each other with SPSS （P＜0.05).5.Detection of the proliferation of B lymphoblast with phosphoryla-tion of NF-kB and NF-kB.Cells of control group had no obviousproliferation observed through confocal immunofluorescence microscopy. LPS group had obvious proliferation of cells, and the LPS+Vitmin D3had a few of proliferating cells. The number of cell proliferation in threegroups were analyzed by SPSS using t-test (P<0.05).6.Western Blot wasused to detect the expression of protein in human B lymphoblast withphosphorylation of NF-kB, NF-kB and TNF-α protein.It was found thatthe stripe of the PBS coutrol group was the finest,the stripe of the LPSgroup was obviouly thick,and that of LPS+Vitmin D3group wasobviously thinner than that of LPS group.The protein concentration ofthree groups were analyzed by SPSS.The difference was statisticallysignificant.(P<0.05). Conclusion:1Vitamin D3can effectively supressoxidative stress and DNA damage in human B lymphoblast induced byLPS.2LPS cannot induce the apoptosis of human B lymphoblast.3Vitamin D3can effectively supress the proliferation of human Blymphoblast,the phosphorylation of NF-kB and increased expression ofNF-kB.4Vitamin D3can effectively supress the phosphorylation of NF-kB,the expression of TNF-a protein and NF-kB... |
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